Transcriptome-wide Regulation of Pre-mRNA Splicing and
mRNA Localization by Muscleblind Proteins
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Citation
Wang, Eric T., Neal A.L. Cody, Sonali Jog, Michela Biancolella,
Thomas T. Wang, Daniel J. Treacy, Shujun Luo, et al.
“Transcriptome-wide Regulation of Pre-mRNA Splicing and
mRNA Localization by Muscleblind Proteins.” Cell 150, no. 4
(August 2012): 710-724. © 2012 Elsevier Inc.
As Published
http://dx.doi.org/10.1016/j.cell.2012.06.041
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Elsevier B.V.
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Final published version
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Thu May 26 20:40:20 EDT 2016
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http://hdl.handle.net/1721.1/84686
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Detailed Terms
Transcriptome-wide Regulation
of Pre-mRNA Splicing and mRNA
Localization by Muscleblind Proteins
Eric T. Wang,1,2,3 Neal A.L. Cody,5 Sonali Jog,6 Michela Biancolella,6 Thomas T. Wang,1,3 Daniel J. Treacy,1 Shujun Luo,7
Gary P. Schroth,7 David E. Housman,1,2,3 Sita Reddy,6 Eric Lécuyer,5,8 and Christopher B. Burge1,2,3,4,*
1Department
of Biology
of Health Sciences and Technology
3Koch Institute for Integrative Cancer Research
4Department of Biological Engineering
Massachusetts Institute of Technology, Cambridge, MA 02142, USA
5Institut de Recherches Cliniques de Montréal (IRCM), Montréal, Québec H2W 1R7, Canada
6University of Southern California, Los Angeles, CA 90033, USA
7Illumina, Inc., Hayward, CA 94545, USA
8Département de Biochimie, Université de Montréal, Québec H3C 3J7, Canada
*Correspondence: cburge@mit.edu
http://dx.doi.org/10.1016/j.cell.2012.06.041
2Division
SUMMARY
The muscleblind-like (Mbnl) family of RNA-binding
proteins plays important roles in muscle and eye
development and in myotonic dystrophy (DM), in
which expanded CUG or CCUG repeats functionally
deplete Mbnl proteins. We identified transcriptomewide functional and biophysical targets of Mbnl
proteins in brain, heart, muscle, and myoblasts by
using RNA-seq and CLIP-seq approaches. This
analysis identified several hundred splicing events
whose regulation depended on Mbnl function in
a pattern indicating functional interchangeability
between Mbnl1 and Mbnl2. A nucleotide resolution
RNA map associated repression or activation of
exon splicing with Mbnl binding near either 30 splice
site or near the downstream 50 splice site, respectively. Transcriptomic analysis of subcellular compartments uncovered a global role for Mbnls in
regulating localization of mRNAs in both mouse
and Drosophila cells, and Mbnl-dependent translation and protein secretion were observed for a
subset of mRNAs with Mbnl-dependent localization.
These findings hold several new implications for DM
pathogenesis.
INTRODUCTION
Muscleblind-like (Mbnl) proteins are a deeply conserved, developmentally regulated family of RNA-binding factors that
contribute to muscle and eye development and have been implicated in the genetic disease myotonic dystrophy (DM) (Artero
et al., 1998; Begemann et al., 1997; Miller et al., 2000). Flies
possess a single gene, mbl, whereas mammals express
710 Cell 150, 710–724, August 17, 2012 ª2012 Elsevier Inc.
three closely related Mbnl genes (Fardaei et al., 2002). In mouse
and human, Mbnl1 and Mbnl2 are expressed across many
tissues, including brain, heart, and muscle, whereas Mbnl3 is
expressed primarily in placenta (Kanadia et al., 2003b; Squillace
et al., 2002). Mammalian Mbnl proteins contain two pairs of
highly conserved zinc fingers, which bind to pre-mRNA to
regulate alternative splicing (Pascual et al., 2006). In DM,
Mbnls are sequestered away from their normal RNA targets
by interaction with expanded CUG or CCUG repeats (Miller
et al., 2000). This reverses the normal developmental accumulation of Mbnls, shifting splicing toward fetal isoforms (Lin
et al., 2006).
The hypothesis that Mbnl proteins are responsible for a large
fraction of the aberrant splicing patterns observed in DM was
supported by a splicing microarray analysis (Du et al., 2010),
which found that 80% of 200 alternative isoform changes
observed in a CUG-expressing mouse model of DM—including
55 cassette exons—were reproduced in mice lacking functional
Mbnl1 protein. These results suggested that CUG-repeatinduced transcriptome changes are largely Mbnl dependent
but did not identify direct targets of Mbnl.
Mbnl proteins also have significant cytoplasmic expression
and have been proposed to contribute to regulation of
messenger RNA (mRNA) stability (Du et al., 2010; Masuda
et al., 2012; Osborne et al., 2009) or localization (Adereth et al.,
2005). Most mRNAs exhibit specific patterns of localization in
the cell, which may be mediated by sequence-specific RNAbinding proteins (RBPs) that interact with cis-regulatory
elements located in 30 untranslated regions (UTRs) (Andreassi
and Riccio, 2009; Lécuyer et al., 2007; Martin and Ephrussi,
2009). Localized translation of mRNAs is thought to play important roles in proper protein localization and assembly of protein
complexes (Besse and Ephrussi, 2008; Lécuyer et al., 2009)
and to affect cellular properties such as synaptic plasticity
(Horne-Badovinac and Bilder, 2008; Kislauskis et al., 1997;
Martin and Zukin, 2006). Localization of mRNAs to the rough
endoplasmic reticulum (ER) via interaction of the signal recognition particle (SRP) with the nascent signal peptide can also
involve recognition of sequences in the mRNA to a substantial
extent (Pyhtila et al., 2008). Thus, sequence-specific RBPs like
Mbnls that have significant expression in both nucleus and cytoplasm may potentially influence diverse aspects of mRNA
biogenesis and function from processing through localization
and translation.
To identify direct regulatory targets of Mbnl1, we analyzed the
transcriptomes of brain, heart, and muscle tissue isolated from
mice lacking functional Mbnl1 protein (Kanadia et al., 2003a) in
conjunction with ultraviolet crosslinking/immunoprecipitation/
sequencing (CLIP-seq) to identify binding targets in the same
tissues and in mouse myoblasts. These data yielded a transcriptome-wide RNA map for Mbnl-dependent splicing regulation.
We observed extensive binding to 30 UTRs of mRNAs encoding
membrane and synaptic proteins and uncovered a widespread,
conserved role for Mbnls in regulating localization. This function
may contribute to the molecular events responsible for DM
pathology.
RESULTS
Identification of Hundreds of Mbnl-Responsive Exons
We performed single-end RNA sequencing (RNA-seq) of
poly(A)+ RNA from brain, heart, and muscle from five
4-month-old Mbnl1DE3/DE3 mice and age-matched controls
(129/SvJ), yielding 247 million uniquely mapped 48-base reads.
Mbnl1DE3/DE3 mice lack Mbnl with RNA-binding activity
(Kanadia et al., 2003a) (Figure S1A available online). MISO
was used to compute percent spliced in (PSI or J) values for
cassette alternative exons, representing the fraction of a gene’s
mRNAs that include the exon (Katz et al., 2010). These values
showed high concordance with J values assessed by
RT-PCR (Figure S1B). We observed hundreds of consistent
changes between wild-type (WT) and knockout animals, with
largest changes in muscle, intermediate changes in heart,
and smallest changes in brain (Figure S1D and Table S1). For
example, exon 8 of the Trim55 gene, which encodes a
muscle-specific really interesting new gene (RING) finger
protein involved in sarcomere assembly (Pizon et al., 2002),
had 59%–72% inclusion in WT heart and 15%–30% inclusion
in knockout heart (Figure 1A). Using J values of widely
expressed exons, the samples clustered first by tissue and
then by genotype, as expected (Figure 1B). Two knockout
brains detected as outliers in this analysis were omitted (data
not shown).
Changes in splicing of cassette exons (DJ values) correlated
well with previous estimates by splicing microarray (Du et al.,
2010) (Figure S1E). However, our analysis identified 199 cassette
exons with significantly altered splicing in muscle (Bayes factor
[BF] > 5 and jDJj > 0.05), which is roughly four times the number
identified by microarray (Figures S1D and S1E), and many more
Mbnl-dependent cassette exons were identified in heart and
brain (Table S1). Alternative 50 and 30 splice sites and other types
of events also exhibited Mbnl dependence, yielding a total of 912
Mbnl-dependent splicing events and 555 Mbnl-dependent alternative 30 UTRs in mouse tissues (Table S1). Together, these
exons provide a resource for analyses of the phenotypic consequences of Mbnl depletion.
Splicing Regulation by Mbnls Is Dependent
on Aggregate Levels of Mbnl Proteins
To supplement our analysis of mouse tissues and to assess the
roles of Mbnl1 relative to Mbnl2 in splicing regulation, we stably
infected C2C12 mouse myoblasts with lentiviral hairpins against
Mbnl1, Mbnl2, or both Mbnls (which did not cause gross
changes in morphology or viability) and conducted RNA-seq.
Western blotting confirmed reduction in protein levels (Figure S1C) and revealed increased levels of a higher molecular
weight isoform of Mbnl2 in Mbnl1-depleted cells and vice versa,
suggesting that these factors cross-regulate each other
posttranscriptionally.
Expression levels of isoforms were comprehensively assessed
(Table S1), and levels of Mbnl1, Mbnl2, and total Mbnl (Mbnl1 +
Mbnl2) were determined (Figure 1C). We also calculated
the mean splicing change as the mean jDJj for cassette exons
that varied in at least one sample relative to control
(jDJj > 0.1, BF > 5, and n = 465 cassette exons). The mean
splicing change was only modestly correlated to the level of
Mbnl1 (Figure 1D) or the level of Mbnl2 (Figure S2A) but showed
very strong correlation with total Mbnl levels (Mbnl1 + Mbnl2)
(Figure 1E). This trend held not only for exon skipping but also
for alternative 30 splice sites (178 events), alternative 50 splice
sites (107 events), and gene expression changes (Figure S2).
Mbnl3 was lowly expressed and did not vary significantly
between treatments, so it was omitted from this analysis.
Together, these data suggested that Mbnl1 and Mbnl2 function
interchangeably in regulation of a large set of splicing targets
and that the net depletion of both factors will be relevant in DM
and genetic models of the disease.
Transcriptome-wide Binding Locations of Mbnl1 in
Brain, Heart, Muscle, and Myoblasts
To distinguish between direct and indirect Mbnl targets and to
study context-dependent rules for Mbnl regulation, we performed CLIP-seq (with random barcodes in some cases) as
described (König et al., 2010; Ule et al., 2005; Wang et al.,
2009), yielding 1.6 million, 250,000, 120,000, 71,000, and 2.06
million reads uniquely mapping to the genome and splice junctions in the 129/SvJ brain, C57BL/6 brain, heart, and muscle,
and myoblast samples, respectively (after collapsing identical
reads). Mapping occurred predominantly to transcribed regions,
with enrichment for introns and/or 30 UTRs (Figure S3).
Mbnl1 CLIP binding locations were consistently observed in
different mouse strains, even when comparing different cell
types (e.g., reticulon 4, Figure 2A). These sites were essentially
uncorrelated with CLIP sites for a different RNA-binding protein,
Nova (Zhang et al., 2010), in this example and overall, supporting
the specificity of each CLIP data set (Figure 2B). Controlling for
effects of pre-mRNA length and gene expression (Yeo et al.,
2009), CLIP clusters were enriched for UGC- and GCU-containing 4-mers in myoblasts (Figure 2C) and other tissues. Alternative
exons with CLIP clusters within 1 kilobase were more highly
conserved than other alternative exons (Figure 2D, controlling
for gene expression), supporting their function.
Cell 150, 710–724, August 17, 2012 ª2012 Elsevier Inc. 711
A
B
WT heart
1.00
brainWT/WT
Pearson Correlation, Ψ
brainΔE3/ΔE3
RPKM
heartWT/WT
KO heart
heartΔE3/ΔE3
muscleWT/WT
0.95
0.90
0.85
0.80
muscle
ΔE3/ΔE3
0.75
Trim55
mm9, chr3 (plus strand)
C
D
E
Figure 1. Dependence of Splicing Changes on Total MBNL Levels by RNA-Seq Analysis
(A) RNA-seq read coverage across Trim55 exon 8 from WT and Mbnl1 knockout heart tissue. MISO J values and 95% confidence intervals shown at right.
(B) Global correlation of J values for cassette exons with dendrogram shown below.
(C) The fraction of MBNL1, MBNL2, or total MBNL remaining in the indicated knockout tissues and knockdown myoblasts relative to control tissues/cells.
(D and E) Mean splicing change (mean jDJj) correlates much better with total MBNL depletion than with MBNL1 depletion.
See also Figures S1 and S2 and Table S1.
Context-Dependent Splicing Regulation by Mbnl
Proteins
Several individual Mbnl-regulated exons have been well characterized, with Mbnl either activating or repressing exon inclusion (Hino et al., 2007; Ho et al., 2004; Warf and Berglund,
2007; Sen et al., 2010). Binding sites upstream or within the
30 splice site generally repress splicing (Warf and Berglund,
2007), and enrichment of Mbnl motifs downstream of activated
exons suggests that Mbnl may activate splicing in this context
(Du et al., 2010; Goers et al., 2010). Our CLIP-seq data
712 Cell 150, 710–724, August 17, 2012 ª2012 Elsevier Inc.
confirmed a number of binding sites discovered in previous
studies, e.g., sites downstream of SercaI exon 22 (Hino et al.,
2007), whose inclusion is highly sensitive to Mbnl levels
(Figure 2F). We also uncovered thousands of new locations
for Mbnl1 binding associated with splicing activity, including
sites in/around exon EIIIB of Fibronectin I (Figure 2E), whose
splicing is regulated during development and wound healing
(Ffrench-Constant and Hynes, 1989; Ffrench-Constant et al.,
1989) and which increased as total Mbnl levels decreased (Figure 1E). Splicing repression was generally associated with
B
40
Myoblasts
20
0
50
Nova 1 CLIP
Adult Cortex1
25
0
40
Adult Brain129SVJ
Myoblasts
Adult Cortex2
Adult Cortex1
P16 Brain
P16 Brain
Adult Cortex2
20
0
80
P16 Brain
Pearson Correlation
30
0
40
0
Reticulon 4 3' UTR
C
0.6
0.2
Adult BrainC57BL/6
Adult BrainC57BL/6
1.0
Adult BrainC57BL/6
Myoblasts
60
Adult Brain129SVJ
120
0
Adult Cortex2
Mbnl1 CLIP
Adult Brain
Nova 1 CLIP Mbnl1 CLIP
240
129SVJ
Adult Cortex1
A
D
CLIP clusters
no CLIP clusters
F
Fibronectin1
RPKM (x 102)
10
5
0
C2C12 CLIP
Atp2a1 (SERCA1)
Mbnl1 & Mbnl2
15
7.5
0
15
7.5
0
Mbnl1
Mbnl2
15
7.5
0
15
7.5
0
RPKM (x 103)
E
40
20
0
40
20
0
muscle CLIP
muscleWT/WT
muscleΔE3/ΔE3
40
20
0
Control
mm9, chr7 (minus strand)
exon EIIIB
mm9, chr1 (minus strand)
G
CLIP density
low
high
Figure 2. MBNL Binding Is Conserved and Associated with Context-Dependent Splicing Activity
(A) Mbnl1 and Nova CLIP tags derived from various tissues and cells in the 30 UTR of reticulon 4.
(B) Correlation of CLIP tag densities in five nucleotide windows across all 30 UTRs expressed in brain and C2C12 myoblasts.
(C) Histogram of Z-scores of 4-mers occurring in Mbnl1 CLIP clusters from C2C12 myoblasts, relative to control regions from the same genes.
(D) Alternative exons with Mbnl1 CLIP clusters upstream or downstream are more conserved than other alternative exons expressed at similar levels (shading
represents SEM).
(E and F) Dependence on Mbnl levels of J values of Fibronectin 1 exon EIIIB and SercaI exon 22, respectively. CLIP data are shown above. MISO J values and
95% confidence intervals are shown at right.
(G) Dependence of change in J value (WT and Mbnl1/Mbnl2 double knockdown) on Mbnl1 binding in the upstream intron (last 300 bases), alternative exon, or
downstream intron (first 300 bases).
See also Figures S3 and S4 and Table S2.
Cell 150, 710–724, August 17, 2012 ª2012 Elsevier Inc. 713
CLIP
reads
CLIPseq
read density
A:
0
C:
52 (74%)
G:
0
T:
18 (26%)
Total: 70
p(CIS): 18 / 70 = 0.26
A:
2 (7%)
C:
18 (60%)
G:
1 (3%)
T:
9 (30%)
Total: 30
p(CIS): 12 / 30 = 0.4
B
Frequency, p(CIS) > 0.3
A
{
mm9, chr8:131,256,618-131,256,697 (Integrin β1 3' UTR)
D
CLIP sites, p(CISC→T) > 0.1
Minimum
p(CIS)
2
1
0
2
1
0
2
1
0
2
1
0
0.3
0.2
0.1
Activated exons
(BF > 5, ΔΨ > 0.05, N = 88)
0.05
-2
0
Z-score
10
20
Mean CLIP density
(arbitrary units)
Information (bits)
C
Z-score
Repressed exons
(BF > 5, ΔΨ < -0.05, N = 87)
10
20
2
Position relative to
substituted C
Distance to nearest splice site (nt)
G
2
0
7B
n C5
H
ai
Myoblasts
Brain129SVJ
BrainC57BL/6
Br
4
n 12
6
ai
9S
VJ
8
ea
L/
6
r
M t C57B
us
L/
6
c
M le C5
7B
yo
L/
bl
as 6
ts
10
Br
Normalized density (x10-4)
E
insoluble fraction
synaptosome
neuron projection
actin cytoskeleton
axon
synapse
F
protein complex
perinuc. region of cytoplasm
membrane fraction
neuronal cell body
Enrichment
CLIP clusters
no CLIP clusters
1
2.5
4
*BF > 5, **BF > 100
Figure 3. Patterns of MBNL Binding and a Nucleotide Resolution Map of Splicing Activity
(A) Examples of substitutions observed in Mbnl1 CLIP tags and calculation of probability of crosslink-induced substitution (p(CIS)).
(B) Cytosine exhibits the greatest p(CIS) within Mbnl1 CLIP clusters and is most often sequenced as thymine.
(C) Information (relative entropy) of positions adjacent to frequently substituted cytosines as a function of substitution frequency.
(D) Mean CLIP density near exons activated (red) and repressed (blue) by Mbnl1, using binding sites with p(CIS)C/T > 0.1. Density around exons unaffected by
Mbnl depletion is shown by dotted line, with 90% confidence intervals shown in gray. Colored dots denote 100 nucleotide-long windows with a significantly
greater density of CLIP sites at regulated than nonregulated exons.
714 Cell 150, 710–724, August 17, 2012 ª2012 Elsevier Inc.
binding in the upstream intron and in the alternative exon, with
increased binding associated with stronger repression (Figure 2G). Splicing activation was associated with binding in
the downstream intron and also increased with increased
binding density (Figure 2G).
Crosslink-Induced Substitutions Locate Precise
Mbnl1 Binding Sites
To define a high-resolution ‘‘RNA map’’ of the regulatory activity
associated with binding at specific distances from splice sites,
we sought to pinpoint Mbnl binding sites by analysis of crosslink-induced mutations (Kishore et al., 2011). The frequency of
crosslink-induced substitutions (CIS) was calculated at each
position as the fraction of reads that cover the position but do
not match the reference base (Figure 3A). The base with highest
CIS frequency was cytosine, and C to T substitutions occurred at
least 5- to 10-fold more often than any other substitution (Figure 3B). Cytosines with high CIS had biases in flanking bases;
the 1 base just preceding the substituted C was increasingly
biased toward guanine (reaching 94%), and the 2, +1, and +2
bases were preferentially uracil (Figure 3C). The resulting motif
closely matched the UGCU motif found in previous studies of
Mbnl1 binding affinity and in Figure 2C, suggesting that
frequently substituted cytosines represent sites of direct crosslinking to Mbnl1 zinc fingers (Teplova and Patel, 2008). Deletions
in CLIP-seq reads were also observed but were rarer and less
frequently associated with Mbnl motifs, and so they were not
separately analyzed (Figure S3).
The overlap between MBNL-regulated and MBNL-bound
exons was quite extensive (Table S2). In all, 31% of regulated
exons (compared to 14% of unregulated exons) were associated
with high-confidence CLIP sites (P(C/T) > 10%), and this is
likely an underestimate, as the fraction increased to 44% in
genes expressed above median levels and exceeded 75%
when considering all CLIP sites.
We first constructed an RNA map for Mbnl-dependent
splicing regulation by using the frequently substituted positions
(P(C/T) > 10%, Figure 3D). Activation- and repressionassociated binding were 4.5-fold and 9-fold above background, respectively (both Z-scores > 10; Experimental Procedures), and occurred in almost completely distinct locations.
A repression-associated peak was centered on the alternative
exon and extended through the 30 splice site 140 bases into
the upstream intron, which is consistent with repression via
direct occlusion of exonic enhancer, 30 splice site, or branch
site elements. Additional repressive peaks occurred 100 bases
50 of the downstream 30 splice site, suggesting that repression of
splicing may occur by different mechanisms (Figure 3D). An activation-associated peak began just 30 of the regulated exon and
extended 120 bases into the downstream intron. These data
establish precise locations at which binding of Mbnl is associated with repression or activation of splicing, enabling prediction
of regulatory consequences. An RNA map constructed by using
all CLIP sites yielded qualitatively similar results but with peaks
that were less consistent and pronounced, suggesting lower
precision (Figure S4).
An RNA map constructed for alternative 30 splice sites revealed significant enrichment of Mbnl binding between the
competing splice sites in cases associated with repression of
the intron-proximal site by Mbnls (Figure S4D). This pattern of
activity is analogous to that observed for other motifs that function as exonic splicing silencers (ESSs) (Wang et al., 2006).
30 UTR Binding Is Associated with Specific Protein
Functions and Localizations
Genes with Mbnl binding in 30 UTRs were most enriched for
‘‘cellular component’’ Gene Ontology categories, as compared
to ‘‘molecular function’’ and ‘‘biological process’’ (Figure S4).
In all tissues and myoblasts, enriched cellular component
categories included synapse, membrane fraction, and insoluble
fraction (Figure 3G). Some of these targeted genes included
mRNAs encoding proteins involved in synaptic vesicle fusion
such as Snap25 and Vamp1, signaling proteins like Camk2a,
the extracellular matrix (ECM)-interacting membrane protein integrin b1, and secreted ECM proteins such as collagens. These
data suggested a functional association between Mbnl1 and
messages encoding proteins with these localizations. Consistent with this interpretation, the cytoplasmic distribution of
Mbnl1 did not appear uniform, with localization at the periphery
of the cell or in structures appearing to anchor cellular projections (Figure S4F). Furthermore, as observed previously in
human cells (Holt et al., 2009), Mbnl protein partially shifts from
the nucleus to cytoplasm following trypsinization and replating
of mouse myoblasts (Figure S4E). Genes containing Mbnl-regulated alternative exons were modestly enriched for some of the
same Gene Ontology categories as above, but none reached
statistical significance (data not shown).
A substantial proportion of Mbnl CLIP reads (sometimes the
majority) were located in 30 UTRs, which is consistent with
a recent study (Masuda et al., 2012), and exhibited enrichment
relative to RNA-seq reads (Figure S3C). Tetramer motifs in
CLIP reads mapping to 30 UTRs and introns (based on Z-scores,
as in Figure 2C) were highly overlapping, suggesting that Mbnl
binds similar motifs in 30 UTRs as in introns (data not shown).
Binding occurred predominantly 50 of the cleavage and polyadenylation site (PAS), which is consistent with interaction in the
cytoplasm, and tended to increase closer to the PAS, suggesting
a function other than splicing regulation (Figure 3E). Those
30 UTRs containing CLIP clusters were more highly conserved
than other 30 UTRs of similar length and expression level,
suggesting enrichment for functional features (Figure 3F).
Mbnl1 Preferentially Binds to Localized mRNAs
Proteins that bind 30 UTRs are associated with specific cellular
and molecular functions, including regulation of mRNA stability,
translation, and localization (Moore and Proudfoot, 2009; Zhao
(E) Mean Mbnl1 CLIP density at positions along 30 UTRs by using three CLIP data sets.
(F) Conservation in 30 UTRs with and without Mbnl1 CLIP clusters with similar expression levels and UTR lengths (shading represents SEM).
(G) Significant Gene Ontology categories of genes with 30 UTRs having Mbnl1 CLIP.
See also Figure S4 and Table S2.
Cell 150, 710–724, August 17, 2012 ª2012 Elsevier Inc. 715
Cytosolic
A
Membrane
Insoluble
+ digitonin,,
elute
+ Triton X-100,
elute
+ high salt,
elute
Homogenize,
remove nuclei
+ Triton X-100,
spin
Harvest remaining
material
Mouse C2C12
myoblasts
high speed spin
Drosophila
S2R+ cells
B
Mouse myoblast protein
Control
knockdown
C
Mouse myoblast mRNA
Membrane
Mbnl1 & 2
knockdown
Nucleosome
Integral to plasma
membrane
Proteinaceous extracellular
matrix
Other genes
Hsp90
Calnexin
cy
to
s
em olic
br
a
in ne
so
lu
bl
e
cy
to
m sol
em ic
br
a
in ne
so
lu
bl
e
Tpm1
m
Insoluble
Cytosolic
D
Cumulative Fraction
Cumulative Fraction
E
log 2 (Insoluble / Cytosolic)
log 2 (Membrane / Cytosolic)
Figure 4. mRNA Localization Is Associated with Binding by Mbnl1
(A) Cellular fractionation scheme in C2C12 mouse myoblasts and fly S2R+ cells. Adherent myoblasts were fractionated on tissue culture plates, whereas
semiadherent S2R+ cells were fractionated by using centrifugation, yielding ‘‘cytosolic,’’ ‘‘membrane,’’ and ‘‘insoluble’’ fractions.
(B) Western blot of Hsp90, Calnexin, and Tropomyosin I proteins for each compartment in control and Mbnl knockdown myoblasts.
(C) Simplex representation of mRNA expression across compartments in control myoblasts, with colored mRNAs belonging to specific functional
categories.
716 Cell 150, 710–724, August 17, 2012 ª2012 Elsevier Inc.
et al., 1999). The strong biases for binding to mRNAs associated
with particular cellular compartments and a previous study
demonstrating that Mbnl2 localizes integrin a3 mRNA to the
plasma membrane for translation (Adereth et al., 2005) suggested that Mbnls might play a general role in localization of
many mRNAs.
To assess mRNA localization in mouse myoblasts, we used
cellular fractionation followed by RNA-seq. Sequential detergent
extractions yielded cytosolic, membrane (including organelles
such as rough ER), and insoluble fractions (Jagannathan et al.,
2011; Vedeler et al., 1991) (Figure 4A). Western analysis of
protein markers Hsp90, calnexin, and tropomyosin 1 in each of
these fractions confirmed separation of the corresponding
cellular compartments (Figure 4B). Hsp90 was exclusively
cytosolic (Csermely et al., 1998); calnexin, an ER resident protein
(Bergeron et al., 1994), was almost exclusively associated
with the membrane fraction; and tropomyosin, which decorates
F-actin filaments (Gunning et al., 2005), was substantially
enriched in the insoluble fraction. In parallel, we performed
similar analysis in Drosophila S2R+ cells to assess potential
phylogenetic conservation of effects on mRNA localization.
Semiadherent S2R+ cells were processed by sequential homogenization/centrifugation to obtain analogous cellular compartments (Figure 4A). Assessment of several marker proteins in
S2R+ cells confirmed successful fractionation (Figure S5B),
and the mRNA content of each fraction was analyzed by
RNA-seq (Table S3).
To visualize differences in mRNA abundance across the three
fractions, we displayed mRNAs as points in a triangular simplex,
where the proximity to each corner of the triangle represents the
relative expression bias toward the corresponding cellular
compartment (cytosolic, membrane, or insoluble). Although
this approach does not provide absolute mRNA abundance in
each compartment, it allows the comparison of relative mRNA
levels across compartments and conditions. mRNAs belonging
to particular functional categories showed compartment-biased
expression consistent with the localization patterns of their encoded proteins (Figure 4C), which is similar to what has been established for some mRNAs (Besse and Ephrussi, 2008). In
myoblasts, mRNAs encoding nucleosomal proteins were enriched in the cytosolic compartment, whereas integral plasma
membrane and proteinaceous ECM proteins were biased
toward the membrane and especially insoluble compartments
relative to the cytosol (Figure 4C). These observations are
consistent with previous studies showing that mRNAs encoding
secreted proteins are biased toward membranous cellular
compartments such as rough ER and nuclear envelope (Pyhtila
et al., 2008) and are also consistent with studies showing that
mRNAs encoding cytoskeletal or ECM proteins are actively
translated on cytoskeletally anchored ribosomes (Hesketh,
1994; Pachter, 1992; Sundell and Singer, 1991). As expected,
mRNAs encoding signal peptides were highly enriched in the
membrane and especially in insoluble compartments (Figure S5G). Similar associations between mRNA localization and
protein function were observed in Drosophila cells (Figure S5D).
To assess the relationship between Mbnl binding and mRNA
localization, we subdivided genes by the number of 30 UTR
CLIP clusters and plotted their relative density within the
simplex. We observed that the proportion of mRNAs biased
toward the ‘‘insoluble’’ corner increased significantly with the
number of CLIP clusters (Figures 4D and 4E). These observations support the association of Mbnl with cytoskeletally trafficked mRNAs and suggest a connection to the secretory
pathway.
Mbnl Depletion Alters Localization of Bound mRNAs
To assess whether Mbnl directly impacts localization of its
binding targets, we performed analogous fractionation/RNAseq analyses of mouse myoblasts depleted of Mbnl1 and
Mbnl2. Comparable separation of cellular compartments in
knockdown cells was confirmed by western analysis (Figure 4B),
and similar associations between protein function and mRNA
localization were observed (Figure S5C), suggesting that depletion of Mbnls did not grossly alter cellular RNA trafficking.
Assessment of mRNA abundance in each compartment in
control and Mbnl-depleted cells enabled analysis of changes in
localization for individual mRNAs. These changes were displayed as vectors in the triangular simplex and were colored to
highlight patterns in direction of change following knockdown
(Figure 5A). We focused our analyses on the subset of genes
that were biased toward the insoluble corner (in which Mbnl
binding was most enriched) and further subdivided these genes
according to CLIP cluster density. Following Mbnl depletion,
these genes tended to have reduced membrane association
and increased bias toward the insoluble compartment (Figure 5C). The extent of relocalization correlated with increasing
CLIP density (Figure 5C), supporting a model in which Mbnl
binding directly affects mRNA trafficking and localization.
Muscleblind proteins are highly conserved across species and
have been shown to be functionally interchangeable between
mammal and fly in certain cases (Goers et al., 2010). To assess
whether the mRNA localization activity of mouse Mbnl1 is
conserved to fly, we depleted mbl mRNA from Drosophila
S2R+ cells (Figure S5A) and performed fractionation/RNA-seq
analysis as above (Figure 5B and Table S4). Again, mbl depletion
did not cause gross changes in cellular morphology, viability, or
bulk mRNA trafficking (Figure S5D). To assess effects of mbl
depletion, we analyzed mRNAs biased toward the insoluble
compartment by using UGCU motif density as a proxy for mbl
binding (Goers et al., 2010). As in mouse, we observed a bias
for relocalization of putative mbl targets away from membrane
and toward the insoluble compartment, and, again, the strength
of this trend increased with motif density (Figure 5D). Thus,
depletion of muscleblind proteins results in similar alterations
(D) Density map of Mbnl1 CLIP targets in control myoblasts. The relative abundance of genes with 0, R4, R6, or R8 CLIP clusters is illustrated within the simplex
and is quantified for areas of the simplex on either side of the dashed line.
(E) Cumulative distribution functions of relative enrichment in insoluble/cytosolic (left) or membrane/cytosolic (right) of genes grouped by number of 30 UTR CLIP
clusters, with significance determined by Kolmogorov-Smirnov test.
See also Figure S5 and Table S3.
Cell 150, 710–724, August 17, 2012 ª2012 Elsevier Inc. 717
A
B
Mouse
Fly
Membrane
Membrane
Change in localization,
Control shRNA →
Mbnl shRNA
Insoluble
Change in localization,
Control RNAi →
Mbl RNAi
Cytosolic
Insoluble
C
Cytosolic
D
Mouse
Fly
M
M
M
M
No. clusters /
kb of 3’ UTR
No. UGCU /
kb of 3’ UTR
0 - 0.5
0-4
0.5 - 2
C
C
C
Insoluble
p = 6e-7
00
0. .5
52
>2
00
0. .5
52
>2
00
0. .5
52
>2
p = 3e-7
Cytosolic
No. UGCU / kb of 3' UTR
Mbnl2
vs. Ctl
Mbnl1 & 2
vs. Ctl
F
M
p = 8e-3
-0.8
-0.8
-0.8
CLIPseq
-0.4
0.8
0.8
0.8
0.4
0.4
0.4
0
0
0
-0.4
-0.4
-0.4
-0.8
-0.8
-0.8
No. clusters / kb of 3' UTR
M
C
I
400
200
0
Fn1 3' UTR
Gaussia / Cypridina (%)
-0.4
00
0. .5
52
>2
0
-0.4
00
0. .5
52
>2
0.4
00
0. .5
52
>2
C
0
00
0. .5
52
>2
I
0.4
0
00
0. .5
52
>2
M
I
0.8
0.4
00
0. .5
52
>2
C
Fold-change, ribosome RPKM (log2)
I
0.8
0.8
Insoluble
p = 1e-6
CLIPseq
Mbnl1
vs. Ctl
Membrane
p = 1e-6
No. clusters / kb of 3' UTR
E
C
04
48
>8
Membrane
I
04
48
>8
Cytosolic
M
>8
04
48
>8
I
Change in localization
rel. to compartment (a. u.)
4-8
I
>2
Change in localization
rel. to compartment (a. u.)
I
Bgn 3' UTR
120
110
C
80
40
0
**
*
***
*
*
100
90
80
70
60
50
Fn1
Bgn
Reporter 3’ UTR
Control
960 CUG
960 CUG + Mbnl1
*** p < 1e-5
** p < 0.01
* p < 0.05
Figure 5. Mbnl Binding Is Associated with Regulation of mRNA Localization in Mouse and Fly and May Contribute to Protein Secretion
(A and B) Change in mRNA localization following depletion of Mbnls in mouse myoblasts and fly S2R+ cells. Arrows represent changes in mRNA localization
where the direction change is encoded by color.
(C and D) The aggregate behavior following Mbnl depletion of the subset of genes biased toward the insoluble compartment in control cells is shown in a polar plot
in which the radial amplitude is the number of genes that relocalized in the given direction following Mbnl depletion, stratified by density of CLIP clusters (for
mouse) or UGCU 4-mers (for fly). The aggregate change in localization toward each compartment is shown in bar plots. p values denote significance when
comparing change in localization of MBNL targets versus nontargets (e.g., >2 versus 0–0.5 for mouse myoblasts).
718 Cell 150, 710–724, August 17, 2012 ª2012 Elsevier Inc.
in the localization of putative mRNA-binding targets (Tables S2
and S3) in both mammalian and fly cells.
Mbnls have also been implicated in regulating mRNA stability
(Du et al., 2010; Masuda et al., 2012; Osborne et al., 2009). To
investigate a potential link between message stability and localization, we analyzed total gene expression of Mbnl 30 UTRbinding targets following Mbnl depletion. Modest increases
(<5%) in mean expression of Mbnl targets following Mbnl depletion were observed overall and in mRNAs localized to particular
subcellular compartments (Figure S6).
Mbnl Function Contributes to mRNA Translation
Because mRNA localization is often coupled to translational
regulation, we sought to assess effects of Mbnl at the translational level by performing ribosome footprint profiling (Ingolia
et al., 2011) using control C2C12 mouse myoblasts and
myoblasts depleted of Mbnl1, Mbnl2, or both Mbnl1 and
Mbnl2. These data, representing millions of ribosome-protected
mRNA fragments mapping primarily to coding and 50 UTR
regions (Figure S6), were used to obtain genome-wide RPKM
(ribosome reads per kilobase of mRNA per million mapped
reads) measurements that are analogous to RNA-seq-based
RPKMs but reflect the ribosomal association of mRNAs
rather than mRNA abundance. We observed that subsets of
Mbnl 30 UTR targets, in particular those messages that had
membrane-biased localization and moved toward the insoluble
compartment following MBNL depletion, had substantially
reduced ribosome RPKMs in myoblasts depleted of both Mbnls
relative to controls or cells depleted of Mbnl1 or Mbnl2 alone
(Figure 5E). By contrast, the overall abundance of these mRNAs
(based on RNA-seq) actually increased slightly following depletion of Mbnls (Figure S6), implying that Mbnl depletion results
in reduced translation of this subset of mRNAs. Alternatively,
these effects could possibly result from changes in mRNA
stability dependent on the translational status of the mRNA.
The analogous subset of messages biased to the insoluble
compartment did not exhibit significantly decreased ribosome
footprint RPKMs (Figure 5E).
Mbnl Function Contributes to Protein Secretion
Many of the mRNAs biased toward the insoluble compartment
that relocalized following Mbnl depletion encode secreted
proteins (Figure S5G). Although, classically, recognition of the
signal peptide by the SRP targets mRNAs to the secretory
pathway (Blobel and Dobberstein, 1975a, 1975b), more recent
studies have demonstrated that RNA elements at the 50 ends
of open reading frames (ORFs) also contribute to targeting of
mRNAs to the rough ER or to efficiency of nuclear export
(Cenik et al., 2011; Palazzo et al., 2007; Pyhtila et al., 2008).
We therefore hypothesized that effects of Mbnl on mRNA
localization might play a role in targeting mRNAs to the secretory pathway.
To test this hypothesis, we performed a dual luciferase assay using two efficiently secreted luciferases, Gaussia and
Cypridina. We cloned the 30 UTRs of Biglycan and Fibronectin
1 onto the 30 end of Gaussia luciferase; each gene has abundant
Mbnl binding in the 30 UTR and substantially changes in localization following Mbnl depletion (Figure 5F). Upon cotransfection
with a plasmid encoding 960 CUG repeats, which mimics DM
via functional inactivation of Mbnls (Cooper et al., 2009), we
observed a 10%–20% reduction in secretion of Gaussia with
the Fn1 or Bgn 30 UTR relative to control (Figure 5F). Overexpression of Mbnl1 coding sequence led to a complete rescue of the
Fn1 30 UTR-mediated secretion defect and a partial rescue of
Bgn 30 UTR-mediated secretion (Figure 5F), suggesting that
the effects of CUG repeats on secretion are attributable to the
depletion of Mbnls. Taken together, these data and the footprinting results support a model in which Mbnl binding to the 30 UTR
of an mRNA promotes productive translation and/or secretion of
the encoded protein.
Mbnl Function Is Associated with Localization
of Alternative 30 UTR Isoforms
Thousands of mammalian genes express mRNA isoforms with
different 30 ends as a result of alternative PAS usage, which
can be quantitated based on RNA-seq data (Katz et al., 2010;
Wang et al., 2008). Alternative 30 UTR isoforms provide natural
‘‘reporters’’ for the function of regulatory elements located in
the distal (long isoform-specific) region. We found hundreds of
genes whose alternative 30 UTR isoforms had differing localization patterns in myoblasts (Table S4). These longer isoforms
tended to be localized toward the insoluble compartment relative to the shorter isoform (Figure 6A). This effect is likely attributable to RNA sequence elements uniquely present in the distal
regions, which were frequently bound by Mbnl1 (Figure 6B).
Depletion of Mbnls led to relocalization of specific subsets of
Mbnl-bound mRNA isoforms. Focusing on mRNA isoforms
biased toward the insoluble compartment as in Figure 5, we
observed that Mbnl1-bound long isoforms shifted strongly
away from cytosol and toward the insoluble compartment
following depletion of Mbnl1 and Mbnl2. More modest shifts
were observed for Mbnl1-bound short isoforms (Figure 6C).
These effects persisted when restricting the analysis to
30 UTRs whose overall isoform abundance did not change
(Figure S7A), suggesting that they resulted from Mbnl activity
outside the nucleus rather than from altered PAS choice in the
nucleus. This analysis suggests that Mbnl may play a role in
regulating isoform-specific subcellular localization of long UTR
isoforms, which are particularly abundant in mammalian brain
and muscle (Ramsköld et al., 2009).
(E) The mean fold change in ribosome footprint density following Mbnl1, Mbnl2, or double mbnl depletion relative to control was quantitated for mRNAs that were
initially biased toward the membrane compartment (top) or the insoluble compartment (bottom) and whose localization shifted toward the insoluble compartment
following mbnl depletion (error bars represent SEM). Genes were further stratified by density of Mbnl CLIP sites in 30 UTR. The p value denotes the significance of
the change in footprint density when comparing MBNL targets versus nontargets (rank-sum test).
(F) Change in mRNA localization of Fibronectin 1 (Fn1) and Biglycan (Bgn) following Mbnl depletion in mouse myoblasts (top), Mbnl1 CLIP tag density in the 30 UTR
of each gene (middle), and results of secreted luciferase assay by using luciferases containing the Bgn 30 UTR or the Fn1 last intron plus 30 UTR (bottom).
Error bars represent SEM. See also Figure S6 and Table S4.
Cell 150, 710–724, August 17, 2012 ª2012 Elsevier Inc. 719
Cumulative Fraction
Genes with localized 3’ UTR isoforms
(BF > 5, |ΔΨ| > 0.05, N=828)
A
distal PAS
proximal PAS
p = 3e-14 (KS test)
Cumulative Fraction
log 2 (Insoluble / Cytosolic)
p = 1e-11 (KS test)
log 2 (Insoluble / Membrane)
C
B
Gtpbp4
8972368
8971845
8971321
Gtpbp4
proximal PAS
distal PAS
I
C
Long, control shRNA
Long, Mbnl shRNAs
Short, control shRNA
Short, Mbnl shRNAs
log2(KD/Control)
M
1
0.5
Change in localization
rel. to compartment (a. u.)
Cytosolic
Mbnl1
300
CLIP-Seq 150
(RPKM)
0
*
**
Membrane
Insoluble
*
*
***
Long
Short
0
* p < 0.05, ** p < 0.01, *** p < 1e-4
-0.5
-1
C
M
I
Figure 6. Mbnl Regulates Isoform-Specific mRNA Localization via Binding to Alternative 30 UTRs
(A) The localization of long and short alternative 30 UTR isoforms is shown at left (as in Figure 4B). Relative enrichment in pairs of cellular compartments is shown
for long and short isoforms at right (as in Figure 4E).
(B) CLIP data for the 30 UTR of Gtpbp4. Localization of the long and short isoforms in normal myoblasts is displayed in solid blue and black circles, respectively,
and in myoblasts depleted of Mbnl in outlined blue and black circles, respectively, with arrows indicating change in localization. Bar plot shows change in RPKM
(Mbnl-depleted versus control myoblasts) for the long and short 30 UTR isoforms of Gtpbp4.
(C) Genes were separated into those with and without CLIP clusters in the core or extended 30 UTR region for short and long isoforms, respectively, and assessed
for direction of relocalization following Mbnl depletion. Bar plot shows the extent of relocalization toward each compartment in which the magnitude of relocalization for CLIP targets is normalized relative to nontargets.
Error bars represent SEM. See also Figure S7 and Table S5.
DISCUSSION
Because of the extreme heterogeneity of clinical symptoms, DM
has been described as one of the most variable disorders known
in medicine (Harper, 1979). Some of the heterogeneity in DM1
undoubtedly results from the variability in inherited triplet repeat
length and from differences in the extent of somatic expansion in
different tissues (Ashizawa et al., 1993). This genomic variability
interacts with the diverse tissue expression of DMPK mRNA and
of Mbnl and CELF family proteins. Thus, even among different
720 Cell 150, 710–724, August 17, 2012 ª2012 Elsevier Inc.
patients with the same inherited repeat length, Mbnl proteins
may be depleted to different extents in skeletal muscle, brain,
or other tissues. A comprehensive understanding of the molecular basis of DM will therefore require not only an understanding
of the normal functions of Mbnl proteins in affected cell and
tissue types but also an appreciation of the extent to which
different functions are impacted by particular degrees of Mbnl
depletion.
Our global analyses yielded several insights into Mbnl
function and established genomic resources for future
Figure 7. A Model for Nuclear and Cytoplasmic Functions of Mbnl
Mbnls repress or activate splicing, depending on binding location. In the cytoplasm, Mbnl binding in 30 UTRs may facilitate targeting of mRNAs with signal
sequences to the rough ER. Alternatively, transcripts may be targeted to membrane-rich organelles for localized translation via actin-, microtubule-, or intermediate filament-based molecular motors. These organelles may include synapses, NMJs, or the plasma membrane, depending on cell type. Mbnl may mediate
isoform-specific mRNA localization in which Mbnl binding sites within distal 30 UTRs are required for targeting to particular compartments. (Model based on data
from previous figures. See also Figure S7.)
functional, modeling, and clinical studies, identifying hundreds of
Mbnl-regulated exons and thousands of Mbnl1 binding sites
across the transcriptome, many of which are likely to be
conserved to human (Figures 2D and 4B). Knowledge of direct
regulatory targets should aid in reconstructing the order of
events that occur during disease pathogenesis as Mbnl levels
are depleted and should provide insights into mechanism.
The transcriptome data indicate that Mbnl1 and Mbnl2
proteins coregulate a set of shared targets and that their splicing
functions may be largely interchangeable. The effects of interventions that alter the levels of Mbnl1 or Mbnl2 are therefore
likely to depend strongly on the level of the other factor, which
will vary substantially depending on the affected tissue. For
example, Mbnl2 depletion might produce greater changes than
Mbnl1 depletion in neurons and vice versa in muscle, whereas
the effects of simultaneous depletion of both factors by CUG
repeats should depend more on the size of the total pool of
Mbnls than on the level of either factor alone.
We present several lines of evidence for extranuclear functions
of Mbnl1 in regulation of mRNA localization and protein expression. A previous study demonstrated that Mbnl2 localizes integrin a3 mRNA to the plasma membrane (Adereth et al., 2005). In
this and many other cases, membrane localization is associated
with efficient mRNA translation. Here, we show that Mbnls
contribute to targeting of hundreds of mRNAs to membranes.
Further, we show that the efficient translation of these
mRNAs—particularly those that are membrane biased—is
dependent on Mbnls. We also observed that Mbnl-bound 30
UTRs contribute to protein secretion (Figure 5F), perhaps by
facilitating targeting to rough ER.
Our results led to a model of the activities of Mbnl proteins in
nucleus and cytoplasm that is illustrated in Figure 7. In this
model, nuclear Mbnls bind introns or exons to activate or
repress splicing, whereas cytoplasmic Mbnls bind 30 UTRs
and facilitate transport along the cytoskeleton to the rough ER
or other membranes, presumably through interaction with
cellular transport machineries. Nuclear and cytoplasmic functions of Mbnls may be related. Indeed, splicing regulation by
Mbnls was associated with 30 UTR binding; genes with evidence
of direct splicing regulation by Mbnl1 were twice as likely to have
CLIP clusters in their 30 UTR (p = 7 3 106, Fisher’s exact test;
Figures S7B and S7C). We observed a positive correlation
between the density of 30 UTR CLIP clusters and proximity to
Mbnl-regulated exons, but whether this correlation reflects a
Cell 150, 710–724, August 17, 2012 ª2012 Elsevier Inc. 721
functional relationship or simply results from the correlation
between high C + G content and short intron length is not clear
(Figure S7D).
Both cytoplasmic and nuclear pools of Mbnl are affected in
DM (Miller et al., 2000), suggesting that functions of Mbnl outside
the nucleus may play a role in DM pathogenesis. Cellular structures particularly sensitive to alterations in mRNA localization,
such as synapses and neuromuscular junctions (NMJs), may
be perturbed in DM as a result of Mbnl sequestration. Indeed,
NMJ degeneration has been observed in diaphragm muscles
in mouse models of DM (Panaite et al., 2008). Additionally, motor
neurons derived from human DM embryonic stem cells fail to
form mature neuromuscular connections with normal muscle
(Marteyn et al., 2011), and worms deficient in neuronal muscleblind expression fail to form distal NMJs, implicating presynaptic
dysregulation (Spilker et al., 2012). Secretion of proteins with
important functions in muscle may also be affected in DM. Biglycan, whose 30 UTR confers Mbnl-dependent secretion, is of
particular interest because its loss leads to NMJ degeneration
and because it has been proposed as a therapy for Duchenne
muscular dystrophy (Amenta et al., 2012). Other relevant
secreted factors include Fibronectin 1 and Thymosin-b 4 (Figure 6E and Table S2). Our findings suggest that downstream
consequences of Mbnl’s cytoplasmic functions, such as altered
levels of extracellular proteins or circulating factors, may play
important roles in disease pathogenesis and could serve as
easily accessible diagnostics for monitoring disease progression
and assessing response to therapy.
EXPERIMENTAL PROCEDURES
RNA-Seq Library Preparation and Sequencing
Five 16-week-old 129/SvJ Mbnl1DE3/DE3 mice and littermate WT mice were
sacrificed for isolation of whole brain, heart, and skeletal muscle (vastus
lateralis). Poly(A)+ RNA was prepared for single-end RNA sequencing
(50 cycles by Illumina GAII). RNA-seq libraries for C2C12 mouse myoblasts
were performed similarly (paired-end 2 3 36 cycles by Illumina GAIIx). For
the cellular fractionation experiments, Ribo-Zero columns (Epicenter Biotechnologies) were used to reduce ribosomal RNA abundance instead of oligo
deoxythymine (dT) beads, and barcodes were used to multiplex three samples
per lane (paired-end 2 3 36 by Illumina HiSeq 2000).
CLIP-Seq Library Preparation and Sequencing
CLIP was performed by using 254 nm UV irradiation as previously described
(Wang et al., 2009) using whole brain, heart, and muscle or C2C12 myoblasts.
Minor modifications to the protocol can be found in Extended Experimental
Procedures. Immunoprecipitation was performed by using a polyclonal
antibody that does not exhibit cross-reactivity with Mbnl2 (T. Cooper, personal
communication).
Cellular Fractionation for C2C12 Mouse Myoblasts
Extraction of cellular compartments from C2C12 monolayers was performed
by using cytosolic lysis buffer with 0.015% digitonin, membrane lysis buffer
with 0.5% Triton X-100, and cytoskeletal lysis buffer with 1 M NaCl. Cells
were washed with PBS between each extraction. Protein and RNA from each
compartment were prepared by heating in SDS page buffer or by guanidinium
isothiocyanate, phenol/chloroform, and RNeasy mini columns, respectively.
0.15 units/ml aprotinin, 20 mM leupeptin, and 40 units/ml RNase-Out
[Invitrogen]), homogenized by Dounce, and spun for 10 min at 1,000 g at
4 C to clear nuclear and cell debris. The lysate was spun to obtain the cytosolic
fraction as the supernatant. The remaining pellet was resuspended in hypotonic buffer containing 1% Triton X-100, homogenized, and spun again to
obtain a membrane fraction as the supernatant and an insoluble fraction as
the pellet. RNA was extracted by using Trizol (Invitrogen).
Ribosome Footprinting
Ribosome footprint profiling of C2C12 mouse myoblasts was performed as
previously described (Ingolia et al., 2011), with several modifications. Instead
of isolating ribosomes from polysome gradients, ribosomes were isolated by
pelleting through a sucrose cushion. Further details can be found in Extended
Experimental Procedures.
Immunoblotting
Bis-tris SDS-PAGE gels (Invitrogen) were used to separate proteins by molecular weight, and protein was transferred to nitrocellulose by using the iBlot
transfer apparatus (Invitrogen). Nitrocellulose blots were blocked in 5% milkPBS with 0.1% Tween-20. Mouse antibodies against Mbnl1 and Mbnl2,
MB1a and MB2a, respectively, were kind gifts from the Wolffson Center for
Inherited Neuromuscular Disease and were used at 1:300 dilutions. Rabbit
Upf1 antibody (Bethyl Laboratories) was used at 1:10 K. Antibodies against
Hsp90 (mouse), Calnexin (rabbit), and Tropomyosin I (mouse) were used at
dilutions of 1:1K. SuperSignal West Femto ECL reagent (ThermoScientific)
was used to detect activity of horseradish peroxidase (HRP)-conjugated
anti-mouse and anti-rabbit secondary antibodies.
Cell Culture, Cloning, and Luciferase Assays
C2C12 mouse myoblasts were cultured in high-glucose Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with 20% fetal bovine serum (FBS)
and penicillin/streptomycin at 37 C and 5% CO2. Knockdowns of Mbnl1 and
Mbnl2 in mouse myoblasts were performed via polybrene-assisted infection
of lentivirus derived from the pLKO.1 vector, followed by puromycin selection
starting 24 hr after infection. RNA and/or protein were isolated from C2C12
mouse myoblasts 7 to 10 days after infection.
Fly S2R+ cells were cultured in 13 Schneider’s Drosophila media (Lonza)
supplemented with 10% FBS, 50 units/ml penicillin, and 50 mg/ml streptomycin at room temperature. Double-stranded RNA (dsRNA) synthesis and
knockdown in S2R+ cells were performed as described previously (Clemens
et al., 2000). DsRNAs were used to knock down cells on days 1, 3, and 4,
and cells were harvested on day 5.
The Infusion system (Clontech) was used to clone fragments of Bgn and Fn1
into the 30 of Gaussia luciferase (pCMV-Gluc, New England Biolabs N8081).
The Fn1 fragment contained the last intron of Fn1, in addition to the 30 UTR.
Liposome mixtures of 400 ng plasmid encoding Gaussia luciferase, 100 ng
plasmid encoding Cypridina luciferase, and either 100 ng of a control
(pLKO.1) plasmid, 100 ng of a plasmid containing 960 CUG repeats (DT960),
or 100 ng DT960 plus 100 ng of a plasmid encoding the Mbnl1 coding
sequence were prepared by using Trans-IT (Mirus). The liposome mixtures
were added to C2C12 cells in 12-well plates, and luciferase assays were
performed 24 hr later. Luciferase assays (Targeting Systems) were used to
assay Gaussia and Cypridina activity. Significance between means was
computed by using the Mann-Whitney U test.
Computational Analysis of RNA-Seq, CLIP-Seq, and Ribosome
Footprinting Data
RNA-seq, CLIP-seq, and ribosome footprinting data have been submitted to
GEO (GSE39911). Computational and statistical methods used to analyze
short read data are described in Extended Experimental Procedures.
ACCESSION NUMBERS
Cellular Fractionation for Fly S2R+ Cells
S2R+ cells were fractionated by using hypotonic lysis and ultracentrifugation.
Cells were lysed in hypotonic buffer (20 mM Tris HCl [pH 7.5], 20 mM NaCl,
5 mM EGTA, 1 mM EDTA, 1 mM dithiothreitol [DTT], 1 mM PMSF,
722 Cell 150, 710–724, August 17, 2012 ª2012 Elsevier Inc.
The GEO accession number for the RNA-seq, CLIP-seq, and ribosome footprinting data is GSE39911. Additional information can be found at http://
genes.mit.edu/burgelab/Supplementary/.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Extended Experimental Procedures,
seven figures, and five tables and can be found with this article online at
http://dx.doi.org/10.1016/j.cell.2012.06.041.
ACKNOWLEDGMENTS
We thank Tom Cooper for the anti-Mbnl1 antibody used to perform CLIP. We
thank Andy Berglund and David Brook for insightful conversations about
muscleblinds and Phil Sharp and Bob Brown Jr. for their comments and
suggestions to perform cellular fractionation experiments. We thank Michael
Zuker for suggestions on depleting ribosomal RNAs during ribosome footprint
library preparation. We thank the MIT Biomicro Center and the IRCM molecular
biology core facility for assistance with deep sequencing and the Koch Institute Swanson Biotechnology Center for microscopy assistance. This work
was supported by a Poitras Fellowship (E.T.W.), by equipment grant
0821391 from the National Science Foundation, and by grants from the
National Institutes of Health to C.B.B. E.T.W. performed CLIP-seq, cellular
fractionation of mouse cells, ribosome footprint profiling, and luciferase assays
and also analyzed RNA-seq, CLIP-seq, and footprint data. N.A.L.C. performed
cellular fractionation of fly cells. S.J., M.B., and S.R. isolated RNA from mouse
tissues and performed splicing RT-PCR assays. T.T.W. performed lentiviral
knockdown of Mbnls in mouse cells. D.J.T. and S.L. prepared RNA-seq
libraries. N.C., E.L., S.R., D.E.H., and G.P.S. contributed to study design.
E.T.W. and C.B.B. designed the study and wrote the paper with contributions
from E.L. and N.C.
Received: February 17, 2012
Revised: April 30, 2012
Accepted: June 20, 2012
Published: August 16, 2012
REFERENCES
Adereth, Y., Dammai, V., Kose, N., Li, R., and Hsu, T. (2005). RNA-dependent
integrin alpha3 protein localization regulated by the Muscleblind-like protein
MLP1. Nat. Cell Biol. 7, 1240–1247.
Amenta, A.R., Creely, H.E., Mercado, M.T., Hagiwara, H., McKechnie, B.A.,
et al. (2012). Biglycan is an extracellular MuSK binding protein important for
synapse stability. J. Neurosci. 32, 2324–2334.
Andreassi, C., and Riccio, A. (2009). To localize or not to localize: mRNA fate is
in 30 UTR ends. Trends Cell Biol. 19, 465–474.
Artero, R., Prokop, A., Paricio, N., Begemann, G., Pueyo, I., Mlodzik, M.,
Perez-Alonso, M., and Baylies, M.K. (1998). The muscleblind gene participates
in the organization of Z-bands and epidermal attachments of Drosophila
muscles and is regulated by Dmef2. Dev. Biol. 195, 131–143.
Ashizawa, T., Dubel, J.R., and Harati, Y. (1993). Somatic instability of CTG
repeat in myotonic dystrophy. Neurology 43, 2674–2678.
Begemann, G., Paricio, N., Artero, R., Kiss, I., Pérez-Alonso, M., and Mlodzik,
M. (1997). muscleblind, a gene required for photoreceptor differentiation in
Drosophila, encodes novel nuclear Cys3His-type zinc-finger-containing
proteins. Development 124, 4321–4331.
Blobel, G., and Dobberstein, B. (1975b). Transfer of proteins across
membranes. II. Reconstitution of functional rough microsomes from heterologous components. J. Cell Biol. 67, 852–862.
Cenik, C., Chua, H.N., Zhang, H., Tarnawsky, S.P., Akef, A., Derti, A., Tasan,
M., Moore, M.J., Palazzo, A.F., and Roth, F.P. (2011). Genome analysis reveals
interplay between 50 UTR introns and nuclear mRNA export for secretory and
mitochondrial genes. PLoS Genet. 7, e1001366.
Clemens, J.C., Worby, C.A., Simonson-Leff, N., Muda, M., Maehama, T.,
Hemmings, B.A., and Dixon, J.E. (2000). Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways.
Proc. Natl. Acad. Sci. USA 97, 6499–6503.
Cooper, T.A., Wan, L., and Dreyfuss, G. (2009). RNA and disease. Cell 136,
777–793.
Csermely, P., Schnaider, T., Soti, C., Prohászka, Z., and Nardai, G. (1998). The
90-kDa molecular chaperone family: structure, function, and clinical applications. A comprehensive review. Pharmacol. Ther. 79, 129–168.
Du, H., Cline, M.S., Osborne, R.J., Tuttle, D.L., Clark, T.A., Donohue, J.P., Hall,
M.P., Shiue, L., Swanson, M.S., Thornton, C.A., and Ares, M., Jr. (2010).
Aberrant alternative splicing and extracellular matrix gene expression in
mouse models of myotonic dystrophy. Nat. Struct. Mol. Biol. 17, 187–193.
Fardaei, M., Rogers, M.T., Thorpe, H.M., Larkin, K., Hamshere, M.G., Harper,
P.S., and Brook, J.D. (2002). Three proteins, MBNL, MBLL and MBXL,
co-localize in vivo with nuclear foci of expanded-repeat transcripts in DM1
and DM2 cells. Hum. Mol. Genet. 11, 805–814.
Ffrench-Constant, C., and Hynes, R.O. (1989). Alternative splicing of fibronectin is temporally and spatially regulated in the chicken embryo. Development 106, 375–388.
Ffrench-Constant, C., Van de Water, L., Dvorak, H.F., and Hynes, R.O. (1989).
Reappearance of an embryonic pattern of fibronectin splicing during wound
healing in the adult rat. J. Cell Biol. 109, 903–914.
Goers, E.S., Purcell, J., Voelker, R.B., Gates, D.P., and Berglund, J.A. (2010).
MBNL1 binds GC motifs embedded in pyrimidines to regulate alternative
splicing. Nucleic Acids Res. 38, 2467–2484.
Gunning, P.W., Schevzov, G., Kee, A.J., and Hardeman, E.C. (2005). Tropomyosin isoforms: divining rods for actin cytoskeleton function. Trends Cell Biol.
15, 333–341.
Harper, P.S. (1979). Myotonic Dystrophy (Philadelphia: W.B. Saunders Co.).
Hesketh, J. (1994). Translation and the cytoskeleton: a mechanism for targeted
protein synthesis. Mol. Biol. Rep. 19, 233–243.
Hino, S.-I., Kondo, S., Sekiya, H., Saito, A., Kanemoto, S., Murakami, T.,
Chihara, K., Aoki, Y., Nakamori, M., Takahashi, M.P., and Imaizumi, K.
(2007). Molecular mechanisms responsible for aberrant splicing of SERCA1
in myotonic dystrophy type 1. Hum. Mol. Genet. 16, 2834–2843.
Ho, T.H., Charlet-B, N., Poulos, M.G., Singh, G., Swanson, M.S., and
Cooper, T.A. (2004). Muscleblind proteins regulate alternative splicing.
EMBO J. 23, 3103–3112.
Holt, I., Jacquemin, V., Fardaei, M., Sewry, C.A., Butler-Browne, G.S., Furling,
D., Brook, J.D., and Morris, G.E. (2009). Muscleblind-like proteins: similarities
and differences in normal and myotonic dystrophy muscle. Am. J. Pathol. 174,
216–227.
Horne-Badovinac, S., and Bilder, D. (2008). Dynein regulates epithelial polarity
and the apical localization of stardust A mRNA. PLoS Genet. 4, e8.
Bergeron, J.J., Brenner, M.B., Thomas, D.Y., and Williams, D.B. (1994).
Calnexin: a membrane-bound chaperone of the endoplasmic reticulum.
Trends Biochem. Sci. 19, 124–128.
Ingolia, N.T., Lareau, L.F., and Weissman, J.S. (2011). Ribosome profiling of
mouse embryonic stem cells reveals the complexity and dynamics of mammalian proteomes. Cell 147, 789–802.
Besse, F., and Ephrussi, A. (2008). Translational control of localized mRNAs:
restricting protein synthesis in space and time. Nat. Rev. Mol. Cell Biol. 9,
971–980.
Jagannathan, S., Nwosu, C., and Nicchitta, C.V. (2011). Analyzing mRNA
localization to the endoplasmic reticulum via cell fractionation. Methods Mol.
Biol. 714, 301–321.
Blobel, G., and Dobberstein, B. (1975a). Transfer of proteins across
membranes. I. Presence of proteolytically processed and unprocessed
nascent immunoglobulin light chains on membrane-bound ribosomes of
murine myeloma. J. Cell Biol. 67, 835–851.
Kanadia, R.N., Johnstone, K.A., Mankodi, A., Lungu, C., Thornton, C.A.,
Esson, D., Timmers, A.M., Hauswirth, W.W., and Swanson, M.S. (2003a). A
muscleblind knockout model for myotonic dystrophy. Science 302, 1978–
1980.
Cell 150, 710–724, August 17, 2012 ª2012 Elsevier Inc. 723
Kanadia, R.N., Urbinati, C.R., Crusselle, V.J., Luo, D., Lee, Y.-J., Harrison, J.K.,
Oh, S.P., and Swanson, M.S. (2003b). Developmental expression of mouse
muscleblind genes Mbnl1, Mbnl2 and Mbnl3. Gene Expr. Patterns 3, 459–462.
Pascual, M., Vicente, M., Monferrer, L., and Artero, R. (2006). The Muscleblind
family of proteins: an emerging class of regulators of developmentally programmed alternative splicing. Differentiation 74, 65–80.
Katz, Y., Wang, E.T., Airoldi, E.M., and Burge, C.B. (2010). Analysis and design
of RNA sequencing experiments for identifying isoform regulation. Nat.
Methods 7, 1009–1015.
Pizon, V., Iakovenko, A., Van Der Ven, P.F., Kelly, R., Fatu, C., Fürst, D.O.,
Karsenti, E., and Gautel, M. (2002). Transient association of titin and myosin
with microtubules in nascent myofibrils directed by the MURF2 RING-finger
protein. J. Cell Sci. 115, 4469–4482.
Kishore, S., Jaskiewicz, L., Burger, L., Hausser, J., Khorshid, M., and Zavolan,
M. (2011). A quantitative analysis of CLIP methods for identifying binding sites
of RNA-binding proteins. Nat. Methods 8, 559–564.
Kislauskis, E.H., Zhu, X., and Singer, R.H. (1997). beta-Actin messenger
RNA localization and protein synthesis augment cell motility. J. Cell Biol.
136, 1263–1270.
König, J., Zarnack, K., Rot, G., Curk, T., Kayikci, M., Zupan, B., Turner, D.J.,
Luscombe, N.M., and Ule, J. (2010). iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution. Nat. Struct. Mol. Biol. 17,
909–915.
Lécuyer, E., Yoshida, H., Parthasarathy, N., Alm, C., Babak, T., Cerovina, T.,
Hughes, T.R., Tomancak, P., and Krause, H.M. (2007). Global analysis of
mRNA localization reveals a prominent role in organizing cellular architecture
and function. Cell 131, 174–187.
Lécuyer, E., Yoshida, H., and Krause, H.M. (2009). Global implications of
mRNA localization pathways in cellular organization. Curr. Opin. Cell Biol.
21, 409–415.
Lin, X., Miller, J.W., Mankodi, A., Kanadia, R.N., Yuan, Y., Moxley, R.T.,
Swanson, M.S., and Thornton, C.A. (2006). Failure of MBNL1-dependent
post-natal splicing transitions in myotonic dystrophy. Hum. Mol. Genet. 15,
2087–2097.
Marteyn, A., Maury, Y., Gauthier, M.M., Lecuyer, C., Vernet, R., Denis, J.A.,
Pietu, G., Peschanski, M., and Martinat, C. (2011). Mutant human embryonic
stem cells reveal neurite and synapse formation defects in type 1 myotonic
dystrophy. Cell Stem Cell 8, 434–444.
Martin, K.C., and Zukin, R.S. (2006). RNA trafficking and local protein synthesis
in dendrites: an overview. J. Neurosci. 26, 7131–7134.
Martin, K.C., and Ephrussi, A. (2009). mRNA localization: gene expression in
the spatial dimension. Cell 136, 719–730.
Masuda, A., Andersen, H.S., Doktor, T.K., Okamoto, T., Ito, M., Andresen,
B.S., and Ohno, K. (2012). CUGBP1 and MBNL1 preferentially bind to
30 UTRs and facilitate mRNA decay. Sci. Rep. Published online January 4,
2012. http://dx.doi.org/10.1038/srep00209.
Miller, J.W., Urbinati, C.R., Teng-Umnuay, P., Stenberg, M.G., Byrne, B.J.,
Thornton, C.A., and Swanson, M.S. (2000). Recruitment of human muscleblind
proteins to (CUG)(n) expansions associated with myotonic dystrophy. EMBO
J. 19, 4439–4448.
Moore, M.J., and Proudfoot, N.J. (2009). Pre-mRNA processing reaches back
to transcription and ahead to translation. Cell 136, 688–700.
Osborne, R.J., Lin, X., Welle, S., Sobczak, K., O’Rourke, J.R., Swanson, M.S.,
and Thornton, C.A. (2009). Transcriptional and post-transcriptional impact of
toxic RNA in myotonic dystrophy. Hum. Mol. Genet. 18, 1471–1481.
Pyhtila, B., Zheng, T., Lager, P.J., Keene, J.D., Reedy, M.C., and Nicchitta,
C.V. (2008). Signal sequence- and translation-independent mRNA localization
to the endoplasmic reticulum. RNA 14, 445–453.
Ramsköld, D., Wang, E.T., Burge, C.B., and Sandberg, R. (2009). An abundance of ubiquitously expressed genes revealed by tissue transcriptome
sequence data. PLoS Comput. Biol. 5, e1000598.
Sen, S., Talukdar, I., Liu, Y., Tam, J., Reddy, S., and Webster, N.J. (2010).
Muscleblind-like 1 (Mbnl1) promotes insulin receptor exon 11 inclusion via
binding to a downstream evolutionarily conserved intronic enhancer. J. Biol.
Chem. 285, 25426–25437.
Spilker, K.A., Wang, G.J., Tugizova, M.S., and Shen, K. (2012). Caenorhabditis
elegans Muscleblind homolog mbl-1 functions in neurons to regulate synapse
formation. Neural Dev. 7, 7.
Squillace, R.M., Chenault, D.M., and Wang, E.H. (2002). Inhibition of muscle
differentiation by the novel muscleblind-related protein CHCR. Dev. Biol.
250, 218–230.
Sundell, C.L., and Singer, R.H. (1991). Requirement of microfilaments in
sorting of actin messenger RNA. Science 253, 1275–1277.
Teplova, M., and Patel, D.J. (2008). Structural insights into RNA recognition by
the alternative-splicing regulator muscleblind-like MBNL1. Nat. Struct. Mol.
Biol. 15, 1343–1351.
Ule, J., Jensen, K., Mele, A., and Darnell, R.B. (2005). CLIP: a method for identifying protein-RNA interaction sites in living cells. Methods 37, 376–386.
Vedeler, A., Pryme, I.F., and Hesketh, J.E. (1991). The characterization of free,
cytoskeletal and membrane-bound polysomes in Krebs II ascites and 3T3
cells. Mol. Cell. Biochem. 100, 183–193.
Wang, Z., Xiao, X., Van Nostrand, E., and Burge, C.B. (2006). General and
specific functions of exonic splicing silencers in splicing control. Mol. Cell
23, 61–70.
Wang, E.T., Sandberg, R., Luo, S., Khrebtukova, I., Zhang, L., Mayr, C.,
Kingsmore, S.F., Schroth, G.P., and Burge, C.B. (2008). Alternative isoform
regulation in human tissue transcriptomes. Nature 456, 470–476.
Wang, Z., Tollervey, J., Briese, M., Turner, D., and Ule, J. (2009). CLIP:
construction of cDNA libraries for high-throughput sequencing from RNAs
cross-linked to proteins in vivo. Methods 48, 287–293.
Warf, M.B., and Berglund, J.A. (2007). MBNL binds similar RNA structures in
the CUG repeats of myotonic dystrophy and its pre-mRNA substrate cardiac
troponin T. RNA 13, 2238–2251.
Pachter, J.S. (1992). Association of mRNA with the cytoskeletal framework:
its role in the regulation of gene expression. Crit. Rev. Eukaryot. Gene Expr.
2, 1–18.
Yeo, G.W., Coufal, N.G., Liang, T.Y., Peng, G.E., Fu, X.-D., and Gage, F.H.
(2009). An RNA code for the FOX2 splicing regulator revealed by mapping
RNA-protein interactions in stem cells. Nat. Struct. Mol. Biol. 16, 130–137.
Palazzo, A.F., Springer, M., Shibata, Y., Lee, C.-S., Dias, A.P., and Rapoport,
T.A. (2007). The signal sequence coding region promotes nuclear export of
mRNA. PLoS Biol. 5, e322.
Zhang, C., Frias, M.A., Mele, A., Ruggiu, M., Eom, T., Marney, C.B., Wang, H.,
Licatalosi, D.D., Fak, J.J., and Darnell, R.B. (2010). Integrative modeling
defines the Nova splicing-regulatory network and its combinatorial controls.
Science 329, 439–443.
Panaite, P.A., Gantelet, E., Kraftsik, R., Gourdon, G., Kuntzer, T., and BarakatWalter, I. (2008). Myotonic dystrophy transgenic mice exhibit pathologic
abnormalities in diaphragm neuromuscular junctions and phrenic nerves.
J. Neuropathol. Exp. Neurol. 67, 763–772.
724 Cell 150, 710–724, August 17, 2012 ª2012 Elsevier Inc.
Zhao, J., Hyman, L., and Moore, C. (1999). Formation of mRNA 30 ends in
eukaryotes: mechanism, regulation, and interrelationships with other steps
in mRNA synthesis. Microbiol. Mol. Biol. Rev. 63, 405–445.