Failure of MBNL1-dependent post-natal splicing transitions in myotonic dystrophy

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Failure of MBNL1-dependent postnatal splicing transitions in myotonic dystrophy

Xiaoyan Lin1, Jill W. Miller2, Ami Mankodi2‡, Rahul N. Kanadia3, Yuan Yuan3,

Richard T. Moxley2, Maurice S. Swanson3, Charles A. Thornton2*

Departments of 1Neuroscience and 2Neurology, University of Rochester Medical Center,

Rochester, New York, 14642, and 3Department of Molecular Genetics and Microbiology and the

Genetics Institute, University of Florida, College of Medicine, Gainesville, FL 32610. ‡Current

address, Department of Neurology, Johns Hopkins University School of Medicine, Baltimore,

MD

*Corresponding Author:

Charles A. Thornton, M.D.

Department of Neurology, Box 673

University of Rochester Medical Center

601 Elmwood Avenue

Rochester, NY 14642

Tel # 585 275 2542

Fax # 585 273 1255

Email: [email protected]

© The Author 2006. Published by Oxford University Press. All rights reserved

HMG Advance Access published May 22, 2006 by guest on February 19, 2016

http://hmg.oxfordjournals.org/

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Abstract

In myotonic dystrophy (DM), expression of RNA containing expanded CUG or CCUG repeats

leads to misregulated alternative splicing of pre-mRNA. The repeat-bearing transcripts

accumulate in nuclear foci, together with proteins in the muscleblind family, MBNL1 and

MBNL2. In transgenic mice that express expanded CUG repeats, we show that the splicing

defect selectively targets a group of exons that share a common temporal pattern of

developmental regulation. These exons undergo a synchronized splicing switch between

postnatal day 2 and 20 in wild-type mice. During this postnatal interval, MBNL1 protein

translocates from a predominantly cytoplasmic to nuclear distribution. In the absence of

MBNL1, these physiological splicing transitions do not occur. The splicing defect induced by

expanded CUG repeats in mature muscle fibers is closely reproduced by deficiency of MBNL1

but not by deficiency of MBNL2. A parallel situation exists in human DM type 1 and type 2.

MBNL1 is depleted from the muscle nucleoplasm due to sequestration in nuclear foci, and the

associated splicing defects are remarkably similar to those observed in MBNL1 knockout mice.

These results indicate that MBNL1 participates in the postnatal remodeling of skeletal muscle by

controlling a key set of developmentally-regulated splicing switches. Sequestration of MBNL1,

and failure to maintain these splicing transitions, has a pivotal role in the pathogenesis of muscle

disease in DM.

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Introduction

Myotonic dystrophy type 1 (DM1) is the most common degenerative disease of skeletal muscle

in adults. This multi-system disorder is characterized by muscle wasting, myotonia,

degeneration of the cardiac conduction system, cataracts, and neuropsychological dysfunction.

DM1 is caused by expansion of a CTG repeat in the 3' untranslated region (UTR) of the

dystrophia myotonica protein kinase (DMPK) gene (1). A second, less common form of

myotonic dystrophy, DM type 2 (DM2), is caused by expansion of a CCTG repeat in intron 1 of

the zinc finger 9 (ZNF9) gene (2). Both mutations are in non-coding sequences, raising

questions about the mechanism of genetic dominance in DM.

A body of work has accumulated indicating that DMPK mRNA containing an abnormally

expanded CUG repeat has a deleterious effect on muscle fibers. For example, expression of

CUG expansion RNA in transgenic mice reproduces characteristic signs of DM1, either when the

CUG tract is expressed in the natural context of the DMPK 3' UTR (3) or when it is inserted in

the 3' UTR of an unrelated transcript (4). In both types of DM, and in transgenic mouse models,

the mutant RNA forms intranuclear (ribonuclear) foci in muscle fibers (2,4,5).

A core mechanism underlying symptoms of DM1 and DM2 is that expanded poly(CUG) or

(CCUG) RNA interferes with the regulated alternative splicing of certain pre-mRNAs. This

effect was first observed for cardiac troponin T (cTnT) (6). In the case of the insulin receptor

(IR), the predominant splice product expressed in DM1 muscle is the exon 11 skipped (non-

muscle) isoform, which may contribute to the insulin resistance in DM1 muscle fibers (7).



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Misregulated splicing of the muscle-specific chloride ion channel (ClC-1) leads to reduced

chloride conductance and repetitive action potentials (myotonia) in DM muscle fibers (8,9).

CUG binding protein 1 (CUG-BP1) and related RNA-binding proteins in the CELF family are

implicated in the misregulated alternative splicing of DM (6). CUG-BP1 binds to UG-rich

sequences in vitro (10,11), including (CUG)8 (12), but it does not colocalize with ribonuclear

foci in DM1 cells (13-15) (for a contrary view, see reference (19)). The RNA binding motifs in

CUG-BP1 are of the type that recognize single stranded RNA, whereas poly(CUG) is stabilized

in a duplex (hairpin) conformation when the repeat is pathologically expanded (16-18).

Irrespective of whether CUG-BP1 directly interacts with the mutant mRNA, this protein may

have a role in the pathogenesis of splicing abnormalities because it is overexpressed in DM1

muscle cells (7,19,20). The mechanism for this increase has not been determined, but

overexpression of CUG-BP1 in striated muscle has been shown to trigger abnormal splicing of

cTnT and IR similar to that observed in DM1 (6,7).

In contrast, proteins in the muscleblind-like (MBNL) family preferentially recognize CUG or

CCUG repeats when they are pathologically expanded (13,21). MBNL proteins colocalize with

ribonuclear foci in DM1 and DM2 cells (13,22,23), which led to the proposal that symptoms of

DM may result from loss of MBNL activity due to protein sequestration on repeat expansion

RNAs. All three family MBNL members, MBNL1, MBNL2, MBNL3, are able to regulate

alternative splicing of cTnT and IR minigenes (24). In support of a sequestration mechanism,

Mbnl1 knockout mice show myotonia, cataracts, and misregulated splicing of ClC-1 and cTnT

that are similar to DM1 (25).



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One putative mechanism for DM pathogenesis involves increased CUG-BP1 activity, another

involves sequestration of MBNL proteins, but presently it is unclear which mechanism is mainly

responsible for the splicing defect, or whether both must operate in concert. Moreover, as

MBNL proteins are abundant in skeletal muscle, it is uncertain whether expanded poly(CUG)

RNA is actually expressed in muscle fibers at levels that are capable of sequestering these factors.

If DM1 cells achieve such levels of expression, it is unknown which members of the MBNL

family are mainly responsible for the misregulation of splicing. To address these questions, we

have compared the developmental regulation of alternative splicing in skeletal muscle in

transgenic and Mbnl1 knockout models of DM1, and we have derived mice that have reduced

expression of Mbnl2. We found that misregulated splicing in a transgenic model that expresses

expanded poly(CUG) RNA is closely reproduced by deficiency of Mbnl1 but not by deficiency

of Mbnl2. Remarkably, every exon that is misregulated in response to expanded poly(CUG),

among the exons that we examined, shows a similar pattern of developmental regulation. In

WT mice, these exons transition from neonatal to adult splice isoforms within the first 3 weeks

of postnatal life, but in the absence of Mbnl1, these transitions do not occur. In both types of

human DM, MBNL1 is recruited into ribonuclear foci so extensively that free MBNL1 is

depleted from the nucleoplasm, and the splicing defects are strikingly similar to those observed

in Mbnl1 knockout mice. By contrast, splicing defects in mice that express expanded

poly(CUG) are not associated with abnormal accumulation or mislocalization of CUG-BP1.

These results point to a pivotal role for MBNL1 in DM pathogenesis, and indicate that this

splicing factor functions in the postnatal remodeling of skeletal muscle by controlling a key set

of developmentally-regulated splicing switches.



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Results

Expanded poly(CUG) RNA and deficiency of Mbnl1 have equivalent effects on alternative

splicing in mouse skeletal muscle. Alternative splicing of fast troponin T (Tnnt3) is abnormally

regulated in DM1 patients and in transgenic mice that express expanded CUG repeats (25). The

isoform of Tnnt3 expressed in DM1 muscle fibers includes an alternative exon that normally is

included in fetal transcripts. In normal adult muscle, however, this exon is skipped. To further

examine the physiological regulation of this exon during muscle development we examined its

splicing in WT mice from embryonic day 18 onward. The transition from inclusion to exclusion

of the “fetal” exon mainly occurred after birth, between postnatal day 2 and day 16 (Fig. 1A).

In an effort to identify other exons that respond to expanded poly(CUG), and assess their

developmental regulation, we selected 54 exons (from 36 genes) known to be alternatively

spliced in skeletal muscle. For each exon selected, the ratio of alternative splice products was

determined in mouse models of DM1, relative to WT mice of the appropriate background strain.

The models we examined were HSALR transgenic mice that express a human skeletal actin

mRNA containing (CUG)250 in the 3' UTR (4), and Mbnl1∆E3/∆E3 mice homozygous for a targeted

allele of Mbnl1 (25). In the initial RT-PCR splicing assays, eleven exons showed abnormal

regulation in HSALR mice. For example, splice products containing Serca1 exon 22 were

decreased in HSALR mice, whereas inclusion of ZASP exon 11 and titin m-line region exon 5

were increased (Fig. 1B). Re-examination of these 11 exons in an independent group confirmed

the abnormality of splicing regulation (n = 6 WT and HSALR mice per group, p < 0.0001 for each

exon, Supplemental Fig. 1, Table 1). The direction and magnitude of the effect varied among

genes. The most extreme example involved Serca1, encoding the calcium re-uptake pump of



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the sarcoplasmic reticulum. The fraction of Serca1 mRNA skipping exon 22 increased from 3 ±

0.7% in WT to 78 ± 4% in HSALR mice. Abnormal splicing of Serca1 in DM1 has recently been

reported (26). Interestingly, the alternative splicing of Mbnl1 was also misregulated in HSALR

mice, showing an increased frequency of exon 7 inclusion (Fig. 1B). Ten of the 11 misregulated

exons were correctly spliced in disease controls having generalized myotonia (ClC-1 null mice)

or dystrophin deficiency (mdx mice), indicating that these abnormalities did not result from non-

specific effects of repetitive action potentials or dystrophic muscle (Fig. 1C and data not shown,

splicing of m-Titin was abnormally regulated in ClC-1 null mice).

The pattern of misregulated alternative splicing in HSALR transgenic mice was remarkably

concordant with Mbnl1∆E3/∆E3 mice across all 54 exons that we examined (Table 1 and

Supplemental Table 1). Furthermore, among exons that were abnormally regulated in DM1

mouse models, the pattern of splicing in adult HSALR or Mbnl1∆E3/∆E3 mice invariably resembled

that seen in WT mice at post-natal day 2 (Fig. 1B, C). We carried out a more detailed

examination of the postnatal splicing switch for 7 of the most-affected exons (Serca1, ZASP, m-

Titin, z-Titin, Nrap, Capn3, Mbnl1). In WT mice, each of these exons showed a splicing

transition between postnatal day 2 and day 20 (Fig. 2 and data not shown). However,

expression of expanded poly(CUG) or loss of Mbnl1 resulted in complete failure of these

splicing transitions. Integrin beta 1 and CapZb also showed a switch in alternative splicing

during this postnatal interval, however, these transitions were properly executed in HSALR and

Mbnl1∆E3/∆E3 mice (Fig. 2, lower panels). Thus, expanded poly(CUG) RNA has not produced a

global defect of developmentally-regulated alternative splicing, instead it has selectively targeted

exons that show Mbnl1-dependent splicing transitions. Furthermore, the postnatal regulation of



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these exons followed the Tnnt3 fetal exon time course, suggesting that all of these splicing

transitions were coordinated by a common triggering event.

Exons misregulated in HSALR transgenic mice are not affected by disruption of Mbnl2. Mbnl1

and Mbnl2 are both expressed in skeletal muscle, and, when overexpressed, either factor is able

to regulate the alternative splicing of IR and cTnT minigenes (24). As a first step to investigate

the respective contributions of Mbnl1 and Mbnl2 in skeletal muscle, we examined the effects of

Mbnl2 deficiency on alternative splicing in adult muscle fibers. Mbnl2 gene trap (GT) mice

were derived from ES cells that have integrated a retroviral gene trap vector in Mbnl2 intron 4

(Fig. 3A). In mice homozygous for the GT allele (Mbnl2GT4/GT4 mice), Mbnl2 mRNA was

reduced by >90% (Fig. 3B), yet muscle histology was normal at 4 months of age and there was

no myotonia. Each of the exons misregulated in HSALR and Mbnl1∆E3/∆E3 mice showed normal

regulation in Mbnl2GT4/GT4 adult mice (Fig. 3C and data not shown, with the exception that

splicing of Mbnl2 itself could not be assessed due to insertion of the gene trap vector).

Mis-splicing in DM1 and DM2 skeletal muscle is similar to mouse models of DM1. To determine

if transgenic and Mbnl1 knockout mouse models predict misregulated splicing in human DM, we

examined splicing of the equivalent exons in skeletal muscle from patients with DM1 or DM2, as

compared to healthy subjects. Each exon misregulated in HSALR mice showed a similar

alteration in both types of DM (n = 3 per group, representative gels are shown in Fig. 4,

summary data are listed in Table 1) with the exception that splicing of GFAT1 exon 10 was not

consistently increased in DM2. Conversely, among the exons that were normally regulated in

HSALR and Mbnl1∆E3/∆E3 mouse models, the 27 that we examined in human DM also were



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correctly regulated. Thus, both of these mouse models accurately reproduce the splicing defect

in human DM1 and DM2 skeletal muscle.

Mbnl1 is sequestered in DM1 and DM2 myonuclei. While MBNL1 protein has been localized

to nuclear foci in DM1 and DM2 muscle (22), the extent of MBNL1 sequestration (i.e., depletion

from the nucleoplasm) was unclear because antibodies used in the previous studies did not

clearly show MBNL1 in normal myonuclei. To re-examine this question, we raised a

polyclonal antibody against a C-terminal peptide that is conserved in human and murine Mbnl1.

Specificity of the antibody was demonstrated by lack of immunoreactivity on immunoblots or

tissue sections from Mbnl1∆E3/∆E3 muscle (see Fig. 6A and 6B). In sections of normal human

muscle, MBNL1 immunofluorescence (IF) was diffusely distributed in myonuclei. In sections

of DM1 and DM2 muscle, MBNL1 was heavily recruited into RNA foci (Supplemental Fig. 2A)

and the mean intensity of MBNL1 IF in nucleoplasm, exclusive of nuclear foci, was reduced by

≥78% (Fig. 4B). Interestingly, the amount of MBNL1 in nuclear foci was greater in DM2 than

in DM1 (Fig. 4B).

Cumulatively, these results suggest that loss of MBNL1 activity due to sequestration on mutant

DMPK and ZNF9 transcripts is the primary determinant of misregulated splicing in DM1 and

DM2. However, CUG-BP1 also plays a role in the developmental regulation of alternative

splicing in striated muscle (27,28), and increased levels of this protein have been linked to

abnormal splicing regulation in DM1 (6,7). Next, we examined the developmental regulation of

CUG-BP1 expression in skeletal muscle and determined if the splicing defect in HSALR mice and

DM2 patients is also associated with increased levels of CUG-BP1 protein.



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CUG-BP1 protein levels are not elevated in HSALR or DM2 skeletal muscle. In WT skeletal

muscle, levels of CUG-BP1 decreased sharply between P2 and P20 (Fig. 5A). Postnatal

downregulation of CUG-BP1 occurred to a similar extent in HSALR transgenic mice. In HSALR

transgenic mice, marked accumulation of expanded poly(CUG), and induction of splicing

abnormalities characteristic of DM1, was not associated with increased steady state levels of

CUG-BP1 protein (Fig. 5B). CUG-BP1 did not colocalize with ribonuclear foci in HSALR

muscle, as visualized by immunofluorescence of the endogenous protein or by expression of

GFP-tagged CUG-BP1 in vivo by electroporation (Fig. 5D and 5E). By comparison, GFP-

tagged Mbnl1 colocalized with nuclear RNA foci under these conditions (Fig. 5F). Interestingly,

while abnormalities of alternative splicing in quadriceps muscle biopsy samples tend to be more

pronounced in DM2 than in DM1 (8), we did not find any consistent increase in the levels of

CUG-BP1 in DM2 muscle tissue compared to healthy controls (Fig. 5C). These observations

argue that misregulated splicing in HSALR transgenic mice and human DM2 does not require

elevated levels or mislocalization of CUG-BP1 protein.

Mbnl1 relocalizes to the nucleus during postnatal development. To investigate the mechanism

for the postnatal splicing transitions pertinent to DM, we also examined the developmental

regulation of Mbnl1. Levels of Mbnl1 protein, as determined by immunoblot of whole muscle

lysates, declined in the interval between P2 and P20 (Fig. 6A). A similar decline occurred in

HSALR mice, and expression of poly(CUG) RNA did not result in abnormal accumulation of

Mbnl1 protein in mature muscle fibers (Fig. 6A, right panel). However, the postnatal decline

occurred during a period of rapid muscle fiber hypertrophy, and consequently these results may



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reflect increasing dilution of nuclear contents by proteins in the muscle cytoplasm. In view of

the difficulty in obtaining nuclear fractions from multinucleate muscle fibers (29), we examined

the distribution of Mbnl1 by IF on muscle sections. Surprisingly, the postnatal splicing

transitions coincided with translocation of Mbnl1 from a predominantly cytoplasmic location at

P2 to a predominantly nuclear location at P20 (Fig. 6B). By comparison, neither hnRNP I nor

hnRNP H showed a parallel shift in cellular distribution during this interval (Suppl. Fig. 2B and

2C). However, Mbnl1 is not entirely excluded from the nucleus in neonatal muscle, as indicated

by its presence in nuclear foci of HSALR mice throughout the postnatal interval (Fig. 6C). This

indicates that CUG expansion RNA is expressed, and available to interact with Mbnl1 in the

nucleus, as early as P2 in HSALR muscle.

Mbnl1 pre-mRNA is alternatively spliced at exons 3, 5, 7, and 9, raising the possibility that

postnatal splicing transitions result from a switch to production of Mbnl1 splice products that

have a different cellular distribution or activity. For example, exclusion of exon 3 (E3)

eliminates one of four zinc finger domains and alters the RNA binding activity of Mbnl1 (21,25).

However, the fraction E3– splice products was similar in neonatal and adult WT and HSALR

mice, and the 30 kD protein encoded by this isoform was not detected in skeletal muscle by

immunoblot or immunoprecipitation (not shown). Exon 5 was included in all Mbnl1 transcripts

in neonatal and adult WT or HSALR muscle (not shown). Exon 9 (E9) was alternatively spliced

in muscle, but the ratio of E9+:E9– splice products was similar in neonatal and adult WT or

HSALR muscle (not shown).



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In contrast, inclusion of exon 7 (E7) was increased in HSALR and Mbnl1∆E3/∆E3 mice, and

alternative splicing of this exon was developmentally regulated in WT mice (Fig. 2). The

function of the 18 amino acid domain encoded by this small exon is unknown. However, E7

and 156 nt of the flanking intronic sequence are ultraconserved regions in vertebrate genomes

(30), suggesting that regulation of E7 splicing is functionally important. Also, the analogous

exon of Mbnl2 shows similar dysregulation in DM (Suppl. Fig. 1). E7– and E7+ Mbnl1 both

retain their splicing and RNA binding activities (refs. (21) and (24), and M. Swanson,

unpublished observations). We postulated that postnatal splicing transitions may result from

preferentially localization of E7+ Mbnl1 to the muscle cytoplasm. We examined this possibility

by using electroporation to express different GFP-tagged Mnbl1 isoforms in skeletal muscle in

vivo. Against our prediction, however, we found that nuclear localization was stronger for E7+

than E7– Mbnl1 (Fig. 6D). Results in transfected COS cells were similar, with or without the

inclusion of exon 9 (Supplemental Fig. 2D). Thus, a functional depletion of MBNL1 has

occurred in DM1 nucleoplasm, despite a shift to production of E7+ isoforms that preferentially

localize to the nucleus. Taken together, these results raise the possibility that the postnatal

splicing transitions are triggered, at least in part, by translocation of Mbnl1 from the cytoplasm

to nucleus. However, alternative splicing of Mbnl is not the main factor driving this

redistribution.



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Discussion

The disease process in myotonic dystrophy involves trans-interference by repeat-containing

transcripts with regulated alternative splicing of select pre-mRNAs (6). Current evidence

indicates that misregulated splicing results from abnormal activity of poly(CUG)/poly(CCUG)

binding proteins. The first RNA binding protein shown to interact with poly(CUG) was CUG-

BP1 (12), but the link between this protein and DM-related splicing abnormalities remains

unclear. Despite the accumulation of expanded poly(CUG) to high levels in HSALR muscle, we

did not see colocalization of endogenous or GFP-tagged CUG-BP1 with RNA foci (Fig. 5),

consistent with previous observations that this protein is not sequestered in ribonuclear foci in

DM1 cells (13,14). One potential explanation for the non-colocalization of CUG-BP1 is that its

affinity for expanded poly(CUG) is lower than that of MBNL1 (17), so that colocalization is

difficult to observe due to occupancy of binding sites by Mbnl1. However, this cannot be the

sole explanation, because CUG-BP1 also does not colocalize when expanded poly(CUG) RNA is

expressed in Mbnl1 knockout mice (X Lin, C Thornton, unpublished). Furthermore,

misregulated alternative splicing has occurred in HSALR transgenic mice and DM2 patients

without any consistent effect on the levels of CUG-BP1 protein in skeletal muscle (Fig. 5).

These results suggest that perturbations of CUG-BP1 distribution or amount are not required to

produce the defect of splicing regulation in DM. These results are consistent with findings that

siRNA-mediated depletion of CUG-BP1 failed to restore normal patterns of alternative splicing

in DM1 myoblasts (20).

By contrast, we found that splicing derangements in Mbnl1 deficient muscle are remarkably

concordant with those induced by CUG- or CCUG-repeat expansion RNA, whether in human



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DM1, DM2, or the HSALR transgenic mouse model. These observations, when coupled with

evidence for depletion of MBNL1 from the nucleoplasm in both types of human DM (Fig. 4),

argue that sequestration of a single splicing factor, MBNL1, is sufficient to explain misregulated

splicing in adult DM skeletal muscle. A similar conclusion was reached in studies of siRNA-

mediated knockdown of MBNL1, MBNL2, or CUG-BP1, individually or in combination, in

myoblasts (20). In the previous studies, knockdown of either MBNL1 or MBNL2 repressed

splicing of IR exon 11 in WT myoblasts, suggesting that IR splicing in myoblasts was

determined by combined action of both MBNL proteins (MBNL3 was not examined because its

expression is mainly restricted to placenta). However, in mature skeletal muscle, we found that

the full extent of poly(CUG)-induced splicing abnormalities, in terms of magnitude and range of

exons affected, was reproduced by loss of Mbnl1 alone, whereas splicing of these same exons in

Mbnl2GT4/GT4 mice remained normal. It is possible that Mbnl2 acts mainly during earlier stages

of muscle development, or that its main function in mature tissue is at the level of cytoplasmic

localization or decay of mRNA (31). Further studies are needed to determine the combined

effects of Mbnl1 and Mbnl2 loss in vivo.

The extent of MBNL1 accumulation in ribonuclear foci was greater in DM2 than in DM1. This

extends our previous finding that levels of repeat expansion RNA in ribonuclear foci are greater

in DM2 than DM1, which likely reflects greater expansion and higher expression of the ZNF9

repeat (15). Together, these results indicate that capacity for MBNL1 sequestration by an

intronic CCUG repeat in DM2 is no less than an exonic CUG repeat in DM1. Despite greater

severity of the muscle degeneration in DM1, our results indicate that depletion of MBNL1 from

nucleoplasm is more extensive in DM2. Thus, while sequestration of MBNL1 evidently has a



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central role in splicing misregulation in both types of DM, it appears likely that CUG-expanded

DMPK mRNA has additional pathogenic consequences in DM1, not shared by CCUG-expanded

ZNF9 RNA or mediated through MBNL1, that lead to greater muscle wasting.

Muscle fibers form during prenatal development but are extensively remodeled after birth. In

rodents, the first three weeks of postnatal life are associated with pruning and stabilization of the

neuromuscular junction, formation of the T tubule system, maturation of excitation-contraction

coupling and the sarcoplasmic reticulum, and restructuring of the sarcomere (32,33). Postnatal

remodeling may require activation of transcriptional programs that were not initially engaged

during muscle differentiation. However, a recent study in cardiac muscle has emphasized the

importance of post-transcriptional regulation, and shown that alternative splicing plays an

essential role in postnatal remodeling. Conditional knockout of splicing factor ASF/SF2 in

cardiac muscle led to failure of a specific set of postnatal alternative splicing transitions (34). In

mice having ASF/SF2 deficiency in heart, persistent expression of neonatal splice isoforms was

closely related to the subsequent development of progressive cardiomyopathy. Thus, a set of

splicing switches during a critical period of postnatal remodeling were required for normal

cardiac function in adults, and these transitions were controlled by a specific splicing factor.

Our results suggest that MBNL1 is such a factor in skeletal muscle, and that symptoms of DM

result, at least in part, from failure to execute or maintain a set of postnatal splicing transitions.

Strikingly, every exon shown to undergo misregulated splicing in DM muscle showed a similar

pattern of developmental regulation between P2 and P20 in mice (Fig. 2). Mbnl1 was required

for these physiological splicing transitions, and the timing of these transitions may depend upon



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translocation of Mbnl1 from the cytoplasm to nucleus, postnatal downregulation of CUG-BP1

(28), or both. The mechanism that controls Mbnl1 translocation to the nucleus has not been

determined, although its presence in the HSALR nucleus at P2 suggests that Mbnl1 shuttles

between cytoplasm and nucleus and has become trapped on the ribonuclear foci. While nuclear

localization is more likely for the E7 inclusion isoforms, alternative splicing is not the main

factor driving the postnatal relocalization of Mbnl1. As yet we have not identified a

posttranslational modification of Mbnl1 that determines its cellular distribution.

The transcripts that undergo Mbnl1-dependent splicing transitions in skeletal muscle encode

proteins involved in excitation and contraction (Serca1, ClC-1, RyR); sarcomere structure (ZASP,

Tnnt2, Tnnt3), and signaling (IR, MTMR1). In the case of ClC-1, the functional implications are

clear: neonatal muscles produce a transcript that encodes a truncated, nonfunctional chloride

channel protein. Reversion to this splicing outcome in adult DM leads to loss of ClC-1 channels

and hyperexcitable muscle fibers (8,9). In the case of other transcripts, the functional

consequences of misregulated splicing are less obvious, but they are likely to impact multiple

pathways. Cumulatively, our results suggest that the complex phenotype of DM may derive, to

a surprising extent, from a single RNA-protein interaction: the recognition of poly(CUG) or

poly(CCUG) RNA by MBNL1. Selective disruption of this interaction may prove to be a

pharmacologically tractable approach for reversing the functional impairments of DM.



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Materials and Methods

Mice. Transgenic mice in HSALR line 20b were maintained as homozygotes on an FVB inbred

background (4). Mbnl1∆E3/∆E3, Mbnl1∆E3/+, and Mbnl+/+ littermates were maintained on a mixed

C57Bl6 • SV129 background (25). adr-mto2J (ClC-1 null mutant) mice with recessive

generalized myotonia were obtained from Jackson Laboratories (Bar Harbor, ME). Hindlimb

muscle from adult mdx mice was kindly provided by Dr Paula Clemens, University of Pittsburgh.

Mice were maintained according to guidelines of the Association for Assessment and

Accreditation of Laboratory Animal Care.

DM muscle tissue. Needle biopsies of vastus lateralis muscle were obtained from patients with

genetically confirmed DM1, DM2, or fascioscapulohumeral (FSH) muscular dystrophy, or

healthy volunteers. All study subjects gave informed consent under protocols approved by the

University of Rochester Human Subjects Review Board. DM1 quadriceps muscle tissue also

was obtained at autopsy. Muscle tissue was flash frozen in liquid nitrogen and stored at -70°C.

Mbnl2 genetrap mice. Clone XB595, derived by integration of genetrap vector pGTOpfs in

sv129/Ola ES cells, was obtained from the Mutant Mouse Regional Resource Center (Davis,

CA). The approximate site of pGTOpfs integration in Mbnl2 was confirmed by sequencing the

Mbnl2-pGTOpfs fusion cDNA, obtained from XB595 cells by RT-PCR. C57Bl6 blastocysts

were injected with XB595 cells and implanted into pseudopregnant females. Chimeric progeny

were bred with C57Bl6 mice, and the agouti offspring were screened by PCR. The exact site of

vector integration in Mbnl2 intron 4, and the integrity of the flanking exons, was determined by

cloning and sequencing the integration site from genomic DNA obtained from homozygous



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Mbnl2GT4/GT4 mice. Splicing of Mbnl2 exon 4 to the engrailed splice acceptor in pGTOpfs was

confirmed in RT-PCR products obtained from Mbnl2GT4/GT4 muscle. The Mbnl2GT4 allele was

maintained on a mixed C57Bl6/129 background. Reduction of Mbnl2 mRNA in skeletal muscle

of Mbnl2GT4/GT4 homozygotes, as compared to WT littermates, was confirmed by Northern blot

using 1.5 ug of polyadenylated RNA probed with a 500 bp fragment of the Mbnl2 3' UTR (nt

2292 – 2793, accession # NM_175341).

Antibodies. CUG-BP1 was detected using monoclonal antibody 3B1 (12). Polyclonal antibody

A2764 directed against the C-terminus of Mbnl1 was raised by immunization of rabbits with

peptide PIISAEHLTSHKYVTQM conjugated to KLH carrier. This sequence is identical in

human and mouse Mbnl1 but not conserved in other Mbnl proteins. Other antibodies used were

anti-GADPH mouse monoclonal (Biogenesis, Brentwood, NH), anti-hnRNP H rabbit polyclonal

(J Wilusz, Ft. Collins, CO), anti-hnRNP I (M Garcia-Blanco, Durham, NC), and secondary

antibodies conjugated with Alexa 488, Alexa 680, or horse radish peroxidase (HRP) (Invitrogen

Molecular Probes, Carlsbad, CA).

RT-PCR analysis of alternative splicing. Total cellular RNA was extracted from tissue using

Tri-reagent (Molecular Research Center). For fetal and neonatal mice, RNA was extracted from

hindlimb muscle. For adult mice, RNA was extracted from vastus (quadriceps) muscle.

Alternative splicing in DM1 muscle was assessed using autopsy samples, because myopathic

abnormality and misregulated splicing is more pronounced than in biopsy tissue (8). All other

analyses of human splicing or tissue sections were performed on quadriceps biopsy samples.

The genes and exons selected for alternative splicing analysis are listed in Supplemental Table 1.



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We selected 54 exons based on (1) previous evidence for alternative splicing in skeletal muscle;

and (2) splicing patterns that were suitable for RT-PCR analysis (alternative exon cassettes or

alternative 5' or 3' splice sites). Primers for these assays are listed in Supplemental Table 2.

RT-PCR splicing assays were carried out using procedures we have previously described (8). In

brief, cDNA synthesis was primed with oligo(dT) (mouse) or oligo(dT) plus random hexamers

(human). All cDNAs were treated with RNase H at 37°C for 35 min. Primers were selected to

give a length difference of >10% and <25% for exon inclusion versus exon exclusion products.

PCR amplification was carried out for 20-24 cycles, within the linear range of amplification for

each gene. PCR products were resolved on agarose gels, stained with SybrGreenII, and scanned

on a laser fluorimager. The density of each band was quantified using ImageQuant software.

Because of the low variability of the RT-PCR splicing assays in inbred mice (within-group

coefficient of variation less than 10%), the initial analysis to identify splicing differences was

carried out on muscle RNA from individual mice in each of the following categories (ages):

wild-type FVB (day 2 and 6 months), HSALR (6 months), and Mbnl1∆E3/∆E3, Mbnl1∆E3/+, or

Mbnl1+/+ littermates (6 months). Apparent differences in alternative splicing were confirmed

and quantified in an independent group of HSALR and WT mice (n = 6 per group, age 6 months).

FISH (fluorescence in situ hybridization) and IF (immunofluorescence). FISH combined with

IF was performed on frozen sections of quadriceps muscle as previously described (22). The

probe for FISH was a CAG-repeat 2-O-methyl oligoribonucleotide, 20 nt in length, conjugated at

the 3' end with Texas Red.



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IF for nuclear Mbnl1. Frozen sections of human or mouse muscle were fixed in 3%

paraformaldehyde for 15 min at room temperature, permeabilized in 2% pre-chilled acetone for 5

min and then soaked in primary antibody (A2764 at 1:10,000) at 4°C overnight. After washing

with PBS, sections were stained with secondary antibody and DAPI at 20°C for 30min. For

quantification of nuclear Mbnl1 in human biopsy samples, sections were stained on the same

slide, imaged at 1000X magnification under identical illumination and exposure settings, and

analyzed using uniform threshold settings. A z-plane stack (15 images, 0.25 μm steps) centered

on the region of maximal MBNL1 staining was deconvolved using Autoquant software v9.3

(Watervliet, NY). The plane with maximum signal intensity was selected for quantification.

The nucleus (defined by DAPI staining), nucleoli, and foci were outlined manually. To estimate

the amount of MBNL1 in foci, the IF signal (area x intensity) in nuclear foci was determined

using Metaview software (Universal Imaging Corp., Downington, PA). We also used Metaview

software to determine the average MBNL1 signal intensity in the nucleoplasm, exclusive of

nuclear foci. Multiple nuclei (42 to 65) were examined in each of the following groups (number

of individuals per group): DM1 (2), DM2 (3), healthy controls (3), FSH (2).

Immunoblot. Mouse quadriceps muscle was pulverized under liquid nitrogen and then

homogenized in lysis buffer (10mM Tris pH 7.6, 2% SDS, 2mM DTT, 1mM PMSF, 2mM

Benzamidine and 1x Protease Arrest). For human biopsy muscles, ten consecutive 10 µm

frozen sections were homogenized in the same lysis buffer. Equal amounts of protein were

resolved on 10% SDS-PAGE gels and then transferred to nitrocellulose (Bio-Rad Laboratories).

After incubation with primary antibody at 4°C overnight, membranes were incubated with

secondary antibodies at room temperature for 1 h. The primary antibodies were anti-MBNL1



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polyclonal A2764 (1:10000), anti-CUG-BP1 mouse monoclonal 3B1 (1:500), or anti-GADPH

mouse monoclonal (1:10,000). Secondary antibodies were conjugated either with HRP or Alexa

680.

Localization of Mbnl1 and CUG-BP1 fusion proteins. MBNL1 cDNAs, including all four

possible combinations of exon 7 or 9 inclusion/exclusion, were cloned in pEGFP-C1 (Clontech)

and verified by sequencing. cDNA constructs were expressed in vivo by electroporation of

muscle tissue. Under general anesthesia, tibialis anterior muscle was prepared for

electroporation by injection of hyaluronidase (25 µl of 0.4 U/µl). Two hours later, 15µl of

pEGFP-CUG-BP1 or pEGFP-MBNL1-41 plasmid (3 µg/µl in normal saline) were injected into

the pretreated muscle followed by electrical field stimulation. The voltage used for

electrotransfer was 175V/cm in eight 20ms square wave pulses at 1 sec intervals (Electro Square

Porator ECM 830, BTX A Division of Genetronics, Inc.). Electrode jelly was applied on the 7

mm circular electrodes to ensure good electrical contact. Muscle was dissected five to seven

days after electroporation and prepared for frozen sectioning. The subcellular localization of

EGFP-Mbnl1 fusion proteins also was determined in COS7 cells by transfection of cDNA

expression constructs using SuperFect (Qiagen, Valencia, CA). Cells were observed 1 or 2 days

following transfection.



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Acknowledgements

The authors thank Matt Krym, Don Henderson, and Bharati Shah for technical assistance and Dr

Thurman Wheeler for assistance with electroporation of skeletal muscle. This work comes from

the University of Rochester Senator Paul D. Wellstone Muscular Dystrophy Cooperative

Research Center (NIH/NS48843) and Wayne C. Gorell Jr. Laboratory with support from

NIH/NIAMS (AR46806, AR48143), the Muscular Dystrophy Association, and the Saunders

Family Neuromuscular Research Fund. Dr Thornton is supported by a Mid-Career Investigator

Award in Patient-Oriented Research (AR48143). Patients were enrolled in the study with the

assistance from the National Registry of Myotonic Dystrophy and Facioscapulohumeral

Dystrophy Patients and Family Members (AR02250).

Conflicts of Interest

No conflict of interest to report.



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27. Ladd,A.N., Charlet,N., and Cooper,T.A. (2001) The CELF family of RNA binding

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29. Edelman,J.C., Edelman,P.M., Kniggee,K.M., and Schwartz,I.L. (1965) Isolation of

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Legends to Figures

Figure 1. Patterns of misregulated alternative splicing in HSALR transgenic and Mbnl1 knockout

mice recapitulate the patterns observed in WT mice at postnatal day 2 (P2) A. In WT mice,

RT-PCR analysis of fast skeletal muscle troponin T (Tnnt3) mRNA showed alternative splicing

of the fetal (F) exon as well as exons 4 through 8. Transition from inclusion to exclusion of the

fetal exon occurred mainly between P2 and P16. Lower panel shows RT-PCR products after

digestion with BsrBI, which cleaved within the fetal exon. B. Compared to WT adult mice of

the same age and strain background, HSALR transgenic and Mbnl1∆E3/ ∆E3 mice had reduced

inclusion of Serca1 exon 22 and increased inclusion of Zasp exon 11, m-line region of titin exon

5 (Mex5), and Mbnl1 exon 7. For each of these exons, the pattern of splicing in DM1 models

was similar to neonatal WT mice at P2. C. Effects on alternative splicing of Serca1 and Zasp

did not occur in adr (ClC-1 null) mice that had severe myotonia or in mdx mice that had

dystrophin deficiency.

Figure 2. Selective failure of Mbnl1-dependent, postnatal splicing transitions in HSALR

transgenic mice. Alternative splicing of Serca1, Zasp, m-Titin, Mbnl1, Itgb1, and CapZb was

developmentally regulated in the interval between P2 and P20. Postnatal splicing transitions for

Serca1, Zasp, and m-Titin failed in Mbnl1∆E3/ ∆E3 mice, whereas transitions for Itgb1 and CapZb

occurred on schedule. Postnatal downregulation of Mbnl1 exon 7 also failed in Mbnl1 knockout

mice, indicating that splicing of this exon was autoregulated. HSALR transgenic mice showed

selective failure of the splicing transitions that were Mbnl1-dependent (Serca1, Zasp, m-Titin,

Mbnl1). Quantification of splicing shows the mean ± standard deviation for fractional inclusion

or exclusion of the specified exon in triplicate assays. Ad indicates adult mice of age 6 months.



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Figure 3. Alternative splicing in mice homozygous for the Mbnl2 genetrap (GT) allele. A. The

gene trap vector is a promoterless construct containing a splice acceptor (SA) site from

engrailed-2, the coding sequence for β-galactosidase fused to neomycin phosphotransferase (β-

Geo), and the SV40 poly-adenylation (pA) signal. In the Mbnl2GT4 allele, the gene trap vector is

integrated in the middle of Mbnl2 intron 4. Transcripts from the Mbnl2GT4 allele encode the N-

terminal and two C3H zinc fingers of Mbnl2, fused in-frame to β-Geo. B. Northern blot of

polyadenylated muscle RNA. Probe from the 3' untranslated region of Mbnl2 shows loss of the

normal Mbnl2 transcript in homozygous Mbnl2GT4/GT4 mice. C. Alternative splicing of indicated

exons in Serca1, Zasp, m-titin, and Mbnl1 is normally regulated in homozygous Mbnl2GT4/GT4

mice.

Figure 4. Splicing defect and nuclear sequestration of MBNL1 protein in human DM1 and

DM2. A. SERCA1, ZASP, m-Titin, and MBNL1 alternative splicing in human DM1 and DM2

skeletal muscle showed abnormal regulation similar to that observed in HSALR transgenic and

Mbnl1 knockout mice. The respective ages of subjects are 47, 55, and 50 years for DM1; 68, 23,

and 24 years for healthy volunteers; and 36, 46, and 33 years for DM2. B. Distribution of

MBNL1 (green) in the nucleus (blue) shown in deconvolved images by immunofluorescence

using anti-MBNL1 polyclonal antibody A2764 on frozen sections of quadriceps biopsy tissue.

In contrast to HSALR transgenic mice (see Fig. 6C), the CUG expansion RNA in DM muscle

nuclei was typically consolidated in a single nuclear focus (see also Supplemental Fig. 2A). In

sections stained on same slide and imaged under the same exposure and threshold settings, the

intensity of MBNL1 staining in nucleoplasm (NP), exclusive of nuclear foci, was reduced in



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DM1 and DM2 compared to healthy controls and patients with fascioscapulohumeral (FSH)

muscular dystrophy. For quantification of MBNL1 in nucleoplasm, the mean

immunofluorescence intensity in the nucleus, exclusive of nuclear foci, was determined on

deconvolved images. The MBNL1 signal within nuclear foci (area x intensity) was 3-fold greater

in DM2 than DM1. Bars indicate mean ± standard deviation. 1000x magnification, bar

indicates 5 µm, sample # indicates number of individuals examined in each category.

Figure 5. Developmental regulation and cellular distribution of CUG-BP1 in skeletal muscle.

A-C. Immunoblot of muscle lysates using anti-CUG-BP1 monoclonal antibody 3B1, with

GAPDH serving as loading control. A. Postnatal downregulation of CUG-BP1 occurred to a

similar extent in hindlimb muscle from WT and HSALR transgenic mice. B. CUG-BP1 protein

did not show a consistent increase in adult skeletal muscle from HSALR transgenic mice as

compared to gender- and age-matched WT mice (representative results from 4 independent

experiments). C. CUG-BP1 protein did not show a consistent increase in quadriceps biopsy

tissue from DM2 patients compared to healthy subjects. D-F. FISH detection of CUG

expansion RNA (left panels, red) combined with fluorescence detection of RNA-binding proteins

(green, center panels) in muscle nuclei (blue, right panels) from HSALR transgenic mice. D.

Endogenous CUG-BP1, revealed by immunofluorescence with antibody 3B1, did not colocalize

with nuclear foci of CUG expansion RNA. GFP-tagged CUG-BP1 also did not colocalize with

nuclear foci of CUG expansion RNA, when expressed by electroporation of skeletal muscle in

vivo (E), whereas MBNL1-GFP did colocalize under these same conditions (F). 1000x

magnification, bar indicates 5 µm.



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Figure 6. Developmental regulation and cellular distribution of Mbnl1 protein in murine

skeletal muscle. A-B. Immunodetection using polyclonal antibody A2764, directed against a

C-terminal peptide of Mbnl1. Specificity of antibody A2764 was demonstrated by lack of

reactivity on immunoblots (A) or tissue sections (B) from Mbnl1∆E3/ ∆E3 mice. A. Immunoblot

of hindlimb muscle showed a slight postnatal reduction of Mbnl1 protein in WT and HSALR

transgenic mice. Levels of Mbnl1 in adult (Ad) muscle were similar in WT and HSALR

transgenic mice. Note that alternative splice isoforms of Mbnl1, including exon 7 exclusion (40

kD), exon 7 inclusion (41kD), and exons 7 + 9 inclusion (42 kD) isoforms, were not resolved on

these gels, but all were recognized by antibody A2764. B. On frozen sections of WT muscle

stained on the same slide and imaged under the same exposure settings, the distribution Mbnl1

was predominantly cytoplasmic at P2 and predominantly nuclear at P20. C. High power views

(1000X) of individual myonuclei showing FISH detection of nuclear foci of CUG expansion

RNA (left panels, red) combined with immunofluorescence detection of Mbnl1 (green, center

panels) in muscle nuclei (blue, right panels) from HSALR transgenic mice. In the transgenic

model, Mbnl1 was not excluded from the nucleus in neonatal mice, as indicated by its presence

in nuclear foci as early as P2. D. Distribution of Mbnl1-GFP fusion protein in vivo after

electroporation of expression construct in WT vastus muscle. As compared to the cytoplasmic

distribution of GFP, the exon 7 exclusion isoform of Mbnl1-GFP was distributed to cytoplasm

and nucleus (left panel), whereas the exon 7 inclusion isoform localized entirely to the nucleus

(middle panel). Scare bar indicates 5 µm in B and C, 10 µm in D.



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Aberrant splicing in DM Cmouse Dhuman

E% neonatal isoform

Gene

accession #

Alt. spliced exon #

ANeonatalisoform

BPost-natal

splicing switch HSALR Mbnl1-/- DM1 DM2 WT HSALR

Serca1 NM_007504 22 exon22- + + + + + 3±0.7 78±3.8

ZASP AY206013 11 exon11+ + + + + + 11±1.6 66±6.3

z-Ttn NT_039207.3 Zr4 Zr4+5+ + + + + + 21±3.7 65±3.2

“ XM_130322 Zr5

m-Ttn XM_130312 Mex5 Mex5+ + + + + + 31±1.2 86±5.4

Nrap AY177622 12 exon12- + + + + + 34±6 59±4

Capn3 X92523 16 exon16- + + + + + 5±0.9 15±0.7

Alp AF002283 5a exon5a+5b- + + + + + 4±0.5 8±0.8

“ NT_039460.3 5b

Fhos NT_078575.3 11a exon11a- + + + + + 6±0.9 18±2

Gfat1 AF334736 10 exon10- + + + + - 7±2.3 22±3.0

Mbnl1 BC060031 7 exon7+ + + + + + 8±0.7 42±2.4 Mbnl2 NM_175341 7 exon7+ + + + + + 31±0.01 41±0.01

Table1. Misregulated alternative splicing in mouse models of DM1 was concordant with human

DM1 and DM2. A indicates the isoform that is preferentially expressed in neonatal muscle at P2

compared to adult WT muscle. B “+” denotes exons that show postnatal splicing transition

between P2 and P20 in WT hindlimb muscle. C “+” denotes exons that show misregulated

alternative splicing in adult (6 month) HSALR transgenic or Mbnl1-/- mice, compared to WT mice

of appropriate background strain. D “+” denotes exons that show misregulated alternative

splicing in quadriceps muscle from DM1 or DM2 patients compared to healthy individuals. E

fraction of splice products, expressed as the % of isoform that was preferentially expressed in

WT neonatal muscle, mean ±S.D for n = 6 per group, see also Suppl. Fig. 1.



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Fig 3



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Failure of MBNL1-dependent post-natal splicing transitions in myotonic dystrophy

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