Function and Regulation of PARP13 Binding to
ARCHE8
Cellular RNA
MASSACHUSETTS INSTITUTE
OF TECHNOLOLGY
By
Tanya Todorova
B.A., Biology
Bowdoin College, 2009
DEC 2 2 2014
LIBRARIES
Submitted to the Department of Biology in Partial Fulfillment of the Requirements
for the Degree of
Doctor of Philosophy
At the
Massachusetts Institute of Technology
February 2015
Massachusetts Institute of Technology, 2015. All rights reserved.
Signature redacted
Signature of Author:............................................
...
Signature redacted
Certified By:..
. Tanya Todorova
Department of Biology
December 15, 2014
.................................
Paul Chang
Assistant Professor of Biology
Thesis Supervisor
Signature redacted
......................................
Michael Hemann
Professor of Biology
Graduate Studies
for
Committee
Chair,
Accepted By:......
1
Table of Contents
Abstract......................................................................................................................................
5
6
C hapter 1. Introd uctio n ....................................................................................................
PA R P B ackground...........................................................................................................................7
PA R Ps and R NA regulation......................................................................................................................9
PA R P 13 - a catalytically inactive R NA -binding PA R P........................................................ 13
Functions of PA R P13 ..................................................................................................................
14
14
A ntiviral functions ........................................................................................................................................
Physiological functions.............................................................................................................................19
PA R P 1 3 regulates the m iR NA silencing pathw ay........................................................... 19
PA R P 13 localization to R NA granules............................................................................... 22
PA R P 13 dom ain structure....................................................................................................
23
23
C C C H -type zinc finger dom ains ..........................................................................................................
25
W W E dom ain ................................................................................................................................................
PA R P dom ain................................................................................................................................................27
28
Low com plexity region ..............................................................................................................................
N uclear-localization and export signals..................................................................................... 28
29
R egulation of PA R P13 activity ...........................................................................................
PA R P 13 is an interferon-stim ulated gene............................................................................... 29
31
Posttranslational regulation of PA R P 1 3.....................................................................................
31
.................................................................................................
R egulation by phosphorylation
31
.................................................
brane
targeting
and
m
em
R egulation by farnesylation
32
........................................
binding
and
ADP-ribose
by
ADP-ribosylation
Regulation
34
C onclusions ....................................................................................................................................
Figures and tables........................................................................................................................
35
R eferences.......................................................................................................................................
40
Chapter 2: PARP13 regulates cellular mRNA posttranscriptionally and
functions as a pro-apoptotic factor by destabilizing TRAILR4 transcript....46
47
A bstract.............................................................................................................................................
47
Introduction .....................................................................................................................................
51
R esults ..............................................................................................................................................
64
D iscussion .......................................................................................................................................
67
M aterials and m ethods ...............................................................................................................
78
Figures and tables........................................................................................................................
S upplem entary figures and tables...................................................................................... 96
S upplem entary data...................................................................................................................105
R eferences.....................................................................................................................................118
Chapter 3. Possible Mechanisms of PARP13 Regulation ................................ 122
A bstract...........................................................................................................................................123
Introduction...................................................................................................................................124
128
R esults ............................................................................................................................................
137
D iscussion .....................................................................................................................................
M aterial and m ethods................................................................................................................139
145
Figures ............................................................................................................................................
R eferences.....................................................................................................................................154
Chapter 4. Conclusions and future directions...................................................... 157
Identifying more endogenous targets of PARP13.........................................................157
2
159
W hat determ ines PARP13 target specificity? .................................................................
Mechanism s of PARP13 regulation.....................................................................................162
PARP13 in hum an health and disease...........................................................................164
References.....................................................................................................................................165
Acknow ledgem ents..........................................................................................................
168
Curriculum Vitae................................................................................................................
169
Appendix 1. Poly(ADP-ribose) regulates post-transcriptional gene
regulation in the cytoplasm ..........................................................................................
171
3
4
Function and Regulation of PARP13 Binding to Cellular RNA
by Tanya Todorova
Submitted to the Department of Biology on December 15, 2014 in
Partial Fulfillment of the Requirements for the Degree of
Doctor of Philosophy in Cell Biology
Abstract
Poly(ADP-ribose) polymerase-1 3 (PARP1 3) is a member of the PARP
family of proteins - enzymes that use NAD+ to synthesize a posttranslational
protein modification called poly(ADP-ribose) (PAR). PARPs function in multiple
cellular pathways, and recently several members of the family have been
implicated in regulating various steps in RNA metabolism, from splicing to
translation and decay. PARP1 3 is the best-understood RNA-regulatory PARP.
Initially discovered as a host immune factor, PARP13 functions by binding viral
transcripts via its four CCCH-type zinc fingers and targeting them for degradation.
In the context of the immune response PARP1 3 can also inhibit the translation of
its viral targets and enhance the activity of other RNA-binding viral receptors,
such as RIG-1.
More recently PARP13 was shown to also indirectly regulate the cellular
transcriptome by inhibiting the activity of Argonaute 2 (Ago2), a member of the
miRNA silencing pathway. While itself catalytically inactive, PARP13 is modified
by PAR and can target Ago2 for modification by a yet unknown PARP. However,
it remains unclear if RNA binding is required for this function of PARP1 3. Indeed,
even though multiple viruses are known to be restricted by PARP13, cellular
mRNA targets of PARP13 binding and regulation have not yet been identified.
Here we show that PARP1 3 binds endogenous RNA and regulates the
cellular transcriptome. We identify TRAILR4 mRNA as the first cellular target of
PARP13 regulation and demonstrate that PARP13 represses TRAILR4
expression posttranscriptionally by binding to a specific region in the 3'
untranslated region of the transcript and targeting it for degradation in a primarily
3'-5' decay mechanism. By inhibiting the expression of TRAILR4 - a decoy prosurvival receptor of the apoptotic ligand TRAIL, PARP1 3 regulates the cellular
response to TRAIL and acts as a pro-apoptotic factor. We also examine possible
mechanisms of regulation of PARP1 3 function. We identify the RNA-helicase
DHX30 as a constitutive PARP1 3-interacting protein and show that the two
proteins co-regulate a subset of cellular transcripts. We further demonstrate that
the PAR-binding domain of PARP1 3 inhibits RNA binding, while PARP1 3
interaction with PARP5a and covalent modification with PAR appear to be
mutually exclusive with RNA binding.
Thesis Supervisor: Dr. Paul Chang
Title: Assistant Professor of Biology
5
Chapter 1. Introduction
To be submitted with some modifications for publication in the journal Trends in
Molecular Medicine under the preliminary title:
PARP13 and RNA stability/turnover in immune system and cancer
Tanya Todorova' and Paul Chang'
2
'Koch Institute for Integrative Cancer Research, 2Department of Biology,
Massachusetts Institute of Technology, Cambridge MA 02139
6
PARP Background
Poly(ADP-ribose) Polymerase-1 3 (PARP1 3), also known as Zinc Finger Antiviral
Protein (ZAP) and ZC3HAV1, is a member of the poly(ADP-ribose) polymerase
(PARP) family of proteins (Ame et al., 2004; Vyas et al., 2013). PARPs are
enzymes that use NAD+ to synthesize a posttranslational protein modification
called ADP-ribose (ADPr) onto target proteins (Ame et al., 2004; Gibson and
Kraus, 2012; Vyas et al., 2013). The addition of a single ADPr is referred to as
mono(ADP-ribos)ylation while poly(ADP-ribos)ylation constitutes the addition of
multiple ADPr moieties in linear or branched chains that can reach up to 200
subunits in length. Both poly(ADP-ribose) (PAR) and mono(ADP-ribose) (MAR)
are reversible modifications: PAR polymers can be hydrolyzed by an enzyme
called poly(ADP-ribose) glycohydrolase (PARG) while MAR can be removed by
the recently identified MacroD1, MacroD2 and terminal ADP-ribose
glycohydrolase (TARG) enzymes (Dunstan et al., 2012; Lin et al., 1997;
Rosenthal et al., 2013; Vyas et al., 2014). ADP-ribose is therefore a very
dynamic modification and changes in expression, activity and localization of
PARPs, PARG, MacroDl/2 and TARG enzymes can rapidly modulate its
amounts in different cellular compartments.
Mono(ADP-ribos)ylation and poly(ADP-ribos)ylation can both function as
traditional posttranslational modifications as they can alter the folding and
structure of their protein targets and thus affect their activity, localization, binding
to other proteins, stability, etc. However, due to its unique size, strong negative
7
charge and complex structure, PAR can also function by recruiting PAR-binding
proteins to the site of its synthesis and thus facilitate the formation of protein
complexes and large macromolecular structures in the cell (Gibson and Kraus,
2012; Leung, 2014) (Fig. 1). Proteins can interact with poly(ADP-ribose) via one
of four known PAR-binding domains -PAR-binding zinc finger (PBZ), Trp-Trp-Glu
(WWE) and macro domains, and the PAR-binding motif (PBM) (Kalisch et al.,
2012; Zaja et al., 2012). More than 800 proteins contain one or more of these
domains (Vyas and Chang, 2014).
All PARPs share the conserved PARP domain, which defines them as members
of the family, but a wide array of additional domains allows them to function in
multiple pathways and to influence a plethora of cellular processes. However, a
common emerging theme that ties these proteins together is the essential roles
they tend to play in the response to various cell stresses, including DNA damage,
heat shock, cytoplasmic stress, viral infections and unfolded protein stress (Di
Giammartino et al., 2013; Gao et al., 2002; Jwa and Chang, 2012; Leung et al.,
2011; Vyas and Chang, 2014). ADP-ribose, therefore, appears to be an
important signaling component in initiating and maintaining stress responses.
Indeed, for some PARPs all known functions occur in response to pathological
conditions, and it remains to be seen if their roles are strictly limited to these
contexts, or if their hyperactivation during stress results in exaggerated outcomes
of normal physiological functions.
8
PARPs and RNA regulation
For a long time PARP1 was the only known member of the PARP family and its
production of poly(ADP-ribose) in the nucleus in response to single-strand DNA
damage remains the best-understood function for PAR in the cell (Ame et al.,
2004; Javle and Curtin, 2011; Krishnakumar and Kraus, 2010). PAR facilitates
DNA repair either by decreasing affinity of modified proteins for DNA thus helping
relax the chromatin (as in the case of histones), or by recruiting PAR-binding
DNA repair factors to the site of damage and facilitating their interaction with the
now accessible DNA (Kraus and Hottiger, 2013; Krishnakumar and Kraus, 2010;
Luo and Kraus, 2012). Thus PAR and the nuclear PARPs that produce it, mainly
PARP1 and 2, regulate DNA-binding proteins on multiple levels and this remains
the best-understood function for the PARP family of proteins.
However, in fact, more PARPs have RNA-binding domains than DNA-binding
domains: PARP7, 12 and 13 all possess one or more CCCH-type RNA-binding
zinc fingers, and are classified as members of the CCCH-PARPs subfamily,
while PARP1 0 contains an RNA-recognition motif (RRM); in contrast, only
PARP1 and PARP2 have a defined DNA-binding domain (Ame et al., 2004; Vyas
et al., 2013). This observation suggests that beyond their role in the regulation of
DNA, PARP family members may have important functions in regulating RNA
metabolism.
9
RNA-binding PARP12 and PARP13, as well as PARPs without known RNAbinding domains including PARP5a, 14 and 15, localize to large cytoplasmic
ribonucleoprotein structures named stress granules (Leung et al., 2011). Stress
granules form in response to translation inhibition triggered by various conditions,
including oxidative stress, hypoxia and heat shock (Kedersha and Anderson,
2007). They represent sites where translation-preinitiation complexes, mRNAs
and RNA-regulatory proteins are sequestered to ensure both that mRNAs that do
not code for stress-response factors are not translated, that they are protected
for the duration of the stress, and that translation can rapidly resume after the
stress is relieved (Anderson and Kedersha, 2008; Kedersha and Anderson,
2007). Poly(ADP-ribose) is essential for the formation and maintenance of stress
granules where it is thought to act as a scaffold recruiting different components
and keeping them together in a large complex structure (Fig. 1) (Leung et al.,
2012; Leung, 2014; Leung et al., 2011). PARP5a and 5b are the only PARPs
capable of poly(ADP-ribose) synthesis in the cytoplasm (Vyas et al., 2014), and
PARP5a localization to stress granules suggests that it may function in
cytoplasmic RNA regulation in a manner similar to the role of PARP1 in nuclear
DNA regulation. PARP5a-mediated modification of RNA-binding proteins with
PAR may change their affinity for RNA targets just as modification of histones by
PARP1 decreases their affinity for DNA. Conversely, stress-induced localized
synthesis of PAR may help recruit RNA-regulatory factors and facilitate their
interaction with target RNAs, similar to PARP1-dependent recruitment of DNArepair factors to sites of DNA damage.
10
In addition to facilitating stress granule assembly, PARP12 acts as an antiviral
factor during infection with certain RNA viruses, such as members of the
alphaviridae family, when it is thought to inhibit the translation of viral RNA
transcripts (Atasheva et al., 2012; Welsby et al., 2014). In addition, PARP12
overexpression leads to global inhibition of cellular mRNA translation, suggesting
that PARP12 may also regulate endogenous transcripts (Atasheva et al., 2014).
Both RNA binding and MAR enzymatic activity are required for PARP12mediated translational repression. Overexpression of PARP7 and PARP10
appears to have a similar inhibitory effect on translation (Atasheva et al., 2014).
However, it is important to note that overexpression of many of these PARPs
results in cellular stress, as evidenced by the formation of stress granules.
Therefore, it remains unclear if the observed effects RNA-binding PARPs have
on translation are physiological functions or whether they represent a stressinduced activity.
PARP14 is a mono(ADP-ribose)-producing PARP that does not have an RNAbinding domain but localizes to stress granules, presumably by binding to PAR
via its three macro domains, suggesting it too may be involved in RNA regulation
(Leung et al., 2011). Indeed, a recent report implicates PARP14 in the
posttranscriptional regulation of tissue factor (TF) in macrophages (lqbal et al.,
2014). PARP14 forms a complex with the RNA-regulatory protein tristetraprolin
(TTP) and assists it in destabilizing TF mRNA specifically, without having an
11
effect on other TTP targets. While the mechanism of this regulation is currently
unclear, an attractive hypothesis is that ADP-ribosylation of TTP by PARP14
changes its affinity for TF mRNA and facilitates binding and subsequent
destabilization.
Finally even the canonical DNA-dependent PARPs, PARP1 and PARP2, appear
to play a role in RNA regulation. For example, during heat shock PARP1 modifies
poly(A) polymerase (PAP), a process that leads to PAP disassociation from
transcripts and inhibition of polyadenylation (Di Giammartino et al., 2013). This
deficient 3' processing results in abrogation of mRNA export to the cytoplasm
and its accumulation in the nucleus - a response thought to limit translation
during stress. Thus, PARP1 has important global effects on posttranscriptional
mRNA processing. On the other hand, PARP2 enzymatic activity was recently
reported to be activated more efficiently by RNA than by DNA during certain
stresses (Leger et al., 2014). It appears that the DNA binding domain of PARP2
recognizes RNA as well. PARP2 may therefore be a sensor of stress-induced
RNA accumulation in the nucleus.
Members of the PARP family of proteins are emerging as important regulators of
RNA metabolism on multiple levels. Given the observation that the activity of
many PARPs is induced by various stresses, it is tantalizing to hypothesize that
PARPs and their enzymatic products PAR and MAR have evolved ways to switch
patterns in posttranscriptional RNA regulation, such as splicing, translation and
12
decay, to quickly modulate gene expression in response to adverse conditions.
Understanding how these proteins control RNA metabolism, how this regulation
changes upon stress signaling, and what RNA targets are affected by their
activity is an essential question in both the RNA and the PARP field.
PARP13 - a catalytically inactive RNA-binding PARP
PARP1 3, also known as Zinc-finger Antiviral Protein (ZAP) and ZC3HAV1, is
unique among the PARPs as it is catalytically inactive, capable of producing
neither PAR nor MAR, but is modified by PAR and can bind PAR (Leung et al.,
2011; Vyas et al., 2014). It is an RNA-binding protein containing four tandem
CCCH-type zinc fingers in its N-terminal domain (Fig. 2). It has two major
isoforms, resulting from alternative splicing - the full-length PARP1 3.1 and
PARP13.2 which lacks the PARP domain (Fig. 2) (Vyas et al., 2013). PARP13 is
thought to have arisen from a duplication of the PARP1 2 gene, its closest relative
(Perina et al., 2014). This event likely allowed PARP13 to diversify its RNAregulatory functions while losing its catalytic activity. Initially discovered as a host
antiviral factor active against murine leukemia virus (MLV), PARP13 has
emerged as an important regulator of exogenous RNAs in the cell during
infections with various viruses primarily through its ability to bind to and
destabilize target transcripts (Bick et al., 2003; Gao et al., 2002; Guo et al., 2007;
Mao et al., 2013). More recent data have also demonstrated that PARP13
functions in the regulation of cellular RNA by controlling the activity of the RNA-
13
regulatory protein Ago2, greatly expanding the potential roles of the PARP family
in RNA metabolism (Leung et al., 2011).
Functions of PARP13
Antiviral functions
PARP1 3 was initially discovered in a screen for host factors that restrict the
retroviral murine leukemia virus (MLV) and was therefore named Zinc Finger
Antiviral Protein (ZAP) (Gao et al., 2002). PARP13 had no effect on viral entry,
reverse transcription or nuclear entry, however it strongly inhibited viral gene
expression and resulted in a decrease in cytoplasmic viral RNA. This, along with
the observation that PARP1 3 had four tandem RNA-binding CCCH-type zinc
fingers, lead to the hypothesis that it functions by binding directly to viral RNA
transcripts in the cytoplasm and leading to their degradation.
The PARP13 function as a host immune factor, first described against MLV, was
later expanded to other retroviruses (Human Immunodeficiency Virus, HIV) (Zhu
et al., 2011) as well as other viral families such as the alphaviruses (Sindbis
Virus, SINV; Semliki Forest Virus, SFV; Ross River Virus, RRV; Venezuelan
Equine Encephalitus Virus, VEEV) (Bick et al., 2003), filoviruses (Ebola Virus,
EBOV; Marburg Virus, MARV) (Muller et al., 2007), herpesviruses (murine
gammaherpesvirus 68, MHV-68) (Xuan et al., 2012) and hepadnaviruses
(Hepatitis B Virus, HBV) (Mao et al., 2013). In each of these cases the activity of
PARP1 3 is thought to be dependent on direct recognition and binding to specific
14
regions of the viral RNA; PARP1 3 is not thought to induce a general antiviral
state, since other viruses, such as the vesicular stimatis virus, the yellow fever
virus, the poliovirus and herpes simplex virus, replicate efficiently in the presence
of PARP13 (Bick et al., 2003). Therefore PARP13 only restricts specific viruses,
presumably by recognizing features in their RNA not present in the viruses that
remain unaffected by PARP13.
Identifying common features among the viral targets of PARP1 3, however, has
remained elusive. Fragment analysis showed that PARP1 3 binds the 3' long
terminal repeat of MLV, the terminal redundancy sequences in HBV, the gene
coding for the filovirus L protein in EBOV, the 5'UTR of multiply spliced HIV and
multiple fragments in the SINV genome, but no sequence similarity among these
regions has been identified (Guo et al., 2004; Mao et al., 2013; Muller et al.,
2007; Zhu et al., 2011). In addition further fragmentation of the PARP13-sensitive
regions of these viruses rendered them resistant to PARP1 3 repression; the
smallest fragment that still remained sensitive to PARP13 in SINV is longer than
500nt (Guo et al., 2004). It has therefore been hypothesized that PARP13
recognition of its viral RNA targets is mediated by RNA secondary and tertiary
structures rather than linear motifs. This idea was supported upon solving the
structure of the N-terminal RNA-binding domain of PARP13, which showed a
large RNA-protein interaction surface with multiple cavities and modeled binding
to two anti-parallel RNA strands, suggesting that the protein may recognize
looped RNA (Chen et al., 2012). Consistent with this hypothesis, a systematic
15
evolution of ligands by exponential enrichment (SELEX) approach identified
aptamers enriched for binding to PARP1 3 (GGGUGG and GAGGG) but those
failed to confer sensitivity to PARP13 when cloned into reporter constructs, and
therefore are not likely to be functional PARP13-recognition motifs (Huang et al.,
2010). Consequently, identifying novel targets of PARP13 remains an empirical
process, with no predictive algorithm developed as of yet.
The initial observation that PARP13 results in decreased cytoplasmic viral RNA
levels and the fact that PARP13 does not possess exo- or endo-nuclease activity,
suggested that PARP13 may function by recruiting cytoplasmic RNA-decay
factors to the viral transcripts it binds to and thus lead to their degradation.
mRNA decay in the cytoplasm is usually initiated by removal of the 3' poly-A tail
by deadenylases such as CCR4-NOT, PAN and PARN, and is then proceeded
by removal of the 5' cap structure by decapping enzymes DCP1 and DCP2 (Fig.
3) (Garneau et al., 2007; Schoenberg and Maquat, 2012). Next, the 5'-3'
exonuclease XRN1 or the 3'-5' exonuclease complex, the RNA exosome,
degrade the transcript (Fig. 3). RNA-binding proteins interacting with ciselements found in mRNAs can facilitate or prevent the binding of these factors,
thus leading to the degradation or stabilization of their targets. Different mRNAdestabilizing factors may specifically recruit some of these factors and not others.
For example, tristetraprolin (TTP), a cellular mRNA-destabilizing factor that
possesses a similar CCCH-zinc-finger RNA-binding domain as the one found in
PARP13, recognizes AU-rich elements in the 3'UTRs of cytoplasmic transcripts
16
and leads to their degradation via a primarily 3'-5'-decay mechanism dependent
on recruitment of the exosome complex (Barreau et al., 2005).
Similarly to TTP, PARP13 also recruits 3'-5'-decay factors to degrade its viral
targets. PARP1 3 interacts directly with two components of the exosome complex,
RRP46/EXOSC5 and RRP42/EXOSC7, and with the poly(A)-specific
ribonuclease PARN (Fig. 3) (Guo et al., 2007; Zhu et al., 2011). Consistent with
this observation, depletion of these factors by RNAi results in inefficient
restriction of viral infections by PARP13, suggesting that they are required for
PARP13 antiviral function. On the contrary, the 5'-3'-decay factors XRN1, DCP1
and DCP2 only co-immunoprecipitate with PARP1 3 under RNAse-free conditions
but not when RNA is degraded, suggesting that while they may be bound to the
same RNA transcripts, they do not undergo direct protein-protein interactions.
However, PARP13 can recruit 5'-3'-decay factors indirectly by first recruiting the
DEAD-box RNA-helicase DDX17 which then binds to DCP2 and XRN1 (Zhu et
al., 2011). Thus, depletion of XRN1 and DCP2 also decreases the efficiency of
PARP13-dependent viral repression, albeit to a lesser degree compared to
depletion of components of the 3'-5' degradation pathway. PARP13 interaction
with another helicase, DHX30, also appears to be important for its function in the
antiviral response (Ye et al., 2010). Although the relevance of this interaction
remains unclear, it is likely that certain helicases help targets assume a fold that
is most efficient for PARP13 binding and thus help recruit the protein to its
cognate RNAs.
17
While degradation of viral RNA constitutes the best-understood function of
PARP13, its role in the antiviral response is not limited to this activity. Early
reports suggested that PARP1 3 may inhibit the translation of SINV RNA, but it
was unclear if this is a direct consequence of decreased RNA amounts or a
separate mechanism (Bick et al., 2003). A later report examined this possibility in
greater detail, demonstrating that PARP13 results in the exclusion of its HIV
target RNA from actively translating polysomes and therefore inhibits its
translation (Zhu et al., 2012). PARP13 achieves this by binding directly to the
translation-initiation factor eIF4A, and preventing it from interacting with another
initiation factor - elF4G. PARP13-dependent translation inhibition is independent
of RNA decay, and may precede recruitment of RNA-decay factors by PARP1 3.
It remains unclear if this is an HIV-specific mechanism, or if translational
inhibition is a common step in PARP1 3-dependent restriction of other viruses.
Another function of PARP1 3 during the antiviral response is to synergize with
and enhance the activity of other host factors. For example the shorter isoform of
PARP13, PARP13.2, enhances signaling by RIG-1, a key host pattern-recognition
receptor that recognizes non-self RNA in the cytoplasm and triggers downstream
antiviral signaling (Hayakawa et al., 2011). PARP13.2 binds the carboxy-terminal
region of RIG-1 and facilitates its oligomerization, a process essential for RIG-I
activity. In this way PARP13.2 enhances RIG-1-mediated initiation of the type I
interferon response, as well as NF-KB and IRF3 signaling. It remains unclear if
18
binding to RNA is required for this function of PARP1 3 or if it presents a
completely separate activity of the protein. Regardless, it is clear that PARP1 3
functions at multiple steps during the antiviral response, directly inhibiting viral
RNA replication and translation, and enhancing the signaling pathways that
establish an antiviral state and promote the immune response.
Physiological functions
While the majority of what we know about PARP1 3 is based on its functions in
the antiviral response, recent research has started to shed light on novel
physiological functions of the protein in the absence of viral infection. These new
data suggest that PARP1 3 is a protein of diverse functions affecting multiple
pathways in the cell.
PARP13 regulates the miRNA silencing pathway
The first described immune-response-independent function of PARP1 3 was its
regulation of Ago2, a key component in the miRNA pathway. In a process
broadly referred to as RNA interference (RNAi), the RNA-induced silencing
complex (RISC) is loaded with small RNA species known as miRNAs that can
recognize specific mRNA molecules through base-pairing with sequences in their
3'UTRs (Bartel, 2009) (Meister, 2013). miRNA-mediated binding of RISC to
target mRNAs results in their silencing through translational inhibition or
destabilization of the transcript. In cases of perfect base-pairing between the
miRNA and the target mRNA, Argonaute 2 (Ago2), the enzymatically active
19
member of the complex, can also act as an endonuclease, cleaving the transcript
directly and causing its degradation. miRNA-mediated silencing is an important
mechanism of posttranscriptional RNA regulation and is essential for optimizing
gene expression programs during development and differentiation (Huang et al.,
2011). It is therefore a tightly regulated process that can be modulated by
multiple mechanisms, including posttranslational modifications of Ago2 (Meister,
2013).
Modification of Ago2 with poly(ADP-ribose) by a yet unknown member of the
PARP family results in the inhibition of its activity and repression of miRNA
silencing (Leung et al., 2011). Such general relief of silencing occurs during
oxidative cytoplasmic stress when cellular PARP activity and PAR modification of
Ago2 increase dramatically. It is unclear why RNA silencing is globally inhibited
during the stress response but it may be a mechanism to allow for the
stabilization of transcripts coding for stress-response factors, which may be
targets of silencing and decay under physiological, non-stress conditions.
Interestingly, while PARP13 is enzymatically inactive, it is essential for targeting
Ago2 for ADP-ribosylation through a currently unknown mechanism (Leung et al.,
2011). Depletion of PARP1 3 inhibits Ago2 modification with poly(ADP-ribose)
and prevents the global repression of miRNA silencing during stress. Conversely,
overexpression of PARP13 results in increased Ago2 PARylation and a decrease
in its activity. Therefore, by regulating Ago2 function, PARP13 emerges as a
20
general regulator of posttranscriptional RNA regulation and may have global
effects on the cellular transcriptome.
The role of PARP1 3 in Ago2 repression, initially identified in the response to
-
oxidative stress, also appears to be relevant for the response to viral infections
a context in which PARP13 seems to function on multiple levels (Seo et al.,
2013). Many immune-response genes, including the interferon-stimulated genes,
produce unstable transcripts with short half-lives, quickly silenced and degraded
in the absence of infection by various mechanisms including the miRNA pathway
(Seo et al., 2013). In this way, the cell ensures the presence of a constant pool of
immune-factor transcripts while preventing the futile synthesis of antiviral proteins
when they are not needed (Seo et al., 2013). Furthermore, many antiviral
transcripts can also be cytotoxic, so miRNA-mediated degradation adds an
additional layer of security by limiting their expression. During infection the
miRNA pathway is globally repressed, resulting in the stabilization of immuneresponse mRNAs and the rapid synthesis of antiviral proteins (Seo et al., 2013).
PARP1 3-dependent targeting of Ago2 for ADP-ribosylation is an essential step in
this process, and ensures proper response to the viral infection.
PARP13 regulation of the miRNA pathway is a constitutive function of PARP13
that is hyperactivated in pathological contexts such as during infection or under
oxidative stress (Leung et al., 2011; Seo et al., 2013). Consequently, it is
possible that other PARP13 functions, previously thought to occur exclusively
21
during the immune response, such as RNA binding, targeting RNAs for decay
and translation inhibition, and facilitating receptor signaling (e.g. RIG-1), are
extensions of normal, albeit currently unknown, physiological functions of the
protein. Indeed, indirect evidence, discussed below, already suggests that
PARP13 binds endogenous RNA.
PARP13 localization to RNA granules
Strong, indirect evidence that PARP13 interacts with cellular RNA is its
localization to stress granules - large ribonucleoprotein structures that form as a
consequence of the regulated aggregation of multiple cellular mRNAs, the 40S
ribosomal subunit, translation initiation factors and RNA-binding proteins, in
response to global repression of translation triggered by phosphorylation of
elF2a (Anderson and Kedersha, 2008; Kedersha and Anderson, 2007). PARP13
localizes to stress granules with kinetics similar to G3BP - an RNA-regulatory
protein used as a stress-granule marker (Lee et al., 2013b; Leung et al., 2011).
PARP1 3 is also poly(ADP-ribos)ylated during stress, similarly to TIA1, G3BP and
Ago2 - RNA-binding proteins found in stress granules, although the PARPs
responsible for these modifications remain unknown (Leung et al., 2011).
Therefore, even though binding of PARP1 3 to cellular RNA has not been directly
addressed or demonstrated, at least during stress conditions PARP13 behaves
very similarly to known cellular-RNA-regulatory proteins and it is thus plausible to
hypothesize that PARP1 3 is itself a cellular-RNA-binding protein. PARP1 3 also
appears in a proteome-wide screen for TDP-43-interacting proteins (Freibaum et
22
al., 2010). Aggregation of TDP-43, an RNA-binding protein, to neuronal RNA
granules is a hallmark of Amyotrophic Lateral Sclerosis (ALS) pathology (LagierTourenne and Cleveland, 2009). PARP13 interaction with TDP-43 under
physiological conditions suggests that these two proteins may function in similar
nodes of cellular RNA metabolism. TDP-43 has been shown to destabilize its
own mRNA in an exosome-dependent manner, a mechanism very similar to that
of viral RNA decay by PARP1 3 (Ayala et al., 2011).
PARP13 domain structure
CCCH-type zinc finger domains
PARP1 3 is a member of the CCCH PARP subfamily, which also includes
PARP12 and PARP7. These proteins share the presence of one or more CCCHtype Zinc Finger domains - RNA-binding modules characterized by the presence
of three cysteine and one histidine amino acid residues, usually forming a CysX 8-Cys-X 5 -Cys-X 3-H sequence (Hall, 2005). CCCH-type zinc finger domains are
found in a number of RNA-binding proteins with diverse functions. The first
CCCH zinc finger was identified in tristetraprolin (TTP) - a protein that
recognizes and binds to AU-rich elements (AREs) found in the 3' untranslated
regions (3'UTRs) of mRNAs (Barreau et al., 2005; Brooks and Blackshear, 2013;
Sanduja et al., 2012). Binding of TTP to AREs leads to the subsequent
degradation of the target RNA, resulting in decreased mRNA half-life and
increased turnover. Well-known mRNA targets of TTP are TNFaC and GM-CSF,
short-lived transcripts that code for cytokines involved in cell growth,
23
differentiation and inflammation (Brooks and Blackshear, 2013; Sanduja et al.,
2012). TTP-null mice suffer from weight loss, arthritis and autoimmunity, thought
to be the result of chronic inflammation due to the increased stability of TNFa
and GM-CSF RNAs in the absence of TTP. This outcome exemplified the
physiological relevance of posttranscriptional gene regulation by RNA decay and
garnered interest in CCCH zinc finger RNA regulation and in characterizing more
RNA-binding proteins containing these domains.
Currently there are 62 human proteins annotated as containing a CCCH-type
zinc finger. They regulate a wide variety of RNA-regulatory processes such as
splicing (MBNL1, U2AF1), deadenylation (CNOT4, PAN3, ZC3H14), RNA decay
(TTP) and viral restriction (MCPIP) (Brooks and Blackshear, 2013; FernandezCosta et al., 2011; Lin et al., 2013; Schoenberg and Maquat, 2012; Soucek et al.,
2012; Wang et al., 2012a). Many of these proteins recognize AREs or poly(A)
stretches and many contain tandem zinc fingers, which, at least in the case of
MBNL1, create increased protein-RNA interaction surface and allow for the
recognition of tri-dimensional RNA fold structures (Chen et al., 2012). Tandem
CCCH zinc finger domains, such as those found in PARP1 3 (Fig. 2), therefore,
present a versatile and multi-faceted RNA-binding module that helps recognize
complex RNA structures.
Indeed, the crystal structure of the N-terminal RNA-binding domain of PARP13,
which contains four CCCH zinc fingers, two more than MBLN1, provided a
24
powerful insight into the mechanism of RNA recognition by PARP13 (Chen et al.,
2012). The four zinc fingers form a large area for RNA interaction defined by the
presence of multiple positively charged residues, thought to facilitate non-specific
interactions with negatively charged RNA, and of two cavities, thought to provide
specificity for target recognition. Binding of target RNA was modeled onto the
PARP13 structure based on its similarities to the structure of MBNL1 and
revealed binding of two anti-parallel RNA chains, suggesting that PARP13, like
MBNL1, binds looped RNA. However, since PARP13 possesses two more zinc
fingers compared to MBNL1, it likely recognizes RNA via a somewhat more
complex mechanism. It is also likely that specific RNA targets bind to PARP1 3 in
different ways with the two cavities contributing to various extents in every case,
or that posttranslational modifications of PARP1 3 give priority to one or the other
feature of the RNA-binding domain.
WWE domain
PARP13, along with the other CCCH PARPs, also contains a WWE domain (Fig.
2). Until recently, little was known about this domain, found primarily in PARPs
and in proteins involved in ubiquitination and protein degradation. However,
recent data identified the WWE domain as an ADP-ribose-binding module,
functioning similarly to the Macro, PBZ and PBM ADP-ribose-binding domains
(He et al., 2012; Wang et al., 2012b). The presence of an ADPr-binding domain
in PARP1 3 gives rise to the intriguing possibility that ADPr binding regulates
PARP13 function, especially its affinity for RNA or specificity for certain RNA
25
targets. ADPr regulation of RNA-binding proteins has a long history. As early as
the 1980s, poly(ADP-ribose) was shown to be associated with free
ribonucleoprotein complexes in the cytoplasm that are enriched in RNAregulatory and decay factors, suggesting that it may have a functional role in
RNA-regulatory processes (Elkaim et al., 1983; Thomassin et al., 1985). hnRNP
proteins, well known splicing regulators, were also shown to be major of
poly(ADP-ribose) binding proteins: poly(ADP-ribose) binding decreases their
RNA binding affinity and inhibits their function (Ji and Tulin, 2013; Kostka and
Schweiger, 1982). More recently Ago2, a mediator of miRNA silencing, as well as
G3BP and TIA1, RNA-regulatory factors, were identified as targets of poly(ADPribos)ylation, with ADP-ribose affecting their function and localization (Isabelle et
al., 2012; Lee et al., 2013a; Leung et al., 2011). Beyond these specific examples,
proteome-wide mass-spectrometry screens for ADPr-associated proteins have
clearly demonstrated a strong enrichment for mRNA-metabolism factors,
indicating that interaction with or modification by poly(ADP-ribose) is common for
RNA-binding proteins and may be a major mechanism of regulation of their
function (Gagne et al., 2008; Jungmichel et al., 2013). PARP13 has already been
shown to be modified with poly(ADP-ribose) and the presence of an ADPrbinding domain strongly suggests that ADPr is an important regulator of its
activity and function in the cell (Leung et al., 2011).
26
PARP domain
The last functional module of PARP13 is the C-terminal PARP domain, present
only in the longer PARP13 isoform - PARP13.1, and removed by splicing in the
other major PARP13 isoform, PARP13.2 (Fig. 2) (Vyas et al., 2013). The PARP
domain of PARP1 3.1 is catalytically inactive due to substitutions of two of the
three amino acid residues that constitute the HYE motif required for PARP
enzymatic activity - PARP1 3 instead contains a YYV motif. While the PARP
domain of PARP13.1 cannot produce mono or poly(ADP-ribose), it appears to be
functionally relevant. The PARP domain, but not the other domains of PARP13.1,
has experienced positive selection, suggesting it may have evolved under an
evolutionary pressure from host-pathogen conflict (Kerns et al., 2008). In addition,
a polymorphism in the PARP domain (Thr851 lie), conserved in humans and
chimpanzees, and maintained under balancing selection, is associated with
susceptibility to multiple sclerosis (Cagliani et al., 2012). Finally, a CaaX motif in
the PARP domains targets PARP1 3.1 for farnesylation - a lipid modification that
increases protein affinity for certain cellular membranes and results in
membranous localization of PARP13.1 (Charron et al., 2013). The PARP domain
may therefore be important for conferring differential localization and proteinbinding partners to PARP13.1 compared to PARP1 3.2, resulting in functional
differences between the two isoforms.
27
Low complexity region
The region between the N-terminal RNA-binding domain of PARP13 and its
WWE domain is a disordered low-complexity region (Fig. 2). Low complexity
regions have recently been identified as important features of RNA-binding
proteins that allow them to reversibly transition from soluble state to insoluble
amyloid-like fibers (Han et al., 2012; Kato et al., 2012; Leung, 2014). It is thought
that this is one mechanism by which RNA-regulatory proteins can assemble
themselves into functional non-membrane-bound structures such as cytoplasmic
RNA granules (e.g. stress granules and neuronal granules), p-bodies, Cajal
bodies, nuclear speckles, etc. These macromolecular complexes allow for certain
RNA-regulatory processes to occur in specialized and dedicated, albeit very
dynamic and reversible, subcellular compartments - for example RNA decay in
p-bodies. The presence of a low complexity region in PARP1 3 is consistent with
its localization to stress granules, and may be relevant to its proper function in
the cell.
Nuclear-localization and export signals
PARP13 harbors a nuclear-localization signal (NLS) in its N-terminus and a
nuclear-export signal within the low complexity region (Fig. 2) (Liu et al., 2004).
Therefore, even though at steady state PARP13 exhibits a cytoplasmic
localization, it actively shuttles between the nucleus and the cytoplasm. PARP13
is exported from the nucleus in a CRMI1-dependent manner (Liu et al., 2004).
28
Regulation of PARP13 activity
As the number of known PARP13 functions is growing, it is becoming
increasingly clear that PARP1 3 has to be tightly regulated to ensure normal
levels of activity during physiological conditions as well as proper switch in
function during pathological conditions, such as cytoplasmic stress or viral
infection. PARP13 expression levels are transcriptionally regulated, and, in
addition, posttranslational modifications and interactions with protein binding
partners modulate its activity.
PARP13 is an interferon-stimulated gene
Phosphorylation of Interferon Regulatory Factor 3 (IRF3) is one of the first
response steps upon detection of foreign elements by toll-like receptors or
related factors in mammalian cells (Akira and Takeda, 2004). Phosphorylation of
IRF3 leads to its dimerization and translocation to the nucleus where it binds to
IRF elements found upstream of the transcription start site of certain immuneresponse genes, most importantly a family of cytokines known as interferons
(IFNs) (Platanias, 2005). IFNs are rapidly induced and secreted, and once in the
extracellular environment they act in an autocrine or paracrine manner to engage
a set of IFN receptors that activate the JAK/STAT signaling pathway.
Phosphorylated STAT dimerizes, localizes to the nucleus and results in the
transcriptional upregulation of hundreds of immune-response factors known as
interferon-stimulated genes (ISGs). IRF3-induced genes are also often
upregulated by interferon signaling, in a positive feed-back-loop mechanism.
29
PARP13 expression levels increase both upon viral infection and upon treatment
with IFNs in the absence of infection, leading to the categorization of PARP13 as
an ISG (MacDonald et al., 2007). A closer examination of the mechanism of
transcriptional activation of PARP13 revealed that PARP13 is a primary response
gene, being induced directly by IRF3 in the very first step of the antiviral
response (Wang et al., 2010). IRF3 is necessary and sufficient for optimal
transcriptional activation of PARP13, independent of the IFN response pathway.
In addition the PARP13 promoter harbors STAT-binding sites, that are occupied
by STAT upon activation of the IFN pathway. It therefore appears that the initial
induction of PARP13 by IRF3 is further supplemented by the IFN signaling
downstream of IRF3 and presents a secondary mode of PARP13 activation.
The finding that PARP13 is among the genes activated by the primary antiviralresponse signaling suggests that its rapid upregulation is essential for the proper
response to infection, perhaps because PARP1 3 in not only a downstream
effector of antiviral signaling, but is itself a receptor for foreign ribonucleic-acid
molecules in the cytoplasm. Also of note is the observation that only the shorter
isoform of PARP13 - PARP13.2, appears to be upregulated upon viral signaling,
even though both isoforms have antiviral activity, suggesting that changes in
splicing may also be a hallmark of the antiviral response.
30
Posttranslational regulation of PARP13
Regulation by phosphorylation
Four serine residues in the N-terminal RNA-binding domain of PARP13 are
phosphorylated by Glycogen Synthase Kinase 3b (GSK3) (Fig. 4, Table 1) (Sun
et al., 2012). Depletion of GSK3P or inhibition of its activity results in inefficient
PARP13-mediated restriction of HIV infection. However, decreased
phosphorylation of PARP1 3 had no effect on its targeting of viral RNA for
degradation; rather, it resulted in a pronounced defect in its ability to repress viral
translation. Therefore, phosphorylation by GSK3P appears to specifically regulate
the translation-inhibition function of PARP13, perhaps by altering its ability to
interact with factors involved in the regulation of translation initiation and
elongation. PARP1 3 has also been reported to interact with the phosphatase
PR65A, depletion of which also interferes with proper antiviral activity of PARP1 3
(Wang et al., 2012c). However, exactly how PR65A regulates PARP13, and
whether it dephosphorylates the same residues that are modified by GSK3P,
remains unclear.
Regulation by farnesylation and membrane targeting
Protein prenylation constitutes the addition of a hydrophobic lipid group onto a
target protein, a modification that facilitates protein interaction with membranes
and may serve as a lipid anchor similarly to GPI anchors. Farnesylation is a type
of prenylation: a 15-carbon farnesyl group is added onto proteins containing a
CaaX motif by an enzyme called farnesyltransferase (Basso et al., 2006; Sebti,
31
2005). PARP13 harbors a CaaX motif in its PARP domain - therefore PARP13.1
but not PARP13.2 was identified as a potential target of prenylation and was later
shown to be farnesylated at Cys899 (Fig. 4, Table 1) (Charron et al., 2013).
Farnesylation targets PARP13.1 to cellular membranes and this membrane
enrichment is lost upon disruption of the CaaX motif. Furthermore, farnesylation
is required for optimal antiviral activity of PARP13.1 against SINV and loss of
membrane targeting results in inefficient restriction of SINV by PARP13.1. It is
currently unknown how membrane targeting facilitates PARP13.1 antiviral activity.
One hypothesis is that localization of PARP13.1 on endocytic membranes may
allow it to more efficiently interact with incoming virus (Charron et al., 2013).
Regulation by ADP-ribosylation and ADP-ribose binding
Another potential mode of regulation of PARP13 is ADP-ribosylation (Fig. 4,
Table 1). PARP13 is poly(ADP-ribos)ylated by a yet unknown PARP, and this
modification increases during stress suggesting that it may have a regulatory
function (Leung et al., 2011). Indeed, ADP-ribosylation modulates the function of
other RNA-binding proteins, such as Ago2 (discussed above), hnRNPs, poly(A)
polymerase (PAP) and others (Di Giammartino et al., 2013; Ji and Tulin, 2013). It
-
is usually thought to affect the RNA-binding affinity of the modified targets
poly(ADP-ribose) is a bulky and strongly negatively charged modification, and
can therefore increase the negative charge of the protein, disrupting its
interaction with an also negatively charged RNA molecule (Ji and Tulin, 2013).
This seems to be the case for Ago2, TIA1 and G3BP, for which the modification
32
occurs in their RNA-binding domains and is therefore predicted to have an
inhibitory effect on RNA binding (Leung et al., 2011). Similarly ADP-ribosylation
of PAP facilitates its dissociation from transcripts (Di Giammartino et al., 2013).
However, the modification site(s) in PARP13 is not known and therefore it is hard
to predict if PARylation would have a negative or positive effect on PARP1 3
RNA-binding affinity.
PARP13 is not only covalently modified by poly(ADP-ribose), but it is also
predicted to bind the polymer via an ADP-ribose-binding domain in its C-terminus
- the WWE domain (Fig. 4, Table 1). It is less clear how non-covalent interaction
of an RNA-binding protein with poly(ADP-ribose) may affect its affinity for RNA.
In some cases RNA-binding proteins have been shown to bind to PAR via the
same domain that binds RNA - in those cases there is a clear binding
competition between the two macromolecules and PAR decreases RNA binding
(Ji and Tulin, 2013). However, this does not always seem to be the case. Indirect
evidence suggests that PAR binding may directly or indirectly facilitate proteinRNA binding. For example, poly(ADP-ribose) is enriched in the nucleoli - sites of
ribosomal RNA biogenesis and ribosome assembly in the nucleus, where it is
thought to have a structural role and help recruit factors involved in ribosomal
RNA synthesis and maturation (Boamah et al., 2012). Inhibition of PARP1 activity
and decrease of poly(ADP-ribose) levels results in disintegration of the nucleoli,
and delocalization of nucleolar proteins to the cytoplasm. In the context of the
nucleolus, poly(ADP-ribose) recruits RNA-regulatory factors and facilitates their
33
interaction with the nascent rRNA, as RNA binding alone is not sufficient to
properly localize these factors to the site of rRNA production. Therefore, it is
possible that binding of PAR to PARP13 at its C-terminus - away from the Nterminal RNA-binding domain, may actually facilitate RNA binding, either by
helping to recruit the protein at subcellular sites enriched for the target RNAs or
by changing the overall structure of the protein and making the N-terminal
domain more exposed and available for interaction with RNA moieties.
Conclusions
RNA regulation by PARPs is an exciting and growing field of research and a
member of the family - PARP1 3, has emerged as an important mediator of decay
and translation inhibition of foreign RNAs during infection with different viral
pathogens. In the decade since the initial discovery of PARP1 3, our
understanding of its viral targets, the mechanisms it employs to lead to their
restriction and the ways in which its activity may be regulated has grown. A major
break-through revealed that PARP1 3 can also regulate the cellular transcriptome
by modulating the activity of Ago2 - a mediator of miRNA silencing. This finding
strongly suggests that PARP13 functions expand beyond the immune response,
and indeed, that its antiviral activity may be an expansion of its roles under
normal cell physiology. However, while indirect evidence that PARP13 binds to
and regulates cellular RNA in the absence of infection has accumulated, this
possibility has not been experimentally addressed, and until now no cellular
targets of PARP13 have been identified.
34
Figures and tables
PARP
PARP
Poly(ADP-ribose)
- Scaffold Model
Mono(ADP-ribose)
- Covalent
Modification Model
Acceptor protein
Mono(ADP.rlbose)
Modified acceptor
Poly(ADP-ribose)
PAR binding proteins
Figure 1. ADP-ribose affects acceptor proteins via two distinct mechanisms
Addition of a single ADP-ribose moiety (Mono(ADP-ribose)) acts as a traditional
posttranslational modification, changing the structure of the modified protein and
affecting its function, activity, localization, stability, etc. Poly(ADP-ribose) may
have these same effects, but in addition can act as a scaffold, recruiting PARbinding proteins to the site of synthesis.
35
CCCH Zinc Fingers
M"
"
PARP13.1
WWE PARP
'
PARP13.2
LOW
Cornp exity Regiori
Figure 2. Domain structure of the two major isoforms of PARPI3 PARP13
harbors four CCCH-type Zinc Fingers that constitute its N-terminal RNA-binding
domain; a WWE PAR-binding domain; a catalytically inactive PARP domain (only
in PARP13.1); and a low complexity region between the RNA-binding domain
and the WWE domain. The locations of a nuclear-localization signal (NLS) and
nuclear-export signal (NES) are also indicated.
36
m 7GpppG
AAAAAAAAAA
Deadenylation
PAN2, PAN3
mGpppG
Decapping
TGpppG
DCP2
NUDT16
Exonuclase
_____
CleavageV~
"
ado W-M-IExosome
Figure 3. Major steps in cytoplasmic mRNA decay, and factors involved
RNA decay starts with deadenylation: the removal of the 3' poly(A) tail by a set of
deadenylases including PARN, PAN2 and PAN3, and the CCR4-NOT complex.
This step is followed by removal of the 5' 7-methylguanylate cap by the
decapping enzymes DCP1, DCP2, NUDT16 and possibly others. Deadenylated
and decapped RNAs are digested either by the 5'-3' exonuclease XRN1 or by the
3'-5' exonuclease complex called the RNA exosome. Factors known to bind to
PARP13 directly are shown in red boxes; factors recruited by PARP13 indirectly
via DDX17 are shown in blue boxes. Figure is adapted from (Schoenberg and
Maquat, 2012).
37
fl-
N0
0
4
WWE
00
*
Phosphorylation
4
Poly(ADP-ribose)
*
Farnesyl group
Figure 4. Posttranslational regulation of PARP13 A schematic of PARP13.1
with known regulatory events shown at the residues where they occur:
phosphorylation by GSK3P at S257, S262, S266, S270; farnesylation at C899;
PAR binding at the WWE domain; and covalent modification with PAR at
unknown residue(s).
38
...
.........
.........
Phosphorylation
Farnesylation
S257, S262,
S266, S270
C899
________________
Covalent
modification with
poly(ADP-ribose)
Non-covalent
interaction with
poly(ADP-ribose)
GSK3P
Farnesyl-
transferase
Unknown
Unknown
WWE domain
Unknown
Unnw
PARR
Table 1. Posttranslational regulation of PARP13
39
Facilitates PARP13dependent translational
repression of target
mRNAs
Membrane targeting
_
_
_
_
_
_
_
_
_
_
_
_
Unknown, but likely an
effect on RNA binding
affinity
Unknown, but likely an
effect on RNA binding and
protein localization
'INA
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45
Chapter 2: PARP13 regulates cellular mRNA posttranscriptionally and
functions as a pro-apoptotic factor by destabilizing TRAILR4 transcript
Tanya Todoroval' 2 , Florian J Bock 2 and Paul Chang 1 2
,
Department of Biology1, Koch Institute for Integrative Cancer Research2
Massachusetts Institute of Technology, USA
500 Main Street, Cambridge MA 02139
Correspondence: P.C. (pchang2@mit.edu)
Published as:
Todorova, T., Bock, F.J. and Chang, P. (2014) PARP13 regulates cellular mRNA
posttranscriptionally and functions as a pro-apoptotic factor by destabilizing
TRAILR4 transcript. Nature communications, 5
46
Abstract
Poly(ADP-ribose) Polymerase-1 3 (PARP1 3/ZAP/ZC3HAV1) is an antiviral factor,
active against specific RNA viruses such as MLV, SINV and HIV. During infection,
PARP1 3 binds viral RNA via its four CCCH-type zinc finger domains and targets
it for degradation by recruiting cellular-mRNA-decay factors such as the exosome
complex and XRN1. Here we show that PARP13 binds to and regulates cellular
mRNAs in the absence of viral infection. Knockdown of PARP13 results in the
misregulation of hundreds of transcripts. Among the most upregulated transcripts
is TRAILR4 that encodes a decoy receptor for TRAIL - a pro-apoptotic cytokine
that is a promising target for the therapeutic inhibition of cancers. PARP1 3
destabilizes TRAILR4 mRNA posttranscriptionally in an exosome-dependent
manner by binding to a region in its 3'UTR. As a consequence, PARP1 3
represses TRAILR4 expression and increases cell sensitivity to TRAIL-mediated
apoptosis, acting as a key regulator of the cellular response to TRAIL.
Introduction
Poly(ADP-ribose) Polymerase-1 3 (PARP1 3), also known as Zinc Finger Antiviral
Protein (ZAP), ARTD1 3, and ZC3HAV1, is a member of the PARP family of
proteins - enzymes that modify target proteins with ADP-ribose using
nicotinamide adenine dinucleotide (NAD') as substrate (Vyas et al., 2013). Two
PARP13 isoforms are expressed constitutively in human cells: PARP13.1 is
targeted to membranes by a C-terminal CaaX motif, whereas PARP13.2 is
cytoplasmic (Charron et al., 2013; Vyas et al., 2013). Both proteins are unable to
47
generate ADP-ribose - PARP13.1 contains a PARP domain lacking key aminoacid residues required for PARP activity whereas the entire PARP domain is
absent in PARP13.2 (Vyas et al., 2013). Both isoforms of PARP13 contain four
N-terminal RNA-binding CCCH-type Zinc Fingers - domains found in proteins
that function in the regulation of RNA stability and splicing such as tristetraprolin
(TTP) and muscleblind-like (MBNL1), respectively (Brooks and Blackshear, 2013;
Chen et al., 2012; Fernandez-Costa et al., 2011; Gao et al., 2002).
PARP1 3 was originally identified in a screen for antiviral factors (Gao et al.,
2002). It binds RNAs of viral origin during infection and targets them for
degradation via the cellular mRNA-decay machinery (Bick et al., 2003; Guo et al.,
2007; Zhu et al., 2011). Several RNA viruses, including MLV, SINV, HIV and
EBV as well as the RNA intermediate of the Hepatitis B DNA virus have been
shown to be targets of PARP13 (Bick et al., 2003; Gao et al., 2002; Mao et al.,
2013; Muller et al., 2007; Xuan et al., 2012; Zhu et al., 2011). How viral RNA is
detected by PARP13 is currently not known, and, although binding to PARP13 is
a requirement for viral RNA degradation, no motifs or structural features common
to the known targets have been identified (Huang et al., 2010). Structural
analysis of the PARP1 3 RNA-binding domain suggests that PARP1 3 binds
looped RNA, therefore target recognition could involve structural features rather
than linear sequence motifs (Chen et al., 2012).
48
PARP1 3 binds to multiple components of the cellular 3'-5' mRNA-decay
machinery including polyA-specific ribonuclease (PARN), and subunits of the
exosome exonuclease complex, RRP46/EXOSC5 and RRP42/EXOSC7 (Guo et
al., 2007; Zhu et al., 2011). Recruitment of these decay factors results in the 3'-5'
degradation of viral RNAs bound to PARP13. Although 5'-3' RNA decay has also
been shown to play a role in PARP13-mediated viral degradation, proteins
involved in this process including the decapping factors DCP1 and DCP2 and the
5'-3' exonuclease XRN1, do not bind to PARP13 directly and are instead
recruited by other PARP13 binding partners such as DDX17 (Zhu et al., 2011).
Whether or not PARP1 3 binds to and modulates cellular RNAs either in the
absence or presence of viral infection is unknown. However, several indications
point towards a role for PARP1 3 in cellular RNA regulation: 1) both PARP1 3
isoforms are expressed at high levels in cells, however only PARP1 3.2
expression is upregulated during viral infection suggesting that PARP13.1 has
functions unrelated to the antiviral response (Hayakawa et al., 2011; Vyas et al.,
2013); 2) even in the absence of viral infection, PARP13 localizes to RNA-rich
stress granules - non-membranous ribonucleoprotein structures that form during
cellular stress in order to sequester mRNAs and inhibit their translation (Leung et
al., 2011 a); 3) PARP13 regulates the miRNA pathway by targeting Argonaute
proteins for ADP-ribosylation and this regulation occurs both in the absence and
in the presence of viral infection (Leung et al., 2011a; Seo et al., 2013). This
suggests that PARP13 targeting of RNA to cellular decay pathways could also
49
occur in the absence of viral infection, and that PARP13 could therefore function
as a general regulator of cellular mRNA.
Here we show that PARP13 binds to and regulates cellular RNA in the absence
of viral infection, and that its depletion results in significant misregulation of the
transcriptome with an enrichment in signal-peptide-containing transcripts and
immune-response genes. From the list of PARP1 3-dependent differentially
expressed genes we focused this study on understanding how PARP13
regulates TRAILR4 - a member of a family of transmembrane receptors
composed of TRAILR1-4 (Degli-Esposti et al., 1997; Johnstone et al., 2008) that
bind to TRAIL, a pro-apoptotic TNF-family cytokine. We show that PARP1 3
destabilizes TRAILR4 mRNA posttranscriptionally but has no effect on the levels
of other TRAIL receptors. It binds to a specific fragment in the 3' untranslated
region (3'UTR) of TRAILR4 mRNA, and leads to its degradation via the RNA
exosome complex.
Primary cells are TRAIL resistant; however, many transformed cells become
sensitive to TRAIL-induced apoptosis, making it an attractive target for the
treatment of cancers (Johnstone et al., 2008). TRAIL binding to TRAILR1 and
TRAILR2 triggers the assembly of the Death-Inducing Signaling Complex (DISC)
(Kischkel et al., 2000; Sprick et al., 2000) leading to the recruitment and
activation of caspase-8 and induction of the extrinsic apoptotic pathway (Kischkel
et al., 2000; Sprick et al., 2000). In contrast TRAILR3 and TRAILR4 act as pro-
50
survival decoy receptors that bind TRAIL but cannot assemble functional DISCs
and therefore cannot signal apoptosis (Marsters et al., 1997; Merino et al., 2006).
The relative expression of each receptor varies in different cancers and tissue
types and is thought to be important for the overall cellular response to TRAIL
(LeBlanc and Ashkenazi, 2003). Accordingly, high levels of these decoy
receptors can prevent TRAIL induced cell death and likely contribute to acquired
TRAIL resistance in cancer cells (Morizot et al., 2011). By repressing TRAILR4
expression PARP1 3 acts a key regulator of the cellular response to TRAIL and
depletion of PARP13 results in TRAILR4 protein accumulation and TRAIL
resistance in multiple cell lines.
Results
PARP13 binds to cellular RNA
To determine if PARP1 3 binds to cellular RNA, we performed crosslinking
immunopreciptation (CLIP) in HeLa cells using affinity-purified PARP13 antibody.
A strong signal from bound, crosslinked RNA that collapsed to two major bands
at high RNase concentrations was identified (Fig. 1a). The collapsed signal
migrated at the molecular weight of PARP13.1 and 13.2, and was PARP13specific since it was not detected in similar purifications performed in PARP1 3'
HeLa cell lines generated using zinc finger nucleases (Fig. 1a, Supplementary
Fig.1). Since PARP13.1 and PARP13.2 are constitutively expressed in HeLa
cells, we compared the binding of cellular RNA to each isoform using N-terminal
streptavidin-binding protein (SBP) fusions. SBP-PARP1 3.1 and SBP-PARP13.2
51
bound similar amounts of RNA and the signal for both was RNAse sensitive
confirming the attached molecules as RNA (Fig. 1b, 1c). For both the
endogenous PARP13 and the SBP precipitations no signal was identified when
UV cross-linking was omitted demonstrating the specificity of the reactions (Fig.
1 d). To further confirm that binding of RNA to PARP1 3 is specific and requires
the CCCH zinc fingers, we generated deletions of these domains from PARP1 3.1
and PARP13.2 and performed CLIP (PARP13.1,ZnF and PARP1 3.2 ^ZnF) (Fig. 1e,
1f). Deletion of these domains resulted in dramatic reduction of signal.
Structural analysis of the PARP13 RNA-binding domain containing four CCCH
zinc fingers identified key amino-acid residues for viral RNA binding (Chen et al.,
2012). Two cavities, defined by V72, Y108, F144 (Cavity 1) and H176, R189
(Cavity 2) are thought to be important for RNA binding. We mutated each residue
of Cavity 1, multiple residues in Cavity 2, and all five residues found in both
cavities to alanine in SBP-PARP13.1 and assayed the mutants for RNA binding
using CLIP (Fig. le, 1g, Table 1). Mutation of all five residues to generate
PARP1 3 .1VYFHR reduced RNA binding to negligible levels and mutation of all
three Cavity 1 residues resulted in a similar decrease in RNA binding as
individual mutations of each Cavity 2 residue (Fig. 1g, 1h).
It is possible that the reduction in RNA binding in the mutants was a result of
aggregation or mislocailzation of the mutant proteins. To test this, we compared
the localization of PARP13.1 AZnF and PARP13.1 VYFHR to wild-type protein in HeLa
52
cells. Both mutants exhibited localization patterns similar to PARP1 3.1 (Fig. 2).
We also examined localization of the mutant proteins to stress granules. We
previously showed that PARP13 is highly enriched in stress granules, structures
that are assembled during cytoplasmic stress and contain high concentrations of
cellular mRNA (Leung et al., 201 1a). PARP1 3.1 properly localized to stress
granules upon sodium arsenite treatment, however both PARP13.1 AZnF and
PARP1 3 .1VYFHR failed to localize to these structures (Lee et al., 2013; Leung et
al., 2011 a) (Fig. 2, Supplementary Fig. 2). This defective targeting was even
more striking for PARP13.2 AZnF and PARP1 3 .2 VYFHR (Fig. 2, Supplementary Fig.
2). These results confirm that the mutants are defective in binding RNA and that
this defect affects cellular function. They further suggest that binding to cellular
RNA is critical for PARP13 localization to stress granules.
PARP13 regulates the transcriptome
To determine if PARP1 3 regulates cellular RNA we analyzed the transcriptome in
the absence of PARP13. We used Agilent microarrays to compare the relative
abundance of transcripts in HeLa cells transfected with control siRNA to cells
transfected with PARP13-specific siRNA (Fig. 3a). Depletion of PARP13 resulted
in significant misregulation of the transcriptome with 1841 out of a total of 36338
transcripts analyzed showing >0.5 Log2 fold change (Log2FC) relative to control
knockdowns (1065 upregulated and 776 downregulated transcripts). Of these, 85
transcripts exhibited Log2FC>1 relative to control siRNAs (66 upregulated and 19
53
downregulated). In total 73 transcripts passed a significance threshold of p<0.05
(moderated t-statistic with Benjamini Hochberg adjustment) (Table 2).
The 50 upregulated transcripts with a p-value <0.05 showed enrichment for
genes containing a signal peptide required for targeting of mRNA for translation
at the endoplasmic reticulum (ER) (analyzed with the Database for Annotation,
Visualization and Integrated Discovery (DAVID) (Huang da et al., 2009),
(Enrichment Score 3.4, p-value<0.0001; Supplementary Data Tablel),
suggesting that PARP13 could regulate transcripts at the ER. The membranous
perinuclear localization we observed for PARP1 3.1 (Fig. 2) and the previously
reported membrane targeting of this protein (Charron et al., 2013) suggested a
potential enrichment at the ER. We therefore costained exogenously expressed
PARP13 isoforms with the ER marker ER Tracker, and observed colocalization
with PARP1 3.1 but not PARP1 3.2. This localization is independent of RNA
binding since PARP1 3 .1VYFHR localized very similarly to the wild-type protein (Fig.
3b). Targeting of PARP13 to the ER may therefore be one mechanism of
regulating its function and its RNA-target specificity. Gene Set Enrichment
Analysis (Subramanian et al., 2005) of the same genes identified enrichment for
members of the interferon immune-response pathway (p-value<0.0001,
Normalized Enrichment Score = 2.22) (Supplementary Fig. 3, Supplementary
Data Table 2).
54
To verify our results, we analyzed 6 of the top 10 most upregulated transcripts
using quantitative real-time reverse-transcription PCR (qRT-PCR) in both
PARP13 knockdowns and PARP13- cells. All 6 transcripts were upregulated
relative to controls upon PARP13 depletion (Fig. 3c). With the exception of
TRAILR4/TNFRSF10D, each of these genes encodes an immune-response gene
and is a member of the interferon-stimulated genes (ISGs), activated in response
to interferon signaling. The upregulation of the five ISGs appears to be specific
and is not the result of a general activation of the interferon response since JAKSTAT signaling was not increased in the knockdown or knockout cells (Fig. 3d)
and since other canonical ISGs (IRFs, TRIMS, IFITMs) were not upregulated in
our transcriptome analysis. Interestingly, like PARP1 3, the upregulated ISGs
(OASL, IFIT2, IFIT3) function in inhibition of viral replication and translation
(Schoggins and Rice, 2011) suggesting that their upregulation could be a
compensatory response to PARP1 3 depletion.
To identify the direct targets of PARP1 3 regulation among the 6 highly
upregulated transcripts, we compared their expression levels in PARP1 3- cells
relative to PARP13- cells expressing wild-type PARP13 or PARP13 RNAbinding mutant. While both PARP13.1 and PARP13.2 are constitutively
expressed in HeLa cells, PARP13.2 expression increases during viral infection in
an interferon-dependent manner, whereas PARP1 3.1 expression does not
(Hayakawa et al., 2011). Therefore, to exclude interferon-related effects we
focused our experiments on PARP13.1. Direct targets of PARP13 binding and
55
regulation would in theory decrease upon PARP1 3.1 but not PARP1 3 VYFHR
expression in PARP13-1- cells. TRAILR4 mRNA clearly behaved in this manner
with a 25% decrease in transcript levels upon PARP1 3.1 expression and no
change upon PARP1 3 .1VYFHR expression (Fig. 3e). This analysis proved difficult
for the remaining 5 transcripts since transient plasmid transfection induced a
transcriptional upregulation of these ISGs as previously shown (Li et al., 2005).
PARP13 represses TRAILR4 mRNA and protein expression
Due to its biological importance and the clinical interest in TRAIL we examined
the role of PARP13 in the regulation of TRAILR4 expression and how that
regulation might impact TRAIL signaling and apoptosis. Upregulation of
TRAILR4 mRNA in PARP13-depleted HeLa cells had a direct effect on TRAILR4
protein expression: TRAILR4 protein levels, barely detectable in wild-type HeLa
cells, increased in PARP13 knockdown cells and in all three independently
isolated PARP13-1- cell lines (Fig. 4a, 4b). In addition, consistent with our results
identifying TRAILR4 as a direct target of PARP13 regulation (Fig. 3e,
Supplementary Fig. 4), expression of PARP13.1, but not PARP13.1 VYFHRin
PARP1
3 /-A
cells was sufficient to reduce TRAILR4 protein expression (Fig. 4c).
PARP13 repression of TRAILR4 mRNA represents a general mechanism of
TRAILR4 regulation in multiple human cell types. In all cell lines tested, including
primary cells such as Tert immortalized Retinal Primary Epithelial (RPE1) cells
and transformed cells such as human colon HCT1 16, human colon
adenocarcinoma SW480 and HeLa cells, TRAILR4 mRNA levels increased upon
56
PARP13 depletion identifying suppression of TRAILR4 expression as a
widespread physiological function of PARP1 3 (Fig. 4d). Under physiological
conditions, the primary isoform of PARP13 that regulates TRAILR4 is PARP13.1
since specific knockdown of PARP13.1 in HeLa cells increased TRAILR4 mRNA
to levels similar to those obtained upon total PARP1 3 depletion (Fig. 4e).
PARP13 inhibits TRAILR4 posttranscriptionally via its 3'UTR
The PARP13.1 and PARP1 3 .1VYFHR rescue assays performed in PARP13-' cells
suggest that TRAILR4 regulation by PARP1 3 is posttranscriptional and requires
RNA binding to PARP13 (Fig. 3e, Supplementary Fig. 4). Posttranscriptional
regulation was confirmed by analyzing the upregulated TRAILR4 transcripts via
qRT-PCR with primers that overlap intron-exon boundaries to identify unspliced
pre-mRNA, and primers that overlap exon-exon boundaries, to identify mature
transcripts. PARP13 knockdown resulted in increased amounts of mature
TRAILR4 mRNA, but had no effect on the amount of pre-mRNA, suggesting that
TRAILR4 transcription is not altered upon PARP13 depletion and that regulation
of TRAILR4 by PARP13 is posttranscriptional (Fig. 5a). Since posttranscriptional
regulation of mRNA often occurs via the 3' untranslated region (3'UTR) we
designed reporter constructs containing the 3'UTR of TRAILR4 or GAPDH (as
negative control) fused to Renilla luciferase in the psiCHECK2 vector. This
vector also encodes Firefly luciferase as a transfection control. Renilla-TRAILR4
3'UTR expression was decreased -20% in HeLa cells relative to PARP13~ cells
whereas no significant difference in Renilla or Renilla-GAPDH 3'UTR expression
57
was detected between the two cell lines (Fig. 5b). Together these results
suggest that PARP13 destabilizes TRAILR4 posttranscriptionally via its 3'UTR.
Computational analysis of the TRAILR4 3'UTR identified 7 putative AU-rich
elements (ARE) known to destabilize RNA (Gruber et al., 2011; Schoenberg and
Maquat, 2012), one conserved miRNA-binding site that is muscle specific (miR133abc) (Lewis et al., 2005) (Luo et al., 2013), and 4 short and poorly
characterized ZAP-responsive elements (ZREs) - GGGUGG and GAGGG,
predicted by SELEX to mediate PARP13 recognition of RNA targets (Huang et
al., 2010) (Fig. 5c, Supplementary Fig. 5). To identify the key PARP13dependent regulatory sequences in the TRAILR4 3'UTR, we designed
truncations of the TRAILR4 3'UTR and fused them to Renilla luciferase in the
psiCHECK2 vector (Fig. 5c). Fragments were designed based on a secondarystructure prediction of the TRAILR4 3'UTR to avoid disturbing high-probability
RNA folds (Lorenz et al., 2011) (Supplementary Fig. 6). The relative PARP13dependent destabilization of each fragment was determined by subtracting
expression in wild-type cells from expression in PARP13- cells (Fig. 5c,
Supplementary Fig. 7). This analysis identified nucleotides 516-1115 of the
3'UTR as necessary for PARP1 3 regulation. Fusion of nucleotides 516-1115
(Fragment E) to Renilla resulted in destabilization of the construct in wild-type
cells, confirming that this sequence contains the relevant signal for PARP13dependent repression (Fig. 5c). This fragment includes 2 ZREs and 2 AREs,
including one that contains multiple overlapping ARE sequences suggesting that
58
PARP1 3 regulation of TRAILR4 mRNA might require ARE and/or ZRE
recognition. Our analysis also suggests that TRAILR4 regulation is likely miRNAindependent since no conserved miRNA binding sites are found in the TRAILR4
regulatory sequence.
PARP13 binds TRAILR4 mRNA
To determine if PARP13 regulation of TRAILR4 occurs via direct binding to
TRAILR4 mRNA, we performed CLIP qRT-PCR in cells expressing SBPPARP13.1, SBP- PARP1 3 .1VYFHR or PARP13.1 AZnF and electrophoretic mobility
shift assays (EMSA) using purified SBP-PARP13.1 or SBP- PARP1 3 .1VYFHR and
32
P labeled Fragment E or Fragment 1 as control. CLIP qRT-PCR analysis
identified significant enrichment of TRAILR4 mRNA in wild-type PARP13.1
precipitations relative to PARP1 3 .1VYFHR or PARP1 3.1 AZnF confirming a direct and
specific binding interaction between TRAILR4 mRNA and PARP13 in vivo (Fig.
5d, 5e). These results were confirmed in vitro by EMSA assays where SBPPARP13.1 bound to Fragment E with high affinity (Kd=123nM) and to Fragment 1
with lower affinity (Kd=508nM). PARP13.1VYFHR failed to bind either fragment
(Fig. 5f). These experiments demonstrate the specificity of TRAILR4 mRNA
binding to PARP1 3 and show that the regulatory region of the TRAILR4 3'UTR
binds directly to PARP13 with good selectivity (Fig. 5f).
PARP13 destabilization of TRAILR4 mRNA is exosome dependent
59
PARP13 regulates viral RNA stability via XRN 1-dependent 5'-3' decay, and
exosome-dependent 3'-5' decay (Zhu et al., 2011). PARP13 can also bind to and
modulate Argonaute (Ago) activity, critical for miRNA-dependent
posttranscriptional regulation of mRNA stability (Leung et al., 2011 a). To
determine if TRAILR4 mRNA stability is regulated through any of these pathways,
TRAILR4 mRNA levels were examined upon knockdown of Ago2, XRN1 or
EXOSC5, an exosome-complex component shown to bind PARP13 (Guo et al.,
2007). Knockdown of EXOSC5, verified by qRT-PCR (antibodies were nonreactive), resulted in stabilization of TRAILR4 mRNA in HeLa cells suggesting
that exosome function is necessary for regulation of TRAILR4 mRNA (Fig. 6a,
6b). In contrast, neither XRN1 knockdown in HeLa cells nor depletion of Ago2 in
HEK293 cells using tetracycline-inducible Ago2 shRNA (Schmitter et al., 2006)
resulted in obvious TRAILR4 mRNA stabilization (Fig. 6a, 6b, 6c). PARP13
depletion in the Ago2 shRNA inducible cell lines resulted in similar levels of
TRAILR4 upregulation regardless of Ago2 depletion, further suggesting that
Ago2 function is not necessary for TRAILR4 regulation by PARP13 (Fig. 6c).
To determine if exosome or XRN1 activity is required for PARP1 3-dependent
destabilization of TRAILR4 mRNA, we examined the expression of psiCHECK2
reporter constructs encoding Renilla and Renilla-TRAILR4 3'UTR in wild-type
and PARP13-'~ cells transfected with control, EXOSC5 or XRNI siRNA (Fig. 6d).
Relative PARP13-dependent destabilization was then calculated by subtracting
the Renilla/Firefly luciferase signal obtained from wild-type cells from the signal
60
obtained from PARP1 3-' cells (Fig. 6d). Knockdown of EXOSC5 resulted in a
pronounced defect in the ability of PARP1 3 to repress TRAILR4 3'UTR with a
15% relative destabilization of the construct compared to 40% in control
knockdowns. Knockdown of XRN1 resulted in milder but potentially relevant
defects with a 30% relative destabilization of the Renilla-TRAILR4 3'UTR
compared to 40% in controls consistent with the observation that exosome
activity was the primary pathway regulating endogenous TRAILR4 mRNA (Fig.
6a). We therefore conclude that PARP13 requires exosome activity, and
potentially XRN1, to destabilize TRAILR4 mRNA (and likely other PARP13
targets).
PARP13 decreases TRAILR4 mRNA half life
Since the exosome complex is a key regulator of mRNA decay, we examined the
TRAILR4 mRNA decay rate in PARP13~1 and wild-type cells. Newly transcribed
RNA was pulse-labeled with 4-thiouridine and labeled transcripts purified at
specific time points after 4-thiouridine removal. qRT-PCR was then performed
on the purified transcripts to quantitate amounts of TRAILR4 mRNA and GAPDH
mRNA. ACTB mRNA was used to normalize inputs. TRAILR4 mRNA decay rates
were significantly higher in wild-type cells (t1/2 =1 .5 h) than in PARP1 3~' cells
(t 1/2 =13 h) whereas GAPDH decay rates were similar in both cell lines (Fig. 6e).
Together our data are consistent with a model in which PARP1 3 facilitates
efficient degradation of TRAILR4 mRNA via the activity of the exosome complex
61
(Fig. 6a, 6d) and suggest that PARP13 functions as a novel RNA binding protein
that regulates cellular RNA stability by binding to the 3'UTR.
PARP13 depletion inhibits TRAIL-induced apoptosis
To investigate the physiological relevance of TRAILR4 regulation by PARP1 3 we
examined TRAIL-induced apoptotic signaling upon PARP13 depletion. TRAILR4
expression levels are a key regulator of TRAIL sensitivity in certain cancers
(Degli-Esposti et al., 1997; Morizot et al., 2011). HeLa cells are TRAIL-sensitive
due to low TRAILR4 expression and exogenous expression of TRAILR4 is
sufficient to confer TRAIL resistance (Merino et al., 2006; Morizot et al., 2011)
(Fig. 7a). Of the four TRAIL receptors, only TRAILR4 expression is regulated by
PARP13 - TRAILR4 mRNA expression, examined by qRT-PCR, and protein
levels, assayed by immunoblot, were increased in PARP1 3-- relative to wild type
cells, whereas no differences in protein and mRNA levels of TRAILR1-R2 were
identified between PARP13-- and wild-type cells, and TRAILR3 protein could not
be detected in this cell type, consistent with previous reports (Supplementary
Fig.8, Fig. 7b) (Merino et al., 2006). These results suggest that by modulating
TRAILR4 expression PARP13 could directly regulate the cellular response to
TRAIL. We examined this possibility by assaying TRAIL-induced apoptosis upon
PARP1 3 depletion in TRAIL-sensitive HCT1 16, SW480 and HeLa cells.
Consistent with the increase in TRAILR4 mRNA levels (Fig. 4d), PARP1 3
knockdown resulted in a pronounced resistance to TRAIL treatment in each of
these cell types identifying PARP1 3 as a key regulator of the TRAIL response in
62
these cell lines (Fig. 7c). The newly acquired TRAIL resistance was a specific
result of increased TRAILR4 expression upon PARP1 3 knockdown since
simultaneous knockdown of PARP13 and TRAILR4 in HeLa cells resulted in wildtype TRAILR4 mRNA levels and TRAIL sensitivity profiles similar to control
knockdowns (Fig. 7c, 7d).
The TRAIL resistance conferred by PARP13 inhibition can be permanently
acquired. PARP1 3'- cells were resistant to both short-term (24 h, Fig. 7e, 7f) and
long-term TRAIL treatment (7 days, Fig. 7g), suggesting that one mechanism of
TRAIL resistance in cancers could be loss of PARP13 function. TRAIL
resistance in PARP13-'- cells was completely reversed by expression of
I31AZnF suggesting that the TRAIL
PA~l.VYFHR oPA
or PARP13.1n,
PARP13.1 but not PARP13.1
resistance in these cells results from the lack of TRAILR4 mRNA regulation by
PARP1 3 (Fig. 7h, 7i). Together these results suggest that PARP1 3 is necessary
and sufficient to regulate the cellular response to TRAIL in cancer cells that are
TRAIL-sensitive in a manner dependent on TRAILR4 expression.
PARP13 depletion abrogates DISC assembly and function
TRAILR4 expression levels are important for TRAIL sensitivity in certain cancers
due to the receptor's ability to sequester TRAIL from TRAILR1 and R2 binding
resulting in decreased DISC assembly and apoptotic signaling at these receptors
upon TRAIL treatment. This apoptotic signaling is mediated by caspase-8, which
is recruited to the DISC where it is activated and autoprocesses itself. Thus
63
caspase-8 cleavage can be used to report directly on caspase-8 enzymatic
activity. To determine if the TRAIL resistance observed in PARP13-'~ cells results
from attenuated apoptotic signaling at the TRAIL receptor level, time-dependent
caspase-8 processing was analyzed in wild-type or PARP1 3 -1-A cells treated with
TRAIL. Whereas caspase-8 was processed in HeLa cells resulting in the
appearance of p43/p41 and p18 fragments, no such processing was observed in
PARP13-'- cells, demonstrating an ablation of DISC signaling (Fig. 8a). A
consistent upregulation of both PARP1 3.1 and PARP1 3.2 was observed upon
TRAIL treatment suggesting positive feedback signaling (Fig. 8a). To determine
if DISC assembly itself is defective in PARP1 3-' cells we compared assembly to
wild-type cells using standard DISC precipitation assays that utilize epitopetagged TRAIL (Walczak and Haas, 2008). Recruitment of TRAILR1, TRAILR2
and caspase-8 to the DISC was greatly diminished in PARP1 3-/- cells relative to
wild-type (Fig. 8b). Together these results suggest that the TRAIL resistance
found upon PARP13 depletion is due to defective DISC assembly, decreased
caspase-8 activation and decreased apoptotic signaling from TRAIL receptors.
Discussion
In this manuscript we show that PARP13 binds cellular RNA and that its
depletion results in significant misregulation of the transcriptome with an
enrichment in signal-peptide-containing transcripts and immune-response genes.
We identify the mRNA encoding TRAILR4 as the first experimentally verified
cellular target of PARP1 3 repression and demonstrate that PARP1 3 destabilizes
64
TRAILR4 mRNA posttranscriptionally by binding to its 3'UTR and targeting it for
degradation via the exosome complex. Consistent with these data, PARP1 3
depletion markedly alters TRAILR4 mRNA decay kinetics. By repressing
TRAILR4 expression in the cell, PARP13 shifts the balance in the TRAIL
signaling pathway towards decreased anti-apoptotic signaling and sensitizes
cells to TRAIL-mediated apoptosis (Fig. 8c).
Expression of decoy receptors has been described as one mechanism of TRAIL
resistance in cancer (Johnstone et al., 2008; Walczak and Haas, 2008). Our
results suggest that PARP13 could have important functions in regulating TRAIL
resistance and that modulation of PARP1 3 may have the potential to overcome
TRAILR4-mediated TRAIL resistance. This approach could improve the efficacy
of TRAIL therapies currently in clinical trials to target multiple cancers (Stuckey
and Shah, 2013). Furthermore, it was recently shown that PARP1/2 inhibitors
result in transcriptional upregulation of TRAILR2 and subsequent apoptosis in
acute myeloid leukemias (Meng et al., 2014). It therefore appears that the cellular
response to TRAIL is regulated by multiple PARPs via transcriptional and
posttranscriptional mechanisms.
Several important questions remain regarding PARP13 function in the regulation
of cellular mRNA - what are the direct targets of regulation, how is target
specificity determined, and does the regulation of cellular targets change upon
viral infection? With the exception of TRAILR4, many of the transcripts
65
misregulated upon PARP13 knockdown identified in this work are likely indirect
targets. To better understand the biology of PARP1 3, identifying additional direct
targets will be critical. Based on previous research on the selectivity of viral RNA
binding to PARP1 3, target recognition of cellular mRNA is more likely to be
mediated by structural features rather than linear sequence motifs, therefore it is
unlikely that a consensus sequence will be identified (Chen et al., 2012; Huang et
al., 2010). Interestingly, the expanded AU-rich element in the TRAILR4 3'UTR is
predicted to form a hairpin with high probability (Supplementary Fig. 6),
suggesting that it may represent a structure recognized by PARP1 3 (Lorenz et al.,
2011).
The highly significant enrichment of signal-peptide-containing transcripts upon
PARP13 depletion (corrected p-value<0.0001) strongly suggests that PARP13
has a specific function in the regulation of transmembrane proteins, or those that
are destined to be secreted. This enrichment might be related to PARP13.1
localization at the ER. Indeed, PARP13.1 has been shown to be farnesylated,
and this modification targets PARP13.1 to membranes and is required for its
antiviral activity (Charron et al., 2013). It is therefore possible that similar
targeting of PARP13.1 to membranes might also regulate its function in
destabilizing cellular transcripts, including those at the ER.
It is important to note that PARP1 3 is just one member of the CCCH PARP
-
subfamily identified based on the presence of CCCH RNA-binding domains
66
PARP12 and PARP7 are the other two members (Vyas et al., 2013). Whereas
PARP7 is poorly studied and little is known about its function, both PARP12 and
PARP13 function in the antiviral response and localize to membraneous
organelles (PARP13 to the ER and PARP12 to the Golgi) (Atasheva et al., 2012;
Vyas et al., 2013; Welsby et al., 2014). In addition, PARP12 and PARP13 exhibit
similar domain structures including the presence of multiple tandem CCCH zinc
fingers (vs only one in PARP7) (Vyas et al., 2013). Therefore, PARP12 might
also regulate cellular RNA in a manner similar to PARP13.
Materials and methods
General
Experiments were performed in HeLa Kyoto cells unless otherwise stated.
Knockdowns were performed using Lipofectamine 2000 as per manufacturer's
instructions with double transfections of 48 h. Exogenously expressed constructs
were transfected using Lipofectamine 2000 for 24 h before the assay. Mutants
were cloned using GeneString technology. TRAILR4 3'UTR was cloned from
Origene clone SC117708 into psiCHECK2 using Gene String technology.
TRAILR4 3'UTR fragments were cloned by PCR amplification of the indicated
regions and cloned into psiCHECK2. Renilla and Firefly luminescence were
measured 48 h post transfection. Crosslinking followed by immunoprecipitation
was performed as previously described (Leung et al., 2011 b). To assess cell
sensitivity to TRAIL-mediated apoptosis, cells were treated with TRAIL for 24 h,
67
and cell viability was assayed by MTT assay (Millipore) or by Annexin V/PI flow
cytometry (Biolegend) as per manufacturer's instructions. Standard Western
Blotting techniques were used, and full scans of important blots along with
protein ladder markers are shown in Supplementary Fig. 9.
Cell culture and transfection
Cells were grown at 37 C and 5% CO 2. HeLa Kyoto (ATCC), SW480 (a gift from
Ryoma Ohi, Vanderbilt), and HEK293 (ATCC) cells were maintained in DMEM
(Invitrogen) supplemented with 10% Fetal Bovine Serum (Life technologies);
hTERT-RPE1 cells (ATCC) in Ham's F12/DMEM (Mediatech) supplemented with
10% Fetal Bovine Serum and HCT1 16 cells (ATCC) were cultured in McCoy's 5A
(ATCC) supplemented with 10% Fetal Bovine Serum (Life technologies). For
expression of recombinant proteins, HeLa cells were transfected with
Lipofectamine 2000 (Life Technologies) 24 h prior to assay. For RNAi, two 48 h
transfections were performed with 20nM siRNA for Stealth siRNAs or 5nM for
Silencer Select siRNAs using Lipofectamine 2000 according to the
manufacturer's protocol. For RPE1 RNAi, 5nM of siRNA was transfected with
Silentfect (BioRad) following manufacturer protocols. IFNy was from R&D
Serotec, JAKi from Calbiochem and Flag-TRAIL from Axxora. His-TRAIL was
purified according to standard procedures (Kim et al., 2004).
PARP13 knockout cell lines
68
Zinc finger nucleases specific to the PARP13 genomic locus were purchased
from Sigma Aldrich and transfected into HeLa Kyoto cells. Monoclonal cell lines
(PARP13-- A/B/C) were generated using serial dilution in 96 well plates, then
tested for PARP13 expression via western blot. Three independent monoclonal
cell lines lacking PARP13 expression were generated.
Cloning
GFP-PARP13 has been described previously (Vyas et al., 2013). To generate
SBP-PARP13, GFP was substituted with streptavidin binding peptide tag using
Nhel and BspEl. PARP13,ZnF and PARP13 RNA binding point mutants were
generated using GeneString (Invitrogen) flanked by Xhol/BstXl, which are
internal sites in PARP13. PARP13,ZnF features a deletion from nt228 to nt669.
The psiCHECK2 vector encoding Renilla and Firefly luciferase genes was
purchased from Promega. TRAILR4 ORF was purchased from Origene
(SC1 17708). A Sall site was introduced after the TRAILR4 stop codon using a
Gene String flanked by PpuMl and Scal, which are internal sites in TRAILR4
cDNA. The 3'UTR of TRAILR4 was then introduced downstream the Renilla
+
luciferase in psiCHECK2 using Sall/Xhol and Notl digestion. psiCHECK2
GAPDH 3'UTR is a kind gift from Dr. Benilde Jimenez. Truncations of TRAILR4
3'UTR were generated by PCR using primers with Xhol/Notl overhangs.
psiCHECK2+TRAILR4 3'UTR was used as a template. Fragments were
designed based on TRAILR4 3'UTR folding prediction (RNAFold) so as to
preserve high-probability folding structures (Lorenz et al., 2011).
69
Total RNA purification and Agilent microarrays
Total RNA purification was performed using Qiagen RNeasy Kit, following
manufacturer instructions. Samples were labeled using the Two Color Quick Amp
Labeling Kit (Agilent) following manufacturer protocol and hybridized on
SurePrint G3 Human Gene Expression v2 8x60 microarray. Microarrays were
scanned on SureScan Microarray Scanner (Agilent) and processed with Feature
Extractor v10.5. Microarrays have been submitted to GEO, NCBI; accession
number GSE56667.
CLIP
HeLa cells were UV crosslinked at 254 nm with 200mJ/cm 2 (Stratagene
Stratalinker). For endogenous PARP1 3 immunoprecipitation, cells were lysed in
CLIP Lysis Buffer (1% NP-40, 0.1% SDS, 150mM NaCl, 1mM EDTA, 50mM
TRIS (pH7.4), 1mM DTT), precleared at 16100 g, treated with RNaseA for 10min
at 37 C, immunoprecipitated overnight with PARP1 3 antibody and washed 2 X in
CLIP Lysis buffer containing 1 M NaCl. Bound RNA was labeled with
32
P and
detected using T4 polynucleotide kinase according to Leung et al (Leung et al.,
2011 b). For SBP-PARP1 3 precipitation, cells were UV crosslinked as described
above, lysed with Cell Lysis Buffer (150mM NaCl, 50mM HEPES (pH7.4), 1mM
MgC 2 , 0.5% Triton, 1mM EGTA, 1mM DTT ), precleared at 16100 g, incubated
with RNase A for 10 min at 37 C and bound to Streptavidin Sepharose beads
70
(GE Healthcare). RNA bound to SBP-PARP13 was labeled according to Leung et
al (Leung et al., 2011 b) and bound protein eluted with 4mM biotin.
CLIP qRT-PCR
Cells were UV-crosslinked at 254nM 200mV/cm 2 and lysed in 1% Triton, 125mM
KCI, 1 mM EDTA, 20mM HEPES pH7.9 under RNase-free conditions. SBPPARP13 and PARP13 mutants were immunoprecipitated using Streptavidin
Sepharose beads. After binding, beads were washed with lysis buffer
supplemented with 10tg/ml tRNA and 250mM KCl. Proteins were eluted in 4mM
Biotin, treated with Proteinase K, and RNA was purified using Trizol, following
manufacturer protocol. Input RNA was collected similarly from total lysate before
the immunoprecipitation step. cDNA was prepared from input and bound RNA as
described below.
qRT-PCR
cDNA was prepared using ViLo First Strand Kit (Life Technologies) and random
primers.1 tg of total RNA or all CLIP-bound RNA was used per reaction. 100ng of
cDNA was used for each qRT-PCR reaction. Sybr Select reagent (Life
Technologies) was used as directed and qRT-PCR was performed on a Roche
480 Light Cycler. Data analysis was performed as previously described (Livak
and Schmittgen, 2001), using the AAcT method. In all cases ACTB was used as
a normalizing control. For gene-specific qRT-PCR primers used in this
manuscript refer to table below.
71
Dual luciferase assays
HeLa cells were transfected with 50ng of psiCHECK2 constructs in 24-well plates.
48 h post transfection cells were lysed and lysates treated with the Pierce
Renilla-Firefly Dual Luciferase Assay Kit as per instructions (Thermo Scientific).
Firefly and Renilla luminescence was measured in white 96-well plates in a
Tecan Plate Reader (Magenta and Green, 1OOOms each). Renilla luminescence
signal was normalized to Firefly signal for each well. For all figures bars
represent averages of three individual 24-well plate wells; error bars represent
standard deviation.
Cell staining and microscopy
Cells were split onto glass coverslips 16 h before treatment. To induce
cytoplasmic stress, cells were incubated with 200M Sodium Arsenite for 45 min
at 37 C; control cells were left untreated. Unstressed cells were fixed in 4%
formaldehyde for 30 min then extracted with Abdil 0.5% Triton for 25 min.
Stressed cells were preextracted with HBS containing 0.1% Triton for 1min, then
fixed in 4% Formaldehyde in HBS for 30min. Blocking and staining was
performed as previously described (Vyas et al., 2013). Fixed cells were blocked
in Abdil (4% BSA, 0.1% Triton in PBS), then incubated with antibodies diluted in
Abdil for 45min each.
Survival assay
72
For proliferation assays, 5000 cells were plated in 96 well plates and incubated
with recombinant TRAIL the following day for 24 h. Proliferation was analyzed
with the Cell Proliferation Kit II (Roche) according to the manufacturer's
instructions and survival was calculated by normalizing treated to untreated cells.
For apoptosis assays, 40,000 cells were plated in 24 well plates and incubated
with recombinant TRAIL for 24 h. Cells were harvested with Trypsin and stained
with Annexin V-488 (Biolegend) and propidium-iodide (Sigma) in Annexin binding
buffer (10mM HEPES, 140mM NaCl, and 2.5mM CaCl
2,
pH 7.4) for 15 min at RT.
FACS analysis was performed on a FACScan instrument (BD) and cells negative
for Annexin V and propidium iodide considered as alive. For colony forming
assays, the indicated numbers of cells were plated in 12 well plates and grown
for 7 days in medium with TRAIL changed every second day. Colonies were
visualized by staining with 0.02% crystal violet (Sigma) in 50% methanol.
Electrophoretic Mobility Shift Assays
SBP-PARP13.1 and SBP-PARP13.1VYFHR were purified from HEK293 cells lysed
2
,
with Cell Lysis Buffer (CLB, 150mM NaCl, 50mM HEPES (pH7.4), 1mM MgC
0.5% Triton, 1 mM EGTA, 1 mM DTT ), precleared at 80000g, bound to
Streptavidin Sepharose beads (GE Healthcare). Beads were washed with CLB
containing 1 M NaCl, and proteins were eluted with 4mM Biotin in CLB, then
dialyzed overnight in 100mM KCI, 50mM TRIS, pH 7.5. Protein concentrations
were determined by Coomassie blue stain by comparison to a dilution series of
BSA, and by UV spectrophotometry.
73
Fragment 1 and Fragment E were PCR-amplified, in-vitro transcribed using T7
RNA polymerase, purified and end-labeled with T4 Polynucleotide Kinase and
32P
yATP as previously described (Huang and Yu, 2013).
EMSA binding reactions were performed for 1 h at 20C in 10mM Tris, pH 7.5,
1mM EDTA, pH 8, 0.1M KCl, 0.1mM DTT, 5% vol/vol Glycerol, 0.01mg/ml BSA,
0.4units/i RNAse inhibitor, 0.1 tg/ml tRNA with 2nM RNA and decreasing
amounts of protein. Reactions were loaded onto 8% TBE Urea gels, and run in
0.5X TBE at room temperature, then exposed to phosphor screen and scanned.
To calculate Kd, bands were quantified using ImageJ, fraction bound was
calculated, and data was fit to Hill's equation using IGOR Pro.
4-thiouridine labeling and mRNA decay measurements
Wild type and PARP13-/-A cells were incubated with 200 M 4-Thioruridine for 2h,
then growth media was changed and cells were collected immediately, and at
two hour intervals for 8 h. Total RNA was Trizol extracted at each time point and
newly transcribed RNA was biotin-labeled and purified as previously described
(Radle et al., 2013). In brief, newly transcribed RNA was labeled with biotinHPDP, RNA was repurified, and newly transcribed RNA was separated on
streptavidin-coated magnetic beads (Miltenyi). RNA was eluted with 100mM DTT,
and purified using MinElute Cleanup Kit (Qiagen).RT-qPCR was performed as
described above. TRAILR4 and GAPDH levels were normalized to ACTB for
74
each sample. Each time point represents an average of three independent
experiments; error bars show the standard deviation. Half life was calculated as
previously described (Chen et al., 2008). Half-life is an underestimate as
expression levels are normalized to ACTB levels, which are also decreasing
within this time-course (ACTB half life in HeLa cells is -8h (Leclerc et al., 2002).
DISC-IP
1x1 0A6 wild type or Parp13-1-A cells each were plated in two 10 cm plates for 2
days. Plates were washed once in DMEM (without FCS) and then incubated for
45 min in 2.5 ml DMEM without FCS and with or without 1 pg/ml Flag-TRAIL
(Axxora). After addition of 15ml cold PBS, cells were washed once with 15 ml
cold PBS and scraped with a rubber policeman in 1 ml lysis buffer (30mM
Tris/HCI pH7.4, 150mM NaCl, 5mM KCI, 10% Glycerol, 2mM EDTA and protease
inhibitors). After addition of 100pl Triton X-100, lysates were rotated 30 min at
40C and harvested by centrifugation (45 min, 40C, 15000g). The supernatant was
removed, added to 20pl magnetic Protein-G beads (Invitrogen), washed three
times in lysis buffer including Triton X-1 00 and rotated at 4 C overnight. After five
washes in lysis buffer including Triton X-100, beads were heated at 75 C for 10
min in 20pl loading buffer, subsequently loaded on a gel and blotted for the
indicated antibodies.
75
Caspase-8 processing
Wild type and PARP13- cells were plated in 6 wells and treated with His-TRAIL
for the indicated time periods. Cells were harvested, lysed and analyzed by
immunoblot with the indicated antibodies.
Reagents used in this manuscript
For reagents used please refer to Supplementary Table 1.
Acknowledgements
We thank Frank Solomon, Phil Sharp and Sejal Vyas for their helpful
comments and discussion about the manuscript and Tenzin Sangpo for technical
assistance. We thank Manlin Luo at the BioMicro Center and Charlie Whittaker of
the Bioinformatics facility in the Swanson Biotechnology Center at the David H.
Koch Institute for Integrative Cancer Research at MIT. We thank Dr. P. Svoboda
for the kind gift of shAgo2 HEK293 cells, and Dr. B. Jimenez for the kind gift of
psiCHECK2-GAPDH 3'UTR. This work was partially supported by Cancer Center
Support (core; grant P30-CA14051) and R01GM087465 from the National
Institutes of Health to PC, and a Frontier Research Program grant from Curt and
Kathy Marble to PC. Additional funding was provided while PC was a Rita Allen
and Sidney Kimmel Cancer Research Foundation scholar. FB was funded by a
Ludwig Postdoctoral Fellowship and TT was funded by an MIT School of Science
Fellowship in Cancer Research.
76
Author contributions
TT performed CLIP experiments, immunofluorescence, microarray analysis, and
assays related to TRAILR4 mRNA regulation by PARP13. FB performed TRAIL
apoptosis assays and DISC precipitations. TT, FB and PC designed experiments.
TT, FB and PC wrote the manuscript.
Statement of competing financial interests
The authors declare no competing financial interests.
Accession codes
Microarray data for control and PARP13 knockdowns has been submitted to
GEO, NCBI; accession number GSE56667.
77
00
r.
a
PARP13.1wvR
PARP13.lvw
PARPI3.R
PARP13.1
PARP13.1A
#
PARPI3.1Azn
0
-L
7
PARP13.1H
0.51 PARP13.IR
0.60 PARP13.1wvP
0.04 PARP13.1"vy"
0.27
0.49
F
(a
. OM
#1.00 PARP13.1
PARP"13
PARP13.2
PARP13.1
PARP13.1
No rmalbed RNA binding
Autoradlagram
PARP13.1
ra
LE
Autoradlogram
C
5.
10
0
0
0
-U
Ci)
ri
-h
('3
-U
-U
-U
cc
z
0*
0
0,
CD
F:*
-
S
Autoradiogram
Autoradlogram
Autoradiogram
Autoradiogram
*
7
+ SBP-PARP
13.2
SBP-PARP
13.1
PARP13
2.
47
+1Endogenous
0.1 gImIl1+
0.1AglmrI
*1gImI
PARP13.2
*IPARP13.1
0.13pglmI
Iligiml
0.13pigimi
CD
(A
-I
Ci.
Figurel. PARP13 binds cellular RNA. a, Autoradiogram of PARP13 CLIP
reactions performed using wild type (+/+) or PARP13-null (-/-) cells treated with
1 tg/ml or 0.1 3ptg/ml RNaseA. Triangle indicates molecular weight (MW) of
PARP13.1, circle indicates MW of PARP13.2. PARP13 immunoblot (IB) shown
below. b, Autoradiogram of CLIP reactions from SBP-PARP1 3.1 and PARP13.2
expressed and purified in wild type cells treated with 1 tg/ml RNaseA. PARP1 3
immunoblots shown below. c, Autoradiogram of SBP-PARP13.1 and SBPPARP13.2 CLIP reactions treated with 1ptg/ml or 0.1 tg/ml RNase A. PARP13
immunoblots are shown below. d, CLIP autoradiograms of endogenous PARP1 3,
SBP-PARP13.1 and SBP-PARP1 3.2 treated with 1 g/ml RNase A with or without
UV crosslinking (254nm, 200mJ). PARP13 immunoblots shown below. e,
Diagram of PARP13 isoforms and mutants. f, CLIP autoradiograms of SBPPARP13.1, PARP1 3 .1AZnF, PARP1 3.2 and PARP13.2AZnF precipitations treated
with 1 kg/ml RNase A. PARP1 3 immunoblot shown below. g, Autoradiogram of
wild type and mutant PARP13.1 CLIP reactions. PARP13 immunoblot shown
below (IB). Numerical values of 32 P signal normalized to protein levels shown
above; PARP1 3.1 RNA binding levels set to 1. h, Graph of 3 2 P signals
normalized to PARP13.1 protein levels for CLIP analysis shown in Fig. 1g.
79
No treatment
200sM sodium arsenite
Figure 2. Localization of PARP13.1, PARP13.2 and RNA binding mutants.
a,Immunofluorescence showing co-staining of exogenously expressed
PARP13.1, PARP13.1
ZnF,
PARP1 3. 1 VYFHR, PARP13.2, PARP13.2 ZnF or
PARP 13.2VYFHR in non-stressed (No Treatment) and stressed (200M Sodium
Arsenite) cells. Scale Bar = 20pm.
80
a
b
3
TNFRSFIOD
C
CCLS
OASL IFIT2
0
RARRFS3
0
"0
G0
0
.2
U
05.2
0.1
0
U.
0.2
0.3 0.4 0.5 0.6 0.7
p-value
0.8 0.9
1
Jaki
C
-
Z -
.
d
PARPI 3 siRNA vs control siRNA
N PARP13-1 vs wild type
-
siRNA:
PARP13
0
m 2>
pSTATI
.
m
aftif mb
116
97
pSTAT1
j
PARP13
5
GAPDH
CCL-5 TRAILR14
e
OASL IFIT2
RARRIES3
IFIT3
UPARP13^
* PARP13-^+ PARP13.1"
8
cc
3
2
U PARP13 A+ GFP
'
,2
"
* PARP13-'^+ PARP 3.1
9
0
TRAILR4
CCL5
OASL
IFIT2
Z%+r
0. V.
-
8
7
6
IFN-y
inhibitor
RARRES3 IFIT3
81
97
116
-97
Figure 3. PARP13 depletion results in misregulation of the transcriptome. a,
Volcano plot showing transcriptome-wide Log2 fold changes in mRNA
expression in PARP1 3 knockdowns relative to control knockdowns obtained via
Agilent array analysis of total mRNA (n=2 independent experiments). 6 of the
Top 10 upregulated transcripts are labeled. The remaining mRNA data shown in
the figure were obtained using qRT-PCR. b, Immunofluorescence of cell
expressing SBP-PARP13.1, SBP-PARP13.1 VYFHR or SBP-PARP13.2, stained
with anti-PARP13 and anti-SBP antibodies, and ER Tracker Red. In merge SBP
signal is in green, ER Tracker signal is in red, PARP13 signal is not shown. Scale
bar = 20im. c, Confirmation of CCL5, TRAILR4, OASL, IFIT2, RARRES3 and
IFIT3 upregulation in PARP13 knockdown relative to control knockdown and in
PARP1
3 /-A
HeLa cells relative to wild type cells (n=3 independent experiments,
bars represent SD) d, Left, Immunoblots of PARP13 and pSTAT1 in
untransfected cells, or cells transfected with control or PARP13-specific siRNA,
untreated or treated with 5 M Jak1 inhibitor; right, immunoblots of PARP1 3 and
pSTAT1 in wild type and PARP13-'- HeLa cells untreated or treated with
100units/ml IFNy. GAPDH shown as loading control. e, mRNA levels of TRAILR4,
CCL5, OASL, IFIT2, RARRES3 and IFIT3 in untransfected PARP13~/~ cells and
PARP13-/- cells expressing PARP13.1, PARP1 3 .1VYFHR or GFP (DNA transfection
control) relative to HeLa cells (n=3 independent experiments, bars represent SD).
82
a
b
6
-
06
3
>
a. a.oC.
4
sIRNA:
zw
c
7
PARP13
-
-
E
2-
2
731
TRAILR4
~0
0
0
GAPDH
PARP13
sIRNA
c
T-
a.
0.
-116
97
PARP13
TRAILR4
m
GAPAH
GAPDH~
31
CL
ix
d
&
SW480
HCT116
RPEI
2.5
+
~444
a siRNA:
i
97
PARP13
2
240.
4
3
2
siRNA:
PARP13
GAPDH ii
45
IJF97 7
5
0
i$
2
PARP13
I
GAPDHf
Ix40
E20
.
sIRNA:
u
1
0
PARPI3
sIRNA
e
*
-
.
GAPDH Wo-70.5 -
31
TRAILR4
0
7
J9
4
5
~ARP3.9
U
83
cr
siRNA:
2.5
2
-c
'0
PARP13
1
97
PARP131
GAPDH
40.5 -
GAPDH
PARP13
siRNA
-
PARP13
siRNA
797
4
Figure 4. PARP13 repression of TRAILR4 mRNA and protein levels is
dependent on its RNA binding. a, TRAILR4 mRNA and protein levels in
PARP13 knockdown relative to control. GAPDH shown as loading control. n=3
independent experiments, error bars show SD. b, TRAILR4 mRNA and protein
levels in PARP1 3-' cell lines relative to wild type cells. GAPDH shown as loading
control. n=3 independent experiments, error bars show SD. c, TRAILR4 protein
levels in wild type, PARP13-1-A, and PARP13-1-A cells expressing PARP13.1 or
PARP13.1
VYFHR.
GAPDH shown as a loading control. d, TRAILR4 mRNA levels
(Log2FC) in PARP13 knockdown relative to control knockdown in RPE1, SW480
and HCT1 16 cells (averages of n=3 parallel reactions (RPE1) or n=3
independent experiments (SW480 and HCT1 16) shown, error bars show SD).
Immunoblots show PARP13 knockdown; GAPDH shown as normalizing control.
e, TRAILR4 mRNA levels in cells treated with PARP13.1-specific and total
PARP13 specific siRNAs relative to control siRNAs (averages of n=3
independent experiments shown, error bars show SD, p>0.05 (n.s.), two-sided ttest comparing the two knockdowns). Immunoblots show PARP13.1 depletion
upon knockdown with PARP1 3.1 specific siRNA. GAPDH shown as loading
control.
84
b
a
0
6
2 5-
Wild type
M PARP13"
1.4
-1.*
1.2-
2
-
M 0.8* 0.6
0
3 .
0.2
-
1
Exon1/
Intron6 Introni
Exon3
Exon7
Exon9
Empty
GAPDH
TRAILR4
vector
3'UTR
3'UTR
C
V AU-rich element
"%P
32208382495744
673
I1
2 u
3
4
0.6
1635
1051-1081
10_
907
1357
185
0.5
0 3
22
0 .21
-!I1331b)
***
0.1
0
.
Fragment A
UT
0.
.
42
Fragment B
Fragment C
Fragment D
>
0
Fragment E
4
M
-w
E
E
E
Uj
-
Fragment
Fragment
Fragment
Fragment
0 ZAP responsive element
mRNA binding site
-
Regulated
Mfwwl Unregulated
IL.
-f
E
E
E
E
E
U..
.
U.
U.
IL.
xuM U.L.
f
d
52.5
aFragment
L4f
S2.5
2
Fragment I (2nM)
E (2nM)
qf 4. 0
1.5
PARP13.1
c .-
PARP1 3.1F
w.
-
-.
>R 3
E
S1.5
S0.5I-
0
Input
Bound
0
Input
Bound
Buffer Only
85
4..
11111 g.
weo
g.66
Figure 5. PARP13 represses TRAILR4 mRNA posttranscriptionally by
binding to a specific region in its 3'UTR. All RNA quantitation performed using
qRT-PCR a, TRAILR4 mature RNA (Exon1/Exon3 primer) and pre-mRNA
(Intron6-Exon7, Intron 8/Exon9 primers) levels in PARP13 knockdown relative to
control knockdown (mean of n=3 independent experiments, error bars show SD).
b, Normalized Renilla/Firefly luminescence for psiCHECK2 empty vector,
psiCHECK2 expressing Renilla-GAPDH 3'UTR (GAPDH 3'UTR), and
psiCHECK2 expressing Renilla-TRAILR4 3'UTR (TRAILR4 3'UTR) expressed in
wild type or PARP1 3 -/-A cells (mean of n=3 independent experiments, error bars
represent SD, p<0.01 (**), two sided t-test). c, Left top, diagram of RenillaTRAILR4 3'UTR construct identifying AU-rich element (ARE), ZAP responsive
element (ZRE), and miRNA binding sites for miR-1 33; triangle shading indicates
relative length of motif- darker shades correspond to longer motifs. Specific ARE
sequences and locations are shown in Supplementary Fig. 5. Left bottom,
fragments used in 3'UTR destabilization assay. Blue fragments exhibited
PARP13-dependent destabilization whereas red fragments were not regulated.
Right, relative PARP13-dependent destabilization for each 3'UTR fragment,
represented by fraction increase of normalized Renilla luminescence in PARP13/-A
cells relative to wild type cells, (means of n=3 independent experiments, error
bars show SD, asterisks represent significance relative to empty vector, p<0.05
(*), p<0.01 (**) and p<0.001 (***), two-sided t-test) d, Fold enrichment (Log2) of
TRAILR4 mRNA in input and bound fraction in PARP1 3.1 CLIP reactions relative
to PARP1 3 .1VYFHR reactions (mean of n=3 independent experiments, error bars
86
show SD, p<0.01 (**), two-sided t-test). PARP1 3 immunoblot shows precipitated
protein levels. e, Relative log2 levels of TRAILR4 mRNA in input and bound
fractions in PARP13.1 CLIP reactions relative to PARP13.1AZnF reactions
(averages of n=3 independent experiments, bars show SD, p<0.05 (*) relative to
PARP13.1^ZnF, two-sided t-test). PARP13 Immunoblot of precipitated protein
shown at right. f, Electrophoretic Mobility Shift Assays (EMSA) of decreasing
amounts of PARP13.1 and PARP1 3 .1VYFHR (from 533nM to 71nM, in 25%
interval decrease) with radiolabeled Fragment E and Fragment 1 (experiment
was repeated 3 times with similar results). Right, Coomassie stain showing equal
protein concentration of PARP13.1 and PARP13.1VYFHR
87
a
C
b
2
0-
0 PARP13
~1 5
.1-2
0.5
E
x
siRNA:
XRNI
4XZ -3
z'
-C
n.s.
x
sIRNA:
sRNA
1
R0A
siRNA:
IX 3
45 J52.5
*1.5
1
.9 0.5
GAPDH
-6
.7
EXOSC5 XRNI
Cr w
E 4
2
r---P2O' 3.5
0
-0.5
PARP13
+Tet
-Tet
EXOSC5
Empty vector
TRAILR4
n.s.
I
0.8
0.6
0.4
0.2
0
sIRNA:
3'UTR
1.2
*
1.2
1
0.8
0.6
0.4
0.2
Control
EXOSCS
XRN1
Control
XRNI
EXOSCS
4-
42
4
S-2E
-
E-2
-4
-t
08
I-
4
2
4
Time (h)
-
-U-PARP13 A
0
- Wild type
4
*PARP13
.
+-Wild type
=4
-8
6
8
2
0
88
2
CL0.U 0
- OM-
97
-66
& 0.4
. Wild typo
MPARP13"
+Tat
-
Ago2
sIRNA
d
.et
--
E Control siRNA
4
Time (h)
6
8
GAPDH
5
Figure 6. PARP13 destabilization of TRAILR4 mRNA is exosome dependent.
a, TRAILR4 mRNA levels in EXOSC5 and XRN1 knockdowns relative to control
knockdown (means of n=3 independent experiments, bars show SD, asterisks
represent significance relative to control siRNA, p<0.05 (*), p>0.05 (n.s.), twosided t-test). b, Left, EXOSC5 mRNA levels in EXOSC5 knockdown relative to
control knockdown (bars show SD, n=3 independent experiments); right,
immunoblot showing XRN1 protein levels in control and XRN1 knockdown.
GAPDH is shown as loading control. c, Relative TRAILR4 mRNA levels in Tettreated or untreated HEK293 cells expressing Tet-inducible Ago2 shRNA, treated
with control or PARP1 3-specific siRNA (averages of n=3 parallel reactions, error
bars show SD). Immunoblots of PARP13, Ago2 and GAPDH (loading control)
shown at right. d, Bar graphs showing normalized Renilla luminescence for
empty vector and Renilla-TRAILR4 3'UTR, expressed in wild type (red) or
PARP13-1-A cells (blue) treated with control siRNA or siRNA specific for EXOSC5
or XRN1. PARP1 3-dependent destabilization levels, calculated by substracting
normalized Renilla luminescence signal in wild type cells from signal in PARP1 3-'cells, is shown at left of the bars. (means of n=3 independent replicates, bars
show SD, asterisks represent significance relative to control siRNA
destabilization levels, p<0.001 (***), p>0.05 (n.s). e, Decay of GAPDH mRNA and
TRAILR4 mRNA is wild type and PARP13- cells measured by qRT-PCR of 4thiouridine incorporated and purified RNA. At each time point GAPDH and
TRAILR4 levels were normalized to ACTB levels. Levels at Time 0 were set as 0.
(means of n=3 independent experiments, error bars show SD, asterisks
89
represent significance relative to wild type levels for each time point, p<0.05(*),
p<0.01(**), two-sided t-test).
90
aE
.a ar.
Wild type
M Wild type + TRAILR4-Flag
,13.
100
PARP13
7
TRAILR4
31
GAPDH
46
7
PARP13
-80
a 60
TRAILRI
t 40
20
TRAILR2
No treatment TRAIL (lpg/ml)
L
*i
45
41
GAPDH -
C
100-
80
-sIRNA:
20
s
40-
Control
20
PARP13
.---
RNA:
z
I
%
.
~
-siRNA:
40
20
ontrol
--
60
4
-Control
-
-- PARP13t-4
TRAILR4+ PARP13
-
I
d
e
%
57*
0 PARP13
MPARP13 4
0 PARP13
2-
100
1
0-
80
-2 J
I
IL
siRNA:
M--I
.
jo, 1
]*
E
L
0.8
0.2
60
0
C1"
No treatment
')
0
ae
(1 lg/mi)
Wild type
PARP13"
0
120
1OOng/mI
-10
0
.Wild type
EPARP13"
PARP13 ^ + PARP13.1
-0PARP13" + PARP13.1"
PARP134 + PARP13.14z*
2000
0
-
1
w~0
l1 igiml TRAIL
>
0
0.8
04
S40
2000 1000
Cells plated
1.
M0
0
-
h
1000
1 glmI TRAIL
1.21
-
~ 0.4
g
TRAIL:
f
.Wild type
4
03E
TRAIL (ng)
TRAIL (ng)
TRAIL (ng)
No E .2
No treatment
TRAIL (I1jg/mI)
0.
M~
CL
91
.
0 40
20
HeLa
100
80
80
60-
, .PARPI
120
HCT116
SW480
-aj100
4.
Figure 7. PARP13 depletion results in resistance to TRAIL-mediated
apoptosis. a, Percent survival of untreated and TRAIL treated wild type cells and
wild type cells expressing TRAILR4-Flag assayed via Annexin-V/PI FACS
(average of n=3 independent experiments, bars represent SEM, p<0.05(*), twosided t-test). b, Immunoblot of TRAILR1-2 and TRAILR4 proteins in wild type and
PARP13-/-A cells. GAPDH is used as loading control. c, Survival assay
measuring proliferation of SW480, HCT1 16 and HeLa cells with or without
PARP13 knockdown after treatment with increasing concentrations of TRAIL for
24 h. Results are shown relative to untreated cells (means of n=3 independent
experiments, error bars show SEM). Results for double knockdown of TRAILR4
and PARP13 are shown for HeLa cells. d, TRAILR4 mRNA levels after PARP13
and PARP13+TRAILR4 knockdown relative to control knockdown (averages of
n=3 independent experiments, error bars show SD). e, Annexin-V/PI apoptosis
assays comparing the percent survival of wild type and three independent
PARP13~/~ cell lines (A, B, C) upon 1 pg/ml TRAIL treatment for 24 h (n=3
independent experiments, bars show SEM, asterisks show significance relative
to wild type, p<0.001 (***), two-sided t-test) f, Normalized survival of wild type
and three PARP13-'~ cell lines treated with 1pg/ml TRAIL relative to untreated
(averages of n=3 independent experiments, bars show SEM, asterisks show
significance relative to wild type, p<0.05 (*) or p<0.01(**), two-sided t-test). g,
Colony formation assay measured by crystal violet staining of wild type or
PARP13-/-A cells treated with or without the indicated amounts of TRAIL for 7
days. h, Annexin-V/PI apoptosis assays comparing the percent survival of wild
92
type, PARP13-/-A or PARP13-/-A cells expressing PARP13.1, PARP1 3 .1VYFHR or
PARP13.1
ZnF
upon treatment with or without 1 pg/ml TRAIL for 24 h. Data is
shown as % survival (means of n=3 independent experiments, bars show SEM,
asterisks show significance relative to wild type, p<0.05 (*), p<0.01 (**), p>0.05
(n.s.), two-sided t-test). i, Normalized survival of wild type, PARP13--A cells and
PARP13-/-A cells expressing PARP13.1, PARP1 3 .1VYFHR or PARP13.1 AZnF treated
with 1 pg/ml TRAIL relative to untreated (n=3 independent experiments, bars
show SEM, asterisks represent significance relative to wild type, p<0.05 (*),
p<0.01 (**), p<0.001 (***), two-sided t-test).
93
a
Wild type
0
PARP13-'A
2 4 8 16 24 0 2 4 8 16 24 Time (h)
b
P431p1-31 Caspae-8
2
p18-
5
TRAILRI
4
TRAILR2
045
Caspaue-6
'014
DISC IP
HeLa PARP131
-+
- + TRAIL (Ipg/mI)
Input
HeLa PARP13
- + - +
PARP13
PARP13
GAPDH
GAPDH
4m
M1iI*!TRAILR1
TRAILR2
Caspa-
66
97
High PARPI3
L;W PARP1,]
TRAILR4 mRNA
TRAIL
TRAIL
TRAIL
V%*
IITRALR2ALIIII
TRALRIWW
A
~,
TRAILRII
J
kev
TRAILR2
I
I
TRAILR4
I
APOPTOSIS
Figure 8.PARP13 depletion inhibits the formation of a functional DISC
complex. a, Immunoblot examining caspase-8 cleavage at various time points
after 1 pg/mI TRAIL treatment in wild type and PARP1 3'- cells. Arrows indicate
full-length (FL) caspase-8 and its cleavage products. GAPDH shown as loading
control. b, Flag-TRAIL pulldown of the TRAIL-receptor complex in wild type and
PARP1 3-/-A cells blotted for TRAILR1, R2 and caspase-8. Inputs for the reaction
are also shown. c, Model of PARP1 3 dependent TRAILR4 mRNA regulation and
its effects on TRAIL mediated apoptosis.
94
Table 1. PARP13 RNA binding mutants used in this manuscript
Mutant
Mutations
PARP13.1zn
Deletion AA 77-223
RP1 3.1H
H176A
PARP13.1R
R189A
PARP13.1vyF
V72A, Y108A, F144A
PARP13.1 VYFHH
V72A, Y1 08A, F144A, H1 76A, R1 89A
Table 2. Number of differentially expressed transcripts upon PARP13.1
depletion
Cut-off
Number of
% of total
Upregulated
Downregulated
genes
Log2FC>0.5
1841
5.066
1065
776
Log2FC>1
85
0.234
66
19
p-value<0.1
134
0.369
84
50
p-value<0.05
73
0.201
50
23
Log2FC>1 /
49
0.135
34
15
p-value<0.05
95
Supplementary figures and tables
PARP13
7
GAPDH
a.
C9
a.
Supplementary Figure 1. Characterization of PARP13~1~ cell lines. PARP13 and
GAPDH immunoblots (top) and PARP13 immunofluorescence staining of wild
type HeLa cells and three independently isolated PARP13~'~ cell lines (PARP13-A
PARP13-/-B and PARP13--c). Scale bar = 50mm.
96
Supplementary Figure 2. PARP13 colocalizes with elF3 at stress granules.
Costaining of exogenously expressed SBP-PARP1 3.1, PARP1 3.1 DZnF,
PARP1 3 .1VFHR, PARP13.2, PARP13.2DZnF or PARP1 3 .2 FHR (SBP, green) with
endogenous PARP13 (red) and the stress granule marker elF3 (blue) in wild type
cells treated with 200mM sodium arsenite. Scale bar = 20mm.
97
kvnn response I
hitsrfsron sgnsig
NE$w222
Supplementary Figure 3. Gene Set Enrichment Analysis (GSEA) plot identifying
enrichment of interferon pathway components among upregulated transcripts
with p<0.05. NES and FDR are reported.
98
4
*
n.s.
3.5
3
ed
2.5
2m
1.5
E
I
0.5
s
I-
0
Vi
4
4
Supplementary Figure 4. PARP1 3.1 but not PARP13.1^ZnF rescues TRAILR4
mRNA levels in PARP13-1-A cells.TRAILR4 mRNA levels in PARP13-' cells and
PARP13-1- cells expressing PARP13.1 and PARP13.1,ZnF relative to wild type
cells (averages of n=3 independent experiments, error bars show SD, asterisks
show significance relative to PARP13-'-, p<0.01 (**), p>0.05 (n.s.), two sided ttest).
99
TRAILR4 3'UTR
V ATMA
V VFrTVrTAWW
T W1TTATMW
V WW~s1#n*WW
V W/tATTTAMWW
VWWWV*ITTAW1W
V TVAlTYT?
Supplementary Figure 5. ARESITE derived schematic of AU-rich elements
present in the TRAILR4 3'UTR.
100
.. . ......
Fragment A
Fragment I
Fragment 2
Fragment B
Fragment 3
TRAILR4 3TUTR
Fragment C
Fragment 4
Fragment D
Fragment E
Supplementary Figure 6. RNAFold derived Minimum Folding Energy (MFE)
predictions of secondary structure for full length TRAILR4 3'UTR and 3'UTR
fragments used in the manuscript. Arrows point to fragment boundaries in the full
length 3'UTR.
101
SWild type
PARP13 4A4
0.8
0*0
-
1.2
-
0.6
0.4
0.2
0
00
0
-e
00
0
-
>
0.
00
00
0
0C
U. U.
U.
W
OE
..
I.
I.
U.
IA
E
U.
LU
Supplementary Figure 7. Normalized Renilla/Firefly luminescence for 3'UTR
fragment constructs in wild type and PARP13-/-A cells (averages of n=3
independent replicates shown, error bars show SD, asterisks represent
significance relative to PARP1 3-' for each fragment, p<0.05 (*), p<0.01(**),
p<0.001 (***), two-sided t-test)
102
6
NPARP13"
*PARP13"'
*PARP13"
5
Se4
%3
2
E
0
-1
-2
TRAILR4
TRAILRI
TRAILR2
TRAILR3
Supplementary Figure 8. Quantitation of TRAILR1 -R4 mRNA levels in the three
PARP134- cell lines relative to wild type cells (averages of n=3 independent
experiments, error bars show SD).
103
IL
-
ii
Figure If Fil" Ig
Figure id
Pigure it
Figure
3d
]I
-
Fgrei
t
IT
IIIJ!
lid
T
3d
41
3,
3d
a
Figue 4b
Figure 4M
TOWAM
P-AM4
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N6
"-a.
4'
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Imos
64
Fymi* b
Figure Sa
PINW. lb
PlAWI
TRAAJV
MI
Wil TYPe
PAAPI3Tu
*a 4 41 StG
110
TUM
tfti- O Ltc I
ThAJLRI
APIS1
Supplementary Figure 9. Full scans and molecular weight markers of important
immunoblots shown in this work.
104
Supplementary data
Supplementary data table 1. DAVID reports
Term
Category
SPPIRKEYWORDS
Secreted
Count
0/
value
20
205.1
5.14
0.00
0.00
Fold
Enrichment
Bonferroni
Benjamini
MATN2, CPA4, FST, MMP7, MGP,
SPOCKI, VIT, CCL5, SPINKS, CALU,
N
PSG8, SERPINE2, ISK5, P13,
CFH, ILB, GDF15, COL8A1, MFAP5
5.06
1.80E-07
1.80E-07
3.29
2.05E-06
1.03E-06
Genes
SPPIRKEYWORDS
signal
25
6.43
0.00
FST, MMP7, SPOCKI, VIT, CCL5,
SPINK5, CALU, NOV, SERPINE2, TEK,
CFH, CD24, GPNMB, COL8A1, NT5E,
MATN2, CPA4, MGP, HCST, PSG8,
TNFRSF10D, P13, RORI, GDF15, MFAP5
UPSEQFEATURE
signal peptide
25
6.43
0.00
FST, MMP7, SPOCKI, VIT, CCL5,
SPINK5, CALU, NOV, SERPINE2, TEK,
CFH, CD24, GPNMB, COL8A1, NT5E,
MATN2, CPA4, MGP, HCST, PSG8,
TNFRSF10D, P13, ROR1, GDF15, MFAP5
3.27
4.94E-06
4.94E-06
GOTERMCCFAT
GO:0044421~extracellular
region part
15
3.86
0.00
MATN2, MMP7, MGP, SPOCK1, VIT,
CCL5, NOV, SERPINE2, ISG15, P13, CFH,
IL1B, GDF15, COL8A1, MFAP5
5.55
8.18E-06
8.18E-06
GOTERMCC-FAT
GO:0005576~extracellular
region
20
5.14
0.00
MATN2, CPA4, FST, MMP7, MGP,
SPOCKI, VIT, CCL5, SPINK5, CALU,
NOV, PSG8, SERPINE2, I5G15, P13,
CFH, ILIB, GDF15, COL8A1, MFAP5
3.53
1.51E-05
7.56E-06
GOTERMCCFAT
GO:0031012~extracellular
matrix
9
2.31
0.00
NOV, MATN2, P13, MMP7, MGP, SPOCK1,
VIT, COL8A1, MFAP5
9.26
0.0003
0.0001
SPPIRKEYWORDS
disulfide bond
20
5.14
0.00
MATN2, CPA4, HS3ST2, FST, MGP,
SPOCK1, VIT, CCL5, SPINK5, HCST,
NOV, PSG8, TNFRSF10D, TEK, P13, CFH,
ROR1, GDF15, MFAP5, NT5E
2.92
0.0012
0.0004
GOTERMCCFAT
GO:0005578~proteinaceou
s extracellular matrix
8
2.06
0.00
MATN2, P13, MMP7, MGP, SPOCK1, VIT,
COL8A1, MFAP5
8.88
0.0020
0.0005
2.28
0.0079
0.0020
SPPIRKEYWORDS
glycoprotein
23
5.91
0.00
MATN2, CPA4, HS3ST2, FST, SPOCK1,
CCL5, VIT, TMEM2, CALU, HCST, NOV,
PSG8, SERPINE2, TNFRSF10D, TPPP,
TEK, CFH, ROR1, CD24, GPNMB, GDF15,
MFAP5, NT5E
UPSEQFEATURE
disulfide bond
17
4.37
0.00
MATN2, CPA4, HS3ST2, FST, MGP,
SPOCKI, VIT, CCL5, SPINK5, NOV,
PSG8, TNFRSF10D, TEK, P13, CFH,
ROR1, GDF15
2.56
0.0933
0.0478
SPPIR KEYWORDS
extracellular matrix
5
1.29
0.00
MMP7, SPOCK1, VIT, COL8A1, MFAP5
8.87
0.2688
0.0607
MMP7, IL1B,
4.15
0.1904
0.0414
14.18
0.8540
0.8540
GOTERMCCFAT
GOTERM
FAT
cGO:0005615-extracellular
utspace
8
82.06
2.0
0.00
0.00
CFH,
ISG15,
SERPINE2,
CCL5
MGP, GDFiS,
GOTERMBP FAT
GOTEMBPFAT
GO: 0009615- response to
virus
4
1.03
0.00
DDX58, ISG15, IFI44, CCL5
UPSEQFEATURE
glycosylation site: N-linked
(GlcNAc...)
19
4.88
0.00
MATN2, CPA4, HS3ST2, FST, VIT,
TMEM2, CALU, NOV, PSG8, SERPINE2,
TNFRSF10D, TEK, CFH, ROR1, CD24,
GPNMB, GDF15, MFAP5, NT5E
1.95
0.5936
0.2593
GOTERM BP FAT
GO:0006952~defense
response
7
1.80
0.00
DDX58, PSG8, HIST1H2BK, CFH, IL1B,
CD24, CCL5
4.40
0.9481
0.7722
UPSEQFEATURE
domain:Ubiquitin-like 2
2
0.51
0.00
OASL, ISG15
424.73
0.7503
0.2931
UPSEQFEATURE
domain:Ubiquitin-like 1
2
0.51
0.00
OASL, ISG15
424.73
0.7503
0.2931
INTERPRO
IPR002350:Proteinase
inhibitor 11, Kazal
3
0.77
0.01
FST, SPOCK1, SPINK5
26.50
0.5134
0.5134
105
TEK, MGP, SPOCK1, CD24, GPNMB,
COL8A1, CCL5
3.87
0.9961
0.8425
1.80
TEK, MGP, SPOCK1, CD24, GPNMB,
COL8A1, CCL5
3.86
0.9962
0.7524
GO:0050900~leukocyte
migration
0.77
IL1B, CD24, CCLS5
20.34
0.9989
0.7446
UPSEQ FEATURE
domain:Kazal-like 3
0.51
FST, SPINK5
212.37
0.9377
0.4259
KEGGPATHWAY
hsa04623:Cytosolic DNAsensing pathway
0.77
DDX58, IL1B, CCLS5
18.49
0.2444
0.2444
GOTERMBP_FAT
GO: 0045785-positive
regulation of cell adhesion
0.77
IL1B, CD24, COL8A1
19.33
0.9995
0.7153
SMART
SM00280:KAZAL
0.77
FST, SPOCK1, SPINK5
18.50
0.3820
0.3820
GOTERMBPFAT
GO:0045765-regulation of
angiogenesis
I
0.77
TEK, ILIB, SPINK5
18.41
0.9997
0.6937
GOTERMBPFAT
GO:0009617~response to
bacterium
1.03
HIST1H2BK, IL1B, CD24, CCL5
8.01
0.9999
0.6918
SPPIRKEYWORDS
Serine protease inhibitor
0.77
SERPINE2, P13, SPINK5
16.03
0.8731
0.2911
GOTERMBPFAT
GO:0002544-chronic
inflammatory response
0.51
ILIB, CCL5
128.84
1.0000
0.7192
GOTERMBPFAT
GO:0050727~regulation of
inflammatory response
0.77
CD24, CCL5, NT5E
15.26
1.0000
0.6949
PIRSUPERFAMILY
PIRSFOO5680: interferoninduced 56K protein
0.51
IFIT3, IFIT2
118.34
0.3657
0.3657
GOTERMBPFAT
GO:0007267~cell-cell
signaling
1.54
ISG15, TEK, IL1B, CD24, GDF15, CCLS5
3.87
1.0000
0.6746
UP SEQFEATURE
short sequence motif:Cell
attachment site
0.77
PSG8, GPNMB, MFAP5
14.48
0.9951
0.5874
UPSEQFEATURE
domain:Kazal-like 2
0.51
FST, SPINK5
106.18
0.9961
0.5475
GOTERMBPFAT
GO:0030005-cellular di-,
tri-valent inorganic cation
homeostasis
1.03
MT2A, ILIB, CD24, CCL5
6.81
1.0000
0.7024
GOTERMBPFAT
GO:0002237biresponse to
molecule of bacterial origin
0.77
IL1B, CD24, CCL5
13.48
1.0000
0.6850
GOTERMMFFAT
GO:0004867~serine-type
endopeptidase inhibitor
activity
0.77
SERPINE2, P13, SPINKS
13.23
0.8676
0.8676
GOTERMBPFAT
GO: 000 1934~ positive
regulation of protein amino
acid phosphorylation
0.77
IL1B,
13.03
1.0000
0.6817
GOTERMBPFAT
GO: 0048584~ positive
regulation of response to
stimulus
1.03
CFH, ILIB, CD24, CCL5
6.55
1.0000
0.6586
GOTERMBPFAT
GO:0055066~di-, trivalent inorganic cation
homeostasis
1.03
MT2A,
CD24, CCL5
6.47
1.0000
0.6472
GOTERMBPFAT
GO: 0051240~ positive
regulation of multicellular
organismal process
1.03
DDX58, FST, IL1B, CCLS5
6.34
1.0000
0.6449
GOTERM.BPFAT
GO:0042327~positive
regulation of
phosphorylation
0.77
ILIB, CD24, FAM129A
11.95
1.0000
0.6492
GOTERMBPFAT
GO:0007155~cell adhesion
GOTERMBPFAT
GO: 0022610~ biological
adhesion
GOTERMBP-FAT
7
1.80
0.01
106
CD24, FAM129A
IL1B,
SPPIR KEYWORDS
protease inhibitor
3
0.77
0.02
SERPINE2, P13, SPINK5
11.98
0.9722
0.4006
GOTERMBPFAT
GO:0030003~cellular
cation homeostasis
4
1.03
0.03
MT2A, IL1B, CD24, CCL5
6.09
1.0000
0.6428
GOTERMBPFAT
GO: 0045937~positive
regulation of phosphate
metabolic process
3
0.77
0.03
IL1B, CD24, FAM129A
11.60
1.0000
0.6315
GOTERM_BP_FAT
GO: 00 10562~ positive
regulation of phosphorus
metabolic process
3
0.77
0.03
IL1B, CD24, FAM129A
11.60
1.0000
0.6315
GOTERM_BP_FAT
GO:0048585~negative
regulation of response to
stimulus
3
0.77
0.03
ILlB, NT5E, SPINK5
11.60
1.0000
0.6315
GOTERMBPFAT
GO:0002697~regulation of
immune effector process
3
0.77
0.03
DDX58, IL1B, CD24
11.48
1.0000
0.6204
GOTERMBPFAT
GO: 0022409~positive
regulation of cell-cell
adhesion
2
0.51
0.03
ILlB, CD24
70.28
1.0000
0.6144
GOTERM_BPFAT
GO:0006955~immune
response
6
1.54
0.03
DDX58, OASL, CFH, ILlB, CD24, CCL5
3.36
1.0000
0.6041
1.26
0.9823
0.3959
SP PIRKEYWORDS
polymorphism
34
8.74
0.03
RARRES3, HS3ST2, FST, MMP7, CCL5,
SPINK5, CALU, NOV, MACF1, ISG15,
SERPINE2, TEK, CFH, CD24, GPNMB,
FAM129A, NT5E, MATN2, CPA4, MGP,
IFI44, TMEM2, DDX58, IFIT2, OASL,
PSG8, TNFRSF10D, DOK7, P13, MT2A,
ROR1, GDF15, MFAP5, GSTP1
GOTERM_BP_FAT
GO:0006928~cell motion
5
1.29
0.03
MACF1, IL1B, SPOCK1, CD24, CCL5
4.07
1.0000
0.6209
GOTERM_BPFAT
GO:0055080~cation
homeostasis
4
1.03
0.03
MT2A, IL1B, CD24, CCL5
5.41
1.0000
0.6550
GOTERM_BP_FAT
GO:0050863~regulation of
T cell activation
3
0.77
0.03
ILlB, CD24, SPINK5
9.91
1.0000
0.6429
UPSEQFEATURE
region of
interest:Substrate binding
3
0.77
0.04
CPA4, HS3ST2, NT5E
9.80
1.0000
0.7489
UPSEQ_FEATURE
domain:VWFA 1
2
0.51
0.04
MATN2, VIT
53.09
1.0000
0.7088
GOTERMBPFAT
GO:0002711~-positive
regulation of T cell
mediated immunity
2
0.51
0.04
ILIB, CD24
51.54
1.0000
0.6532
UPSEQFEATURE
domain:VWFA 2
2
0.51
0.04
MATN2, VIT
49.97
1.0000
0.6927
SPPIRKEYWORDS
blocked carboxyl end
2
0.51
0.04
CD24, NT5E
47.49
0.9971
0.4783
GOTERM_BP_FAT
GO:0042127~regulation of
cell proliferation
6
1.54
0.05
RARRES3, TEK, MMP7, IL1B, CD24,
GPNMB
2.95
1.0000
0.7131
GOTERMBPFAT
GO:0030155-regulation of
cell adhesion
3
0.77
0.05
IL1B, CD24, COL8A1
8.46
1.0000
0.7095
GOTERM_MFFAT
GO:0004866~endopeptida
se inhibitor activity
3
0.77
0.05
SERPINE2, P13, SPINK5
8.39
0.9912
0.9060
GOTERMBP_FAT
GO:0006954~inflammator
y response
4
1.03
0.05
CFH, ILB, CD24, CCL5
4.76
1.0000
0.7073
SPPIRKEYWORDS
serine proteinase inhibitor
2
0.51
0.05
SERPINE2, P13
38.86
0.9992
0.5112
107
GOTERM_BP_FAT
GO:0050778~positive
regulation of immune
response
3
0.77
0.05
CFH, IL1B, CD24
8.00
1.0000
0.7229
GOTERMMFFAT
GO:0030414-'peptidase
inhibitor activity
3
0.77
0.05
SERPINE2, P13, SPINK5
7.96
0.9946
0.8244
GOTERM BP.FAT
GO:0002709~regulation of
T cell mediated immunity
2
0.51
0.05
IL1B, CD24
36.81
1.0000
0.7138
GOTERM_BPFAT
GO:0051249~regulation of
lymphocyte activation
3
0.77
0.05
IL1B, CD24, SPINKS
7.83
1.0000
0.7140
GOTERM_BPFAT
GO: 0007167~ enzyme
linked receptor protein
signaling pathway
4
1.03
0.05
FST, TEK, ROR1, GDF15
4.52
1.0000
0.7083
GOTERM_BPFAT
GO:0022407~regulation of
cell-cell adhesion
2
0.51
0.05
IL1B, CD24
35.14
1.0000
0.6983
SPPIRKEYWORDS
inflammation
2
0.51
0.06
ILIB, CCL5
34.20
0.9997
0.5227
GOTERMMF_FAT
GO:0008083~growth
factor activity
3
0.77
0.06
NOV, IL1B, GDF15
7.56
0.9967
0.7612
GOTERM_BP_FAT
GO:0032101~"regulation of
response to external
stimulus
3
0.77
0.06
CD24, CCL5, NT5E
7.29
1.0000
0.7295
1.20
1.0000
0.8185
34
8.74
0.06
RARRES3, HS3ST2, FST, MMP7, CCL5,
SPINK5, CALU, NOV, MACF1, ISG15,
SERPINE2, TEK, CFH, CD24, GPNMB,
FAM129A, NT5E, MATN2, CPA4, MGP,
IFI44, TMEM2, DDX58, IFIT2, OASL,
PSG8, TNFRSF10D, DOK7, P13, MT2A,
ROR1, GDF15, MFAP5, GSTP1
GO: 0008285~ negative
regulation of cell
proliferation
4
1.03
0.06
RARRES3, ILIB, CD24, GPNMB
4.28
1.0000
0.7262
INTERPRO
IPRO11497:Protease
inhibitor, Kazal-type
2
0.51
0.06
SPOCK1, SPINK5
30.10
0.9998
0.9863
GOTERMBPFAT
GO: 0046888~negative
regulation of hormone
secretion
2
0.51
0.06
FST, IL1B
29.73
1.0000
0.7287
GOTERM_BPFAT
GO:0009725~response to
hormone stimulus
4
1.03
0.06
IL1B, MGP, CD24, CCL5
4.21
1.0000
0.7220
SPPIR.KEYWORDS
cytokine
3
0.77
0.06
ILIB, GDF15, CCL5
7.08
0.9999
0.5451
GOTERM_MF_FAT
GO:0005509~calcium ion
binding
6
1.54
0.06
MATN2, MACF1, MMP7, MGP, SPOCKI,
CALU
2.65
0.9986
0.7327
GOTERMBP.FAT
GO:0002694~regulation of
leukocyte activation
3
0.77
0.07
IL1B,
6.99
1.0000
0.7198
GOTERM_BP_FAT
GO:0006873~cellular ion
homeostasis
4
1.03
0.07
MT2A, IL1B, CD24, CCL5
4.13
1.0000
0.7208
SPPIRKEYWORDS
phosphatidylinositol linkage
2
0.51
0.07
CD24, NT5E
27.58
1.0000
0.5398
GOTERM_BP_FAT
GO:0055082-cellular
chemical homeostasis
4
1.03
0.07
MT2A, IL1B, CD24, CCL5
4.07
1.0000
0.7262
GOTERM_BP_FAT
GO:0001932-regulation of
protein amino acid
phosphorylation
3
0.77
0.07
IL1B, CD24, FAM129A
6.70
1.0000
0.7207
GOTERMBPFAT
GO:0002831~-regulation of
response to biotic stimulus
2
0.51
0.07
DDX58, CD24
26.66
1.0000
0.7155
GOTERMBPFAT
GO:0050865~regulation of
cell activation
0.77
0.07
IL1B, CD24, SPINK5
6.63
1.0000
0.7118
UPSEQFEATURE
sequence variant
GOTERM_BP_FAT
108
CD24, SPINK5
GOTERMBP_FAT
GOTERM_.BP_FAT
GO:0050729~positive
regulation of inflammatory
response
GO: 0002824~ positive
regulation of adaptive
immune response based on
somatic recombination of
immune receptors built
from immunoglobulin
superfamily domains
2
0.51
0.07
CD24, CCL5
25.77
1.0000
0.7117
2
0.51
0.07
IL1B, CD24
25.77
1.0000
0.7117
UPSEQ_FEATURE
Aregion:
compositionally biased
Poly-Val
0.51
0.07
TNFRSF10D, VIT
25.74
1.0000
0.8518
GOTERM_BP_FAT
GO:0002821~positive
regulation of adaptive
immune response
0.51
0.08
IL1B, CD24
24.94
1.0000
0.7157
GOTERMBPFAT
GO:0001817~regulation of
cytokine production
0.77
0.08
DDX58, IL1B, CD24
6.41
1.0000
0.7104
GOTERM_BP_FAT
GO:0051130-positive
regulation of cellular
component organization
0.77
0.08
TPPP, IL1l, CD24
6.41
1.0000
0.7104
GOTERMBP_ FAT
GO:0006874~cellular
calcium ion homeostasis
0.77
0.08
IL1B, CD24, CCL5
6.34
1.0000
0.7101
GOTERM_MF FAT
GO:0005125~cytokine
activity
0.77
0.08
IL1B, GDF15, CCL5
6.24
0.9997
0.7408
GOTERM_BP_FAT
GO:0031401~positive
regulation of protein
modification process
0.77
0.08
IL1B, CD24, FAM129A
6.20
1.0000
0.7168
GOTERM_BP_FAT
GO:0009719~response to
endogenous stimulus
1.03
0.08
IL1B, MGP, CD24, CCL5
3.82
1.0000
0.7125
GOTERMBPFAT
GOTEMBPFAT
GO:0055074~calcium ion
homeostasis
0.77
0.08
IL1B, CD24, CCL5
6.17
1.0000
0.7061
GOTERM_BP_FAT
GO: 0002708~ positive
regulation of lymphocyte
mediated immunity
0.51
0.08
IL1B, CD24
22.74
1.0000
0.7058
GOTERMBP_FAT
GO: 0002705~positive
regulation of leukocyte
mediated immunity
0.51
0.08
IL1B, CD24
22.74
1.0000
0.7058
GOTERMBPFAT
GO:0050801~ion
homeostasis
1.03
0.08
MT2A, ILIB, CD24, CCL5
3.78
1.0000
0.7005
GOTERM BPFAT
GO:0048545~response to
steroid hormone stimulus
0.77
0.08
ILlB, CD24, CCL5
6.04
1.0000
0.6995
GOTERM_BPFAT
GO: 0031334~ positive
regulation of protein
complex assembly
0.51
0.08
TPPP, CD24
22.09
1.0000
0.6964
GOTERM BPFAT
GO:0006875~cellular
metal ion homeostasis
0.77
0.09
IL1B, CD24, CCL5
5.92
1.0000
0.6999
GOTERMBP FAT
GO:0030595~leukocyte
chemotaxis
0.51
0.09
IL1B, CCL5
20.89
1.0000
0.7038
KEGG PATHWAY
hsa05020:Prion diseases
0.51
0.09
IL1B, CCL5
19.37
0.9397
0.7544
GOTERMBPFAT
GO:0055065~ metal ion
homeostasis
0.77
0.09
ILlB, CD24, CCL5
5.66
1.0000
0.7162
GOTERMBPFAT
GO:0060326~cell
chemotaxis
0.51
0.09
ILIB, CCL5
19.82
1.0000
0.7106
GOTERM_BPFAT
GO: 0042102~ positive
regulation of T cell
proliferation
0.51
0.09
IL1B, CD24
19.82
1.0000
0.7106
GOTERMBPFAT
GO:0006916~antiapoptosis
0.77
0.09
TNFRSF10D, ILUB, GSTP1
5.63
1.0000
0.7074
109
SPPIRKEYWORDS
duplication
3
0.77
0.10
PSG8, FST, TEK
5.62
1.0000
0.6397
UPSEQFEATURE
propeptide: Removed in
mature form
3
0.77
0.10
ISG15, CD24, NT5E
5.56
1.0000
0.9070
UPSEQFEATURE
site: Reactive bond
2
0.51
0.10
SERPINE2, SPINK5
18.88
1.0000
0.8927
110
Supplementary data table 2. GSEA reports
NAME
SIZE
ES
NES
NOM
p-val
FDR
q-vat
FWER
p-val
RANK
AX
SEITZNEOPLASTICTRANSFORMATIONBY_8 P-DELETIONU P
18
0.81
3.58
0.000
0.000
0.000
120
NUYTTEN-NIPP1_TARGETSUP
55
0.47
3.09
0.000
0.000
0.000
441
ZHANGRESPONSETOIKKINHIBITORANDTNFUP
35
0.54
3.04
0.000
0.000
139
REACTOMECYTOKINESIGNALINGINIMMUNE_.SYSTEM
20
0.64
2.95
0.000
0.000
0.000
0.000
REACTOMEMEIOTICSYNAPSIS
20
0.64
2.93
0.000
0.000
0.000
441
NUYTTEN EZH2_TARGETSUP
90
21
17
0.36
0.61
0.65
2.89
2.87
0.000
0.000
0.000
0.000
0.000
0.000
451
441
2.87
0.000
0.000
0.000
320
19
0.64
2.86
0.000
0.000
0.000
441
24
0.59
2.85
0.000
0.000
0.001
162
19
18
0.64
0.65
2.85
2.83
0.000
0.000
0.000
0.000
0.001
0.002
441
120
REACTOMECHROMOSOMEMAINTENANCE
JOHNSTON EPARVBTARGETS_2_UP
REACTOMEDEPOSITIONOFNEWCENPACONTAININGNUCLEOSOMESA
TTHECENTROMERE
TAKEDATARGETSOFN UP98-HOXA9_FUSION_10DUP
REACTOMEPACKAGINGOFTELOMEREEN DS
LIANGSILENCED_BY_METHYLATION_2
120
53
0.41
2.75
0.000
0.000
0.004
274
REACTOMEMEIOSIS
REACTOMEAMYLOIDS
26
24
0.54
0.000
0.000
0.004
0.000
0.000
0.004
441
441
REACTOMETELOMEREMAINTENANCE
REACTOMERNAPOLI TRANSCRIPTION
20
0.55
0.60
2.75
2.74
2.69
0.000
0.000
0.005
441
25
35
0.53
0.48
2.68
2.67
0.000
0.000
0.000
0.000
0.007
0.007
441
244
24
0.55
2.67
0.000
0.000
0.007
441
0.53
2.66
0.000
0.000
0.008
441
REACTOMEMEIOTICRECOMBINATION
0.53
2.65
0.000
0.000
0.008
441
KINSEYTARGETSOFEWSR1_FLIIFUSIONDN
0.46
2.62
0.000
0.001
0.010
372
TAKEDATARGETSOFNUP98_HOXA9_FUSION_3DUP
0.53
2.62
0.000
0.001
0.011
120
SENGUPTANASOPHARYNGEALCARCINOMAWITHLMPlUP
0.47
2.62
0.000
0.001
0.012
163
FORTSCHEGGERPHF8_TARGETSDN
0.34
2.61
0.000
0.001
222
REACTOMETRANSCRIPTION
0.53
2.60
0.000
0.001
0.013
0.014
REACTOMEIMMUNESYSTEM
0.40
2.57
0.000
0.001
0.016
229
FURUKAWADUSP6_TARGETSPCI35_UP
TAKEDATARGETS_.OFN UP98_HOXA9_FUSION_8DU P
0.64
0.54
2.56
0.000
0.001
2.56
0.000
0.001
0.018
0.018
432
120
CHARAFEBREASTCANCERLUMINAL
VS-BASAL_DN
ZHOUINFLAMMATORYRESPONSELPSUP
REACTOMERNAPOLI PROMOTEROPENING
REACTOMERNAPOLIRNAPOLIIIANDMITOCHONDRIALTRANSCRIPT
ION
25
441
DEBIASIAPOPTOSISBYREOVIRUSINFECTIONUP
0.50
2.54
0.000
0.001
0.020
231
GRAESSMANN _APOPTOSIS BY SERUMDEPRIVATIONUP
SWEETLUNGCANCERKRASDN
0.44
2.53
0.000
0.001
0.022
0.39
2.51
0.000
0.001
0.028
183
435
COLINATARGETSOF4EBP1_AND_4EBP2
0.48
2.50
0.000
0.001
0.029
229
KEGGSYSTEMICLUPUSERYTHEMATOSUS
0.48
2.49
0.000
0.001
0.033
486
SENESEHDACiANDHDAC2_TARGETSDN
0.46
2.46
0.000
0.001
0.042
262
MAHAJANRESPONSETOIL1A_UP
0.59
2.42
0.000
0.002
0.060
174
HOSHIDALIVERCANCERSUBCLASSS3
2.38
0.000
0.003
REACTOMECELL.CYCLE
0.52
0.45
2.36
0.003
0.003
0.084
0.098
441
RODWELLAGINGKIDNEYUP
0.37
2.35
0.000
0.003
0.101
139
BOQUESTSTEMCELLUP
WANGSMARCE1_TARGETS-UP
0.42
2.35
0.002
0.003
0.102
0.41
2.35
0.000
0.003
0.107
199
145
0.52
2.32
0.000
0.003
0.119
292
TONKSTARGETSOFRUNX1_RUNX1T1_FUSIONMONOCYTE_UP
0.52
0.53
2.31
2.30
0.000
0.002
0.004
0.004
0.136
0.142
415
120
SENESEHDAC2_TARGETSDN
0.52
2.30
0.000
0.004
0.150
374
SABATES-COLORECTALADENOMADN
0.44
2.29
0.000
0.004
HELLERSILENCEDBYMETHYLATIONUP
0.40
2.29
0.000
0.004
0.161
0.165
420
325
SCHUETZBREASTCANCERDUCTALINVASIVEUP
0.37
2.27
0.000
0.005
0.179
246
BAELDEDIABETICNEPHROPATHYDN
0.39
2.27
0.37
2.26
0.005
0.005
0.187
SENESEHDAC3_TARGETSUP
MCLACHLAN DENTALCARIESDN
0.000
0.000
0.188
835
140
0.40
2.23
0.002
0.006
0.238
175
MISSIAGLIAREGU LATEDBYMETHYLATIONU P
0.49
0.35
2.23
2.20
0.002
0.000
0.006
0.008
0.243
0.300
139
121
REACTOMEINNATEIMMUNESYSTEM
MONNIER-POSTRADIATIONTUMORESCAPE
UP
FULCHER-INFLAMMATORYRESPONSELECTINVSLPSDN
TAKEDATARGETSOF NUP98_HOXA9_FUSION_16DUP
139
0.52
2.20
0.000
0.008
0.307
20
CHARAFEBREASTCANCERLUMINALVSMESENCHYMALDN
MARKEYRB1_ACUTELOFU P
0.36
0.40
2.19
0.002
0.008
889
0.38
0.000
0.000
0.009
WALLACEPROSTATECANCERRACEUP
2.17
2.16
0.309
0.367
0.009
0.380
175
111
229
HORIUCHI-WTAPTARGETSUP
31
0.39
2.15
0.000
0.009
0.386
127
KIMGLIS2_TARGETSUP
KUMARTARGETSOFMLLAF9-FUSION
16
31
0.52
0.39
2.14
2.14
0.000
0.002
0.010
0.010
0.409
0.414
316
187
GRAESSMANNRESPONSETOMCANDSERUMDEPRIVATION
UP
UP
ACEVEDOLIVERCANCER
JISON-SICKLE_CELLDISEASE_.UP
DEURIGTCELL PROLYMPHOCYTICLEUKEMIADN
15
0.54
2.14
0.000
0.010
0.419
129
42
19
0.35
0.48
2.13
2.13
0.003
0.002
0.011
0.011
0.436
0.439
189
231
17
0.48
2.08
0.005
0.015
0.564
246
KRIEGKDM3ATARGETSNOTHYPOXIA
17
0.49
2.08
0.000
0.015
0.570
570
KONDOEZH2_TARGETS
24
0.41
2.06
0.006
0.017
0.617
153
HANSATB1_TARGETSUP
MCLACHLAN DENTALCARIESUP
40
0.34
2.06
0.006
0.017
0.625
703
30
0.38
2.05
0.002
0.017
0.639
175
LUEZH2_TARGETSUP
22
0.44
2.05
0.002
0.018
0.655
236
RIGGIEWINGSARCOMAPROGENITORUP
38
0.35
2.04
0.003
0.019
0.679
445
OKUMURAINFLAMMATORYRESPONSELPS
18
0.45
2.04
0.005
0.018
HANSATBiTARGETSDN
48
0.33
2.03
0.002
0.020
0.679
0.719
227
140
HOSHIDALIVERCANCERSUBCLASSSi
19
0.45
2.01
0.007
0.022
0.762
168
16
0.49
2.00
0.005
0.023
0.776
96
17
0.47
1.99
0.005
0.024
0.788
144
HELLERHDACTARGETSSILENCEDBYMETHYLATIONDN
RODRIGUESTHYROI DCARCINOMAANAPLASTICUP
GAUSSMANNMLLAF4_FUSIONTARGETSF UP
20
0.43
1.99
0.003
0.023
0.791
455
30
0.36
1.95
0.010
0.030
0.866
127
23
0.41
1.95
0.010
0.030
0.866
161
DELYSTHYROIDCANCERUP
44
0.31
1.92
0.014
0.036
0.924
435
TAKEDATARGETSOFNUP98-HOXA9_FUSION_10DDN
ACEVEDOLIVER_.CANCERWITHH3K27ME3_DN
18
25
0.44
0.38
1.92
1.90
0.002
0.013
0.037
0.041
0.926
0.947
502
500
ALTEMEIERRESPONSETOLPSWITH_MECHANICALVENTILATION
18
0.43
1.90
0.011
0.041
0.950
388
GOZGITESR1_TARGETSDN
71
0.26
1.89
0.004
0.042
0.955
297
YAGIAMLWITH_T_8_21_TRANSLOCATION
FOSTERTOLERANTMACROPHAGEDN
26
0.37
1.88
0.010
0.044
0.963
144
32
0.34
1.88
0.013
0.045
0.966
435
TAKEDATARGETSOFNUP98_HOXA9_FUSION_8D.DN
PEDRIOLIMIR31_TARGETS-DN
16
39
0.44
0.31
1.86
1.83
0.007
0.006
0.051
0.060
0.980
0.990
456
537
FLECHNERBIOPSYKIDNEYTRANSPLANTOKVSDONORUP
33
0.32
1.82
0.008
0.063
0.991
428
SCHAEFFERPROSTATE_.DEVELOPMENT-48HRDN
33
0.33
1.81
0.015
0.065
0.993
715
DOUGLASBMI1_TARGETS_.DN
21
0.39
1.80
0.013
0.067
0.995
229
18
0.41
1.80
0.015
0.069
0.995
13
0.41
ENKUVRESPONSEKERATINOCYTEDN
18
23
0.36
1.79
1.79
0.015
0.015
0.071
0.071
0.997
0.997
435
246
GRAHAMCMLDIVIDINGVSNORMALQUIESCENTDN
15
0.43
1.78
0.018
0.073
0.998
144
CHICASRB1_TARGETSCONFLUENT
61
0.26
1.77
0.009
0.076
0.999
320
IWANAGACARCINOGENESISBYKRASPTENDN
26
0.34
1.76
0.019
0.083
1.000
325
JAEGERMETASTASISDN
31
0.32
1.72
0.028
0.098
1.000
109
HIRSCHCELLULARTRANSFORMATIONSIGNATUREUP
TONKSTARGETSOFRUNX1_RUNX1T1_FUSIONERYTHROCYTEUP
27
0.33
1.72
0.043
0.101
1.000
188
19
0.38
1.71
0.038
0.104
1.000
623
BOQUESTSTEMCELLCULTUREDVSFRESHUP
58
0.25
1.71
0.024
0.104
1.000
317
LINDGRENBLADDERCANCERCLUSTER_2B
ACEVEDOMETHYLATEDINLIVERCANCERDN
34
49
0.30
0.27
1.71
1.70
0.026
0.021
0.103
0.105
1.000
1.000
558
797
YANGBCL3_TARGETSUP
34
0.31
1.70
0.028
0.105
1.000
144
LEIMYBTARGETS
SATOSILENCEDBY-METHYLATIONINPANCREATIC-CANCER_1
36
0.30
1.69
0.113
1.000
97
48
0.26
1.68
0.039
0.031
0.114
1.000
345
SERVITJAISLETHNF1A_TARGETSUP
15
0.42
1.68
0.039
0.117
17
0.39
1.67
0.045
0.117
1.000
1.000
754
HOLLMANNAPOPTOSIS_VIACD40_DN
BLALOCKALZHEIMERSDISEASEINCIPIENTUP
21
0.36
1.67
0.037
0.117
1.000
106
GABRIELYMIR21_TARGETS
19
0.36
1.67
0.035
0.119
1.000
607
GRAESSMANNAPOPTOSIS_.BYDOXORUBICINUP
82
0.22
1.67
0.032
0.119
1.000
CHENMETABOLICSYNDROMNETWORK
90
0.21
1.66
0.019
0.119
1.000
138
75
KEGG CYTOKINECYTOKINERECEPTORINTERACTION
30
0.31
1.65
0.035
0.125
1.000
608
PHONG TNFRESPONSENOTVIAP38
29
0.32
1.65
0.043
0.124
1.000
1029
425
ONDERCDH 1TARGETS
1_DN
GRAHAMNORMALQUIESCENTVSNORMALDIVIDINGU
DACOSTAUVRESPONSEVIA
P
ERCC3_UP
HELLERHDACTARGETSDN
479
35
0.29
1.65
0.031
0.124
1.000
KATSANOUELAVL1_TARGETSUP
17
0.38
1.64
0.042
0.128
1.000
98
SMIDBREASTCANCERLUMINALBDN
65
0.23
1.63
0.029
0.133
1.000
326
MARSONBOUNDBYE2F4_UNSTIMULATED
38
0.28
1.63
0.031
0.134
1.000
441
ONKENUVEAL_.MELANOMADN
26
0.33
1.63
0.030
0.134
1.000
325
HATADAMETHYLATEDINLUNGCANCERUP
26
26
0.33
1.63
0.034
0.32
1.62
0.057
0.133
0.136
1.000
1.000
517
858
22
0.35
1.62
0.048
0.137
1.000
576
ACEVEDOLIVERTUMORVSNORMALADJACENT
TISSUEUP
LUAGINGBRAINUP
GHANDHIDIRECTIRRADIATIONUP
112
MASSARWEHTAMOXIFENRESISTANCED N
19
0.35
1.61
0.058
0.141
1.000
379
WAMUNYOKOLIOVARIANCANCERLMPDN
17
0.38
1.61
0.059
0.143
1.000
317
WESTADRENOCORTICALTUMORDN
50
0.24
1.59
0.045
0.153
1.000
199
HELLER_HDACTARGETSSILENCEDBYMETHYLATIONUP
CHICASRB1_TARGETS SENESCENT
51
0.25
1.59
0.055
0.158
1.000
445
45
0.060
0.159
1.000
227
31
0.25
0.29
1.58
H ELLERH DACTARGETSUP
1.58
0.064
0.160
1.000
445
VARTKSHVINFECTIONANGIOGENICMARKERSUP
JAATINENHEMATOPOIETICSTEMCELLD N
17
0.37
1.58
0.061
0.160
1.000
30
18
0.36
1.58
0.047
0.158
1.000
LIMMAMMARYSTEMCELL_UP
50
0.24
1.57
0.056
0.163
1.000
516
385
PLASARITGFB1 TARGETS_10HRD N
24
0.33
1.56
0.050
0.170
1.000
539
SMID BREASTCANCERNORMALLIKEUP
DURANDSTROMAMAXUP
40
31
0.26
0.045
0.052
0.183
0.192
1.000
0.29
1.54
1.53
1.000
325
161
LIWILMSTUMOR VSFETALKIDNEY_1_.UP
DOUGLASBMI 1_TARGETSUP
17
0.35
1.53
0.070
0.191
1.000
743
26
0.29
1.52
0.063
0.197
1.000
268
ZWANGEGFINTERVALDN
15
0.37
1.52
0.071
0.196
1.000
215
KRIGERESPONSETOTOSEDOSTAT_24HRUP
41
0.24
1.52
0.070
0.197
1.000
94
MIYAGAWATARGETSOFEWSR1_ETSFUSIONSUP
19
0.35
JECHLINGEREPITHELIALTOMESENCHYMALTRANSITIONUP
15
0.37
1.52
1.52
0.069
0.068
0.196
0.196
1.000
1.000
483
120
RODWELLAGINGKIDNEYNOBLOODUP
26
0.30
1.51
0.075
0.204
1.000
138
ZHENG BOUNDBYFOXP3
MARTINEZRB1_AND_TP53_TARGETSUP
30
0.28
0.073
0.207
1.000
435
48
0.23
1.50
1.50
0.078
0.209
1.000
288
THUMSYSTOLICHEARTFAILUREUP
30
0.28
1.50
0.077
0.209
1.000
346
MARTIN EZRB1_TARGETSDN
CUI TCF21_TARGETS_2_DN
36
63
0.26
0.21
1.48
1.48
0.077
0.087
0.221
0.220
1.000
1.000
310
425
16
0.35
1.48
0.085
0.222
1.000
897
PEREZTP53_ANDTP63_TARGETS
RODRIGUESTHYROID CARCINOMAPOORLYDIFFERENTIATEDUP
19
24
0.32
0.30
1.48
0.222
0.229
1.000
1.000
417
1.47
0.088
0.081
MULLIGHANMLLSIGNATURE_1_DN
17
0.34
1.47
0.079
0.231
1.000
161
TONKSTARGETSOFRUNX1_RUNX1T1_FUSIONHSCUP
19
0.33
1.47
0.102
0.230
1.000
460
LIINDUCED_T_TONATURALKILLERUP
19
0.33
1.46
0.100
0.230
1.000
42
PETROVAENDOTHELIUMLYMPHATICVSBLOODDN
20
0.33
1.46
0.086
0.231
1.000
790
VERHAAKAMLWITHNPM1_MUTATEDDN
29
0.28
1.45
0.092
0.242
1.000
165
BILDHRASONCOGENICSIGNATURE
MARTIN EZTP5 3TARGETSU P
28
0.28
1.45
0.078
0.243
1.000
82
50
0.22
1.45
0.092
0.243
1.000
313
VECCHIGASTRICCANCERADVANCEDVSEARLYUP
KRIGERESPONSETOTOSEDOSTAT 6HR UP
26
0.29
1.44
0.099
0.243
1.000
85
51
0.22
1.44
0.089
0.245
1.000
435
VERHAAKAMLWITHNPM1_MUTATEDUP
19
0.32
1.41
0.115
0.283
1.000
576
PEREZTP63_TARGETS
32
0.26
1.40
0.116
0.283
1.000
156
LEEBMP2_TARGETSUP
63
0.20
1.40
0.281
BERENJENOTRANSFORMEDBYRHOADN
ONDERCDH1_TARGETS_2 DN
39
0.23
1.40
0.113
0.110
0.281
1.000
1.000
326
168
MIYAGAWATARGETSOFEWSR1_ETSFUSIONSDN
58
25
0.20
0.28
1.40
1.40
0.119
0.120
0.281
0.280
1.000
1.000
127
602
RIZKITUMORINVASIVENESS_3D
DN
62
LEENEURAL CRESTSTEMCELLUP
19
0.32
1.40
0.122
0.280
1.000
852
ONKENUVEALMELANOMAUP
51
0.21
1.40
0.117
0.281
1.000
175
OSWALDHEMATOPOIETICSTEMCELLINCOLLAGENGELDN
20
0.31
1.39
0.132
0.288
JAATINENHEMATOPOIETICSTEMCELLUP
22
0.30
1.39
0.121
0.287
1.000
1.000
98
717
570
ONDERCDH1_TARGETS_2-UP
24
0.29
1.39
0.125
0.287
1.000
WONGADULTTISSUESTEMMODULE
71
0.19
1.38
0.125
0.291
1.000
790
ACEVEDOLIVERCANCERWITHH3K9ME3_DN
16
0.33
1.37
0.136
0.300
1.000
930
KOINUMATARGETSOFSMAD2_ORSMAD3
59
0.20
1.37
0.129
0.300
1.000
316
POOLAINVASIVEBREASTCANCERUP
21
0.29
1.37
0.126
0.298
1.000
332
RUTELLARESPONSETO-CSF2RBANDIL4.UP
21
0.29
1.37
0.149
0.298
1.000
423
MARSONBOUNDBYFOXP3_UNSTIMULATED
80
0.18
1.37
0.128
0.303
1.000
444
GOLDRATHANTIGENRESPONSE
19
0.30
1.37
0.140
0.301
1.000
68
VECCHIGASTRICCANCEREARLYUP
28
0.26
1.36
0.130
0.302
1.000
76
GALLEUKEMICSTEMCELL.DN
19
0.30
1.36
0.301
XUGH1_AUTOCRINETARGETSUP
18
0.31
1.35
0.129
0.156
0.318
1.000
1.000
244
530
MEISSNERBRAINHCPWITHH3K4ME3_ANDH3K27ME3
88
0.17
1.34
0.153
0.325
1.000
516
NAKAMURATUMORZONEPERIPHERALVSCENTRALUP
17
0.31
0.158
0.326
ACEVEDOLIVERCANCERWITHH3K27ME3_UP
17
0.31
1.34
1.33
0.125
0.331
1.000
1.000
444
763
FEVRCTNNB1_TARGETSUP
54
0.20
1.33
0.160
0.334
1.000
95
PUJANAATMPCCNETWORK
54
0.20
1.33
0.150
0.336
1.000
212
JACKSONDNMT1_TARGETSUP
15
0.32
1.32
23
0.27
1.31
0.183
0.184
0.346
YAGIAML-WITHINV_16_TRANSLOCATION
1.000
1.000
156
174
113
0.349
ACEVEDONORMALTISSUE.ADJACENTTOLIVERTUMORDN
24
0.27
1.30
0.174
0.363
1.000
630
PENGRAPAMYCINRESPONSEUP
DELYSTHYROIDCANCERDN
15
23
0.32
0.27
1.30
1.30
0.178
0.156
0.365
838
0.364
1.000
1.000
EBAUERTARGETSOFPAX3_FOXO1_FUSION.UP
17
0.30
1.29
0.172
0.367
1.000
324
SMIDBREASTCANCERRELAPSE_1NBONEDN
BLALOCKALZHEIMERSDISEASEUP
32
96
0.23
0.16
1.29
1.28
0.174
0.176
0.369
0.376
1.000
1.000
310
236
FARMERBREASTCANCER_BASALVSLULMINAL
ZWANGTRANSIENTLYUPBY_2N DEGFPULSEONLY
28
0.25
1.28
0.183
0.378
1.000
174
KAABHEARTATRIUMVSVENTRICLEUP
96
17
0.16
0.30
1.28
1.28
0.174
0.189
0.380
0.382
1.000
1.000
486
116
BUYTAERTPHOTODYNAMICTHERAPYSTRESSDN
26
0.217
0.381
1.000
16
0.25
0.31
1.28
BROWNEHCMVINFECTION_14HRDN
1.27
0.207
0.380
1.000
1043
539
TARTEPLASMACELLVSPLASMABLASTLUP
29
0.24
1.27
0.188
0.387
1.000
227
ODON NELLTFRCTARGETSUP
21
0.27
1.27
0.199
0.386
1.000
8
RUTELLARESPONSETOHGF UP
36
0.22
0.191
0.384
28
0.24
0.192
0.387
1.000
1.000
501
ACEVEDOFGFR1_TARGETS_1NPROSTATECANCERMODELDN
1.27
1.26
HSIAOLIVERSPECIFICGENES
CUITCF2 1TARGETS2_U P
18
0.29
1.26
0.200
0.386
1.000
169
28
15
0.24
0.31
1.26
0.391
0.390
1.000
1.000
568
1.26
0.188
0.218
CHIANGLIVERCANCERSUBCLASSCTNNB1_DN
MOHANKUMARTLX1 TARGETSDN
17
0.30
1.26
0.196
0.389
1.000
144
15
0.32
1.26
0.204
0.387
297
SENESE HDAC1_TARGETSUP
34
0.22
1.25
0.215
0.389
1.000
1.000
SARRIO EPITHELIALMESENCHYMALTRANSITION_DN
MULLIGHANMLL SIGNATURE_2_DN
19
0.28
1.25
0.210
0.388
1.000
274
0.26
0.16
1.25
1.25
0.217
0.206
0.389
PEREZTP53_TARGETS
22
84
0.394
1.000
1.000
161
423
CHIANGLIVERCANCERSUBCLASSPROLIFERATIONDN
19
0.28
1.25
0.202
0.393
1.000
212
KOKKINAKISMETHIONINEDEPRIVATION_48HRUP
17
0.29
1.24
0.205
0.395
1.000
790
GOBERTOLIGODENDROCYTEDIFFERENTIATION_DN
ICHIBAGRAFTVERSUSH OSTDISEASE3 5D UP
75
0.17
1.24
0.209
0.398
1.000
488
15
0.31
1.23
0.205
0.403
1.000
326
ENKUVRESPONSEEPIDERMISUP
17
0.28
1.23
0.242
0.409
1.000
662
HSIAOHOUSEKEEPING_.GENES
20
0.27
1.23
0.231
0.413
1.000
140
HADDADBLYMPHOCYTEPROGENITOR
23
0.25
1.22
0.218
0.416
367
SENGUPTANASOPHARYNGEALCARCINOMAWITHLMP1_DN
20
0.27
1.22
0.242
0.421
1.000
1.000
PASINISUZ12_TARGETSDN
21
0.26
1.22
0.236
0.420
1.000
505
WUCELLMIGRATION
23
0.25
1.22
0.242
0.420
1.000
97
ZHANGTLXTARGETSDN
16
0.29
1.21
0.248
0.421
1.000
246
WHITFIELDCELLCYCLEG2_M
751
421
972
324
326
116
0.14
1.21
0.237
342
22
0.25
1.20
0.245
0.421
0.438
1.000
MCBRYANPUBERTALBREAST_4_5WKUP
1.000
326
ENK_UVRESPONSEKERATINOCYTEUP
43
0.20
1.20
0.233
0.437
1.000
227
VERHAAKGLIOBLASTOMANEURAL
24
0.24
1.19
0.266
0.439
1.000
456
232
ZWANGTRANSIENTLYUP_BY_1STEGFPULSEONLY
BERTUCCIMEDULLARYVSDUCTALBREASTCANCERDN
15
0.29
1.19
0.235
0.445
1.000
PURBEYTARGETS_0 FCTBP1_NOTSATB1_UP
ACEVEDOLIVERCANCERDN
18
0.27
1.19
0.267
0.443
1.000
67
43
0.20
1.19
0.266
0.446
1.000
255
RUTELLA-RESPONSETOHGFVSCSF2RBAN DI L4UP
33
0.21
1.19
0.245
0.446
1.000
68
RENALVEOLARRHABDOMYOSARCOMADN
32
0.22
1.19
0.263
0.444
1.000
779
DANGBOUNDBYMYC
63
0.17
1.18
0.261
0.445
1.000
444
MARTORIATIMDM4_TARGETS_NEUROEPITHELIUMUP
15
0.29
1.18
0.248
0.445
1.000
138
PI LONKLF1_TARGETSDN
90
0.15
1.18
0.250
0.450
BENPORATHCYCLINGGENES
33
0.21
1.17
0.281
0.461
1.000
1.000
502
142
RUIZTNCTARGETSUP
18
0.27
1.17
0.282
0.459
WANGSMARCEITARGETSDN
DUTERTREESTRADIOLRESPONSE_6HR-UP
15
0.29
1.17
0.278
0.462
1.000
1.000
313
19
0.26
1.16
0.286
0.468
38
0.20
1.16
0.283
0.467
1.000
1.000
845
BONOME _OVARIANCANCERSURVIVALSUBOPTIMALDEBULKING
IVANOVAHEMATOPOIESISSTEMCELLANDPROGENITOR
MITSIADESRESPONSETOAPLIDINUP
42
0.19
1.15
0.275
0.479
1.000
690
23
0.24
1.15
0.291
0.478
1.000
120
WIERENGASTAT5AJTARGETSUP
FEVRCTNNB1_TARGETS DN
26
0.23
1.14
0.294
0.482
1.000
74
21
0.25
1.14
0.485
KOYAMASEMA3BTARGETSUP
25
0.23
1.14
0.298
0.283
1.000
1.000
568
329
CREIGHTONENDOCRINETHERAPYRESISTANCE_5
RIGGINSTAMOXIFEN RESISTANCED N
38
0.20
1.14
0.313
0.491
19
0.25
1.13
0.311
0.494
1.000
1.000
483
526
BENPORATH SUZ 1 2_TARGETS
83
0.15
1.13
0.314
0.495
1.000
469
FARMERBREASTCANCERAPOCRINEVSLUMINAL
26
0.22
1.13
0.301
0.496
1.000
174
DACOSTAUVRESPONSEVIAERCC3_DN
MARTIN EZTP53 TARGETSDN
42
0.18
1.12
0.313
0.501
1.000
317
35
0.20
1.12
0.327
0.502
1.000
145
MIKKELSENMCV6_HCPWITH_H3K27ME3
37
0.19
1.12
0.301
0.502
1.000
392
114
0.486
67
727
59
0.16
1.12
0.325
0.504
1.000
109
37
38
0.19
0.19
1.11
0.334
0.513
702
1.10
0.346
0.531
1.000
1.000
0.22
0.18
0.22
1.10
1.10
0.355
0.342
0.531
PICCALUGAANGIOIMMUNOBLASTICLYMPHOMAUP
26
40
25
1.09
LINDGRENBLADDERCANCERCLUSTER_2ADN
16
0.26
1.09
VANTVEERBREAST CANCERESRIDN
20
0.23
RODRIGUESTHYROIDCARCINOMAPOORLYDIFFERENTIATEDDN
ZHANGTLXTARGETS_60HR UP
50
YAGIAMLWITH_11Q23_REARRANGED
BUYTAERT PHOTODYNAMICTHERAPYSTRESSUP
ROZANOV.M M P14_TARG ETSU P
FULCHERINFLAMMATORYRESPONSELECTINVSLPSUP
QIPLASMACYTOMAUP
BEN PORATHMYCMAXTARGETS
127
229
0.530
1.000
1.000
424
0.354
0.537
1.000
809
0.341
0.540
1.000
790
1.08
0.359
0.541
390
0.17
1.08
0.363
0.548
1.000
1.000
28
0.21
1.08
0.360
0.551
1.000
246
19
0.24
1.07
0.375
0.554
1.000
719
346
33
0.20
1.07
0.375
0.563
1.000
42
IVANOVAHEMATOPOIESISSTEM_CELLLONGTERM
18
0.25
1.06
0.380
0.562
1.000
479
GRAESSMANNRESPONSETOMCANDDOXORUBICIN
DN
PEDERSENMETASTASISBYERBB2_ISOFORM_7
27
0.21
1.06
0.382
0.562
1.000
800
DANGREGU LATEDBYMYCDN
22
0.22
1.06
0.374
0.560
1.000
267
DODDNASOPHARYNGEALCARCINOMADN
CH ENH OXA5_TARGETS 9HRU P
69
0.15
1.06
0.384
0.571
1.000
92
16
0.25
1.05
0.393
0.575
1.000
233
LIUPROSTATECANCERDN
38
0.18
1.04
0.416
0.583
1.000
268
SCHAEFFERPROSTATEDEVELOPMENT_48HR_UP
40
0.17
1.04
0.405
0.581
1.000
212
RUTELLARESPONSETO_HGF_VSCSF2RBANDIL4_DN
SENESEHDAC1_AND_HDAC2_TARGETSUP
20
0.23
1.04
0.397
0.580
1.000
423
19
0.23
1.04
0.407
0.578
1.000
313
MIKKELSENNPC HCPWITHH3K27ME3
28
0.20
1.04
0.402
0.578
1.000
445
CHYLA-CBFA2T3_TARGETSUP
ONDERCDH1_SIGNALINGVIACTNNB1
30
15
0.19
0.25
1.04
1.04
0.411
0.407
0.578
0.584
1.000
1.000
790
378
WANGTUMORINVASIVENESSUP
BENPORATH ESWITH H3K27ME3
18
0.24
1.04
0.414
0.582
1.000
1206
CHIARADONNANEOPLASTICTRANSFORMATIONKRASDN
87
20
0.13
0.22
1.03
1.03
0.418
0.404
0.586
0.592
1.000
1.000
217
417
MARTINEZRB1_AND_TP53_TARGETSDN
37
0.18
1.03
0.420
0.593
1.000
145
MARSONBOUNDBYFOXP3STIMULATED
66
0.14
1.02
0.424
0.592
1.000
236
DODDNASOPHARYNGEALCARCINOMAUP
MARTIN EZRB1_TARGETSU P
126
0.12
1.02
0.448
0.591
1.000
429
50
0.16
1.02
0.444
0.602
1.000
140
RAYTUMORIGENESISBYERBB2CDC25A_DN
REACTOMEN EURONALSYSTEM
15
0.25
1.01
0.427
0.607
1.000
546
19
0.22
1.01
0.466
0.612
1.000
941
BENPORATHEEDTARGETS
80
0.13
1.00
0.464
0.620
1.000
501
CAIROHEPATOBLASTOMACLASSESDN
17
0.23
1.00
0.439
0.623
1.000
KIMRESPONSETOTSAANDDECITABINEUP
WANGRESPONSETOGSK3_INHIBITORS B216763_UP
17
0.23
1.00
0.433
0.624
1.000
447
442
23
0.21
0.99
0.452
0.636
1.000
325
MIKKELSENMEFHCPWITHH3K27ME3
41
0.16
0.99
0.480
0.637
1.000
238
WAKABAYASHIADIPOGENESISPPARGRXRABOUND_8D
51
0.15
0.98
0.473
0.636
1.000
607
GOZGITESR1_TARGETSUP
17
0.23
0.98
0.458
0.641
1.000
539
VECCHIGASTRICCANCER_EARLYDN
33
0.18
0.98
0.489
0.642
1.000
18
ZWANGCLASS_1_TRANSIENTLY_INDUCED_BYEGF
36
0.17
0.98
0.469
0.640
1.000
220
VARTKSHVINFECTIONANGIOGENICMARKERSDN
18
0.22
0.97
0.490
0.648
1.000
336
MOREAUXMULTIPLEMYELOMABYTACIUP
OSWALDHEMATOPOIETICSTEMCELL. NCOLLAGENGELUP
26
26
0.19
0.19
0.97
0.97
0.497
0.493
0.649
0.648
1.000
22
1.000
501
17
0.22
0.97
0.478
0.647
1.000
66
RODRIGUESTHYROIDCARCINOMAANAPLASTIC_DN
JOHNSTONEPARVBTARGETS_3_UP
42
0.16
0.96
0.497
0.652
1.000
140
38
0.16
0.96
0.494
0.651
1.000
447
PATILLIVERCANCER
40
0.16
0.96
0.651
LINDGRENBLADDERCANCERCLUSTER1_DN
REACTOME OLFACTORYSIGNALINGPATHWAY
DAVICIONIMOLECULARARMS VSERMSDN
23
0.20
0.96
0.501
0.511
0.655
1.000
1.000
82
139
15
0.23
0.96
0.512
0.653
1.000
605
17
0.23
0.96
0.507
0.653
1.000
632
CAIROH EPATOBLASTOMAUP
23
0.19
0.95
0.508
0.655
1.000
815
RUTELLARESPONSETOCSF2RBAND_IL4_DN
32
0.18
0.95
0.519
0.653
1.000
85
REACTOMEPEPTIDE_LIGANDBINDINGRECEPTORS
19
0.21
0.95
0.487
0.651
1.000
169
BEN PORATHPRC2_TARGETS
52
0.14
0.95
0.489
0.651
1.000
433
DURANDSTROMAMAXDN
19
0.21
0.95
0.530
0.652
1.000
252
ZWANGCLASS_3_TRANSIENTLYIN DUCEDBYEGF
BHATESRiTARGETSNOT VIAAKT1_UP
22
0.20
0.95
0.495
0.651
1.000
297
17
0.22
0.94
SHETHLIVERCANCERVSTXNIPLOSSPAM1
19
0.21
0.93
0.518
0.540
0.672
0.681
1.000
1.000
743
488
BRUINSUVCRESPONSEVIATP53_GROUPB
45
0.15
0.93
0.548
0.680
1.000
236
IVANOVAHEMATOPOIESISLATEPROGENITOR
25
0.19
0.93
0.557
LIAOMETASTASIS
MCCABEBOUNDBYHOXC6
28
0.18
0.93
0.541
0.678
0.679
1.000
1.000
94
771
23
0.19
0.92
0.538
0.679
1.000
763
WAMUNYOKOLIOVARIANCANCERLMP
UP
115
ACEVEDOFGFR1
TARGETSIN
PROSTATECANCERMODELUP
19
0.20
0.92
0.559
0.679
1.000
20
BOYLAN-MULTIPLEMYELOMAC_D_DN
19
0.21
0.92
16
0.23
0.92
0.678
0.678
1.000
HOELZELN FlTARGETSDN
0.533
0.556
1.000
0
1001
LIM_MAMMARYSTEM
CELLDN
CREIGHTONENDOCRINETHERAPYRESISTANCE_1
29
0.17
0.91
0.554
0.689
1.000
470
33
0.16
0.91
0.562
0.691
1.000
134
HUTTMANN_B_CLLPOORSURVIVALUP
24
0.19
0.91
0.562
0.689
1.000
82
REACTOMEHEMOSTASIS
35
0.16
0.91
0.563
0.689
1.000
790
SENESEHDAC3_TARGETSDN
HAMAI-APOPTOSISVIATRAILUP
43
0.15
0.90
0.572
0.702
1.000
490
35
0.16
0.90
0.585
0.700
1.000
444
ZHOUINFLAMMATORYRESPONSEFIMAUP
30
0.17
0.90
0.601
0.701
1.000
141
NUYTTENNIPP1_TARGETSDN
GHANDHIBYSTANDERIRRADIATIONUP
51
0.14
0.90
0.597
0.699
1.000
622
19
0.19
0.89
0.578
0.701
1.000
558
DAZARDRESPONSETOUVNHEKDN
15
0.22
0.89
0.593
0.708
1.000
412
18
0.20
0.89
0.575
0.709
1.000
297
DEBIASIAPOPTOSISBYREOVIRUSINFECTIONDN
17
0.21
0.88
0.587
0.709
1.000
564
SENGUPTANASOPHARYNGEALCARCINOMADN
33
0.16
0.88
0.588
0.708
1.000
326
GRUETZMANNPANCREATICCANCERDN
17
18
0.21
0.21
0.88
0.88
0.597
0.707
0.588
0.708
1.000
1.000
31
0
35
0.15
0.87
0.618
0.720
1.000
30
34
29
0.16
0.16
0.87
0.87
0.598
0.600
0.719
0.719
1.000
1.000
330
451
REACTOMEGENERICTRANSCRIPTIONPATHWAY
18
0.20
0.86
0.624
0.732
1.000
442
REACTOMEDEVELOPMENTAL BIOLOGY
GEORGESTARGETSOFMIR192_ANDMIR215
27
52
0.17
0.13
0.86
0.85
0.611
0.657
0.733
0.736
1.000
1.000
142
164
LOPEZMBDTARGETS
55
0.13
0.85
0.621
0.743
1.000
127
23
0.17
0.85
0.742
24
0.17
0.85
0.612
0.632
0.740
1.000
1.000
169
68
LINDGRENBLADDERCANCERCLUSTER
3_DN
SANSOMAPCTARGETSDN
MATSUDANATURALKILLERDIFFERENTIATION
RICKMANTUMOR DIFFERENTIATEDWELLVSPOORLYD N
BERENJENOTRANSFORMED
REACTOME_CLASSA
BYRHOAUP
_RHODOPSIN_LIKE_RECEPTORS
CREIGHTONENDOCRINE THERAPYRESISTANCE_4
GAVINFOXP3_TARGETSCLUSTER
P3
15
0.20
0.83
0.659
0.756
1.000
743
24
0.16
0.82
0.675
0.769
1.000
274
KEGGOLFACTORYTRANSDUCTION
20
0.18
0.82
0.656
0.767
1.000
738
WANGCISPLATINRESPONSEANDXPCUP
20
0.18
0.82
0.692
0.765
1.000
336
FLECHNERBIOPSYKIDNEYTRANSPLANTREJECTEDVSOKDN
27
0.16
0.82
0.671
0.772
1.000
488
KIMWT1_TARGETS_12HRUP
17
0.19
0.81
0.682
0.773
1.000
120
SENESEHDAC1_TARGETSDN
16
0.20
0.81
0.676
0.777
1.000
1248
CAIROLIVERDEVELOPMENTDN
29
0.15
0.80
0.706
0.783
1.000
212
SPIELMANLYMPHOBLASTEUROPEANVSASIANDN
27
0.16
0.80
0.685
0.784
1.000
24
VERHAAKGLIOBLASTOMACLASSICAL
18
0.18
0.80
0.678
0.782
1.000
828
REACTOMEGPCR
45
TURASHVILIBREAST DUCTALCARCINOMAVSDUCTAL
NORMALDN
DOWNSTREAMSIGNALING
0.713
0.80
0.722
0.787
0.786
1.000
1.000
621
17
0.13
0.19
0.80
KIMWT1_TARGETSUP
REACTOMEAXONGUIDANCE
17
0.18
0.79
0.709
0.787
1.000
428
LIUSOX4_TARGETSDN
H ESSTARGETSOFHOXA9_ANDM EIS1_DN
15
0.19
0.77
0.714
0.812
1.000
950
15
0.19
0.77
0.734
0.810
1.000
306
MIKKELSENNPCICPWITHH3K4ME3
29
0.15
0.77
0.725
0.811
1.000
444
PLASARITGFB1_TARGETS_10HRUP
BOQUESTSTEMCELLDN
22
0.16
0.76
0.755
0.818
1.000
313
21
0.16
0.76
0.752
0.820
1.000
1058
GAUSSMANNMLLAF4_FUSIONTARGETSGUP
22
0.16
0.75
0.747
0.828
1.000
537
SHEDDENLUNGCANCERPOORSURVIVALA6
GINESTIERBREASTCANCERZNF217_AMPLIFIEDDN
16
0.18
0.75
0.772
0.834
1.000
607
17
0.17
0.74
0.778
0.834
1.000
1303
DAVICIONITARGETS_OF_PAXFOXO1_FUSIONSUP
28
0.14
0.74
0.772
0.833
1.000
539
NIKOLSKYBREASTCANCER_17Q21_Q25_AMPLICON
CHIARADONNA_NEOPLASTICTRANSFORMATIONCDC25_UP
21
18
0.16
0.17
0.74
0.73
0.780
0.782
0.834
0.844
1.000
1.000
495
602
412
MARTORIATIMDM4_TARGETSFETALLIVERDN
27
0.14
0.72
0.802
0.852
1.000
209
JOHNSTONEPARVB TARGETS_3_DN
34
0.13
0.71
0.801
0.862
1.000
163
RATTENBACHERBOUNDBYCELFI
32
0.13
0.70
0.825
0.871
1.000
1367
DELACROIXRARGBOUNDMEF
28
0.14
0.70
0.803
0.871
1.000
189
BYSTRYKHHEMATOPOIESISSTEMCELLQTLTRANS
52
0.10
0.69
21
0.14
0.69
0.882
0.880
1.000
1.000
1223
HAMAIAPOPTOSISVIATRAILDN
0.843
0.815
DEURIGTCELLPROLYMPHOCYTICLEUKEMIAUP
28
0.13
0.69
0.806
0.878
1.000
1186
TIENINTESTINE-PROBIOTICS_24HRUP
19
0.15
0.68
CREIGHTONENDOCRINETHERAPYRESISTANCE_3
50
0.10
0.67
0.815
0.849
0.881
0.898
1.000
1.000
139
197
SMIDBREASTCANCERBASALDN
51
0.10
0.67
0.854
0.896
1.000
113
CHICASRB1_TARGETSGROWING
23
0.14
0.67
0.838
0.894
KEGGPATHWAYSINCANCER
33
0.12
LINSILENCEDBYTUMOR_MICROENVIRONMENT
16
0.15
0.66
0.66
0.863
0.862
0.893
0.896
1.000
1.000
279
576
444
116
1.000
67
0.894
1.000
1490
1.000
1.000
1040
0.867
0.905
0.910
0.64
0.881
0.908
1.000
41
0.09
0.10
0.61
0.905
0.60
0.900
0.933
0.934
1.000
1.000
414
479
16
0.15
0.60
0.918
0.933
1.000
969
REACTOMESIGNALING-BYGPCR
MIKKELSENMEF_LCPWITH_H3K4ME3
DAZARD RESPONSE TOUVNHEKUP
54
16
0.09
0.59
0.919
0.13
0.53
0.968
0.937
0.973
1.000
1.000
621
1608
18
0.12
0.985
0.984
1.000
0.11
0.09
0.50
0.50
0.973
WANGLMO4_TARGETSDN
576
292
858
YOSHIMURAMAPK8JTARGETSDN
22
0.14
0.66
TORCHIATARGETSOFEWSR1FLI1_FUSION _DN
24
0.13
0.65
0.889
0.875
RAOBOUND.BYSALL4_ISOFORMB
33
0.12
0.64
PURBEYTARGETSOFCTBP1_NOTSATB1_DN
28
0.12
DUTERTREESTRADIOLRESPONSE24HR_DN
MASSARWEHTAMOXIFENRESISTANCEUP
51
38
KYNGDNADAMAGEUP
419
FARMERBREASTCANCERAPOCRINE
VS BASAL
18
26
0.45
0.974
0.981
0.995
1.000
1.000
DACOSTAUVRESPONSEVIAERCC3
COMMONDN
17
0.10
0.44
0.995
0.995
1.000
1652
16
0.10
0.42
0.997
0.996
1.000
956
RIGGIEWINGSARCOMAPROGENITORDN
117
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121
Chapter 3. Possible Mechanisms of PARP13 Regulation
Tanya Todorova 1 ,2 and Paul Chang1' 2
'Koch Institute for Integrative Cancer Research, 2Department of Biology,
Massachusetts Institute of Technology, Cambridge MA 02139
122
Abstract
Poly(ADP-ribose) polymerase-13 (PARP13) is an RNA-binding protein that plays
important roles both in the antiviral response, when it targets viral RNA
transcripts for degradation, and in the cytoplasmic stress response, when it helps
repress miRNA silencing. The observation that PARP1 3 acts in vastly different
cellular contexts suggests that its functions are regulated. Here we focus on
potential mechanisms of PARP13 regulation in physiological conditions and
during cytoplasmic stress. We identify DHX30, a previously known PARP1 3
interactor during the immune response, as a PARP1 3-binding protein in normal
conditions, and show that DHX30 and PARP1 3 synergize to co-regulate a subset
of cellular transcripts. We also report that an ADP-ribose-recognition module in
PARP13, the WWE domain, inhibits RNA binding and that covalent modification
of PARP13 with poly(ADP-ribose) by PARP5a appears to be mutually exclusive
with RNA binding.
123
Introduction
Poly(ADP-ribose) polymerase-1 3 (PARP1 3/Zinc-finger Antiviral Protein
(ZAP)/ZC3HAV1) is a member of the PARP family of proteins - enzymes that
use NAD* to synthesize a posttranslational protein modification called ADPribose onto target proteins (Ame et al., 2004; Gibson and Kraus, 2012; Vyas et
al., 2013). PARP13 has two major isoforms: PARP13.1, which contains a
catalytically inactive PARP domain, and PARP1 3.2, which lacks the PARP
domain due to alternative splicing (Vyas et al., 2013). Both PARP13 isoforms
lack enzymatic activity and cannot synthesize ADP-ribose. In addition PARP13
contains four CCCH-type RNA-binding zinc fingers and a WWE ADP-ribosebinding domain. It also has nuclear-import and export signals that enable
shuttling between the nucleus and the cytoplasm, even though at steady state it
has a mostly cytoplasmic localization (Liu et al., 2004; Vyas et al., 2013).
PARP13 is an RNA-binding protein initially identified as an immune-response
host factor that restricts proliferation of various viruses such as Sindbis virus,
Human Immunodeficiency virus, Ebola virus, Hepatitis B virus, and others (Bick
et al., 2003; Gao et al., 2002; Mao et al., 2013; Muller et al., 2007; Zhu et al.,
2011). During infection PARP1 3 binds specific target elements in viral RNA
transcripts via its four N-terminal CCCH-type zinc fingers (Chen et al., 2012; Guo
et al., 2004). Upon binding, PARP1 3 recruits mRNA-decay factors: the
deadenylase PARN and the 3-5' exonuclease called the exosome complex are
engaged through a direct interaction with PARP13, while the decapping factors
124
DCP1 and DCP2 and the 5'-3' exonuclease XRN1 are recruited indirectly through
the adaptor protein DDX17 (Guo et al., 2007; Zhu et al., 2011). The recruitment
of these decay factors results in the degradation of target messages, repressing
viral replication and improving cellular outcomes during infection.
More recently, PARP1 3 was also shown to play a role in the cellular response to
cytoplasmic stress when it functions in a very different manner (Leung et al.,
2011). During stress PARP13 inhibits the activity of Argonaute 2 (Ago2), a key
catalytic component of the miRNA silencing pathway, by targeting it for
modification with poly(ADP-ribose) (PAR) - a surprising finding given that
PARP1 3 itself is catalytically inactive. PARP1 3 is therefore thought to recruit an
active PARP and to target it to modify Ago2 via a yet unknown mechanism
(Leung et al., 2011). The resulting repression of Ago2 activity results in stressinduced relief of miRNA silencing. This PARP13-dependent mechanism of Ago2
inhibition has since been expanded to another cellular context - the antiviral
response (Seo et al., 2013).
The observation that PARP1 3 functions in multiple cellular contexts suggests that
it needs to be tightly regulated in order to ensure a proper response. There are a
few known mechanisms of PARP13 regulation First, PARP13 is
posttranslationally modified by phosphorylation on four serine residues by
GSK3P, a process required for PARP1 3-dependent translational repression of
target mRNAs during the immune response against HIV (Sun et al., 2012).
125
PARP1 3 is also targeted for ADP-ribosylation during the cytoplasmic stress
response; however the functional significance of this modification remains
unclear. In other RNA-binding proteins, including poly(A) polymerase, PARylation
inhibits RNA binding (Di Giammartino et al., 2013; Ji and Tulin, 2013). Finally,
PARP13.1, the longer protein isoform, is farnesylated at its C-terminus, resulting
in its localization to membrane compartments (Charron et al., 2013).
Second, protein binding partners may be essential for modulating PARP13
function. For example, during cytoplasmic stress PARP13 binds other members
of the PARP family - PARP5a, PARP1 2 and PARP15, and these interactions,
resulting in the PARylation of PARP13, are likely regulatory in nature (Leung et
al., 2011). During the antiviral response PARP1 3 interacts with two helicases,
DDX17 and DHX30 (Chen et al., 2008; Ye et al., 2010). DDX17 is an adaptor
protein, helping PARP13 recruit 5' decay factors, whereas the exact purpose of
the PARP13-DHX30 interaction, although essential for PARP13's antiviral activity,
is unknown and may potentially present a regulatory step in PARP13-RNA
binding (Chen et al., 2008; Ye et al., 2010). Importantly, it is currently unclear if
these PARP1 3 interactions are restricted to the specific context in which they
were discovered - binding to PARPs during stress, and to DDX17/DHX30 during
the immune response, or if they are general mechanisms of PARP1 3 regulation.
Finally, PARP13 contains a PAR-binding WWE domain (He et al., 2012; Vyas et
al., 2013), raising the possibility that non-covalent interactions with the polymer
126
may also regulate its function. PAR binding modulates the function of several
RNA-regulatory proteins including the splicing factors hnRNPs and ASF/SF2 (Ji
and Tulin, 2013); PAR is also thought to act as a structural component of certain
non-membrane-bound RNA-rich subcellular compartments, including the nucleoli
and stress granules, where interactions with the polymer ensure proper
recruitment, localization, and maintenance of RNA-regulatory factors (Boamah et
al., 2012; Leung, 2014; Leung et al., 2011). Therefore PAR binding to the WWE
domain may modulate both RNA binding and localization of PARP1 3.
Here we examine potential mechanisms of PARP13 regulation in the absence of
viral infection. First, we employ a proteomic screen to identify binding partners
that may modulate PARP13 function. Second, we investigate the contribution of
the WWE domain to PARP13-RNA binding. Finally, we analyze in more detail the
covalent modification of PARP13 with PAR.
We show that DHX30 is the most enriched PARP1 3 binding partner under
physiological conditions, suggesting that the interaction of these two proteins is
not limited to the immune response. PARP13 and DHX30 bind in an RNAindependent manner and co-regulate a subset of cellular transcripts. Furthermore
we report that the WWE domain of PARP1 3 inhibits RNA binding and that
covalent poly(ADP-ribos)ylation of PARP1 3 by PARP5a appears to be mutually
exclusive with RNA binding. These findings reveal a complex regulation of
127
PARP13 by PAR, in which both covalent and non-covalent interactions with the
polymer may have consequences for its function.
Results
PARP13 interacts with ribosomal proteins and RNA-regulatory proteins
To identify PARP13 binding partners that may help regulate its activity we
immunoprecipitated PARP1 3 using an antibody specific for the endogenous
protein from wild-type and PARP13-deficient HeLa cells, used as a negative
control for non-specific binding (Fig. 1 a). Lysates were split in two, left untreated
or treated with RNase A to eliminate non-specific binding due to interaction with a
common RNA molecule, and immunoprecipitates were subjected to mass
spectrometry. Proteins found in the negative control reactions and exclusively in
the absence of RNase treatment were subtracted as non-specific binders, and
only proteins that were detected both with and without RNase treatment were
further analyzed. In total 24 proteins satisfied these criteria (Fig. 1 a). 19 of those
were ribosomal proteins, suggesting that PARP1 3 may interact directly with the
ribosome in an RNA-independent manner. Among the non-ribosomal hits were
Galectin-7, a protein involved in cell-cell adhesion (Saussez and Kiss, 2006), and
two nuclear proteins: CENPJ, a centromere protein that helps to establish normal
spindle morphology during cell division (Tang et al., 2009), and nucleophosmin
(NPM1), a protein associated with nucleolar ribonucleoprotein structures involved
in rRNA biogenesis (Naoe et al., 2006). The identification of nuclear proteins
interacting with PARP1 3 is consistent with PARP1 3 shuttling between the
128
nucleus and the cytoplasm, and suggests that its functions may not be restricted
to the cytoplasm.
Finally, our screen identified two DEAD/H Box helicases, DHX30 and DDX5 as
PARP13 interactors (Fig. 1a). Helicases are important regulators of RNA
metabolism, as they use ATP to unwind secondary structures within their target
RNAs, thus stabilizing specific folds and potentially restricting or facilitating
binding of other RNA-regulatory proteins (Linder and Jankowsky, 2011). DDX5
has known functions in splicing and transcriptional activation or repression of
gene expression and is a predominantly nuclear protein (Linder and Jankowsky,
2011). On the other hand, DHX30 is an antiviral helicase that restricts HIV
infection and has been previously shown to interact with PARP1 3 and to facilitate
its antiviral function (Ye et al., 2010). DHX30 was the most enriched coimmunoprecipitated protein in our screen, suggesting that its interaction with
PARP1 3 is not restricted to the immune response, but may also be relevant to
PARP1 3 function during physiological conditions. For this reason we decided to
focus on characterizing the PARP13-DHX30 interaction in more detail.
PARP13 binds DHX30 directly
First we confirmed the interaction of PARP1 3 and DHX30 by immunoblot. We
immunoprecipitated SBP-tagged PARP1 3.2, PARP13.2 RNA binding mutants
(PARP13.2R 18
9
Aand
PARP13.2,ZnF) and a WWE domain deletion mutant,
PARP13.2^WWE, along with PARP16 used as a negative control (Fig. 1b). RNA-
129
binding deficiency of PARP13.2R 189A and PARP13.2,ZnF was confirmed by CLIP
(Fig. 1c). DHX30 co-immunoprecipitated with PARP1 3.2, PARP13.2R 1 89Aand
PARP13.2,WWE, but not with PARP13.2^ZnF and PARP16. This result suggests
that the region within PARP13 required for DHX30 binding is within the RNAbinding N-terminus, consistent with previous reports (Ye et al., 2010). RNase
treatment decreased the interaction but did not eliminate it, consistent with our
mass spectrometry results (Fig. 1a, 1b), suggesting that while there is a pool of
PARP13 that is directly bound to DHX30, the two proteins can also bind to the
same RNA transcript without interacting with each other. A reciprocal
immunoprecipitation of GFP-DHX30 and GFP alone (negative control) confirmed
these observations - PARP13 co-immunoprecipitated with DHX30, while RNase
treatment resulted in decreased interaction between the two proteins (Fig. 1d).
PARP13 decreases DHX30 binding to RNA
Next we determined whether PARP1 3 and DHX30 modulate each other's ability
to bind RNA by CLIP assays. While DHX30 depletion by siRNA had no effect on
PARP13 RNA binding (Fig. le), DHX30 bound more RNA in PARP13 knock-out
cells compared to wild-type cells (Fig. 3f). Rescue of PARP1 3 expression in
these cells decreased DHX30 RNA binding to wild-type levels (Fig. 1f). This
result suggests that PARP13 negatively regulates DHX30 binding to RNA and
that the two proteins may indeed regulate each other's functions.
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PARP13 and DHX30 depletion results in decreased proliferation
PARP13 depletion has been previously shown to result in decreased proliferation
(Vyas et al., 2013). To confirm this observation and determine if DHX30 may
have a similar phenotype, which would be consistent with genetic interaction
between the two proteins, we depleted PARP13 and DHX30 separately or
together in HeLa cells and assayed proliferation at 24 and 72 hrs after seeding
(Fig. 2a, left). Protein amounts at each time point were assayed by immunoblot
(Fig. 2a, right). Compared to control knock-downs, PARP13 and DHX30 siRNAtreated cells showed significantly decreased proliferation at both time points, but
the effect was more obvious at 72hrs, when proliferation of PARP1 3-depleted
cells was 62% of controls and proliferation of DHX30-depleted cells was 23% of
controls (Fig. 2a). Interestingly depleting both PARP13 and DHX30 resulted in
increased proliferation compared to depletion of DHX30 alone - 46% vs. 23% of
controls, respectively. This result suggests that PARP13 misregulation in the
absence of DHX30 is one reason for the proliferative defect we observed, and is
consistent with DHX30 regulation of PARP13 functions. Therefore, removal of
PARP13 in the double knock-down leads to better cellular outcomes than
maintaining normal PARP13 levels in the absence of DHX30 in the cell.
PARP13 and DHX30 co-regulate a subset of transcripts
To determine if PARP1 3 and DHX30 function together in regulating RNA
metabolism in the cell, we depleted each protein by siRNA and identified
differentially expressed genes compared to control siRNA-treated cells by Agilent
131
microarrays (n=2). More than 500 genes showed an average of log2 fold change
(log2FC) > 1 between the two PARP13 siRNA replicates; for DHX30-depleted
samples the number was more than 1800 (Fig. 2b). More transcripts were
misregulated upon DHX30 depletion compared to PARP13 depletion, consistent
with the hypothesis that DHX30-mediated modulation of RNA structure affects
transcriptome regulation by multiple RNA-regulatory proteins, and therefore has
more global effects (Fig. 2b). 103 genes were misregulated at log2FC>1 in both
PARP13 and DHX30 depletions (Fig. 2c), identifying a subset of RNAs that may
be co-regulated by the two proteins.
To assay the functional outcomes of PARP1 3 and DHX30 depletion we analyzed
the upregulated and downregulated differentially expressed transcripts for gene
ontology (GO) category enrichment. Surprisingly, no categories were significantly
enriched among the downregulated genes. On the other hand, upregulated
genes in both PARP13 and DHX30 knock-downs were enriched for immuneresponse factors and secreted/extracellular factors, and some of the specific
enriched GO categories were shared between the two (Fig. 2d). Genes
upregulated in DHX30 also showed enrichment for factors involved in cell
signaling, adhesion and motility, and angiogenesis. This analysis gives rise to a
couple of conclusions: 1) DHX30 and PARP1 3 co-regulate a subset of transcripts
enriched for secreted factors and genes that function in the immune response; 2)
DHX30 has PARP1 3-independent functions that affect other cellular processes
and likely rely on interactions with other RNA-regulatory proteins; 3) Functional-
132
category enrichment only among the upregulated but not among the
downregulated genes suggests that the upregulated genes may represent direct
targets of regulation by DHX30 and PARP1 3, consistent with previously reported
functions of PARP1 3 in RNA degradation (Gao et al., 2002; Guo et al., 2007; Zhu
et al., 2011).
If PARP13 and DHX30 bind and form a complex in order to co-regulate a subset
of transcripts, the expectation would be that the depletion of either protein would
cause a similar change in the expression of these transcripts as the other - for
example a co-regulated transcript upregulated upon PARP1 3 depletion should
also be upregulated upon DHX30 depletion. We tested this hypothesis by first
identifying transcripts with log2FC>1 in PARP13 knock-down (both upregulated
and downregulated) and examined how these transcripts behave upon DHX30
depletion (Fig. 2e). Consistent with our hypothesis, transcripts upregulated upon
PARP13 depletion tended to be either upregulated or unchanged upon DHX30
depletion; conversely transcripts downregulated upon PARP13 depletion tended
to be either downregulated or unchanged upon DHX30 depletion. We then
focused on the transcripts that showed log2FC>1 in both PARP13 and DHX30
depletions (103 genes, overlap shown in Fig. 2c) and examined how their
expression changed in each knock-down. Expression changes showed strong
positive correlation (R 2=0.7) suggesting that PARP1 3 and DHX30 regulate this
overlapping set of transcripts in a similar manner, consistent with a model in
133
which PARP1 3 and DHX30 function together to modulate the expression of these
genes.
The WWE domain of PARP13 inhibits RNA binding
The WWE domain at the C-terminus of PARP1 3 is a poly(ADP-ribose)-binding
module (He et al., 2012; Wang et al., 2012). To test if this domain may function in
regulating RNA binding, we assayed RNA binding of a WWE domain deletion
construct, PARP13.2AWWE, via two different assays. First we analyzed its
localization to RNA-rich stress granules. PARP1 3 localization to stress granules
is mediated by RNA binding (Lee et al., 2013); therefore the ability to localize to
these structures can be utilized as an assay for PARP1 3-RNA binding. An RNA
binding mutant of PARP1 3.2 - PARP13.2R 189A, shows a more diffuse cytoplasmic
signal and decreased concentration in stress granule foci during stress compared
to the wild-type protein (Fig. 3a). On the other hand, PARP1 3 .2
WWE
localized
very strongly to stress granules. Indeed, its enrichment in foci and depletion from
the cytoplasm was more intense than that of the wild-type protein, suggesting
that it is targeted to stress granules more efficiently. One hypothesis to explain
this observation is that PARP1 3 AWWE binds RNA with higher affinity than the wildtype protein, thus exhibiting stronger stress-granule localization (Fig. 3a).
To test this hypothesis, we assayed PARP1 3.2 and PARP1 3 .2 AWWE RNA binding
by CLIP. Consistent with our immunofluorescence data, we observed that
PARP1 3.2AWWE binds more RNA than the wild-type protein (Fig. 3b). Increased
134
RNA binding of PARP13.2AWWE can be explained by two hypotheses: 1) WWE
domain interaction with poly(ADP-ribose) inhibits RNA binding, thus abrogating
this interaction by deleting the WWE domain improves RNA binding; or 2) the
WWE domain itself inhibits RNA binding to the N-terminal domain, perhaps by
folding over it and decreasing its access to target transcripts, a model that would
be consistent with poly(ADP-ribose) binding alleviating this inhibitory effect in a
manner similar to the deletion of the domain (Fig. 3c).
Covalent modification of PARP13 by PAR
PARP13 is modified by PAR and this modification increases upon stress (Leung
et al., 2011). To test if RNA binding is required for modification and if the
modification occurs in the RNA-binding domain, as reported for other RNAregulatory proteins (for example Ago2, TIA1, and G3BP) (Leung et al., 2011), we
immunoprecipitated PARP13.2 and the N-terminal-deletion mutant
PARP1 3.2AZnF, and immunoblotted for PAR. A smear originating at the molecular
weights of PARP1 3.2 and PARP1 3.2AZnF suggested that both proteins are
modified (Fig. 4a); furthermore, the modification increased upon stress as
previously reported (Fig. 4a). Interestingly, PARP1 3.2AZnF was not only modified
with PAR, suggesting that the PAR acceptor site is not in the N-terminal RNAbinding domain, but it was modified more than the wild-type protein under both
physiological conditions and stress. This observation suggests that the zincfinger domain inhibits modification, consistent with a model in which being bound
to RNA and being modified with PAR are mutually exclusive PARP13 states.
135
The only cytoplasmic PARPs capable of synthesizing PAR are PARP5a and
PARP5b. Since both PARP5a and PARP13 localize to stress granules, whereas
PARP5b does not, we focused on identifying a possible interaction between
PARP5a and PARP1 3. Consistent with our previous observations, PARP1 3. 2AZnF
which is highly modified with PAR, co-immunoprecipitated PARP5a protein while
PARP13.2 did not (Fig. 4b). These data suggest that RNA-bound PARP13
cannot interact with PARP5a - in contrast, abolishing RNA binding by deleting
the N-terminal domain results in strong binding between PARP13 and PARP5a
and increased PAR modification.
To determine if RNA binding may indeed play a role in restricting PARP13
interaction with PARP5a, we fractionated HeLa cell lysate on a sucrose gradient
and analyzed the fractionation patterns of PARP13, PARP5a and ribosomal RNA.
The vast majority of PARP1 3 protein occupied the heavier fractions following a
pattern similar to ribosomal RNA, consistent with it binding to actively translated
RNAs and migrating with polysomes (Fig. 4c). This observation also strongly
suggests that in physiological conditions most PARP13 is RNA bound. On the
other hand, PARP5a was exclusively found in the light soluble fractions. The lack
of co-migrations is consistent with no or little stable interaction between RNAbound PARP13 and PARP5a.
136
Discussion
PARP1 3 binds both endogenous and foreign RNA, and has important functions
in regulating RNA targets and the activity of other RNA-binding proteins during
normal cell physiology, stress and viral infections (Guo et al., 2004; Hayakawa et
al., 2011; Leung et al., 2012; Zhu et al., 2011). It therefore appears essential that
PARP13 functions be tightly regulated. Here we explore two potential
mechanisms of PARP13 regulation - by protein binding partners and by the
posttranslational protein modification poly(ADP-ribose).
One potential mechanism of differential regulation of PARP13 during specific
cellular conditions is through interaction with different regulatory proteins and
adaptors that can potentially modulate PARP13 localization, target specificity or
the outcome of PARP13-RNA binding. Therefore, the identification of DHX30 as
the most enriched PARP13 interactor in our proteomics screen was somewhat
surprising as DHX30 was previously reported as a PARP13-binding protein
during the immune response, and as a factor that facilitates PARP1 3 antiviral
activity (Ye et al., 2010). However, we show that PARP1 3 binds DHX30 in the
absence of viral infection, and the two proteins synergize to co-regulate a subset
of transcripts enriched for secreted factors and immune-response factors. This
result suggests that binding of PARP13 to DHX30 may present a fundamental
step in the mechanism of PARP1 3-mediated RNA regulation independent of
context, rather than a specific regulatory event during the immune/antiviral
response. This observation does not, however, undermine the fact that DHX30
137
appears to be essential for proper PARP13 function - PARP13 co-depletion
improves the DHX30-knock-down proliferation-deficiency phenotype suggesting
that at least part of the negative cellular outcomes that result from DHX30
depletion can be explained by misregulated PARP1 3 functions.
PARP13 contains a WWE domain which can bind to poly(ADP-ribose) (He et al.,
2012; Vyas et al., 2013; Wang et al., 2012), and in addition was previously
reported to be covalently modified by PAR (Leung et al., 2011). It is thus possible
that PAR regulates PARP13 function both through a covalent and a non-covalent
mechanism. To address this we tested whether the WWE domain helps to
regulate PARP13-RNA binding, and, indeed, we observed that deletion of the
WWE domain increases RNA binding. This opens the question of the effect that
PAR binding to the WWE domain would have on RNA affinity. One possibility is
that ADP-ribose binding constitutively represses RNA binding, and thus
abrogation of the interaction with the polymer by deleting the WWE domain
increases PARP13 affinity for RNA. Another hypothesis is that the WWE domain
itself is inhibitory in nature and that WWE interaction with PAR relieves this
inhibitory effect and increases PARP1 3 binding to RNA. Although further
research is needed to fully address this issue, given that PAR often acts as a
stress-response signaling molecule it is unlikely that it regulates PARP13 RNA
binding constitutively.
138
Covalent modification of PARP1 3 with PAR, on the other hand, may be mutually
exclusive with RNA binding. The PARP13 RNA-binding mutant, PARP13.2AznF
forms a strong interaction with PARP5a, a cytoplasmic catalytically active PARP.
This observation correlates with increased PARylation of PARP1 3.2AZnF
compared to the wild-type protein, which does not bind PARP5a during
physiological conditions. These results also suggest that the inability to bind RNA
makes PARP1 3 a better PARP5a target, and that RNA-bound PARP1 3 may be
somehow inaccessible for modification and regulation by PARP5a. Since the
majority of PARP13 appears to be RNA-bound under physiological conditions,
and PARP13 and PARP5a do not co-migrate in sucrose-gradient fractionation,
their interaction is likely restricted during normal cell physiology. PARP13
modification with PAR, however, increases during stress. It is unclear if this
inhibits RNA binding, but if PARylation and RNA binding are indeed mutually
exclusive, PARP13 modification with PAR may present a mechanism to restrict
its RNA-degradation activity during stress, in a manner similar to PARylation of
Ago2 inhibiting its miRNA silencing functions (Leung et al., 2011).
Material and methods
Cell lines and transfections
All experiments were performed with HeLa Kyoto cells (ATCC) unless otherwise
specified. HeLa Kyoto cells were grown at 370C, 5% CO 2 , in Dulbecco's Modified
Eagle's Medium, DMEM (Invitrogen), supplemented with 10% Fetal Bovine
Serum. For siRNA transfections, 150,000 cells were seeded and transfected with
139
20nM siRNA using Lipofectamine 2000 (Invitrogen); for siRNA sequences refer
to table below. Two consecutive 48 h transfections were performed. DNA
transfections were performed for 24 h using Lipofectamine 2000 (Invitrogen).
DNA constructs and cloning
GFP-PARP1 3.2 was previously described (Leung et al., 2011; Vyas et al., 2013).
ZTS- and SBP-tagged versions were cloned by replacing the GFP tag in the
eGFPC1 vector using Nhel and BspEl. DHX30 cDNA was synthesized by Epoch
Life Science Inc. and inserted into the eGFPC1 vector. PARP1 3.2 RNA-binding
mutants were generated using Gene String technology (Invitrogen); strings
harboring the desired mutations were inserted with Xhol/BstXI, which are internal
sites within PARP13 cDNA. Deletion of the WWE domain was generated with a
gene string inserted with BstXI / BamHI.
Immunoprecipitations and immunoblotting
Cells were lysed in Cell Lysis Buffer (CLB, 150mM NaCl, 50mM HEPES (pH 7.4),
1mM MgC 2 , 0.5% Triton, 1 mM EGTA and 1mM DTT, supplemented with
protease inhibitor) for 15 min / 4 0C and lysates were precleared at
16,000g/l5min/4*C. Streptavidin Binding Protien (SBP) and ZZ-TEVStreptavidin-Binding Protein (ZTS) tagged proteins were precipitated using
streptavidin beads (GE Healthcare); GFP-tagged and endogenous proteins were
precipitated using protein A magnetic beads (Millipore) and specific antibodies.
For list of antibodies used refer to table below. Lysates and beads were
140
incubated at 40C for 1.5 h with agitation. Beads were collected, washed 3X with
CLB and resolved on SDS-PAGE gels following standard protocols.
Crosslinking Immunoprecipitation (CLIP)
Cells were lysed and precleared as above. Lysates were treated with 1 tg/ml
RNAse A (10min/37*C), precleared again, and incubated with beads as
described above. Beads were collected, washed 1X with CLB, 3X with High Salt
CLB (1M NaCl, 50mM HEPES (pH 7.4), 1mM MgC 2 , 0.5% Triton, 1mM EGTA
and 1mM DTT, supplemented with protease inhibitor) and incubated with 1X
PNK reaction buffer (NEB) supplemented with 0.5 units/ d T4 Polynucleotide
Kinase and 2.5%
P yATP (v/v). Beads were incubated at 370C / 10 min and
32
washed 3X with CLB; SBP and ZTS-tagged proteins were eluted with 1 mg/ml
biotin in CLB, 1 h / RT. Samples were loaded on 10% Bis-TRIS gels (Invitrogen);
gels were transferred onto nitrocellulose membrane and membranes were
exposed to phosphor screen to detect bound RNA signal.
Mass Spectrometry
LC/MS/MS mass spectrometry on Coomassie-stained PARP1 3
immunoprecipitated samples was performed by the Taplin Mass Spectrometry
Facility, Harvard Medical School, on Orbitrap mass spectrometer (Thermo
Scientific).
141
Proliferation assays
Cells were treated with control, PARP13, DHX30 or PARP13+DHX30 siRNAs as
described above. After 2X48h transfections 250,000 cells were seeded at Day 0
in 6-well plates. At 24 h (Day 1) and 72 h (Day 3) cells were trypsinized and
recounted. All cell counting was performed on a Coulter Counter (Beckman).
Agilent microarrays and data analysis
Cells were treated with control, PARP1 3 and DHX30 siRNAs. After 2X 48 h
transfections cells were collected and total RNA was extracted using RNeasy
columns (Qiagen). Samples were labeled using the Two Color Quick Amp
Labeling Kit (Agilent), and hybridized on a SurePrint G3 Human Expression v2
8X60 microarray. Microarrays were scanned on the Sure Scan Microarray
Scanner (Agilent) and processed using the Feature Extractor v1 0.5. All data was
analyzed using GeneSpring Software (Agilent).
Immunofluorescence and microscopy
Cells were split onto glass coverslips 16h before the experiment. Cells were
treated with 200 M sodium arsenite for 45 min, then fixed in 4% formaldehyde
for 30 min, extracted with 0.5% Triton X in Abdil (4% BSA in PBS) for 25min, and
blocked in 0.1% Triton-X Abdil for 30 min. All antibody dilutions were in 0.1%
Triton-X Abdil. Fixed cells were incubated with antibody dilutions for 45 min,
stained with 1 tg/ml Hoechst and mounted on glass slides with SlowFade
mounting media (Life Technologies). Stress granule localization quantification
142
was performed on NIS-Elements software (Nikon). In short a small Region of
Interest (ROt) was created and placed in the middle of a stress granule (for
stress granule signal) and right next to the stress granule (for a corresponding
cytoplasmic/background signal) for each stress granule in each channel. The
ratio between SG and background signal was calculated for each stress granule
in each channel. More than 20 stress granules in multiple cells and fields were
analyzed for each transfection. Data shown is representative of multiple
independent experiments.
Sucrose gradients and fractionation
10-40% sucrose gradient in CLB was poured manually using sucrose dilutions in
5% intervals and incubated overnight at 4C. HeLa Kyoto cells were lysed in CLB
as described above, precleared, loaded onto the gradient and centrifuged at
1 00,000g for 1 h. 19 fractions were collected manually from top to bottom. Total
RNA was extracted from half of each fraction using Trizol (Invitrogen), resolved
on an agarose gel and stained with ethidium bromide. The rest was used for
SDS-PAGE gel electrophoresis and immunoblotting.
143
Reagents used in manuscript
Antibody/dilution used
Catalog number
Supplier
PARP13 (1:1000 for IB,
1 g/immunoprecipitation)
In house
Described in (Leung
et al., 2011; Vyas et
PARP13 (1:100 for
immunofluorescence)
DHX30 (1:1000 for IB)
SBP (1:1000 for IB, 1:100
GTX120134
Genetex
A302-219A
Mab10764
Bethyl
Millipore
551813
BD Pharmingen
PARP5a (1:500 for IB)
Sc-22855
GAPDH (1:1000 for IB)
GTX28245
Santa Cruz
Biotechnology
Genetex
siRNA
PARP1 3
Catalog number
GCUCACGGAACUAUGAGCUGA
Supplier
Life technologies
al., 2013)
for IF)
Poly(ADP-ribose) (1:500
for
IB)
GUUU/AAACUCAGCUCAUAGUU
CCGUGAGC
DHX30
Ambion/ Life
S22645
Technologies
144
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145
IB:PARP13
Figure 1. PARP13 binds DHX30 and regulates its RNA binding a) Left,
immunoblot showing input and immunoprecipitated PARP13 from PARP13
knock-out HeLa cells (-/-, negative control) and wild type HeLa cells (+/+)
untreated or treated with 1 tg/ml RNase A, used in mass spectrometry screen for
PARP13 binding partners. Right, list of proteins identified in mass spectrometry
screen for proteins co-immunoprecipitating with PARP1 3 in the presence or
absence of RNaseA treatment. Only proteins that appear in both conditions are
shown. b) SBP-tagged PARP16 (negative control), PARP13, PARP13R18 9 A
PARP13,ZnF and PARP13 AWWE were immunoprecipitated from HeLa Kyoto cells,
untreated or treated with 1 g/ml RNaseA, and immunoblots were performed with
antibodies against SBP, PARP13 and DHX30. Inputs shown to the left. c) An
autoradiogram of CLIP reactions of ZTS (negative control), ZTS-PARP1 3.2, and
RNA binding mutants PARP1 3 2 H176A, PARP1 3 . 2 R189A, and PARP13. 2 V72A, Y1O8A,
F144A.
Protein levels shown below. d) Immunoblots showing GFP-DHX30 and
GFP (negative control) immunoprecipitations untreated or treated with 1 tg/ml
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shown in left lane of each blot. e) An autoradiogram of endogenous PARP1 3
CLIP reactions from cells treated with control or DHX30-specific siRNA. PARP13
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control) or ZTS-DHX30 CLIP reactions from wild type (+/+) or PARP13 knock-out
cells (-/-). In rightmost lanes GFP-PARP13.2 was cotransfected with ZTS-DHX30.
146
Immunoprecipitated ZTS/ZTS-DHX30 protein levels and PARP13 input protein
levels shown below.
147
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IL
p.
DHX30 siRNA (log2FC)
148
Figure 2. PARP13 and DHX30 co-regulate a subset of transcripts a) Top,
proliferation of HeLa cells treated with Control, PARP13, DHX30 and
PARP13+DHX30-specific siRNAs expressed as fold increase over day 0.
Averages of n=4 independent experiments shown; error bars show s.d.; p<0.05
(*), p<0.01(**), p<0.001 (***), significance compared to control siRNA, two sided
t-test. Bottom, immunoblots showing PARP13 and DHX30 protein levels for each
condition and timepoint. GAPDH used as loading control. b) Number of genes
misregulated at log2FC>1 in PARP1 3 and DHX30 knockdowns. c) A Venn
diagram showing overlap between genes misregulated in PARP13 and DHX30
knock-downs. d) A bubble chart displaying types of functional categories
enriched in genes upregulated in PARP13 and DHX30 knockdowns and overlap
between them. Each circle represents one functional category; categories that
connect to both PARP1 3 and DHX30 represent categories that are enriched in
both knockdowns; categories are color-coded based on description; length of line
connectors is random. e) Heat map of genes misregulated at log2FC>1 (top) and
log2FC<1 (bottom) in PARP13 knock-downs (left), and the level of misregulation
of these genes in DHX30 knock-down (right). Each line represents a separate
gene. Color indicates magnitude of fold change - red represents highest positive
increase, blue represents lowest negative decrease. f) A scatter plot of the
overlap of genes misregulated in DHX30 and PARP13 knock-downs (103 genes,
see Venn Diagram in Fig. 2c). Color indicates magnitude of fold change - red
represents highest positive increase, blue represents lowest negative decrease.
149
IL
b
a
PARPIO
1 .2
PARP13.2
E
-5
PARP13. 2 R189A
0
4I
A
PARP13. 2 AWWE
PARP1 31
135.
4.
0
E
SBP staining
PARP13 staining
0)3U
M2
PARP13.2 PARP13.2RMAPARP13.2AWWE
PARP10
C
SBP-fusion constructs
Wild type PARP13.2
Wild type PARP13.2 + Poly(ADP-ribose)
Wild type PARP13.2
Baseline RNA
binding
Baseline RNA
binding
Increased
RNA
binding
PARP13.2AWWE
150
Increased RNA
binding
Figure 3. Deletion of WWE domain results in increased RNA binding of
PARP13.2 a) Left, representative images of immunofluorescence showing
colocalization of SBP-tagged PARP10 (negative control), PARP13.2,
PARP13.2R 1 89A and PARP1 3 .2
WWE
(SBP antibody, green in merge), with
endogenous PARP13 (PARP13 antibody, red in merge) and poly(A) mRNA (594Alexa Oligo-dT, blue in merge) in HeLa cells treated with 200 tM sodium arsenite
for 45 min. Right, intensity ratio of stress granule signal to background
cytoplasmic signal was calculated for the SBP and PARP1 3 channels for each
transfection. n>20 stress granules and cytoplasmic regions counted for each
transfection. Bars show average intensity ratios, error bars show s.d., asterisks
show significant difference between SBP constructs and SBP-PARP13.2 (twosided t-test, p<0.01 (*), p<0.0001 (***). b) Autoradiogram of SBP-PARP1 3.2 and
SBP-PARP1 3.2 AWWE CLIP reactions. Triangle indicates molecular weight of
SBP-PARP13.2AWWE. Immunoblots shown below. Numerical ratio of bound RNA
signal normalized to proteins amounts shown on top of lanes; PARP13.2 ratio set
as 1. c) Models for WWE-domain dependent inhibition of RNA binding. Purple
box: WWE domain is itself inhibitory in nature; its deletion or binding of PAR to it
results in relief of RNA binding inhibition. Orange box: binding of PAR to the
WWE domain inhibits RNA binding. Deletion of the WWE domain abrogates
interactions with PAR and thus removes its inhibitory effects on RNA binding.
151
a
PARP13.2
Str*s
No Str*
PARPI3.2**
NO W ies 8bs
b
IC
CL
0.
IB:PAR
IB: PAR
IS: GFP&*p
IB: GFP
C
Fraction
IB: PARP5a
RNA
PARP 13
PARP5
0
152
Figure 4. PARP13.2 modification with PAR and interaction with other
PARPs a) Immunoblot of input, supernatant and bound (IP) GFP-PARP13.2 and
GFP-PARP13.2ZnF, immunoprecipitated from HeLa cells treated (stress) or not
(no stress) with 20OM sodium arsenite for 45min using antibodies against PAR
and GFP. Triangle indicates molecular weight of GFP-PARP1 3.2, circle indicates
molecular weight of PARP1 3 .2AZnF. b) Immunoblots of GFP-PARP13.2 and GFPPARP13.2AZnF immunoprecipitations using antibodies against PAR, GFP and
PARP5a. Triangle indicates molecular weight of GFP-PARP1 3.2, circle indicates
molecular weight of PARP1 3. 2 AZnF. c) HeLa cells lysate was subjected to 1040% sucrose gradient centrifugation (200,000g/1 h) and fractions were stained for
RNA (ethidium bromide stained agarose gel, above), and blotted with antibodies
against PARP1 3 and PARP5a (immunoblots, below).
153
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156
Chapter 4. Conclusions and future directions
For many years PARP1 3 was thought to function exclusively during the immune
response through its ability to recognize, bind to and lead to the degradation of
certain viral transcripts (Gao et al., 2002; Guo et al., 2004; Guo et al., 2007; Zhu
et al., 2011). We have now shown that PARP13 activity is not restricted to the
context of viral infection - PARP1 3 binds endogenous RNA during physiological
conditions, in the absence of pathogens, and regulates the cellular transcriptome.
An in-depth examination of the regulation of a specific mRNA, TRAILR4, by
PARP1 3 serves as a proof of concept to demonstrate that PARP1 3 employs
many of the same mechanisms it utilizes to destabilize viral RNAs for the
degradation of cellular transcripts. PARP1 3 binding to a specific region in the
3'UTR of TRAILR4 mRNA leads to its destabilization in an exosome-dependent
manner. The resulting decrease of TRAILR4 protein levels sensitizes cells to
TRAIL-mediated cell death, demonstrating that PARP13 regulates apoptotic
signaling and its function has important consequences for the cell, independent
of its role in the immune response. This finding opens up the field to explore what
additional cellular transcripts and pathways are controlled by PARP1 3, and is
likely to initiate the discovery of multiple new functions of this protein.
Identifying more endogenous targets of PARP13
An important step to further understand the cellular functions of PARP1 3 would
be to identify additional transcripts regulated by PARP1 3 binding. Our screen of
157
mRNAs differentially expressed upon PARP1 3 depletion revealed a large
number of transcripts affected by changes in PARP1 3 levels. However, it is
currently unknown which of these represent direct PARP13 targets and which are
indirect targets - genes downstream of nodes modulated by PARP13 whose
changes in expression are a response to misregulated signaling pathways in the
cell rather than direct regulation by PARP1 3. CLIPseq - high-throughput
sequencing of RNA fragments precipitated in CLIP reactions, is a technique that
not only allows for the identification of transcripts directly bound by a specific
protein, but can also pinpoint the exact binding site on the RNA - a piece of
information that may help elucidate the exact mechanism of PARP13 target
recognition (Murigneux et al., 2013; Ule et al., 2005). This approach has been
used successfully to identify RNA targets of multiple RNA-binding proteins,
including NOVA, Ago2, MBNL1 and many others (Leung et al., 2011; Ule et al.,
2003; Wang et al., 2012), and has greatly contributed to our understanding of
their physiological roles. PARP13 CLIPseq would therefore provide invaluable
insights into novel cellular PARP13 functions.
However, a CLIPseq approach provides little functional information - while it may
demonstrate direct interactions between a protein and a (usually large) set of
transcripts, the effect of these interactions may not be obvious. In the case of
PARP1 3 this limitation is especially relevant, since PARP1 3 can affect multiple
aspects of RNA biology - both transcript destabilization and translational
repression are known outcomes of PARP1 3 regulation. It would therefore be
158
important to pair CLIPseq data with high-throughput screens that evaluate
functional consequences. For example, ribosome profiling measures changes in
ribosome occupancy on an mRNA, and is a proxy for translational efficiency
(Ingolia, 2014), while sequencing of a time course of 4-thiouridine-labeled
transcripts provides an estimate of mRNA half-life (Rabani et al., 2011).
Performing these screens on PARP1 3-null or knock-down cells in comparison to
controls, in parallel to CLIPseq, would allow for a more complete understanding
of which PARP1 3-bound transcripts represent functionally relevant targets and
what aspect of their metabolism - stability, translation or both - is regulated by
PARP13.
What determines PARP13 target specificity?
It is now clear that PARP1 3 functions both during physiological conditions and
during infection. This discovery gives rise to a few important questions. First, can
PARP13 distinguish between foreign and endogenous RNA species, and does
affinity for cellular RNA change during infection? Little research is available into
RNA-binding proteins that regulate both exogenous and endogenous transcripts.
For example, CLIPseq data for DDX17, an antiviral helicase that also regulates
cellular-RNA splicing and miRNA biogenesis, identified a sequence motif
enriched in mRNA targets of DDX1 7, but not in pri-miRNAs or viral targets (Moy
et al., 2014). Instead, binding to pri-miRNAs and foreign transcripts appears to be
mediated by a stem-loop structure and does not require sequence conservation.
It is therefore plausible that specific features within target RNAs mediate binding
159
to PARP1 3 via different mechanisms. Indeed, we observed that the VYF cavity
within the N-terminal domain of PARP13, essential for binding to certain viral
-
sequences (Chen et al., 2012), is not as critical for binding to cellular RNA
instead, the cavity defined by the HR residues appears more important in this
context.
It is further possible that posttranslational modifications make certain features of
the RNA-binding domain more accessible and more likely to participate in a
binding interaction. While this field remains mostly unexplored, there are
instances of posttranslational modifications changing specificity of binding. For
example unphosphorylated AUF1, an ARE-binding protein, compacts AU-rich
elements upon binding while phosphorylated AUF1 preferentially binds to
extended AREs and maintains them in that state, an outcome that correlates with
decreased stability of the target transcript (Zucconi and Wilson, 2011).
Conversely, posttranslational modifications can change affinity for certain targets:
phosphorylation, ADP-ribosylation and methylation have all been shown to affect
the RNA-binding affinity of RNA regulatory proteins (Di Giammartino et al., 2013;
Jammi and Beal, 2001; Yu et al., 2004).
It also remains unclear why viruses trying to evade the immune system would
preserve RNA structures that facilitate binding of a destabilizing factor such as
PARP13. While PARP13 has experienced positive selection, suggesting that it
has evolved under pressure of host-pathogen conflict, it is only the PARP domain
160
and not the RNA-binding domain that shows such rapid evolution, consistent with
PARP13 having to preserve its ability to bind to endogenous RNAs (Kerns et al.,
2008). Then how is the binding between a host factor, that is well conserved, and
viral genomes, that are evolving to escape immune response, maintained? One
possibility is that PARP1 3 recognizes features in the viral genome that are
conserved due to their functional importance; however, this does not appear very
likely as PARP13 often binds in non-protein coding regions of viral genomes,
which are likely to be less evolutionarily constrained (Guo et al., 2004; Muller et
al., 2007; Zhu et al., 2011). A second hypothesis is that another virus-specific
RNA-binding protein facilitates PARP13 recruitment and binding to viral RNA
targets - this may explain why the smallest region identified in viruses that allows
for PARP13-dependent regulation is longer than 500nt (Guo et al., 2004). Mass
spectrometry analysis of PARP13-bound proteins in infected and uninfected cells
would help determine if context-dependent adapters assist PARP13 with binding
the correct targets.
Finally, it is worthwhile to examine what happens to PARP1 3 cellular targets in
the presence of exogenous targets. One possibility is that foreign RNA acts as a
sponge titrating PARP1 3 away from endogenous targets, which therefore
become deregulated. This mechanism is used by adenovirus Ad5 to decrease
Dicer levels. Viral RNAs compete with Dicer mRNA for binding to exportin-5
(XPO5), a protein required to export Dicer mRNA from the nucleus (Bennasser et
al., 2011). During infection, decreased binding of XPO5 results in Dicer mRNA
161
retention in the nucleus and poor expression of the protein. In a similar
mechanism endogenous targets of PARP1 3 may be stabilized in the presence of
viral transcripts that titrate the protein away. Conversely, the transcriptional
upregulation of PARP13 upon infection may eliminate the possibility of
competition (Hayakawa et al., 2011; Wang et al., 2010). Destabilization of cellular
targets, therefore, may remain unchanged or may even be stimulated during the
immune response. Interestingly, interferon treatment leads to increased
sensitivity to TRAIL-mediated apoptosis, a mechanism that may help eliminate
infected cells (Sedger et al., 1999). This observation is consistent with interferondependent increase in PARP13 levels resulting in decrease in TRAILR4
expression and facilitating pro-apoptotic signaling.
Mechanisms of PARP13 regulation
We examined two potential mechanisms of PARP1 3 regulation - by interaction
with specific protein binding partners and by covalent and non-covalent
interaction with poly(ADP-ribose). We identified DHX30 as a constitutive PARP13
binding partner - it was the most enriched protein in our proteomics screen, and
it was previously identified as a PARP13 interactor in the response to HIV
infection (Ye et al., 2010). DHX30 and PARP13 co-regulate both cellular and
foreign transcripts and, therefore, DHX30-binding does not appear to provide
PARP13 with target specificity. This does not exclude the possibility that there
are context-specific binding interactions between PARP1 3 and adaptor proteins
that maximize its recruitment to specific messages. Proteomic screens
162
comparing PARP1 3 interacting proteins during different conditions would be
critical in establishing context-specific binding events that may provide an
additional level of regulation to PARP13-RNA binding.
Both non-covalent interaction with poly(ADP-ribose) via the WWE domain and
covalent modification with poly(ADP-ribose) appear to be important mechanisms
of regulation of PARP13 function that may potentially regulate its localization and
its RNA-binding affinity and specificity. We showed that the WWE domain inhibits
RNA binding, and that PARP13 interaction with PARP5a and perhaps
modification with poly(ADP-ribose) may be mutually exclusive with RNA binding.
In vitro biochemical analysis of these mechanisms has the potential to provide
the details that are currently missing. Measurement of PARP1 3 affinity for foreign
(viral RNA sequences) and cellular (TRAILR4) targets before and after in vitro
modification of PARP13 with PAR or in the presence of soluble polymer in the
reaction would provide a direct evidence for any contribution of covalent and noncovalent PAR interactions to RNA binding. A cell biological approach of
modulating PAR levels in the cells, by overexpressing or depleting catalytically
active PARPs and PARG, or by inducing stresses that trigger PAR synthesis,
and observing changes in PARP13-dependent mRNA-target regulation can point
to the physiological relevance of such regulation. Such approaches have already
been used successfully to assay changes in DNA or RNA-binding affinities of
proteins upon modification by PARP1 or upon interaction with a PAR polymer in
the nucleus (Di Giammartino et al., 2013; Ji, 2011; Malanga et al., 1998).
163
PARP13 in human health and disease
As a host antiviral factor, PARP13 functions at the forefront of the response to
infectious disease, causing the degradation of foreign transcripts and preventing
viral replication (Bick et al., 2003; Gao et al., 2002; Mao et al., 2013; Muller et al.,
2007; Zhu et al., 2011). We now show that PARP1 3 also acts to repress the
decoy receptor TRAILR4, and thus increases cell sensitivity to TRAIL, a
proapoptotic cytokine that specifically targets transformed cells and is a
promising agent for the development of anti-cancer therapeutics (Degli-Esposti et
al., 1997; LeBlanc and Ashkenazi, 2003; Stuckey and Shah, 2013; Vindrieux et
al., 2011). PARP13 therefore emerges as an important pro-apoptotic factor that
may be essential for TRAIL-dependent elimination of cancerous cells. Stabilizing
the interactions of PARP13 with its known cognate RNAs, either during viral
infection, or in the context of malignancies, has the potential to improve
outcomes in the fight against infections and cancer, respectively. It is therefore
imperative that we better understand how PARP1 3 recognizes its targets and
how its binding affinity and specificity for different RNAs is regulated. This
information would not only elucidate how PARP13 regulates various disease and
stress-response pathways but can also help develop targeted therapeutics that
selectively affect PARP13 function during infections or in cancer.
164
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167
Acknowledgements
I would like to thank my advisor, Dr. Paul Chang, for his continuous guidance and
encouragement during my PhD, and the members of my committee, Dr. Richard
Hynes and Dr. David Bartel, for their support and thoughtful advice through the
years.
I also thank all the members of the Chang lab I worked with during my PhD, Sejal
Vyas, Jenny Rood, Lilen Uchima, Robert Dorkin, Florian Bock, Melissa
Chaserone-Cataldo, Miri Jwa and Tenzin Sangpo, for their help and advice, and
for creating a supportive working environment. I would like to specifically
acknowledge Tenzin for her assistance with cloning a lot of the DNA constructs
described in this work, and Sejal and Florian for helping me edit and proofread
this thesis.
I thank people in the Koch Institute for Integrative Cancer Research, especially
members of the Houseman lab (Theresa and Hilary), Amon lab (Luke) and Sharp
lab (Andrew and Jesse), for helping me learn new techniques and for useful
advice.
Finally I would like to thank my parents, Todor Todorov and Dafinka Todorova,
my brother, Dimitar Todorov, and my fianc6, Matthew Kwan, for their continuous
and unyielding support during my PhD and for giving me the strength and
motivation to keep on working and doing my best.
168
Curriculum Vitae
TANYA TODOROVA
Koch Institute for Integrative Cancer Research, MIT
todorova(almit.edu,
617-324-4057
Education
2009-2014
(expected)
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
Ph.D, Department of Biology
2005-2009
BOWDOIN COLLEGE
Cambridge, MA
Brunswick, ME
Bachelor of Arts, Biology, Summa cum laude
Research
2009-2014
2007-2009
Cambridge, MA
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
PhD Candidate, Laboratory of Dr. Paul Chang
Thesis: PARP1 3 is a post-transcriptional regulator of cellular mRNA and functions as a
pro-apoptotic factor by destabilizing TRAILR4 transcript
Performed CLIP to show PARP13 binds cellular RNA
Designed a high-throughput screen to identify transcripts misregulated upon PARP13
knockdown
Identified TRAILR4 mRNA as a direct target of PARP13 regulation
Developed biochemical and cellular assays to establish the mechanism of PARP1 3
protein-TRAILR4 mRNA binding and regulation
Brunswick, ME
BOWDOIN COLLEGE
Research Assistant, Laboratory of Dr. Bruce Kohorn
Project: Role of Wall-Associated Kinases in the Arabidopsis defense response
Examined the Arabidopsis transcriptional response to pectin-induced stress to
identify wak-2 dependent effects
Identified a requirement for wak-2 for the regulation of wall synthesis genes
- Assayed reactive oxygen species production in wak-2 knockout to demonstrate
increased stress levels in the mutant
Publications
Todorova T, Bock F and Chang P. (2014) PARP13 is a post-transcriptional regulator of cellular mRNA and
functions as a pro-apoptotic factor by destabilizing TRAILR4 transcript. Nature Communications, 5
Vyas S, Chessarone-Cataldo M, Todorova T, Huang YH and Chang P. (2013) A systematic analysis of the
PARP protein family identifies new functions critical for cell physiology. Nature Communications, 4
Leung A, Todorova T, Ando Y and Chang P. (2012) PolyADP-ribose regulates post- transcriptional gene
regulation in the cytoplasm. RNA Biology, 9: 542-548
Kohorn BD, Kohorn SL, Todorova T, Baptiste G, Stansky K, McCullough M. (2012) A dominant allele of
Arabidopsis pectin-binding wall-associated kinase induces a stress response suppressed by MPK6 but not
MPK3 mutations. Molecular Plant, 5: 841-851
Kohorn BD, Johansen S, Shishido A, Todorova T, Martinez R, Defeo E and Obregon P. (2009) Pectin
activation of MAP kinase and gene expression is WAK2 dependent. The Plant Journal, 60: 974-982
169
Skills
*
-
General: Mammalian cell culture, protein expression, protein purification, cloning
RNA biology: Transcriptome screens, crosslinking immunoprecipitation (CLIP), RNA
immunoprecipitation (RIP), QPCR, electrophoretic mobility shift assays (EMSA), Northern blotting
Protein biology: Protein coimmunoprecipitation, in-vitro enzymatic assays, poly(ADP-rybos)ilation
Cell biology: immunofluorescence, fixed cell imaging, live-cell imaging, viability assays, apoptosis
assays, FACS
Teaching
Spring 2013
Spring 2011
2005-2009
MASSACHUSETTS INSTUTUTE OF TECHNOLOGY
Teaching Assistant
* Assisted teaching Genetics (7.03) and Introduction to Biology (7.01)
Lead review sessions three times a week
Designed study and test materials
-
BOWDOIN COLLEGE
Tutor, study group leader, senior mentor
* Tutored Chemistry and Biology
Helped students develop study skills
Mentored first year minority students interested in STEM subjects
-
Conferences and presentations
Apr. 2014
The PARP Family and Friends, Cold Springs Harbor Laboratories
Poster: PARP13 Binds Cellular RNA and Destabilizes TRAILR4 mRNA
Posttranscriptionally by Targeting it for Decay
Jan. 2014
The Sigma-Aldrich Symposium on RNA Science and its Application
Talk: PARP13 Regulation of Cellular RNA
Dec. 2013
American Society for Cell Biology Annual Meeting
Poster: PARP13 Binds and Regulates Cellular RNA
Oct. 2013
Koch Institute for Integrative Cancer Research Annual Retreat
Talk: PARP13 Regulation of Cellular RNA
Mar. 2013
Koch Institute for Integrative Cancer Research Seminar
Talk: Time to Unwind with PARP13 and DHX30
Mar. 2012
Keystone Symposium: RNA Protein Interactions in Physiology and Human Disease
Poster: The CCCH-PARPs -12 and-13 Are Novel RNA Binding Proteins That Function
in the Cytoplasmic Stress Response
Honors and awards
Marlena Felter Bradford Research Travel Fellowship
2014
School of Science Graduate Student Fellowship for Cancer Research
2013
MIT Presidential Graduate Fellowship Recipient
2009
Phi Beta Kappa, Chapter of Maine
2009
References
Dr. Paul Chang, Assistant Professor, MIT
Dr. David Bartel, Professor, MIT
Dr. Richard Hynes, Professor, MIT
170
pchang2@mit.edu, 617-324-3879
dbartel@wi.mit.edu 617-258-5287
rohynes@mit.edu, 617-253-6422
Appendix 1. Poly(ADP-ribose) regulates post-transcriptional gene
regulation in the cytoplasm.
.
Leung A.', Todorova T. 2 , Ando Y. 1, and Chang P2
'Department of Biochemistry and Molecular Biology, Bloomberg School of
Public Health, Johns Hopkins University, Baltimore, MD; 2 Koch Institute for
Integrative Cancer Research, Department of Biology, Massachusetts Institute of
Technology, Cambridge MA 02139
Published as:
Leung, A., T. Todorova, Y. Ando and P. Chang (2012). "Poly(ADP-ribose)
regulates post-transcriptional gene regulation in the cytoplasm." RNA Biol 9(5):
542-548.
171
RNA Biology 9:5, 542-548; May 2012; ( 2012 Landes Bioscience
Poly(ADP-ribose) regulates post-transcriptional gene regulation
in the cytoplasm
Anthony K.L. Leung,,* Tanya Todorova,2 Yoshinari Ando' and Paul Chang"*
2
'Department of Biochemistry and Molecular Biology; Bloomberg School of Public Health; Johns Hopkins University; Baltimore, MD USA; Koch Institute for
Integrative Cancer Research and Department of Biology; Massachusetts Institute of Technology; Cambridge, MA USA
12
poly(ADP- linear or branched chains. It is a postshown translational modification whose function
to play important functions in the is best understood in the context of the
nucleus, where its synthesis is highly
nucleus of multicellular eukaryotes.
Each of these functions centers upon upregulated during DNA damage.' The
DNA metabolism, including DNA- - polymer acts as a scaffold to recruit repair
damage repair, chromatin remodeling, factors to the sites of damage and helps
transcription and telomere functions. We relax chromatin structure to facilitate
recently described two novel functions DNA repair.4 It was later recognized that
for pADPr in the cytoplasm, both of pADPr has additional nuclear functions
which involve RNA metabolism- . during non-stress conditions where it is
(1) the assembly of cytoplasmic stress involved in transcriptional regulation,
granules, cellular macrostructures that chromatin remodeling and telomere
aggregate translationally stalled mRNA/ functions.3
The poly(ADP-ribose) polymerase
protein complexes and (2) modulation
of microRNA activities. Multiple stress (PARP) family of proteins uses NAD' as
post-transcriptional substrate to synthesize ADPr modifications
granule-localized,
gene regulators, including microRNA- onto acceptor proteins.' 2 5 6 PARP-1,which
binding argonaute family members, are functions during DNA damage, is the
substrates for pADPr modification and founding member of the family. Using the
are increasingly modified bypADPr upon PARP-1 catalytic domain, bioinformatics
stress. Interestingly, the cytoplasmic analyses identified 16 additional PARPs in
RNA regulatory functions for PARPs the human genome, many with unknown
are likely mediated through activities function.5 A new nomenclature for this
PARP family was thus recently proposed
of catalytically inactive PARP-13/
(Table 1),' but their conventional names
ARTD13/ZC3HAV1/ZAP and mono/
poly(ADP-ribose)-synthesizing enzymes, are used here for the sake of familiarity.
A detailed examination of their conserved
including
PARP-5a/ARTD5/TNKS1,
and catalytic residues revealed that 11 out
PARP-12/ARTD12/ZC3HDC1
PARP-15/ARTD7/BAL3. These data are of 17 PARPs lack one or more of three
consistent with other recent work, which critical residues-histidine, tyrosine and
suggests that mono (ADP-ribosyl) ated glutamate (or HYE triad)-required
residues can be poly(ADP-ribosyl)ated for pADPr polymerization activities.
Two PARPs, PARP-9 and -13, lack
by different enzymes.
the histidine and glutamate, which are
important for binding NAD+ and pADPr
Introduction
elongation, respectively, and were thus
8
is
(pADPr)
a predicted to be catalytically inactive. On
Poly(ADP-ribose)
macromolecular polymer consisting of the other hand, the remaining 9 PARPs
2-200 ADP-ribose subunits organized in are predicted to transfer single ADP-ribose
in 1963,
its discovery
ribose)
(pADPr) has been
Since
Key words: stress, stress granule,
microRNA, PARP, PARG,
PARP-13, ZAP
Submitted: 08/14/11
Revised: 02/16/12
Accepted: 03/05/12
http://dx.doi.org/10.4161/rna.19899
*Correspondence to: Anthony K.L. Leung and
Paul Chang; Email: anleung@jhsph.edu and
pchang2@mit.edu
542
RNA Biology
Volume 9 Issue
5
POINT-OF-VIEW
Table 1.PARP family
ARTD1
PARP-2
ARTD2
PARP-3
ARTD3
PARP-4
ARTD4, vPARP
PARP-5a
ARTD5, TNKS1
PARP-5b
ARTD6, TNKS2
PARP-6
ARTD17
PARP-7
ARTD14, tiPARP
PARP-8
ARTD16
PARP-9
ARTD9, BALl
PARP-10
ARTD10
PARP-11
ARTD11
PARP-12
ARTD12,ZC3HDC1
PARP-13.1
ARTD13, ZC3HAV1
PARP-13.2
ARTD13, ZC3HAV1, ZAP
PARP-14
ARTD8, BAL2
PARP-15
ARTD7, BAL3
PARP-16
ARTD15
+
PARP-1
Stress Granule Localization
+
Alternative Names
+
(+)
"+" denotes stress granule localization determined by antibody staining against endogenous
proteins and GFP-tagging. The antibody staining of PARP-14 has yet to be verified by GFP-tagging.
units, i.e., mono(ADP-ribose) or mADPr,
onto acceptor proteins. Such mono (ADPribosyl)ating activities were confirmed
recently for PARP-10, PARP-12, PARP-14
and PARP-15.' 9
The presence of PARPs with distinct
catalytic activities-synthesis of mADPr
or pADPr-suggests new possibilities.
First, the effect of mADPr modification of an acceptor is likely quite different from addition of a long pADPr chain.
Second, PARPs of distinct activities could
potentially cooperate in order to modify
a common acceptor protein.8 The physical association of mADPr- and pADPrsynthesizing enzymes was demonstrated
by nuclear-localized PARP-1 and PARP3,10 and, from our recent work, PARP-5a
and PARP-12 in the cytoplasm.9 These
observations suggest a novel mechanism
of pADPr synthesis in which the initiation
and elongation steps can be catalyzed by
different PARPs.
The possibility that poly(ADP-ribosyl)
ation can be mediated by multiple
enzymes is further supported by in vitro
and "in-cell" experiments. For example,
an existing single ADP-ribose could be
poly(ADP-ribosyl)ated by incubating with
NAD+ and bacterially expressed PARP-1
www.landesbioscience.com
in vitro, where the initial and elongation
steps were separated. In these cases, the
initial ADP-ribose could be derived from
the PARP-1 E988K mutant that can
only auto-mono(ADP-ribosyl)-ate itself,
or the ADP-ribose could be chemically
synthesized and conjugated to peptides or
agarose beads.10- 2 Notably, mono(ADPribosyl)ated residues can be derived in cells
from another family of NAD+-consuming
enzymes known as sirtuins.3 This family
is exemplified by the yeast member Sir2p,
which catalyzes the removal of acetyl
groups from histones and is involved in
gene silencing, chromosomal stability and
aging. However, quite a few members in
yeast, Trypanosomes and mammals have
also demonstrated mono (ADP-ribosyl)
ating activities." For example, yeast Sir2p
can catalyze the transfer of ADP-ribose
to itself and histones. In mammals, two
substrates were identified to date-SIRT4
ribosylates glutamate dehydrogenase to
suppress insulin signaling in pancreatic
P cells and, recently, SIRT6 mono(ADPribosyl)ates PARP-1, which then promotes
PARP-1 automodification with further
ADP-ribose units, upon DNA damage."
This latter observation suggests that
not only the two NAD+-dependent
RNA Biology
signaling pathways can crosstalk and
cooperate, but also highlights the
possibility
that
mono(ADP-ribosyl)
ated residues can be poly(ADP-ribosyl)
ated by different enzymes in cells.
Similar sequential modification was
also proposed for other macromolecular
post-translational modifications, such
as poly(ubiquitin)ation.' 6 Potentially,
there are several advantages conferred
by this type of conserved mechanism in
post-translationally modifying substrates
sequentially using different enzymes-(1)
define specificity of substrates to poise for
poly(ADP-ribosyl)-ation only at certain
cellular conditions; (2) increase substrate
diversity given a limited repertoire of
enzymes; or (3) serve as a safeguard/
crosstalk mechanism by adding an extra
layer of regulation. So far, the functions
of mono (ADP-ribosyl)-ation conferred
by these subclasses of PARPs and SIRTs
remain unclear. Given that there are
-1,000-fold more amino acid residues that
are mono(ADP-ribosyl)-ated than being
poly(ADP-ribosyl)ated (as estimated in the
rat liver), 17 it is formally possible that these
mono(ADP-ribosyl)-ated residues could
serve as unique signals or, alternatively,
as starting points for poly(ADP-ribosyl)
ation.
PARP Functions Beyond
the Nucleus
As more was discovered about the biology
of these recently identified PARPs, it
became clear that pADPr does have
cellular functions beyond the nucleus.
Most
PARPs
exhibit
cytoplasmic
localizations, 18-22 yet relatively little is
known about the function ofpADPr in the
cytoplasm. Early works by the founders
of the field identified PARP activities
in
cytoplasmic,
post-mitochondrial
fractions. Such activities accounted for
-25% of total PARP activities in the cell.
These cytoplasmic PARP activities were
identified in multiple mammalian cell
types (HeLa, macrophages, erythroblasts,
plasma cells and neurons) and tissues
(brain and liver extracts) suggesting that
the modification is ubiquitous.2 ' 2 5 Further
functions for cytoplasmic pADPr were
suggested by the activities of its degradative
enzyme, pADPr glycohydrolase (PARG).
543
More than 50% of total PARG activities
are concentrated in the cytosol,23
suggesting that pADPr synthesis and
function in the cytoplasm are tightly
regulated. Hints regarding the functions
of cytoplasmic pADPr came from an early
observation of its activities enriched in
free mRNP fractions in sucrose gradients.
Such fractions were enriched in factors
that regulate translation potential and
decay of mRNAs.24 2, 628 Such correlation
led to the hypothesis that pADPr may be
involved in post-transcriptional mRNA
regulation. This hypothesis was further
supported by the recent discovery of two
PARPs, PARP-12 and -13, that localize to
the cytoplasm and contain CCCH type
zinc finger RNA-binding domains.'2
While the biological function of
PARP-12 remains poorly understood,
PARP-13 is known for its roles in binding
and regulating viral RNA transcripts. 2931
It was initially identified as Zinc finger
Anti-viral Protein (ZAP) in a screen for
host factors that confer resistance to the
retrovirus moloney murine leukemia
virus infection,2 9 and its anti-viral activities were later confirmed in other retroviruses such as HIV-1, or other RNA virus
families, including Alphaviruses and
Filoviruses. 303 2 PARP-13 binding to viral
RNAs correlates with their subsequent
degradation and the inability of the virus
to replicate efficiently. 9 PARP-13 coimmunoprecipitates with two RNA helicases (p7 2 and DHX30) and the exosome
complex, thought to be responsible for
viral RNA unwinding and degradation,
33
respectively. -
Knockdown of specific
exosome components decreased the antiviral activities of PARP-13, suggesting that
PARP-13 mediates viral RNA degradation
3 3
via an exosome dependent pathway. -
Moreover, PARP-13 has two isoforms;
the shorter PARP-13.2 isoform has recently
been shown to directly bind to RIG-I, a
key factor of innate immune response,
after viral infection. 36 RIG-I itself is an
RNA helicase and associates with viral
RNAs. The PARP-13.2 binding to RIG-1
activates the type-I interferon signaling.
As both RIG-1 and PARP-13.2 bind viral
RNAs, such PARP-13.2-to-RIG-1 association stabilizes their binding to viral
RNAs,3 which might further strengthen
the anti-viral signaling pathway.
544
However, these anti-viral properties are
not due to the ADP-ribosylating activities
of PARP-13 because PARP-13 proteins
are catalytically inactive, either lacking a
PARP domain (PARP-13.2 isoform), or
the critical HYE triad residues (PARP13.1 contains YYV),' 8 where their lack
of activities were confirmed by in vitro
ADP-ribosylating assays.89 Yet the longer isoform, PARP-13.1, which contains
the "catalytically inactive" PARP-like
domain, confers stronger anti-viral activi37
ties. Despite the lack of catalytic activities, PARP-13 function may require ADPr
modification by other PARPs instead, and
we found that the modifications of these
two isoforms change upon stress differently-pADPr modification of PARP13.2 increases whereas PARP-13.1 remains
unchanged.9 Such trans-modification is
common among PARPs, including modification between PARP-1 and PARP-2,"
and between PARP-5a and PARP-5b.
Thus, it is possible that trans-modification
of PARP-13 by other cytoplasmic PARPs
is important for mediating two novel
roles of pADPr in the cytoplasm (Fig. 1):
assembly of stress granules and regulating
microRNA activities. 9
Multiple Cytoplasmic PARPs
Regulate SG Assembly
Cells respond to physiological stresses,
such as heat shock, oxidative stress,
ischemia and viral infection by assembling
large multi-protein complexes in the
cytoplasm called stress granules (SGs). 9
SGs are found in many pathological
conditions, including hypoxic tumor
cores49 and neurons in animal models of
ischemia stroke'" and can be induced by
anti-cancer agents, such as arsenite" and
pateamine A." SGs contain poly(A)'
mRNAs, stalled translation initiation
complexes and multiple RNA-binding
proteins, and are thought to regulate
the stability and translation potential
of mRNAs. We have recently shown
that SGs also comprise pADPr, 2 PARG
isoforms (PARG99 and PARG102) and
5 PARPs (PARP-5a, -12, -13.1, -13.2 and
-15; localization of PARP-14 was also
identified by antibody staining but yet
to be confirmed by GFP tagging; Table
1 and Fig. 2).9 Overexpression of these
RNA Biology
Relief of
rmicroRNA silencing
Covalent
Model
.SG
1
proteins
SGs
Scaffold
Model
SG formation
Keys'
Acceptor
pAD~r
pADPr
Protein
Figure 1. Two novel functions for pADPr in
the cytoplasm. The covalent model indicates
the alteration of protein properties inherently
(highlighted with a star in the figure) whereas
the scaffold model indicates that the change
of protein function relies on new non-covalent protein associations through pADPr.
PARPs resulted in the de novo nucleation
of SGs, identifying them as core SG
components. In contrast, overexpression
of the PARG isoforms result in inhibition
of SG assembly and knockdown of PARG
9
delays disassembly of SG. Together, these
data suggest that pADPr concentration
in the cytoplasm is locally regulated for
the assembly and maintenance of SG
structure.
How does pADPr mediate the
assembly and disassembly of the mRNPenriched SG? We propose that pADPr
functions as a scaffold to bridge diverse
mRNA/protein complexes together. Such
scaffolding properties of pADPr have also
been observed in DNA repair complexes,'
4
the mitotic spindle "' and another RNA
organelle Cajal bodies." To scaffold, two
classes of proteins appear to be required:
proteins that are covalently modified by
pADPr and proteins that bind to pADPr
non-covalently. During stress, select
sets of RNA-binding SG components,
AGO1-4,
TIA-1, G3BP1
and PARP-
13.1/2 complex are increasingly modified
by pADPr.9 On the other hand, poly(A)binding protein PABP and heterogeneous
ribonucleoprotein hnRNP Al both bind
to but are not modified by pADPr.47 In
addition, a subset of proteins such as the
endoribonuclease G3BP1, both bind to and
are modified by pADPr.9,47 In assembling
Volume 9 Issue 5
Ankyrin-repeat (ANK)
HPS
PAR?
PARP-5a
CCCH-Zinc finger
PAPPARP- 12
NLS
(dat n
NFS
PAPPARP- 13.1
[IDO
PARP-13.2
PARP
PARP-14
SPAR?
PARP-I S
Domains by functional categories
pADPr binding:ws
Localization targeting:
[]Protein-Protein Interaction:Aw Amw,,.W4&M
&.A.a..
. n[-Gw] PARP catalytic domain
...
.
..
Unknown function: wsmw ..
'
RNA-binding: cc.,.
Figure 2. Domain structure of PARPs localized in SGs.
SGs, proteins that are modified by pADPr
become cross-linked to pADPr binding
proteins via protein-pADPr binding
interactions. The increasing chains of
pADPr emanating from modified RNAbinding proteins then recruit additional
RNA binding proteins via non-covalent
pADPr-protein interactions. Thus pADPr
could function as a focal nucleator of SGs
through multiple cycles of modification,
recruitment and cross-linking (Fig. 3).
Consistent with this hypothesis, previously
reported dominant negative mutants of
G3BP1 4 and TIA-1 9 that disrupt SG
assembly are not modified by pADPr in
stressed cells.9
In our working model, SGs are a
physical manifestation of the extensive
networks formed by interactions between
pADPr modification on RNA-binding
proteins, proteins that bind pADPr and
proteins that bind to RNA. In fact, those
PARPs localized in SGs, including the
CCCH zinc finger PARPs, could initiate
part of this interaction network through
their various binding domains for protein,
RNA and pADPr (Fig. 2). The pADPr-SG
scaffold appears to be highly dynamic and,
depending on cell stress state, disassembled
or assembled by regulating PARP/PARG
activities. These opposing enzymatic
activities could thus provide a rapid,
regulatory mechanism for SG assembly
or disassembly, while keeping individual
www.landesbioscience.com
RNA/protein complexes and their
functions intact (Fig. 4). The preservation
of RNA/protein complex function could
be critical to a rapid cellular recovery
from stress. Furthermore, one notable
characteristic of pADPr, distinctive from
other macromolecular post-translational
modifications, such as ubiquitin and
SUMO, is its non-proteinaceous nature,
which theoretically renders the pADPr
scaffold resistant to the activities of
proteases, such as caspases, during stress.
PARP-13 Together
with Other PARPs Regulate
MicroRNA Silencing
In addition to a function in the assembly
of macrostructures, pADPr also plays
important roles in regulating mRNA/
protein interactions at the molecular scale.
To better understand this role, we focused
on examining pADPr modification of
AGO2 and its effect on microRNA
silencing.' This was due to its pervasive
roles in regulating > 60% of mammalian
mRNAs. 50 AGO2
modification
is
mediated by multiple PARPs that
bind to AGO2, each of which exhibits
distinct catalytic activities: PARP-13
is catalytically inactive, PARP-5a has
demonstrated
poly(ADP-ribosyl)ating
activities and PARP-12 has mono(ADPribosyl)ating activities. We also observed
RNA Biology
that other family members of argonaute
(AGO1-4) are modified by pADPr. Not
surprisingly, AGO2 immunoprecipitates
exhibited ADP-ribosylating activities upon
NAD' addition. Addition of the general
PARP
inhibitor
3-aminobenzamide,
which can potentially bind to PARP
domains of all catalytically active PARPs,
completely abrogated ADPr synthesis
in the AGO2 immunoprecipitates. The
pattern of AGO2 modification and the
concentration-dependent
incorporation
of NAD' confirmed that the AGO2
precipitates contain pADPr synthesizing
activities and that it is directly modified
by mADPr and/or pADPr. However, it
remains to be investigated whether the
poly(ADP-ribosyl)ation is mediated in
a sequential manner-as in the case of
SIRT-6 mediated poly(ADP-ribosyl)ation of PARP-11-that is, first by the
mono(ADP-ribosyl)ating
PARP-12,
followed
by
poly(ADP-ribosyl)ating
PARP-5a, or directly through PARP-5a
alone.
What is the effect of ADPr modification on argonaute activities? The
amount of pADPr modification of argonaute was inversely correlated with
microRNA silencing. Upon stress, argonaute modification by pADPr increased,
and microRNA silencing was relieved.
Notably, under the same conditions, the
global translation rate was reduced; however the expression of microRNA targets
decreased to a lesser extent than other
mRNAs, consistent with selective regulation of argonaute activities. Similarly,
we observed an increase in pADPr modification of argonaute and a decrease in
microRNA silencing when PARG was
knocked down, consistent with a function
for pADPr modification of argonaute in
regulating microRNA activities.
How is argonaute modification by
pADPr regulated? We are just beginning
to study this aspect of microRNA/
ADPr function, however critical data
has emerged. Poly(ADP-ribosyl)ation of
AGO2 is dependent on its ability to bind
RNA. AGO2 mutants lacking the mRNA
binding PIWI domain are unable to be
poly(ADP-ribosyl)ated. Interestingly, a
similar phenomenon was observed for
other post-transcriptional
regulators
TIA-1 and G3BP1, where their respective
545
IMj
Extensive network of interactions between protein,
RNA and pAOPr
SG formation
y
z
z
zy
7ansmodification
pADPRacceptorprotei
pADPr nucleates
of RNA binding protens
swa
Us
pADr bindg pmtcns that bind MNA
At
..,Op
35%
binding of other AMA binding proteins
pADPr
(tGt8,. G Ma
SpAOAWbiNtt paoten that bin otw~protefts
Figure 3. Proposed model of SG assembly.
dppp,
r"McbW
-prow"i
PARG
SG assembly
SG disassembly
PARP
Figure 4. Proposed model of SG disassembly.
mutants lacking RNA-binding motifs
were also not poly(ADP-ribosyl)-ated.
These data suggest that either RNAbinding is required for modification, or
that the RNA-binding domain is the site
of pADPr modification.
546
PARP-13 appears to be critical for
microRNA silencing. Upon stress,
modification ofargonaute family members
and PARP-13.1/2 complex by pADPr
significantly increases. Overexpression of
the catalytically inactive PARP-13.1 or
RNA Biology
PARP-13.2 relieves microRNA silencing.
While the mechanism of PARP-13
function in AGO2 regulation is not
currently clear, one possibility is that the
poly(ADP-ribosyl)ation of argonaute is
mediated by catalytically active PARPs
Volume 9
Issue 5
(PARP-5a and PARP-12) via PARP-13.1/2.
In fact, all of these PARPs bind to PARP13 family members. This is reminiscent of
the activation of tyrosine kinase pathway
where erbB-3, which, though itself has
no active kinase domain, can mediate
signaling through heterodimerization
with active EGF family kinases like
Her2." Consistent with this, AGO2
associates with PARP-13 in an RNAdependent manner. Perhaps, because of
their ability to bind mRNA, the function
of the inactive PARP-13 isoforms is to
anchor the activities of the catalytically
active PARPs to the mRNP complex. This
might partially explain why the mRNAbinding domain of AGO2 is required for
its poly(ADP-ribosyl)ation.
Based on these observations, we propose that the relief of microRNA silencing
occurs due to a decrease in the accessibility
of the argonaute/microRNA complex to
target mRNAs. This decrease likely results
from the increased pADPr modification of
proteins within the complex, such as those
microRNA-binding argonaute family
members and PARP-13. Such modification could change the protein structure of
target proteins resulting in altered accessibility to mRNA. Alternatively, considering that one ADP-ribose unit is -0.5
kDa in size (equivalent to -5 amino acids)
and has two negatively-charged phosphate groups, increased concentrations
of pADPr near the RNA binding site in
the argonaute/microRNA complex could
disrupt electrostatic interactions or present steric hindrances between argonaute/
microRNA and mRNA target. Finally,
the pADPr might provide a scaffold for
recruiting repressor(s) of microRNA activities to target mRNAs.
Conclusion
In summary, we propose that pADPr
functions as a general mediator of
stress and can specifically regulate posttranscriptional gene expression in the
cytoplasm: stress granule assembly and
microRNA activities. These results
further extend its well-characterized DNA
metabolism roles mediated by PARP-1
in the nucleus. Small molecule inhibitors
targeted against PARP-1, which mediates
www.landesbioscience.com
DNA damage repair in the nucleus, are
already in phase II/III human trials for
cancer therapy.1 2 These inhibitors have
also shown promise for treatment of
inflammation, ischemia and degenerative
vascular diseases."" Our results suggest
that these therapeutic effects may be due
to partial inhibition of PARPs in the
cytoplasm.
8.
9.
10.
Note added in proof
Wahlberg et al." recently profiled 185
existing PARP inhibitors and found that
some existing PARP-1 inhibitors can also
bind catalytic domains of other PARPs
with comparable affinity. Therefore,
designing specific inhibitors to PARPs
may potentially result in highly effective
treatments for these stress-related diseases.
11.
12.
13.
Acknowledgements
P.C. is a Rita Allen Foundation Scholar
and a Kimmel Foundation for Cancer
Research Scholar. This work is supported
by NIH Award R01-GM087465-01A2 to
P.C. and DOD Idea Award BC101881 to
A.K.L.L. We thank Phillip A. Sharp for
his insights on the project and comments
on this manuscript.
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