Reduced Expression of Ribosomal Proteins Relieves
MicroRNA-Mediated Repression
The MIT Faculty has made this article openly available. Please share
how this access benefits you. Your story matters.
Citation
Janas, Maja M., Eric Wang, Tara Love, Abigail S. Harris, Kristen
Stevenson, Karlheinz Semmelmann, Jonathan M. Shaffer, et al.
“Reduced Expression of Ribosomal Proteins Relieves
MicroRNA-Mediated Repression.” Molecular Cell 46, no. 2 (April
2012): 171-186. © 2012 Elsevier.
As Published
http://dx.doi.org/10.1016/j.molcel.2012.04.008
Publisher
Elsevier B.V.
Version
Final published version
Accessed
Thu May 26 01:57:55 EDT 2016
Citable Link
http://hdl.handle.net/1721.1/83590
Terms of Use
Article is made available in accordance with the publisher's policy
and may be subject to US copyright law. Please refer to the
publisher's site for terms of use.
Detailed Terms
Molecular Cell
Article
Reduced Expression of Ribosomal Proteins
Relieves MicroRNA-Mediated Repression
Maja M. Janas,1,3,4 Eric Wang,5,6 Tara Love,1,3,4,8 Abigail S. Harris,7 Kristen Stevenson,2 Karlheinz Semmelmann,7
Jonathan M. Shaffer,7 Po-Hao Chen,1,3,4 John G. Doench,4 Subrahmanyam V.B.K. Yerramilli,7 Donna S. Neuberg,2
Dimitrios Iliopoulos,1,3 David E. Housman,5,6 Christopher B. Burge,5 and Carl D. Novina1,3,4,*
1Department
of Cancer Immunology and AIDS, Dana-Farber Cancer Institute
of Biostatistics and Computational Biology, Dana-Farber Cancer Institute
3Department of Microbiology and Immunobiology
Harvard Medical School, Boston, MA 02115, USA
4Broad Institute of Harvard and MIT, Cambridge, MA 02141, USA
5Department of Biology
6The Koch Institute
Massachusetts Institute of Technology, Cambridge MA 02139, USA
7Qiagen, Frederick, MD 21703, USA
8Present address: Correlagen Diagnostics, 307 Waverly Oaks Road, Suite 101, Waltham, MA 02452, USA
*Correspondence: carl_novina@dfci.harvard.edu
DOI 10.1016/j.molcel.2012.04.008
2Department
SUMMARY
MicroRNAs (miRNAs) regulate physiological and
pathological processes by inducing posttranscriptional repression of target messenger RNAs (mRNAs)
via incompletely understood mechanisms. To discover factors required for human miRNA activity, we
performed an RNAi screen using a reporter cell line
of miRNA-mediated repression of translation initiation. We report that reduced expression of ribosomal
protein genes (RPGs) dissociated miRNA complexes
from target mRNAs, leading to increased polysome
association, translation, and stability of miRNA-targeted mRNAs relative to untargeted mRNAs. RNA
sequencing of polysomes indicated substantial overlap in sets of genes exhibiting increased or decreased
polysomal association after Argonaute or RPG
knockdowns, suggesting similarity in affected pathways. miRNA profiling of monosomes and polysomes
demonstrated that miRNAs cosediment with ribosomes. RPG knockdowns decreased miRNAs in
monosomes and increased their target mRNAs in
polysomes. Our data show that most miRNAs repress
translation and that the levels of RPGs modulate
miRNA-mediated repression of translation initiation.
INTRODUCTION
MicroRNA (miRNA)-mediated translational repression of
messenger RNA (mRNA) targets was first described in
C. elegans, where lin-4 and let-7 were reported to reduce protein levels without affecting mRNA stability (Olsen and Ambros,
1999; Wightman et al., 1993). Untargeted and miRNA-targeted
mRNAs demonstrated similar polysomal association as as-
sessed by density gradient centrifugation, suggesting that
miRNAs repressed translation after initiation (Olsen and Ambros, 1999; Seggerson et al., 2002). In mammals, polysome
profiling studies led to contradictory models of miRNAmediated translational repression. Although miRNA-targeted
mRNAs had reduced polysomal association compared to
unrepressed mRNAs in some studies, suggesting repression
of initiation (Bhattacharyya et al., 2006a; Pillai et al., 2005),
miRNAs and their mRNA targets had robust polysomal association in other studies, suggesting repression after initiation
(Maroney et al., 2006; Nelson et al., 2004; Nottrott et al.,
2006; Petersen et al., 2006). Additionally, treatment with translation elongation inhibitors shifted miRNAs and miRNA-targeted
mRNAs from polysomes toward monosomes, indicating that
miRNAs target actively translating mRNAs (Maroney et al.,
2006; Nottrott et al., 2006).
Subsequent studies demonstrated that miRNAs could also
reduce mRNA stability. In C. elegans, lin-4 and let-7 decreased
target mRNA levels (Bagga et al., 2005). In both C. elegans and
mammals, miRNAs were shown to promote deadenylation,
leading to reduced target mRNA stability (Behm-Ansmant
et al., 2006; Wu and Belasco, 2005; Wu et al., 2006). In
mammals, transfected miRNAs globally reduced levels of target
mRNAs (Lim et al., 2005), and ribosome footprinting determined
that miRNA targeting resulted in a decrease in ribosome density
that was only modestly (15%) greater than the decrease in
mRNA level, suggesting that miRNAs act primarily to destabilize
mRNAs (Guo et al., 2010). More recent ribosome footprinting
analysis at earlier time points demonstrated that repression of
translation initiation precedes deadenylation for the majority of
miR-430-targeted mRNAs in zebrafish and that mRNA decay is
likely a consequence of these earlier activities (Bazzini et al.,
2012).
Here we report that 70% of all miRNA species expressed
in HeLa cells are detected in monosomal and polysomal fractions, implying that the majority of miRNA species can repress
translation. We also demonstrate that ribosomal protein genes
Molecular Cell 46, 171–186, April 27, 2012 ª2012 Elsevier Inc. 171
Molecular Cell
Ribosomal Proteins Modulate miRNA Activity
(RPGs) preferentially regulate miRNA-mediated repression of
translation initiation. Specifically, RPG depletion led to
decreased monosomal association of a subset of miRNAs and
increased polysomal association of mRNA targets of those
miRNAs, indicating relief of translation initiation block. This
study establishes a framework for understanding ribosomes as
global regulators of miRNA-mediated repression of translation
initiation.
RESULTS
A Reporter of miRNA-Mediated Repression of
Translation Initiation
We engineered a clonal HeLa cell line we called D8 that stably
expresses two reporter genes: Firefly Luciferase (FL) with six
binding sites for endogenous miR-21 in its 30 untranslated region
(30 UTR), and Renilla Luciferase (RL) lacking miRNA binding sites
(Figure 1A). Inhibition of miR-21 with a miR-21-specific antagomir resulted in up to 7-fold increase in FL relative to RL as
assessed by the dual luciferase assay (Figure 1B). The miR-21specific antagomir increased FL protein levels more than FL
mRNA levels, even after 144 hr of antagomir treatment (Figure S1A available online). These results show that in D8, FL
was strongly repressed by miR-21 and suggest that the mechanism involved was repression of translation.
Additionally, knockdown of miRNA effector genes (Argonautes [Agos] or eIF6) yielded a 2- to 10-fold increase in FL
protein levels accompanied by a proportional yet smaller
increase in FL mRNA levels (Figure 1C). Knockdown of miRNA
biogenesis (Drosha and DGCR8) or mRNA turnover (DCP2 and
PARN) genes also increased FL levels in a small interfering
RNA (siRNA) dose-dependent manner (Figure S1B). These
data show that D8 has a large dynamic range and high sensitivity
to reduced levels of miRNA pathway proteins and thus is
an appropriate tool to discover new factors and regulators of
miRNA-mediated repression by gain-of-signal RNA interference
(RNAi) screening.
To determine which step of FL translation is inhibited by
miR-21 in D8, we performed polysome profiling of control,
Ago1- or eIF6-depleted cells (Figures 1D and 1E). In control
cells, the majority of miR-21-targeted FL mRNA was in monosomes, and the majority of untargeted RL mRNA was in polysomes. Knockdown of Ago1 or eIF6 led to a striking shift of
FL mRNA from monosomes to polysomes, while RL mRNA
exhibited only a modest shift toward heavier polysome fractions but remained polysomal in all conditions. Consistent
with RT-qPCR data in Figure 1C and with previously reported
miRNA effects on mRNA stability (Bagga et al., 2005; Guo
et al., 2010; Behm-Ansmant et al., 2006; Wu and Belasco,
2005; Wu et al., 2006), knockdown of Ago1 or eIF6 stabilized
miR-21-targeted FL mRNA, leading to a higher combined signal
for FL but not RL mRNA across gradients relative to the Scr
control. Consistent with its role in 60S ribosomal subunit
biogenesis (Basu et al., 2001), eIF6 knockdown strongly
reduced the levels of 60S ribosomal subunits without affecting
the levels of 40S subunits as assessed by A254 absorbance
traces (Figure 1D). These data demonstrate that in D8, miR21 repressed initiation of FL translation.
172 Molecular Cell 46, 171–186, April 27, 2012 ª2012 Elsevier Inc.
An RNAi Screen Identifies RPGs as Regulators
of the miRNA Pathway
RNAi screens for RNAi pathway genes have been used effectively in simpler eukaryotes (Dorner et al., 2006; Eulalio et al.,
2007; Kim et al., 2005; Parry et al., 2007; Saleh et al., 2006; Ulvila
et al., 2006), leading to identification of many effectors of small
RNA function, such as RNA binding proteins, cytoskeletal
proteins, and one RPG in C. elegans (Parry et al., 2007), as
well as several RPGs and proteasomal components in
D. melanogaster (Zhou et al., 2008). However, similar screens
have not yet been reported in mammals. Therefore, we used
D8 to perform an RNAi screen for effectors and regulators of
the human miRNA pathway (Figure 2A). In all, 7,000 genes
were targeted with four individual siRNAs per gene from the
Druggable Genome Library of siRNAs (QIAGEN), and effects of
knockdowns were assessed with the dual luciferase assay.
Using stringent criteria (at least two siRNAs per gene causing
derepression of FL R eIF6 knockdown), 314 genes (4.3%)
scored as hits (Table S1, part A). RPGs were enriched more
than 13-fold in this screen: seven out of 12 RPGs (58%) scored
as hits. Closer inspection of the screening data showed that all
12 RPG knockdowns derepressed FL to some extent. Gene
ontology analysis revealed that the ribosomal pathway was
one of the most significantly enriched pathways in the screen
(Figure 2B and Table S1, part B). Thus, we hypothesized that
as a class RPGs might regulate miRNA repression.
To determine whether depletion of every RPG inhibits
miRNA-mediated repression, we targeted all 79 RPGs with
four individual siRNAs per gene in D8. Knockdown of virtually
all 40S (Figure 2C) and 60S (Figure 2D) RPGs with at least
one siRNA led to derepression of FL that was greater than
derepression after eIF6 knockdown. After three days (the point
of strongest FL derepression), most RPG knockdowns reduced
cell numbers by 20%–50%. The Y chromosome-expressed
S4Y1 and S4Y2 provide natural negative controls in female
cells and did not score in our HeLa cell validation screen (Figure 2C). The consistent FL derepression after depletion of
almost every 40S and 60S RPG suggests that altered ribosomal integrity or altered ribosomal subunit stoichiometry
and not extraribosomal functions of RPGs led to decreased
miRNA activity. Supporting this conclusion, knockdown of
factors involved in either 40S (Bms1 and Tsr1) or 60S (Bop1
and Nip7) subunit biogenesis also led to derepression of
miRNA-targeted mRNAs (Figure S2A), and double knockdown
of 40S and 60S RPGs led to greater derepression of miRNAtargeted mRNAs compared to single RPG knockdowns
(Figure S2B).
To validate the screening results, we chose five representative
40S (S5, S11, S12, S15, and S18) and 60S (L5, L11, L12, L35A,
and LP2) RPGs for detailed biochemical analysis. Knockdown
of each RPG in D8 (Figure S2C) derepressed FL mostly at the
protein levels, although proportional increases in FL mRNA
levels were also observed (Figure S2D), consistent with knockdowns of known miRNA pathway genes (Figure 1C). In the luciferase screens, which were not normalized to total protein, depletion of RPGs increased FL levels and decreased RL levels
(Figure S2E). These trends were affected by reduced cell
numbers with, on average, half the number of cells after 72 hr
Molecular Cell
Ribosomal Proteins Modulate miRNA Activity
A
D
miR-21
CMV
Firefly
CMV
B
Fold Derepression
8
7
Scr
Renilla
Protein
mRNA
FL
6
RL
5
4
28S
3
18S
40S 60S 80S
Ago1
1
0
0
12
Fold Derepression
Polysomes
A254
2
C
40S 60S 80S
10
Polysomes
A254
5
20
100
miR-21 antagomir [nM]
0 5 20 100
miR-21
FL
tRNA
RL
28S
Protein
mRNA
18S
8
eIF6
6
40S 60S 80S
Polysomes
A254
4
2
FL
RL
28S
eIF6
Actin
18S
70
Scr
Ago1
eIF6
% RL mRNA
% FL mRNA
90
80
70
60
50
40
30
20
10
0
Ago2
Actin
eIF6
eIF6
Scr
Ago1
Actin
E
Ago2
Scr
Ago1
Ago1
Scr
Scr
Ago2
0
Scr
Ago1
eIF6
60
50
40
30
20
10
1
2
3
4
5
6
7
8
Fraction number
9
10
11
12
0
1
2
3
4
5
6
7
8
Fraction number
9
10
11
12
Figure 1. D8 Is a Highly Sensitive Reporter of miR-21-Mediated Repression of Translation Initiation
(A) Schematic representation of CMV-driven luciferase reporters stably expressed in D8. Firefly Luciferase (FL) possesses six imperfect miR-21 binding sites in its
30 UTR, while Renilla Luciferase (RL) lacks miRNA binding sites.
(B) D8 reports on miR-21-mediated repression. A miR-21-specific antagomir was transfected into D8 at indicated concentrations, and FL and RL protein and
mRNA levels were assessed after 24 hr by the dual luciferase assay and RT-qPCR, respectively. Fold derepression was calculated as (FLtest/RLtest)/(FLcontrol/
RLcontrol). The antagomir reduced miR-21 levels in a dose-dependent fashion as assessed by northern blotting.
(C) D8 reports on miRNA effector function. D8 was transfected with Scrambled (Scr), Ago1-, Ago2-, or eIF6-specific siRNAs, and FL and RL protein and mRNA
levels were assessed after 72 hr. Knockdowns of Ago1, Ago2, and eIF6 were confirmed by western blotting.
(D) Polysome profiling demonstrates that knockdown of Ago1 or eIF6 shifts FL mRNA from monosomes to polysomes. Lysates from D8 transfected either with Scr
or with Ago1- or eIF6-specific siRNAs were fractionated while absorbance was monitored at 254 nm (A254). FL and RL mRNAs were assessed by northern
blotting, and 28S and 18S rRNAs were assessed by ethidium bromide staining.
(E) Quantification of northern blots with ImageQuant. FL and RL mRNA detected in each fraction is represented as the percent of the total mRNA detected in all
fractions across the gradient.
Bar graphs show the mean ± SD from three independent experiments. See also Figure S1.
Molecular Cell 46, 171–186, April 27, 2012 ª2012 Elsevier Inc. 173
Molecular Cell
Ribosomal Proteins Modulate miRNA Activity
A
siRNA library
TRANSFECT
B
+FL substrate
+RL substrate
D8 cell line
LUCIFERASE
ASSAY
72 HRS
Gene ontology
p-value Corrected p-value
GPCR protein signaling pathway 6.20E-09
4.80E-06
Protein metabolic processing
8.70E-08
5.20E-07
Inflammatory signaling pathway 4.70E-06
6.50E-05
Ribosomal pathway
2.80E-04
1.70E-03
Proteolysis
1.10E-04
7.50E-03
PDGF signaling pathway
4.50E-03
2.50E-02
C
D
9
8
7
6
5
4
3
2
eIF6
1
0
Scr
Scr
eIF6
SA
S2
S3
S3A
S4X
S4Y1
S4Y2
S5
S6
S7
S8
S9
S10
S11
S12
S13
S14
S15
S15A
S16
S17
S18
S19
S20
S21
S23
S24
S25
S26
S27
S27A
S27L
S28
S29
S30
Fold Derepression
11
10
9
8
7
6
5
4
3
2
eIF6
1
0
Scr
eIF6
Ago1
L35A
L12
S15
RAS
HMGA2 mRNA
L5
S18
S15
S12
S11
S5
4
eIF6
Scr
5
Ago1
GAPDH
PTEN
3
Scr
Ago2
Ago1
eIF6
LP2
L12
L11
S18
S15
S12
S11
S5
1
L35A
Actin
2
Scr
Fold Derepression
S12
F
6
S5
E
Scr
Scr
eIF6
L3
L4
L5
L6
L7
L7A
L8
L9
L10
L11
L12
L13
L13A
L14
L15
L17
L18
L18A
L19
L21
L22
L23A
L24
L26
L27
L27A
L28
L29
L31
L32
L34
L35
L35A
L36
L36A
L37
L37A
L38
L39
L40
L41
LP0
LP1
LP2
Fold Derepression
11
10
L35A
L15
L11
L12
L5
S18
S15
S12
S11
Ago1
Scr
IMP-1
0
PDCD4
GAPDH
Figure 2. RNAi Screening for Effectors and Regulators of the miRNA Pathway Identifies RPGs
(A) Schematic representation of the RNAi screen. D8 was transfected with siRNAs against 7,000 genes, and FL and RL protein levels were assessed by the dual
luciferase assay after 72 hr.
(B) Ribosomal pathway was significantly enriched among hits from the screen as assessed by gene ontology analysis. The p values were calculated with Fisher
exact test analysis and corrected p values were calculated with the Bonferroni method.
174 Molecular Cell 46, 171–186, April 27, 2012 ª2012 Elsevier Inc.
Molecular Cell
Ribosomal Proteins Modulate miRNA Activity
of RPG knockdown, presumably because RPG depletion
reduced cellular proliferation rates as previously reported (Flygare et al., 2005; Kirn-Safran et al., 2007; Miller et al., 2003; Oliver
et al., 2007; Volarevic
et al., 2004; Oristian et al., 2009; Panic
et al., 2000). When normalized to total protein by western blotting, we observed increased FL and unchanged RL levels (Figure S2F). To further validate these findings and to control for
possible siRNA off-target effects, we expressed five siRNAresistant isoforms of RPGs (S5, S12, S15, L12, and LP2) in D8
cells knocking down these RPGs. In all cases, transfection of
constructs expressing RPGs with silent mutations in regions
complementary to siRNAs partially rescued the FL derepression
(Figure S2G), demonstrating that RPG depletion directly led to
miRNA-targeted mRNA derepression.
We next assessed the effect of RPG depletion on various
reporter and endogenous miRNA-targeted mRNAs. FL and RL
reporters containing six imperfectly complementary binding
sites for the artificial CXCR4 miRNA were derepressed in RPG
knockdowns only in the presence of the CXCR4 miRNA (Figures
S2H and S2I). However, an FL reporter containing one perfectly
complementary site for the CXCR4 miRNA was only modestly
affected by RPG knockdowns (Figure S2J), indicating that
RPGs preferentially regulate miRNA-mediated translational
repression and not siRNA-mediated mRNA cleavage. The RL
reporter containing HMGA2 30 UTR with seven seed matches
to the let-7 miRNA (Mayr and Bartel, 2009) was derepressed
upon RPG knockdowns (Figure S2K). Consistent with previous
studies showing that repression of HMGA2 correlates with its
mRNA level (Lee and Dutta, 2007), endogenous HMGA2
mRNA levels increased after RPG knockdowns (Figure 2E). Similarly, an RL reporter containing the KRAS 30 UTR with seven let-7
sites (Johnson et al., 2005) was derepressed in HeLa cells upon
RPG knockdowns (Figure S2L), as was endogenous RAS protein
(Figure 2F). Other validated endogenous miRNA-targeted
mRNAs, such as miR-21-targeted PTEN (Lewis et al., 2003;
Talotta et al., 2009), let-7-targeted IMP-1 (Boyerinas et al.,
2008), and miR-21-targeted PDCD4 (Asangani et al., 2008)
were all derepressed upon RPG knockdowns (Figure 2F).
Although the degree and pattern of derepression varied between
different miRNA-targeted mRNAs, as a class RPGs regulated
miRNA repression.
RPG Knockdowns Relieve Repression of Translation
Initiation
To explore the mechanism of derepression of miRNA-targeted
mRNAs in RPG knockdown cells, we first examined miRNA
biogenesis. We did not detect any defects in the biogenesis of
any miRNAs tested (Figures S3A and S3B). Additionally, RPG
knockdowns did not decrease the levels of Ago1 or Ago2
proteins (Figure S3C). Polysome profiling of D8 cells knocking
down S15 (Figures 3A and 3C) or L12 (Figures 3B and 3C)
demonstrated a strong shift of FL mRNA but not RL mRNA
from monosomes to polysomes, indicating that RPG knockdowns relieve miRNA-mediated repression of translation initiation. Consistent with knockdowns of miRNA pathway genes
(Figure 1D), RPG knockdowns increased the combined signal
for FL but not RL mRNA across gradients relative to the Scr
control, indicating preferential stabilization of FL mRNA. Heavy
complexes formed on miRNA-targeted mRNAs after RPG
depletion were actively translating polysomes because FL
mRNA shifted to lighter fractions after puromycin treatment
which dissociates only ribosomes actively translocating on
mRNAs (Figures S3D and S3E). Thus, reduced levels of RPGs
increased loading of active ribosomes onto miRNA-targeted FL
mRNA but not untargeted RL mRNA.
Importantly, the effects of RPG knockdowns on ribosomal
subunit levels were similar among all tested 40S and 60S
RPGs. Knockdown of 40S RPGs consistently decreased the
levels of free 40S subunits and 18S ribosomal RNA (rRNA) and
increased the levels of free 60S subunits (Figures 3A, S4A, and
S4B). On the other hand, knockdown of 60S RPGs or eIF6
consistently decreased the levels of free 60S subunits and 28S
rRNA (Figures 3B, S4A, and S4B). These data are consistent
with established contributions of 40S and 60S RPGs to biogenesis and/or stability of ribosomal subunits (Lempiäinen and
Shore, 2009) and further indicate that defects in ribosome
biogenesis rather than extraribosomal functions of RPGs led to
reduced miRNA-mediated repression.
To resolve the 40S, 60S, and 80S peaks, polysome profiling
was performed with lower-density gradients, higher-speed
spins, and longer times, which resulted in reduced resolution
of the highest-density complexes (Figures 3A and 3B). These
conditions enabled detection of reduced 40S and 60S peaks
and thus verified efficient and functional RPG knockdowns. To
resolve the highest density polysomes and prevent pelletting
of the heaviest complexes, we performed polysome profiling
using higher-density gradients, lower-speed spins, and shorter
times. Under these conditions, RPG knockdowns led to a consistent shift of FL but not RL mRNAs to polysomes (Figure S4C).
Together, these data show that RPG knockdowns inhibited
miRNA-mediated repression of translation initiation and that
miRNA-targeted mRNA derepression could be a result of
perturbed stoichiometry between 40S and 60S ribosomal
subunits, either directly through physical interactions with
miRNP complexes or indirectly through signaling pathways
that sense ribosome biogenesis.
(C and D) Subgenomic RNAi screen in D8 identifies almost all RPGs as hits in the miRNA pathway. Small and large ribosomal subunit RPGs were knocked down in
D8 and the dual luciferase assay was performed after 72 hr. Each point represents an individual siRNA. RPGs included in the original screen are highlighted.
Dotted horizontal gray lines represent derepression of FL in the negative control (Scr) and the positive control (eIF6) transfections.
(E) Knockdown of RPGs derepresses endogenous HMGA2 at the mRNA level. HeLa was transfected with siRNAs against indicated genes, and after 72 hr,
endogenous HMGA2 mRNA levels were assessed by RT-qPCR and normalized to GAPDH mRNA levels. Bar graphs show the mean ± SD from three independent
experiments.
(F) Knockdown of RPGs derepresses endogenous miRNA targets at the protein level. HeLa was transfected with siRNAs against the indicated genes, and RAS,
PTEN, IMP-1, PDCD4, and GAPDH protein levels were assessed by western blotting after 72 hr.
See also Figure S2.
Molecular Cell 46, 171–186, April 27, 2012 ª2012 Elsevier Inc. 175
Molecular Cell
Ribosomal Proteins Modulate miRNA Activity
L12
S15
Scr
FLAG-FL6X
SCR
CXCR4
D
L12
40S 60S 80S Polysomes
S15
Scr
Scr
S15
40S 60S 80S Polysomes
A
A254
Input
FL
35
RL
35
S-FLAG IP
S-FLAG IP/Input 1.0 1.3 1.5 0.3 0.8 0.9
E
18S
B
40S 60S 80S Polysomes
Scr
40S 60S 80S Polysomes
L12
Scr
Scr+CHX
S5
S11
S12
S15
S18
L5
L11
L12
L35A
LP2
eIF6
Ago1
28S
A254
35S
FL
RL
Coomassie
28S
18S
% RL mRNA
% FL mRNA
C
80
70
60
50
40
30
20
10
0
70
Scr
S15
Scr
L12
35
S/Coomassie 1.0 0.2 1.1 1.1 1.2 1.2 1.0 0.9 0.7 0.9 0.9 0.9 1.1 1.1
60
50
40
30
20
10
0
1
2
3
4
5
6
7
Fraction number
8
9
10
Figure 3. RPG Knockdowns Selectively Increase Translation of miRNA-Targeted mRNAs
(A and B) RPG knockdowns relieve miR-21-mediated repression of translation initiation. Polysome profiles demonstrate that knockdown of S15 (A) and L12 (B)
shift miR-21-targeted FL mRNA but not untargeted RL mRNA from monosomes to polysomes.
(C) Quantification of northern blots with ImageQuant. FL and RL mRNA detected in each fraction is represented as the percent of the total mRNA detected in all
fractions across the gradient.
(D) Translation rates of CXCR4 miRNA-targeted FL6X are increased after RPG knockdowns only in the presence of the targeting CXCR4 miRNA. HeLa was
transfected with Scr or with S15- or L12-specific siRNAs, and after 48 hr FLAG-tagged FL6X was transfected either with nontargeting Scr (control) or targeting
CXCR4 (test) miRNAs. After 24 hr, cells were 35S-pulse-labeled and FL was IP-ed with anti-FLAG agarose. Inputs and precipitates were resolved by PAGE, and
the amount of 35S incorporation into FL6X was quantified with ImageQuant and normalized to the Scr transfection.
(E) Overall translation rates are not reduced by depletion of RPGs as assessed by 35S-pulse-metabolic-labeling. HeLa transfected with indicated siRNAs was 35Spulse-labeled, resolved by PAGE, and either stained with Coomassie as a loading control or visualized with a phosphorimager. As a positive control, pulse
labeling was performed in the presence of a known translation inhibitor cycloheximide (CHX).
See also Figures S3 and S4.
176 Molecular Cell 46, 171–186, April 27, 2012 ª2012 Elsevier Inc.
Molecular Cell
Ribosomal Proteins Modulate miRNA Activity
IP
Scr
Scr
S15
FLAG-Ago2
S15
FLAG-Ago1
S15
Scr
Scr
Protein
S15
Input
FLAG-Ago1
FLAG-Ago2
A
FLAG
Actin
FL
RNA
miR-21
let-7a
Figure 4. RPG Knockdown Reduces Ago Association with miRNA-Targeted mRNAs and Ribosomes
(A) Knockdown of S15 decreases association of Ago1
and Ago2 with target mRNAs but not miRNAs. D8 was
transfected with Scr or with S15-specific siRNAs, and after
24 hr, with vectors expressing FLAG-tagged Ago proteins.
After 48 hr, inputs and anti-FLAG IPs were blotted for
proteins (FLAG-Ago and Actin) and RNAs (FL, miR-21, let7a, and transfer RNA) and quantified with ImageQuant.
(B) Knockdown of S15 or L12 dissociates endogenous
Agos from ribosomes. HeLa was transfected with indicated siRNAs and ribosomes were pelleted and western
blotted for endogenous Ago1 and Ago2 proteins. The
efficiency and specificity of ribosomal recovery was
assessed by western blotting for Actin, S6, and L7a. Ago1
and Ago2 in inputs and ribosomal pellets was quantified
with ImageQuant.
Bar graphs show the mean ± SD from three independent
experiments. *p < 0.05, **p < 0.01. See also Figure S4.
FL
miR-21
let-7a *
*
**
Scr
eIF6
Ribosomal Pellet
eIF6
L12
Scr
S15
Input
B
1.4
1.2
1
0.8
0.6
0.4
0.2
0
*
L12
3.5
3
2.5
2
1.5
1
0.5
0
S15
Fold Change
tRNA
Ago1
Ago2
S6
L7a
Fold Change
Actin
3
2
Ago1
Ago2
*
*
4
3
2
1
1
0
0
** ** ** **
To further validate that RPG knockdowns increased translation rates of miRNA-targeted but not untargeted mRNAs, we
performed 35S pulse labeling followed by immunoprecipitation
(IP) of FLAG-tagged FL protein targeted by the CXCR4 miRNA.
RPG knockdowns resulted in up to 3-fold
increased incorporation of the 35S label into
FL protein in the presence of the targeting
CXCR4 miRNA, while RPG knockdowns did
not significantly affect 35S incorporation into FL
protein in the absence of the CXCR4 miRNA
(Figure 3D), demonstrating increased translation
rates of a miRNA-targeted but not an untargeted
mRNA. Indeed, gross effects on overall translation rates were not observed after RPG knockdowns (Lai et al., 2009; Volarevic et al., 2000),
as indicated by unchanged polysome peak
heights in A254 absorbance traces (Figures 3A,
3B, and S4A) and pulse 35S labeling of bulk
proteins (Figure 3E). Therefore, depletion of individual RPGs specifically increased translation
rates of miRNA-targeted mRNAs but not untargeted mRNAs.
RPG Knockdowns Reduce Ago
Association with miRNA-Targeted
mRNAs
To address the mechanism of RPG knockdowns
leading to derepression of miRNA-targeted
mRNAs, we assessed Ago1 and Ago2 association with miRNAs and target mRNAs before
and after RPG depletion. RPG knockdown
followed by IP of FLAG-tagged Ago1 or Ago2
did not affect Ago association with miR-21 or
let-7a miRNAs but significantly decreased Ago
association with miR-21-targeted FL mRNA
relative to control cells (Figure 4A). These data
indicate that Ago association with miRNAs
remains stable and that intact miRNPs dissociate from miRNA-targeted mRNAs. As observed for FL mRNA
in D8, RPG knockdowns reduced association of Ago1 and
Ago2 with endogenous miRNA-targeted mRNAs (KRAS and
PTEN) while increasing their stability (Figure S4D). Supporting
Molecular Cell 46, 171–186, April 27, 2012 ª2012 Elsevier Inc. 177
Molecular Cell
Ribosomal Proteins Modulate miRNA Activity
Polysome / Input Ratio
No. of genes up-regulated > 2-fold
A
Polysome / Input Ratio
No. of genes down-regulated > 2-fold
log2 (Observed/Expected)
eIF6
L11
vs. Scr1
L12
S15
Ago1
Ago2
Ago3
1.5
1.2
0.9
0.6
0.3
0.0
-0.3
-0.6
-0.9
-1.2
-1.5
eIF6
L11
L12
S15
Ago1
Ago2
Ago3
Ago4
eIF6
L11
L12
S15
Ago1
Ago2
Ago3
Ago4
Ago4
vs. Scr2
8
6
4
2
0
3
4
5
6
7
No. of knockdowns
8
10
1800
1600
1400
1200
1000
800
600
400
200
0
Observed
Expected
Observed/Expected
8
6
4
2
2
3
4
5
6
0
8
7
log2 (Observed/Expected)
10
Observed
Expected
Observed/Expected
No. of polysomedown-regulated genes
1800
1600
1400
1200
1000
800
600
400
200
0
2
log2 (Observed/Expected)
No. of polysomeup-regulated genes
B
No. of knockdowns
60
No. of shuffled trials
No. of shuffled trials
C
Observed
Expected
50
40
30
20
10
0
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
90
80
70
60
50
40
30
20
10
0
0.00
Fraction of polysome up-regulated genes with
HeLa miRNA sites (conserved 8mers)
Observed
Expected
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
Fraction of polysome down-regulated genes with
HeLa miRNA sites (conserved 8mers)
0.35
Observed
Expected
0.30
0.25
0.20
0.15
0.10
2
3
4
5
6
7
8
No. of knockdowns
Fraction of polysome
down-regulated genes with
HeLa miRNA sites
Fraction of polysome
up-regulated genes with
HeLa miRNA sites
D
0.35
Observed
Expected
0.30
0.25
0.20
0.15
0.10
2
3
4
5
6
No. of knockdowns
7
8
Figure 5. Reduced RPG Expression Upregulates Polysomal Association of a Consistent Set of Genes that Is Enriched in miRNA-Targeted
mRNAs
(A) Genes whose polysome/input ratios change after knockdown of Ago, RPGs, or eIF6 are shared. The numbers of genes whose polysome/input ratios are
increased or decreased more than 2-fold in pairs of knockdowns were counted and are displayed in matrix format. The numbers of genes expected to be
regulated in the same manner by chance are shown in parentheses. The observed/expected ratio for each comparison is displayed in color.
178 Molecular Cell 46, 171–186, April 27, 2012 ª2012 Elsevier Inc.
Molecular Cell
Ribosomal Proteins Modulate miRNA Activity
the model of miRNP dissociation from miRNA-targeted mRNAs,
RPG depletions reduced Ago1 and Ago2 association with ribosomes (Figure 4B). Together, these data demonstrate that RPG
knockdown dissociates miRNPs from miRNA-targeted mRNAs
and does not affect miRNP assembly or stability.
RPG, eIF6, and Ago Knockdowns Alter Polysome
Association of Common mRNAs
To globally assess effects on translation of endogenous mRNAs,
we conducted RNA sequencing (RNA-Seq) analysis of polysomal fractions and total RNA (‘‘input’’ to the gradient) before
and after knockdowns of RPGs, Agos, or eIF6. A remarkably
large number of common genes demonstrated a 2-fold or
greater increase in polysome association (polysome/input ratio)
after knockdown of RPGs, Agos, or eIF6 (Figure 5A). Many
common genes also demonstrated a 2-fold or greater decrease
in polysome association for many pairs of knockdowns, particularly between knockdowns of different Agos. These observations
suggested the surprising conclusion that large and small subunit
RPGs, Agos, and eIF6 have related effects on translational
regulation globally, perhaps through common regulatory pathways. This idea was further supported by analysis of mRNAs
impacted in three or more of these knockdowns. Instead of
decreasing rapidly to zero, as expected if the knockdowns
affected independent sets of genes, the numbers of mRNAs
with at least 2-fold increased polysome association remained
in the several hundred range, even as the number of compared
knockdowns increased from three to eight, with 479 mRNAs
exhibiting an increase in all eight knockdowns (Figure 5B and
Table S2, part A). A smaller but still highly significant set of 57
mRNAs with consistently decreased polysome association in
all knockdowns was also observed (Figure 5B and Table S2,
part B). Thus RPGs, Agos, and eIF6 inhibited polysome association of a large common cohort of mRNAs, and promoted polysome association of a smaller common cohort of mRNAs.
miRNA Target Sites Are Enriched in Polysome-Shifted
mRNAs
To explore potential connections to the miRNA pathway, we
analyzed the fraction of mRNAs containing conserved 8-mer
seed matches to HeLa-expressed miRNAs, a relatively stringent
set of putative targets that likely excludes many authentic targets
with weaker or less stringently conserved seed matches (Friedman et al., 2009). Significant enrichment of conserved target
sites relative to controls was observed in the set of mRNAs
with consistently increased polysomal association in all knockdowns (Figure 5C). The extent of target site enrichment
increased as the number of intersected knockdowns increased
(Figure 5D). The observed enrichment for miRNA target sites in
this common set of polysome-increased mRNAs suggested
that miRNAs are involved in repression of this gene set via
a mechanism involving translation, and that the perturbations
introduced by RPG, Ago, and eIF6 knockdowns alter ribosome
loading onto a cohort of mRNAs that is strongly enriched
for miRNA-targeted mRNAs. Mechanisms involving miRNAdirected changes in stability of polysome-associated versus
non-polysome-associated mRNAs are also possible. In contrast,
the set of genes with consistently decreased polysomal association in the knockdowns was not enriched for conserved miRNA
target sites (Figures 5C and 5D), suggesting that this set of
mRNAs is less often directly impacted by miRNAs. Although
RPG, Ago, and eIF6 knockdowns also altered mRNA levels of
a common cohort of genes, these sets of genes were not enriched for conserved 8-mer seed matches to HeLa-expressed
miRNAs (Figure S5). Together, these data show that RPGs,
Agos, and eIF6 contributed to reducing polysome association
of a common subset of mRNAs enriched for miRNA-targeted
mRNAs. Because miRNA-targeted mRNAs were not enriched
in a common subset of mRNAs exhibiting increased mRNA
levels after knockdowns, we observed translational derepression and not stabilization of miRNA-targeted mRNAs.
miRNAs Predominantly Cosediment with Polysomes
The preceding studies demonstrated that miRNA-targeted
mRNAs associate with monosomes and that RPG knockdowns
increase association of miRNA-targeted mRNAs with polysomes
without changing miRNA levels or miRNA association with Ago
proteins. To gain additional insights into RPG regulation of
miRNA-mediated repression, we performed global miRNA
profiling of total, monosomal, and polysomal RNA after sucrose
gradient fractionation of HeLa cells before and after RPG
knockdown. Out of 669 unique miRNA species expressed in
HeLa cells, 462 miRNAs (70%) were reliably detected in
monosomal and polysomal fractions at steady state (Table S3,
part A). The majority of miRNAs were equally abundant in
monosomal and polysomal fractions. Specifically, out of 462
miRNAs detected in monosomes and polysomes, 265 miRNAs
(57%) had no statistically significant enrichment in either fraction. Thus, the majority of miRNAs are equally distributed across
monosomal and polysomal fractions and may contribute to
(B) A common set of mRNAs shifts to and away from polysomes in Ago, RPG, and eIF6 knockdowns. The numbers of genes whose polysome/input ratios are
increased or decreased more than 2-fold in two-way, three-way, . eight-way comparisons of knockdowns are displayed in red or blue, respectively. The
numbers of genes expected to be regulated in the same manner by chance are shown in black. Ninety percent confidence intervals are shown in pale red, blue,
and black.
(C) The upregulated genes have a significantly increased proportion of HeLa-expressed miRNA binding sites (p < 0.003). The fraction of genes with conserved 8mer miRNA sites was obtained for genes whose polysome expression is upregulated (red) or downregulated (blue) in all eight knockdowns (Ago1–Ago4, L11, L12,
S15, and eIF6). For assessment of significance, the same fraction was determined for 1,000 sets of control genes that matched the original up- or downregulated
sets in 7-mer conservation and UTR length (black histogram).
(D) The proportion of upregulated genes with HeLa-expressed miRNA-targeted mRNAs increases as the number of knockdowns used to define the set of
coregulated genes increases. The fraction of genes with HeLa-expressed conserved 8-mer miRNA sites was obtained for those genes whose polysome
expression is upregulated (red) or downregulated (blue) in two, three, . eight knockdowns. Control genes (black) were selected to match the up- or downregulated genes.
See also Figure S5.
Molecular Cell 46, 171–186, April 27, 2012 ª2012 Elsevier Inc. 179
Molecular Cell
Ribosomal Proteins Modulate miRNA Activity
C
A
hsa−miR−505*
hsa−miR−30d
hsa−miR−1180
hsa−miR−10a
hsa−miR−421
hsa−miR−128
hsa−miR−320b
hsa−miR−335*
hsa−miR−942
hsa−miR−10b
hsa−miR−126*
hsa−miR−342−3p
hsa−miR−532−3p
hsa−miR−99a
hsa−miR−103a
hsa−miR−629
hsa−miR−106b
hsa−miR−423−5p
hsa−miR−151−3p
hsa−miR−30e*
hsa−let−7d*
hsa−miR−26b
hsa−miR−151−5p
hsa−miR−31
hsa−miR−93
hsa−miR−221
hsa−let−7d
hsa−miR−320a
hsa−let−7b
hsa−let−7i
hsa−miR−188−3p
hsa−miR−30a*
hsa−let−7f
hsa−miR−23a*
hsa−miR−500a*
hsa−miR−185
hsa−miR−221*
hsa−miR−324−3p
hsa−let−7f−1*
hsa−miR−378b
hsa−miR−30b*
hsa−miR−200c
hsa−miR−192
hsa−miR−193b*
hsa−miR−1203
hsa−miR−422a
hsa−miR−342−5p
hsa−miR−502−3p
hsa−miR−576−5p
hsa−miR−29b
hsa−miR−590−3p
hsa−miR−3647−3p
hsa−miR−455−3p
hsa−miR−340*
hsa−miR−126
hsa−miR−452
hsa−miR−34a
hsa−miR−26a
hsa−miR−25
hsa−miR−1260
hsa−miR−196b
hsa−let−7a
hsa−let−7e
hsa−miR−23b
hsa−miR−24
hsa−miR−720
hsa−miR−23a
hsa−miR−501−5p
hsa−miR−370
hsa−miR−190
hsa−miR−135a
hsa−miR−596
hsa−miR−326
hsa−miR−93*
hsa−miR−17*
hsa−miR−1238
hsa−miR−135b
hsa−miR−362−5p
hsa−miR−532−5p
hsa−miR−105
hsa−miR−149
hsa−miR−224*
hsa−miR−582−5p
hsa−miR−301b
hsa−miR−330−5p
hsa−miR−15a
hsa−miR−331−3p
hsa−miR−615−3p
hsa−miR−22*
hsa−miR−3676
hsa−miR−181c
hsa−miR−181a
hsa−miR−99b*
hsa−miR−874
hsa−miR−491−5p
hsa−miR−1231
hsa−miR−193a−3p
hsa−miR−361−3p
hsa−miR−340
hsa−miR−146b−5p
hsa−miR−1285
hsa−miR−662
hsa−miR−660
hsa−miR−140−5p
hsa−miR−502−5p
hsa−miR−920
hsa−miR−188−5p
hsa−miR−96
hsa−miR−1296
hsa−miR−497
hsa−miR−24−2*
hsa−miR−31*
hsa−miR−378b
hsa−miR−195*
hsa−miR−675*
hsa−miR−20a*
hsa−miR−28−3p
hsa−miR−3607−5p
hsa−miR−130b
hsa−miR−455−3p
hsa−miR−1247
hsa−miR−629*
hsa−miR−194
hsa−miR−140−3p
hsa−miR−1301
hsa−miR−221*
hsa−miR−1226
hsa−miR−941
hsa−miR−3131
hsa−miR−1287
hsa−miR−486−5p
hsa−miR−92b*
hsa−miR−422a
hsa−miR−877*
hsa−miR−200c
hsa−miR−130a
hsa−miR−148a
hsa−miR−3651
hsa−miR−10a
hsa−miR−193a−5p
hsa−miR−374a
hsa−miR−1180
hsa−miR−532−3p
hsa−miR−1233
hsa−miR−28−5p
hsa−miR−452
hsa−miR−1271
hsa−miR−1281
hsa−miR−126
hsa−miR−942
hsa−miR−183
hsa−let−7a*
hsa−miR−335*
hsa−miR−132
hsa−miR−10b
hsa−miR−126*
hsa−miR−101
hsa−miR−185
hsa−miR−29b
hsa−miR−500a
hsa−miR−324−5p
hsa−miR−301a
hsa−miR−378*
hsa−miR−324−3p
hsa−miR−107
hsa−miR−4289
hsa−miR−330−3p
hsa−miR−339−5p
hsa−miR−18a*
hsa−miR−22
hsa−miR−1260b
hsa−miR−3618
hsa−miR−450a
hsa−miR−33a*
hsa−miR−27b*
hsa−miR−550a
hsa−miR−4323
hsa−miR−3200−5p
hsa−miR−29a*
hsa−miR−425*
hsa−miR−7−1*
hsa−miR−183*
hsa−miR−3909
hsa−miR−1249
hsa−miR−196b*
hsa−miR−296−3p
hsa−miR−3916
hsa−miR−1227
hsa−miR−589
hsa−miR−1909*
hsa−miR−132*
hsa−miR−19b−1*
hsa−miR−1255b
hsa−miR−335
hsa−miR−16−1*
hsa−miR−876−3p
hsa−miR−200b
hsa−miR−99a
hsa−miR−30d
hsa−miR−30b
hsa−miR−424
hsa−miR−342−3p
hsa−miR−1307
hsa−miR−378
hsa−miR−128
hsa−miR−574−3p
hsa−miR−505
hsa−miR−4286
hsa−miR−4291
hsa−miR−484
hsa−miR−34a
hsa−miR−425
hsa−miR−193b
hsa−miR−186
hsa−miR−100
hsa−miR−99b
hsa−miR−93
hsa−miR−195
hsa−miR−30e
hsa−miR−30a
hsa−miR−125b
hsa−miR−19a
hsa−miR−106b
hsa−miR−20b
hsa−miR−18a
hsa−miR−423−3p
hsa−miR−103a
hsa−miR−29a
hsa−miR−29c
hsa−miR−19b
hsa−miR−423−5p
hsa−miR−182
hsa−let−7c
hsa−miR−151−3p
hsa−miR−27b
hsa−miR−26b
hsa−miR−148b
hsa−let−7b
hsa−miR−197
hsa−miR−454
hsa−miR−27a
hsa−let−7i
hsa−miR−92b
hsa−miR−365
hsa−miR−221
hsa−let−7d
hsa−miR−17 // hsa
hsa−miR−31
hsa−miR−16
hsa−miR−20a
hsa−miR−222
hsa−miR−191
hsa−let−7g
hsa−miR−26a
hsa−miR−25
hsa−miR−224
hsa−miR−125a−5p
hsa−miR−30c
hsa−miR−1260
hsa−miR−24
hsa−miR−1280
hsa−miR−92a
hsa−miR−7
hsa−miR−21
R1
R2
R3
R4
R1
Polysomes
R2
R3
R1
R4
Monosomes
R2
R3
R1
R2
S15
R3
R4
Scr
Monosomes
Ct
B
−1
0
1
2
Ct
−1.5
−1.0
−0.5
0.0
0.5
1.0
1.5
22
Ct miR-126*
Ct miR-21
31
21
20
19
29
27
25
18
Monosomes Polysomes
Monosomes Polysomes
Figure 6. miRNAs Cosediment with Ribosomes and Dissociate from Monosomes after RPG Knockdowns
(A) Supervised hierarchical clustering of the 197 HeLa miRNAs differentially expressed in monosomal versus polysomal fractions demonstrates preferential
polysomal cosedimentation of miRNAs. One hundred ninety-five miRNAs were preferentially present in polysomal fractions.
(B) Dot plots of miR-21 and miR-126*, the only two miRNAs that are enriched in monosomes.
(C) Supervised hierarchical clustering of the 67 miRNAs differentially expressed in monosomes in HeLa transfected with control (Scr) versus S15 siRNA
demonstrates that S15 knockdown preferentially dissociates monosomal miRNAs. Out of 462 miRNAs detected in HeLa monosomes and polysomes, 67 miRNAs
(15%) were reduced in monosomes after S15 knockdown, suggesting that RPG depletion relieves miRNA-mediated repression of translation initiation.
Comparisons are based on Ct values (1.5-fold or 0.6 Ct differences across quadruplicate experiments based on RT-qPCR Ct values). p values were calculated
significant at 0.05 using a two-sided Welch t test adjusted using the Benjamini and Hochberg method.
See also Figure S6.
multiple modes of translational repression, suggesting mechanistic variation for molecules of the same miRNA species.
Interestingly, 197 of these miRNAs (43%) were differentially
present in monosomal versus polysomal fractions (Figure 6A
and Table S3, part B). Strikingly, 195 of those 197 miRNAs
(99%) were enriched in polysomal fractions, while only two
miRNAs (miR-21 and miR-126*) were enriched in monosomal
fractions (Figure 6B and Table S3, part B). These data demonstrate that (1) the majority of HeLa miRNA species cofractionate
with ribosomes and thus can repress translation; (2) miR-21
predominantly represses translation initiation, consistent with
biochemical characterization of the D8 cell line where miR-21repressed FL mRNA accumulated in monosomes (Figures 1D,
3A, and 3B); (3) individual miRNA species can be found preferentially in polysomal relative to monosomal fractions, indicating
180 Molecular Cell 46, 171–186, April 27, 2012 ª2012 Elsevier Inc.
that miRNA or target mRNA identity can affect the mode of
translational repression; and (4) most miRNAs preferentially cosediment with polysomal fractions. Because puromycin treatment did not significantly affect sedimentation of the 195 miRNAs
enriched in polysomal fractions (Figure S6A), these miRNAs were
either associated with heavy processing body (P body) aggregates that cosedimented with polysomes or with ribosomes
stalled on target mRNAs, suggesting a postinitiation block.
RPG Knockdowns Decrease miRNA Association
with Monosomes
To assess RPG regulation of miRNA-mediated repression, we
performed global miRNA profiling of total, monosomal, and
polysomal RNA after S15 knockdown in HeLa cells (Figure 6C
and Table S3, part C). S15 knockdown did not globally alter
Molecular Cell
Ribosomal Proteins Modulate miRNA Activity
2.5
B
Scr
p53
3.5
3
2
Fold Change
Fold Derepression
A
1.5
1
FL
RL
2.5
2
1.5
1
0.5
0.5
0
Scr
S5
S15
L11
0
L12
DMSO
0.0005
0.001
0.002
2.5
5.0
ActD
p53
p53
GAPDH
GAPDH
C
Normal
5.0
10
DRB
0.002
ActD
0.001
DMSO
L12
L11
S15
S5
Scr
L12
p53
L11
S15
S5
Scr
Scr
Nucleolar Stress
pre-60S
AAAAAAA
60S
miRNP
10
DRB
miRNP
p53/63/73
p53/63/73
AUG
40S
pre-40S
AAAAAAA
60S
60S
60S
40S
40S
40S
Figure 7. Reduced RPGs Regulate miRNA Function through p53 Pathways
(A) Upregulation of p53 mediates derepression of miRNA-targeted mRNAs after RPG knockdowns. A549 was transfected with either control (Scr) or RPG-specific
siRNAs and with either Scr (black) or p53 (gray) siRNAs. After 48 hr, vectors expressing FL6X reporter with six imperfect CXCR4 miRNA binding sites and RL
reporter with no miRNA sites were transfected along with the CXCR4 miRNA. Dual luciferase assays (top) and western blotting for p53 and GAPDH (bottom) were
performed after 24 hr.
(B) Chemical induction of nucleolar stress phenocopies RPG knockdowns. A549 expressing FL6X and RL reporters was treated with DMSO (control) or indicated
concentrations (mg/ml) of Actinomycin D (ActD) or 5,6-dichloro-1-b-D-ribofuranosylbenzimidazole (DRB). After 24 hr, dual luciferase assays (top) and western
blotting for p53 and GAPDH (bottom) were performed. Bar graphs show the mean ±SD from three independent experiments.
(C) A model of translational derepression of miRNA-targeted mRNAs resulting from altered ribosome subunit biogenesis. When normal RPG expression
generates stoichiometric 40S and 60S subunits, the p53 stress response pathway is not activated and miRNAs repress translation (left). Reduced expression of
RPGs leads to perturbed biogenesis of 40S and 60S ribosomal subunits, leading to nucleolar stress, activation of the p53 pathway, and dissociation of miRNPs
from miRNA-targeted mRNAs (right). While the translation of untargeted mRNAs remains unchanged, miRNA-mediated repression of translation initiation is
relieved, resulting in increased ribosome loading specifically onto miRNA-targeted mRNAs.
See also Figure S7.
miRNA levels (Figure S6B), consistent with results from northern
blotting experiments (Figure S3B). S15 knockdown also did not
affect miRNA abundance in polysomes, where the average
mean fold change (0.99 with a range 0.95–1.06) was not statistically significant according to the p values adjusted for multiple
comparisons. These data indicate that RPG depletion does not
affect miRNA-mediated repression of translation after initiation
or the abundance of miRNAs in P bodies.
In contrast, S15 knockdown reduced monosome association
of 67 out of 462 (15%) miRNAs detected in monosomes
and polysomes, demonstrating that RPGs regulate miRNAmediated repression of translation initiation (Figure 6C). Importantly, all 67 of these miRNAs were reduced in monosomes after
S15 knockdown and none of these miRNAs were increasingly
associated with polysomes. These data suggest that miRNPs
containing these miRNAs dissociated from target mRNAs,
consistent with reduced association of miRNA-targeted
mRNAs with Agos after RPG knockdowns (Figure 4A, 4B,
and S4D). Notably, multiple let-7 family members were among
the 67 miRNAs with reduced expression in monosomes
after S15 knockdown, consistent with derepression of validated
let-7 targets (Figures 2E and 2F). miR-21 was consistently
reduced in monosomes after S15 knockdown (average 3.5fold decrease) and was not affected in polysomes (average
1.1-fold increase). However, we note that the decreased
association of miR-21 with monosomes was not considered
Molecular Cell 46, 171–186, April 27, 2012 ª2012 Elsevier Inc. 181
Molecular Cell
Ribosomal Proteins Modulate miRNA Activity
statistically significant using strict limits (the raw p value for Scr
versus S15 siRNA was 0.03, but the FDR p value [q value] was
0.07).
To determine whether dissociation of the 67 miRNAs from
monosomes after S15 knockdown affected repression, we performed miRNA target prediction analysis using TargetScan on
the set of 479 mRNAs that commonly shift toward polysomes
after RPG, Ago, or eIF6 knockdowns as assessed by RNA-Seq
(Figure 5B and Table S2, part A). Only favorable targets, defined
as mRNAs with TargetScan context score in the top two quartiles
for each miRNA, were considered. We observed a highly significant enrichment of mRNA targets of those 67 miRNAs (Chisquare value of 103.107; p value of 1.97 3 1024) among mRNAs
that shifted to polysomes following RPG knockdowns (Table S3,
part D). These data indicate that RPG knockdowns relieve
repression of translation initiation by decreasing miRNP association and increasing ribosome association of these miRNAtargeted mRNAs.
p53 Pathway Activation Relieves miRNA-Mediated
Repression
Global analysis of transcriptome changes (Figure S5) identified
robust effects on p53 pathway genes after RPG (S15, L11, and
L12) but not Ago knockdowns (Figures S7A and S7B and Table
S4). Specifically, gene network analysis of mRNAs commonly
dysregulated in RPG knockdowns revealed enrichment for
a DNA repair network (p value of 6.2 3 1041; Figure S7B) and
a cell-cycle network (p value of 8.1 1028; Figure S7B). Because
perturbation of ribosome subunit biogenesis induces nucleolar
stress and leads to p53 pathway activation (Deisenroth and
et al., 2007; Rudra and
Zhang, 2010; Hölzel et al., 2010; Panic
Warner, 2004; Volarevic et al., 2000), we hypothesized that
reduced RPG expression led to p53 pathway activation, which,
in turn, reduced repression of translation initiation of miRNAtargeted mRNAs. Notably, the human papillomavirus 16/18 E6
protein degrades endogenous p53 in HeLa cells (Scheffner
et al., 1990; Werness et al., 1990). Thus, RPG depletion activates
p53 pathway genes even in the absence of the p53 protein,
possibly by activating other p53 family members (such as p63
and p73).
To test the hypothesis that reduced RPG expression induces
p53 pathways that inhibit miRNA-mediated repression, we
used the A549 human lung cancer cell line which expresses
wild-type p53. RPG knockdowns upregulated p53 at the protein
level (Figures 7A and S7C), suggesting induction of the nucleolar
stress response. Importantly, simultaneous knockdown of RPG
and p53 reversed derepression of miRNA-targeted mRNAs
(Figure 7A), directly implicating the p53 pathway in RPG regulation of miRNA activity.
To test the effects of nucleolar stress on miRNA activity
independently of RPG knockdowns, we chemically induced
nucleolar stress with low concentrations of Actinomycin
D (ActD) or 5,6-dichloro-1-b-D-ribofuranosylbenzimidazole
(DRB) (David-Pfeuty et al., 2001; Hölzel et al., 2010). Similar to
RPG knockdowns, ActD and DRB treatments led to dose-dependent p53 activation, reduced cell numbers, and specifically
increased the expression of miRNA-targeted mRNAs without
affecting miRNA levels (Figure 7B and S7D). Derepression of
182 Molecular Cell 46, 171–186, April 27, 2012 ª2012 Elsevier Inc.
CXCR4 miRNA-targeted FL mRNA was observed at 8 hr after
treatment (Figure S7E), suggesting that decreased miRNA
activity is a direct result of nucleolar stress induction and not an
indirect result of altered cell metabolism. Taken together, our
data demonstrate that induction of nucleolar stress (triggered
by RPG knockdowns or by small molecules) activates the p53
pathway, which leads to decreased miRNA activity (Figure 7C).
DISCUSSION
RPGs Regulate miRNA-Mediated Repression
of Translation Initiation
An RNAi screen for effectors and regulators of miRNA function
identified an unexpected role for RPGs in regulating miRNAmediated repression of translation initiation. Previous reports
have implicated RPGs in small RNA pathways in other organisms, suggesting RPG regulation of small RNA pathways may
be conserved across phyla. In Drosophila, 11 RPGs scored in
a screen of small RNA pathways, constituting 6% of all 177
hits (Zhou et al., 2008). However, RPG knockdown did not affect
miRNA-mediated repression but instead increased endo-siRNAmediated repression. Depending upon the RPG tested, siRNAmediated repression increased in some cases but decreased
in others. In C. elegans, a genome-wide RNAi screen for miRNA
pathway genes identified one RPG, though this hit was not
confirmed (Parry et al., 2007). In contrast to these model organisms, our data functionally implicates RPGs (as a class) as regulators of miRNA-mediated repression of translation initiation in
human cells.
We showed that RPG depletion reduced the association of 67
miRNAs with monosomes without increasing miRNA association
with polysomes or reducing total miRNA levels. Importantly,
RPG depletion specifically increased polysomal association
of mRNAs targeted by these miRNAs. Together, these highthroughput data independently confirm the biochemical data
and support a model in which RPG depletion inhibits repression
of translation initiation mediated by miRNAs. Moreover, we
propose that parallel miRNA and mRNA expression profiling
from monosomes and polysomes could be an accurate method
of target mRNA identification.
miRNAs have been shown to activate translation under certain
stress conditions. Steitz and colleagues reported that cells
forced into quiescence can switch miRNA activity from translational repression to translational activation (Vasudevan et al.,
2007). It is important to note that RPG knockdowns did not activate translation but rather derepressed miRNA-targeted
mRNAs. In D8, it was impossible to distinguish derepression
from activation because FL was constitutively repressed by
endogenous miR-21, and thus the expression level of unrepressed FL was unknown. Therefore, we knocked down RPGs
in HeLa cells transiently transfected with luciferase reporters
with imperfect binding sites to the artificial CXCR4 miRNA
(Figures S2H and S2I). The expression of unrepressed luciferase
(in the absence of CXCR4 miRNA) was consistently higher than
the expression of derepressed luciferase (in the presence of
CXCR4 miRNA) after RPG knockdowns. These data demonstrate that RPG depletion derepressed but did not activate translation of miRNA-targeted mRNAs.
Molecular Cell
Ribosomal Proteins Modulate miRNA Activity
p53 Rathways Regulate miRNA Function
RPG and eIF6 knockdowns led to substantial changes in abundance of ribosomal subunits. The p53 pathway and nucleolar
stress have been implicated in sensing perturbed ribosomal
subunit stoichiometry, leading to cell-cycle arrest (Bachand
et al., 2006; Deisenroth and Zhang, 2010; Hölzel et al., 2010;
et al., 2007; Rudra and Warner, 2004; Volarevic et al.,
Panic
2000). Interestingly, gene ontology analysis of transcriptional
changes revealed that cell cycle, DNA replication, and p53
signaling were affected in all RPG knockdowns but not Ago
knockdowns (Figures S7A and S7B and Table S4). Although
the connection between ribosomal subunit imbalance, nucleolar
stress, and the p53 pathway activation was already known, the
connection between ribosomal subunit imbalance and global
miRNA activity has not been established prior to this study.
We propose that global reduction in miRNA-mediated repression may be an adaptive response allowing cells to increase
translation of subsets of mRNAs in response to nucleolar stress.
miRNA-mediated repression has been linked to oxidative-,
endoplasmic reticulum-, and nutrient deprivation-stress responses (Bhattacharyya et al., 2006a, 2006b). In contrast to
nucleolar stress, these stress responses do not induce the
p53 pathway and are distinguished by their effects on general
translation. For instance, amino acid starvation increases eIF2a
phosphorylation and leads to global translation inhibition (Bhattacharyya et al., 2006a, 2006b), which we did not observe in
RPG knockdowns (Figure S6C). Amino acid starvation also
does not affect miRNA activity unless an AU-rich element is
present. In contrast, RPG knockdowns derepress both reporter
and endogenous miRNA-targeted mRNAs even in the absence
of AU-rich elements.
The Majority of miRNA Species Can Repress Translation
Previous studies have identified miRNA-targeted mRNAs cosedimenting with polysomes suggesting repression of translation
postinitiation (Kim et al., 2004; Maroney et al., 2006; Nelson
et al., 2004; Nottrott et al., 2006; Olsen and Ambros, 1999;
Petersen et al., 2006; Seggerson et al., 2002). In agreement
with more recent studies (Bhattacharyya et al., 2006a; Pillai
et al., 2005), we detected miR-21-targeted FL mRNAs predominantly in monosomes, suggesting repression of translation
initiation. There has been much speculation about the causes
and implications of these different results (reviewed in Filipowicz
et al., 2008). One possible explanation may be the efficiency of
repression. Less efficient repression of translation initiation
may result in a larger population of miRNA-targeted mRNAs in
polysomes, whereas more efficient repression of translation
initiation might result in a larger population of miRNA-targeted
mRNAs in monosomes. In D8, where FL is repressed by miR21 via six target sites, strong repression and monosome
association was observed.
We report global miRNA expression profiles from HeLa
monosomal and polysomal fractions. Detection of all miRNAs
in both monosomal and polysomal fractions suggests that the
mechanisms of miRNA-mediated translational repression are
not uniform. Differential sedimentation of particular miRNAs
implies that miRNA and/or mRNA identity may affect the mechanism of translational repression. miRNAs cosedimenting with
polysomes did not appear to associate with actively translating
ribosomes. Puromycin treatment reduced polysomes (Figure S3D) but did not affect sedimentation of these miRNAs (Figure S6A), suggesting that miRNAs may associate with stalled
ribosomes or non-translating multimegadalton complexes
(e.g., P bodies).
eIF6 Indirectly Affects the Human miRNA Pathway
Our screen identified eIF6, a eukaryotic translation initiation
factor that has been implicated in growth and transformation
(Gandin et al., 2008). eIF6 binds to 60S subunits and functions
as an antiassociation factor by preventing 60S subunits from
joining with 40S subunits (Ceci et al., 2003). eIF6 has also been
implicated in miRNA-mediated translational repression in worms
and human cells (Chendrimada et al., 2007). Human eIF6 has
been shown to associate with Agos, miRNAs, and 60S subunits
(Chendrimada et al., 2007). In contrast to these observations, we
did not detect any interaction between eIF6 and Agos, miRNAs,
or miRNA-targeted mRNAs (data not shown). Indeed, we did not
detect any association of eIF6 with Agos even by the highly
sensitive Multidimensional Protein Identification Technology
analysis (data not shown). Our data suggest that eIF6 may affect
miRNA-targeted mRNA repression indirectly by altering ribosome subunit stoichiometry. eIF6 is required for 18S and 5.8S
rRNA maturation which, in turn, is required for generating the
60S ribosomal subunit (Basu et al., 2001). Indeed, depletion of
eIF6 reduced 60S ribosomal subunits (Figure 1D), the same
phenotype as in 60S RPG knockdowns.
Reduced RPGs in Ribosomopathies and Cancers
Cancer pathway genes were significantly enriched in polysomes
after RPG, Ago, and eIF6 knockdowns (Table S4). Downregulation of RPGs has been identified in precancerous states, cancers
in situ, and metastatic cancers (van Riggelen et al., 2010). Additionally, several genetic diseases that predispose patients to
cancers (Avondo et al., 2009; Campagnoli et al., 2008; Gazda
et al., 2008) are characterized by mutations in or reduced
expression of RPGs, including dyskeratosis congenita (DKC1),
cartilage-hair hypoplasia (RMRP), Shwachman-Diamond
syndrome (SBDS), Turner syndrome (S4X), Noonan syndrome
(L6), Camurati-Englemann disease (S18), and, most notably,
Diamond-Blackfan anemia (S7, S15, S17, S19, S24, S27A, L5,
L11, L35A, and L36). Interestingly, decreased expression of
individual RPGs in zebrafish (Amsterdam et al., 2004; Lai et al.,
2009; MacInnes et al., 2008) and flies (Stewart and Denell,
1993; Watson et al., 1992) promotes tumorigenesis, suggesting
that RPGs may act as haploinsufficient tumor suppressors.
This relationship is counterintuitive because rapid growth and
proliferation of tumors must require robust translational activity.
Our data offer a possible resolution in that reduced levels of
RPGs may preferentially derepress protein production from a
cohort of miRNA-targeted messages, many of which contribute
to cellular proliferation and oncogenesis.
EXPERIMENTAL PROCEDURES
Detailed experimental procedures can be found in the Supplemental Experimental Procedures.
Molecular Cell 46, 171–186, April 27, 2012 ª2012 Elsevier Inc. 183
Molecular Cell
Ribosomal Proteins Modulate miRNA Activity
Polysome Profiling
HeLa cells were incubated with 100 mg/ml cycloheximide (CHX) for 5 min at
37 C and washed on ice twice with 5 ml cold PBS containing 100 ug/ml
CHX. Cells were scraped in 500 ml lysis buffer (15 mM Tris [pH 7.4], 15 mM
MgCl2, 150 mM NaCl, 1% Triton X-100, 100 mg/ml CHX, and 1 mg/ml heparin)
and centrifuged at 12,000 g for 5 min at 4 C. Supernatant was loaded onto
12 ml 4.5%–45% sucrose gradients in 15 mM Tris (pH 7.4), 15 mM MgCl2,
150 mM NaCl, and 100 mg/ml CHX. Gradients were centrifuged in SW41Ti rotor
at 39,000 rpm for 2.5 hr at 4 C and 1 ml fractions were collected. RNA was
extracted with Trizol LS (Invitrogen) or by addition of 750 ml guanidinium
hydrochloride and 800 ml isopropanol to 500 ml of a fraction and incubation
at 20 C overnight. Samples were centrifuged at 10,000 rpm for 25 min at
4 C, pellets washed with 70% ethanol, and resuspended in 180 ml TE buffer.
After addition of 20 ml 3 M sodium acetate (pH 5.2) and 600 ml 100% ethanol,
RNA was precipitated at 80 C overnight. Samples were centrifuged at
10,000 rpm for 25 min at 4 C, pellets washed with 70% ethanol, and RNA
was resuspended in water.
RNA-Seq Read Mapping and RPKM Calculation
Short reads were mapped to the human genome and a precomputed set of
splice junctions using Bowtie (Langmead et al., 2009). Reads per kilobase of
exon model per million mapped reads (RPKM) for each Entrez gene was
computed by using all reads mapping to constitutive Refseq exons.
Global miRNA Expression Profiling
RNA from unfractionated HeLa cells and from fractionated monosomes and
polysomes was extracted with Trizol LS (Invitrogen). Complementary DNA
(cDNA) synthesis, cDNA preamplification, and real-time PCR were performed
with the miScript PCR System (QIAGEN). miRNA Ct measures were compared
between monosomes and polysomes over all samples and separately for Scr
and S15 knockdown groups using a two-sided Welch t test. p values were
adjusted with the method of Benjamini and Hochberg and considered significant at the 0.05 level. Supervised hierarchical clustering was performed
based on the Euclidean distance function with the complete linkage agglomeration method for the miRNAs found to be differentially expressed between
groups. Only comparisons based on Ct were used, rather than DCt, because
expression of the 5S control varies between monosomes and polysomes,
and is altered in S15 knockdown.
SUPPLEMENTAL INFORMATION
Supplemental Information includes four tables, Supplemental Experimental
Procedures, and seven figures and can be found with this article online at
doi:10.1016/j.molcel.2012.04.008.
ACKNOWLEDGMENTS
We would like to thank Dr. Steffen Schubert and The Institute of Chemistry and
Cell Biology (ICCB) Screening Facility at Harvard Medical School for helping
with designing and performing the RNAi screen. We also thank Dr. Stephen
Buratowski, Dr. Danesh Moazed, Dr. Kai Wucherpfennig, and Dustin Griesemer for insightful discussions. This work was supported by a Distinguished
Young Scholars Award from the W.M. Keck Foundation to C.D.N.
Received: March 17, 2011
Revised: February 16, 2012
Accepted: April 5, 2012
Published online: April 26, 2012
REFERENCES
downregulates tumor suppressor Pdcd4 and stimulates invasion, intravasation and metastasis in colorectal cancer. Oncogene 27, 2128–2136.
Avondo, F., Roncaglia, P., Crescenzio, N., Krmac, H., Garelli, E., Armiraglio,
M., Castagnoli, C., Campagnoli, M.F., Ramenghi, U., Gustincich, S., et al.
(2009). Fibroblasts from patients with Diamond-Blackfan anaemia show
abnormal expression of genes involved in protein synthesis, amino acid
metabolism and cancer. BMC Genomics 10, 442.
Bachand, F., Lackner, D.H., Bähler, J., and Silver, P.A. (2006). Autoregulation
of ribosome biosynthesis by a translational response in fission yeast. Mol. Cell.
Biol. 26, 1731–1742.
Bagga, S., Bracht, J., Hunter, S., Massirer, K., Holtz, J., Eachus, R., and
Pasquinelli, A.E. (2005). Regulation by let-7 and lin-4 miRNAs results in target
mRNA degradation. Cell 122, 553–563.
Basu, U., Si, K., Warner, J.R., and Maitra, U. (2001). The Saccharomyces
cerevisiae TIF6 gene encoding translation initiation factor 6 is required for
60S ribosomal subunit biogenesis. Mol. Cell. Biol. 21, 1453–1462.
Bazzini, A.A., Lee, M.T., and Giraldez, A.J. (2012). Ribosome Profiling Shows
That miR-430 Reduces Translation Before Causing mRNA Decay in
Zebrafish. Science, in press. Published online March 15, 2012.
Behm-Ansmant, I., Rehwinkel, J., Doerks, T., Stark, A., Bork, P., and
Izaurralde, E. (2006). mRNA degradation by miRNAs and GW182 requires
both CCR4:NOT deadenylase and DCP1:DCP2 decapping complexes.
Genes Dev. 20, 1885–1898.
Bhattacharyya, S.N., Habermacher, R., Martine, U., Closs, E.I., and Filipowicz,
W. (2006a). Relief of microRNA-mediated translational repression in human
cells subjected to stress. Cell 125, 1111–1124.
Bhattacharyya, S.N., Habermacher, R., Martine, U., Closs, E.I., and Filipowicz,
W. (2006b). Stress-induced reversal of microRNA repression and mRNA
P-body localization in human cells. Cold Spring Harb. Symp. Quant. Biol. 71,
513–521.
Boyerinas, B., Park, S.M., Shomron, N., Hedegaard, M.M., Vinther, J.,
Andersen, J.S., Feig, C., Xu, J., Burge, C.B., and Peter, M.E. (2008).
Identification of let-7-regulated oncofetal genes. Cancer Res. 68, 2587–
2591.
Campagnoli, M.F., Ramenghi, U., Armiraglio, M., Quarello, P., Garelli, E.,
Carando, A., Avondo, F., Pavesi, E., Fribourg, S., Gleizes, P.E., et al. (2008).
RPS19 mutations in patients with Diamond-Blackfan anemia. Hum. Mutat.
29, 911–920.
Ceci, M., Gaviraghi, C., Gorrini, C., Sala, L.A., Offenhäuser, N., Marchisio, P.C.,
and Biffo, S. (2003). Release of eIF6 (p27BBP) from the 60S subunit allows 80S
ribosome assembly. Nature 426, 579–584.
Chendrimada, T.P., Finn, K.J., Ji, X., Baillat, D., Gregory, R.I., Liebhaber, S.A.,
Pasquinelli, A.E., and Shiekhattar, R. (2007). MicroRNA silencing through
RISC recruitment of eIF6. Nature 447, 823–828.
David-Pfeuty, T., Nouvian-Dooghe, Y., Sirri, V., Roussel, P., and HernandezVerdun, D. (2001). Common and reversible regulation of wild-type p53 function
and of ribosomal biogenesis by protein kinases in human cells. Oncogene 20,
5951–5963.
Deisenroth, C., and Zhang, Y. (2010). Ribosome biogenesis surveillance:
probing the ribosomal protein-Mdm2-p53 pathway. Oncogene 29, 4253–
4260.
Dorner, S., Lum, L., Kim, M., Paro, R., Beachy, P.A., and Green, R. (2006). A
genomewide screen for components of the RNAi pathway in Drosophila
cultured cells. Proc. Natl. Acad. Sci. USA 103, 11880–11885.
Eulalio, A., Rehwinkel, J., Stricker, M., Huntzinger, E., Yang, S.F., Doerks, T.,
Dorner, S., Bork, P., Boutros, M., and Izaurralde, E. (2007). Target-specific
requirements for enhancers of decapping in miRNA-mediated gene silencing.
Genes Dev. 21, 2558–2570.
Amsterdam, A., Sadler, K.C., Lai, K., Farrington, S., Bronson, R.T., Lees, J.A.,
and Hopkins, N. (2004). Many ribosomal protein genes are cancer genes in
zebrafish. PLoS Biol. 2, E139.
Filipowicz, W., Bhattacharyya, S.N., and Sonenberg, N. (2008). Mechanisms of
post-transcriptional regulation by microRNAs: are the answers in sight? Nat.
Rev. Genet. 9, 102–114.
Asangani, I.A., Rasheed, S.A., Nikolova, D.A., Leupold, J.H., Colburn, N.H.,
Post, S., and Allgayer, H. (2008). MicroRNA-21 (miR-21) post-transcriptionally
Flygare, J., Kiefer, T., Miyake, K., Utsugisawa, T., Hamaguchi, I., Da Costa, L.,
Richter, J., Davey, E.J., Matsson, H., Dahl, N., et al. (2005). Deficiency of
184 Molecular Cell 46, 171–186, April 27, 2012 ª2012 Elsevier Inc.
Molecular Cell
Ribosomal Proteins Modulate miRNA Activity
ribosomal protein S19 in CD34+ cells generated by siRNA blocks erythroid
development and mimics defects seen in Diamond-Blackfan anemia. Blood
105, 4627–4634.
Nelson, P.T., Hatzigeorgiou, A.G., and Mourelatos, Z. (2004). miRNP:mRNA
association in polyribosomes in a human neuronal cell line. RNA 10,
387–394.
Friedman, R.C., Farh, K.K., Burge, C.B., and Bartel, D.P. (2009). Most
mammalian mRNAs are conserved targets of microRNAs. Genome Res. 19,
92–105.
Nottrott, S., Simard, M.J., and Richter, J.D. (2006). Human let-7a miRNA
blocks protein production on actively translating polyribosomes. Nat. Struct.
Mol. Biol. 13, 1108–1114.
Gandin, V., Miluzio, A., Barbieri, A.M., Beugnet, A., Kiyokawa, H., Marchisio,
P.C., and Biffo, S. (2008). Eukaryotic initiation factor 6 is rate-limiting in
translation, growth and transformation. Nature 455, 684–688.
Oliver, E.R., Saunders, T.L., Tarlé, S.A., and Glaser, T. (2004). Ribosomal
protein L24 defect in belly spot and tail (Bst), a mouse Minute. Development
131, 3907–3920.
Gazda, H.T., Sheen, M.R., Vlachos, A., Choesmel, V., O’Donohue, M.F.,
Schneider, H., Darras, N., Hasman, C., Sieff, C.A., Newburger, P.E., et al.
(2008). Ribosomal protein L5 and L11 mutations are associated with cleft
palate and abnormal thumbs in Diamond-Blackfan anemia patients. Am. J.
Hum. Genet. 83, 769–780.
Olsen, P.H., and Ambros, V. (1999). The lin-4 regulatory RNA controls developmental timing in Caenorhabditis elegans by blocking LIN-14 protein synthesis
after the initiation of translation. Dev. Biol. 216, 671–680.
Guo, H., Ingolia, N.T., Weissman, J.S., and Bartel, D.P. (2010). Mammalian
microRNAs predominantly act to decrease target mRNA levels. Nature 466,
835–840.
Hölzel, M., Orban, M., Hochstatter, J., Rohrmoser, M., Harasim, T.,
Malamoussi, A., Kremmer, E., Längst, G., and Eick, D. (2010). Defects in
18 S or 28 S rRNA processing activate the p53 pathway. J. Biol. Chem. 285,
6364–6370.
Johnson, S.M., Grosshans, H., Shingara, J., Byrom, M., Jarvis, R., Cheng, A.,
Labourier, E., Reinert, K.L., Brown, D., and Slack, F.J. (2005). RAS is regulated
by the let-7 microRNA family. Cell 120, 635–647.
Kim, J., Krichevsky, A., Grad, Y., Hayes, G.D., Kosik, K.S., Church, G.M., and
Ruvkun, G. (2004). Identification of many microRNAs that copurify with
polyribosomes in mammalian neurons. Proc. Natl. Acad. Sci. USA 101,
360–365.
Kim, J.K., Gabel, H.W., Kamath, R.S., Tewari, M., Pasquinelli, A., Rual, J.F.,
Kennedy, S., Dybbs, M., Bertin, N., Kaplan, J.M., et al. (2005). Functional
genomic analysis of RNA interference in C. elegans. Science 308, 1164–
1167.
Oristian, D.S., Sloofman, L.G., Zhou, X., Wang, L., Farach-Carson, M.C., and
Kirn-Safran, C.B. (2009). Ribosomal protein L29/HIP deficiency delays
osteogenesis and increases fragility of adult bone in mice. J. Orthop. Res.
27, 28–35.
, L., Montagne, J., Cokaric
, M., and Volarevic
, S. (2007). S6-haploinsufPanic
ficiency activates the p53 tumor suppressor. Cell Cycle 6, 20–24.
Parry, D.H., Xu, J., and Ruvkun, G. (2007). A whole-genome RNAi Screen for
C. elegans miRNA pathway genes. Curr. Biol. 17, 2013–2022.
Petersen, C.P., Bordeleau, M.E., Pelletier, J., and Sharp, P.A. (2006). Short
RNAs repress translation after initiation in mammalian cells. Mol. Cell 21,
533–542.
Pillai, R.S., Bhattacharyya, S.N., Artus, C.G., Zoller, T., Cougot, N., Basyuk, E.,
Bertrand, E., and Filipowicz, W. (2005). Inhibition of translational initiation by
Let-7 MicroRNA in human cells. Science 309, 1573–1576.
Rudra, D., and Warner, J.R. (2004). What better measure than ribosome
synthesis? Genes Dev. 18, 2431–2436.
Saleh, M.C., van Rij, R.P., Hekele, A., Gillis, A., Foley, E., O’Farrell, P.H., and
Andino, R. (2006). The endocytic pathway mediates cell entry of dsRNA to
induce RNAi silencing. Nat. Cell Biol. 8, 793–802.
Kirn-Safran, C.B., Oristian, D.S., Focht, R.J., Parker, S.G., Vivian, J.L., and
Carson, D.D. (2007). Global growth deficiencies in mice lacking the ribosomal
protein HIP/RPL29. Dev. Dyn. 236, 447–460.
Scheffner, M., Werness, B.A., Huibregtse, J.M., Levine, A.J., and Howley, P.M.
(1990). The E6 oncoprotein encoded by human papillomavirus types 16 and
18 promotes the degradation of p53. Cell 63, 1129–1136.
Lai, K., Amsterdam, A., Farrington, S., Bronson, R.T., Hopkins, N., and Lees,
J.A. (2009). Many ribosomal protein mutations are associated with growth
impairment and tumor predisposition in zebrafish. Dev. Dyn. 238, 76–85.
Seggerson, K., Tang, L., and Moss, E.G. (2002). Two genetic circuits repress
the Caenorhabditis elegans heterochronic gene lin-28 after translation initiation. Dev. Biol. 243, 215–225.
Langmead, B., Trapnell, C., Pop, M., and Salzberg, S.L. (2009). Ultrafast and
memory-efficient alignment of short DNA sequences to the human genome.
Genome Biol. 10, R25.
Stewart, M.J., and Denell, R. (1993). Mutations in the Drosophila gene encoding ribosomal protein S6 cause tissue overgrowth. Mol. Cell Biol. 13, 2524–
2535.
Lee, Y.S., and Dutta, A. (2007). The tumor suppressor microRNA let-7
represses the HMGA2 oncogene. Genes Dev. 21, 1025–1030.
Talotta, F., Cimmino, A., Matarazzo, M.R., Casalino, L., De Vita, G.,
D’Esposito, M., Di Lauro, R., and Verde, P. (2009). An autoregulatory loop
mediated by miR-21 and PDCD4 controls the AP-1 activity in RAS transformation. Oncogene 28, 73–84.
Lempiäinen, H., and Shore, D. (2009). Growth control and ribosome biogenesis. Curr. Opin. Cell Biol. 21, 855–863.
Lewis, B.P., Shih, I.H., Jones-Rhoades, M.W., Bartel, D.P., and Burge, C.B.
(2003). Prediction of mammalian microRNA targets. Cell 115, 787–798.
Lim, L.P., Lau, N.C., Garrett-Engele, P., Grimson, A., Schelter, J.M., Castle, J.,
Bartel, D.P., Linsley, P.S., and Johnson, J.M. (2005). Microarray analysis
shows that some microRNAs downregulate large numbers of target mRNAs.
Nature 433, 769–773.
Ulvila, J., Parikka, M., Kleino, A., Sormunen, R., Ezekowitz, R.A., Kocks, C.,
and Rämet, M. (2006). Double-stranded RNA is internalized by scavenger
receptor-mediated endocytosis in Drosophila S2 cells. J. Biol. Chem. 281,
14370–14375.
van Riggelen, J., Yetil, A., and Felsher, D.W. (2010). MYC as a regulator of
ribosome biogenesis and protein synthesis. Nat. Rev. Cancer 10, 301–309.
MacInnes, A.W., Amsterdam, A., Whittaker, C.A., Hopkins, N., and Lees, J.A.
(2008). Loss of p53 synthesis in zebrafish tumors with ribosomal protein gene
mutations. Proc. Natl. Acad. Sci. USA 105, 10408–10413.
Vasudevan, S., Tong, Y., and Steitz, J.A. (2007). Switching from repression
to activation: microRNAs can up-regulate translation. Science 318, 1931–
1934.
Maroney, P.A., Yu, Y., Fisher, J., and Nilsen, T.W. (2006). Evidence that
microRNAs are associated with translating messenger RNAs in human cells.
Nat. Struct. Mol. Biol. 13, 1102–1107.
Volarevic, S., Stewart, M.J., Ledermann, B., Zilberman, F., Terracciano, L.,
Montini, E., Grompe, M., Kozma, S.C., and Thomas, G. (2000). Proliferation,
but not growth, blocked by conditional deletion of 40S ribosomal protein S6.
Science 288, 2045–2047.
Mayr, C., and Bartel, D.P. (2009). Widespread shortening of 30 UTRs by
alternative cleavage and polyadenylation activates oncogenes in cancer cells.
Cell 138, 673–684.
Miller, S.A., Brown, A.J., Farach-Carson, M.C., and Kirn-Safran, C.B. (2003).
HIP/RPL29 down-regulation accompanies terminal chondrocyte differentiation. Differentiation 71, 322–336.
Watson, K.L., Konrad, K.D., Woods, D.F., and Bryant, P.J. (1992). Drosophila
homolog of the human S6 ribosomal protein is required for tumor suppression
in the hematopoietic system. Proc. Natl. Acad. Sci. USA 89, 11302–11306.
Werness, B.A., Levine, A.J., and Howley, P.M. (1990). Association of human
papillomavirus types 16 and 18 E6 proteins with p53. Science 248, 76–79.
Molecular Cell 46, 171–186, April 27, 2012 ª2012 Elsevier Inc. 185
Molecular Cell
Ribosomal Proteins Modulate miRNA Activity
Wightman, B., Ha, I., and Ruvkun, G. (1993). Posttranscriptional regulation of
the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in
C. elegans. Cell 75, 855–862.
Wu, L., Fan, J., and Belasco, J.G. (2006). MicroRNAs direct rapid deadenylation of mRNA. Proc. Natl. Acad. Sci. USA 103, 4034–4039.
Wu, L., and Belasco, J.G. (2005). Micro-RNA regulation of the mammalian lin28 gene during neuronal differentiation of embryonal carcinoma cells. Mol. Cell
Biol. 25, 9198–9208.
Zhou, R., Hotta, I., Denli, A.M., Hong, P., Perrimon, N., and Hannon, G.J.
(2008). Comparative analysis of argonaute-dependent small RNA pathways
in Drosophila. Mol. Cell 32, 592–599.
186 Molecular Cell 46, 171–186, April 27, 2012 ª2012 Elsevier Inc.