Human Molecular Genetics

Human Molecular Genetics Advance Access originally published online on May 22, 2006
Human Molecular Genetics 2006 15(13):2087-2097; doi:10.1093/hmg/ddl132

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

Xiaoyan Lin¹, Jill W. Miller², Ami Mankodi^2,, Rahul N. Kanadia³, Yuan Yuan³, Richard T. Moxley², Maurice S. Swanson³ and Charles A. Thornton^2,*

¹ Department of Neuroscience and ² Department of Neurology, Box 673, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642, USA and ³ Department of Molecular Genetics and Microbiology and the Genetics Institute, University of Florida, College of Medicine, Gainesville, FL 32610, USA

^* To whom correspondence should be addressed. Tel: +1 5852752542; fax: +1 5852731255; Email: charles_thornton{at}urmc.rochester.edu

Received April 11, 2006; Accepted May 14, 2006

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 
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 post-natal day 2 and 20 in wild-type mice. During this post-natal 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 because of 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 post-natal 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 
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). 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 (16)]. 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 (17–19). 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 (7,16,20). 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 CIC-1 and cTnT that are similar to DM1 (25).

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 post-natal 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. In 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 post-natal remodeling of skeletal muscle by controlling a key set of developmentally regulated splicing switches.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 
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 post-natal day 2 and day 16 (Fig. 1A).

View larger version (72K): [in this window] [in a new window]   Figure 1. Patterns of misregulated alternative splicing in HSALR transgenic and Mbnl1 knockout mice recapitulate the patterns observed in WT mice at post-natal 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 with WT adult mice of the same age and strain background, HSALR transgenic and Mbnl1E3/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.

 
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 HSA^LR 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, 11 exons showed abnormal regulation in HSA^LR mice. For example, splice products containing Serca1 exon 22 were decreased in HSA^LR 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 HSA^LR mice per group, P<0.0001 for each exon, Supplementary Material, Fig. S1, 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 the sarcoplasmic reticulum. The fraction of Serca1 mRNA skipping exon 22 increased from 3±0.7% in WT to 78±4% in HSA^LR mice. Abnormal splicing of Serca1 in DM1 has recently been reported (26). Interestingly, the alternative splicing of Mbnl1 was also misregulated in HSA^LR 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 (CIC-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 CIC-1 null mice).

View this table: [in this window] [in a new window]   Table 1. Misregulated alternative splicing in mouse models of DM1 was concordant with human DM1 and DM2

 
The pattern of misregulated alternative splicing in HSA^LR transgenic mice was remarkably concordant with Mbnl1^E3/E3 mice across all 54 exons that we examined (Table 1 and Supplementary Material, Table S1). Furthermore, among exons that were abnormally regulated in DM1 mouse models, the pattern of splicing in adult HSA^LR or Mbnl1^E3/E3 mice invariably resembled that seen in WT mice at post-natal day 2 (Fig. 1B and C). We carried out a more detailed examination of the post-natal splicing switch for seven 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 post-natal 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 post-natal interval, however, these transitions were properly executed in HSA^LR 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 post-natal regulation of these exons followed the Tnnt3 fetal exon time course, suggesting that all of these splicing transitions were coordinated by a common triggering event.

View larger version (73K): [in this window] [in a new window]   Figure 2. Selective failure of Mbnl1-dependent, post-natal 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. Post-natal splicing transitions for Serca1, Zasp and m-Titin failed in Mbnl1E3/E3 mice, whereas transitions for Itgb1 and CapZb occurred on schedule. Post-natal 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±SD for fractional inclusion or exclusion of the specified exon in triplicate assays. ‘Ad’ indicates adult mice of age 6 months.

 
Exons misregulated in HSA^LR 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 GT vector in Mbnl2 intron 4 (Fig. 3A). In mice homozygous for the GT allele (Mbnl2^GT4/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 HSA^LR and Mbnl1^E3/E3 mice showed normal regulation in Mbnl2^GT4/GT4 adult mice (Fig. 3C and data not shown, with the exception that splicing of Mbnl2 itself could not be assessed because of insertion of the GT vector).

View larger version (48K): [in this window] [in a new window]   Figure 3. Alternative splicing in mice homozygous for the Mbnl2 GT allele. (A) The GT vector is a promoter-less construct containing a splice acceptor (SA) site from engrailed-2, the coding sequence for ß-galactosidase fused to neomycin phosphotransferase (ß-Geo), and the SV40 poly-adenylation (pA) signal. In the Mbnl2GT4 allele, the GT 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 ß-Geo. (B) Northern blot of polyadenylated muscle RNA. Probe from the 3' UTR 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.

 
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, when compared with healthy subjects. Each exon misregulated in HSA^LR 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 HSA^LR and Mbnl1^E3/E3 mouse models, the 27 that we examined in human DM also were correctly regulated. Thus, both of these mouse models accurately reproduce the splicing defect in human DM1 and DM2 skeletal muscle.

View larger version (49K): [in this window] [in a new window]   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 IF 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 Supplementary Material, Fig. S2A). 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 DM1 and DM2 compared to healthy controls and patients with fascio scapulo numeral (FSH) muscular dystrophy. For quantification of MBNL1 in nucleoplasm, the mean IF intensity in the nucleus, exclusive of nuclear foci, was determined on deconvolved images. The MBNL1 signal within nuclear foci (areaxintensity) was 3-fold greater in DM2 than DM1. Bars indicate mean±SD, 1000x magnification, bar indicates 5 µm, sample number indicates number of individuals examined in each category.

 

View larger version (54K): [in this window] [in a new window]   Figure 6. Developmental regulation and cellular distribution of Mbnl1 protein in murine skeletal muscle. (A and 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 Mbnl1E3/E3 mice. (A) Immunoblot of hind limb muscle showed a slight post-natal 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 kDa), exon 7 inclusion (41 kDa), and exons 7+9 inclusion (42 kDa) 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 IF 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. When compared with 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 (C), 10 µm in (D).

 
Mbnl1 is sequestered in DM1 and DM2 myonuclei
Although 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 below). 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 (Supplementary Material, Fig. S2A) 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 HSA^LR mice and DM2 patients is also associated with increased levels of CUG-BP1 protein.

CUG-BP1 protein levels are not elevated in HSA^LR or DM2 skeletal muscle
In WT skeletal muscle, levels of CUG-BP1 decreased sharply between P2 and P20 (Fig. 5A). Post-natal downregulation of CUG-BP1 occurred to a similar extent in HSA^LR transgenic mice. In HSA^LR 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 HSA^LR muscle, as visualized by IF of the endogenous protein or by expression of GFP-tagged CUG-BP1 in vivo by electroporation (Fig. 5D and E). By comparison, GFP-tagged Mbnl1 colocalized with nuclear RNA foci under these conditions (Fig. 5F). Interestingly, although 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 when compared with healthy controls (Fig. 5C). These observations argue that misregulated splicing in HSA^LR transgenic mice and human DM2 does not require elevated levels or mislocalization of CUG-BP1 protein.

View larger version (61K): [in this window] [in a new window]   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) Post-natal downregulation of CUG-BP1 occurred to a similar extent in hind limb 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 when compared with gender- and age-matched WT mice (representative results from four independent experiments). (C) CUG-BP1 protein did not show a consistent increase in quadriceps biopsy tissue from DM2 patients when compared with 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 IF 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.

 
Mbnl1 relocalizes to the nucleus during post-natal development
To investigate the mechanism for the post-natal 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 HSA^LR 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 post-natal decline occurred during a period of rapid muscle fiber hypertrophy, and consequently these results may 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 post-natal 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 (Supplementary Material, Fig. S2B and S2C). However, Mbnl1 is not entirely excluded from the nucleus in neonatal muscle, as indicated by its presence in nuclear foci of HSA^LR mice throughout the post-natal 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 HSA^LR muscle.

Mbnl1 pre-mRNA is alternatively spliced at exons 3, 5, 7 and 9, raising the possibility that post-natal 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 HSA^LR mice, and the 30 kDa 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 HSA^LR 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 HSA^LR muscle (not shown).

In contrast, inclusion of exon 7 (E7) was increased in HSA^LR 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 (Supplementary Material, Fig. S1). E7^– and E7⁺ Mbnl1 both retain their splicing and RNA-binding activities [(21) and (24), and M. Swanson, unpublished observations]. We postulated that post-natal splicing transitions may result from preferential localization of E7⁺ Mbnl1 to the muscle cytoplasm. We examined this possibility 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 (Supplementary Material, Fig. S2D). 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 post-natal 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.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 
The disease process in DM 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 HSA^LR 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 (18), so that colocalization is difficult to observe because of 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 data). Furthermore, misregulated alternative splicing has occurred in HSA^LR 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).

In 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 DM1, DM2 or the HSA^LR 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 Mbnl2^GT4/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, although sequestration of MBNL1 evidently has a 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 3 weeks of post-natal 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). Post-natal 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 post-natal remodeling. Conditional knockout of splicing factor ASF/SF2 in cardiac muscle led to failure of a specific set of post-natal 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 post-natal 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 post-natal 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 translocation of Mbnl1 from the cytoplasm to nucleus, post-natal 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 HSA^LR nucleus at P2 suggests that Mbnl1 shuttles between cytoplasm and nucleus and has become trapped in the ribonuclear foci. Although nuclear localization is more likely for the E7 inclusion isoforms, alternative splicing is not the main factor driving the post-natal relocalization of Mbnl1. As yet we have not identified a post-translational 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 CIC-1, the functional implications are clear: neonatal muscles produce a transcript that encodes a truncated, non-functional chloride channel protein. Reversion to this splicing outcome in adult DM leads to loss of CIC-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.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 
Mice
Transgenic mice in HSA^LR 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, USA). Hind limb 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 GT vector pGTOpfs in sv129/Ola ES cells, was obtained from the Mutant Mouse Regional Resource Center (Davis, CA, USA). 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 Mbnl2^GT4/GT4 mice. Splicing of Mbnl2 exon 4 to the engrailed splice acceptor in pGTOpfs was confirmed in RT–PCR products obtained from Mbnl2^GT4/GT4 muscle. The Mbnl2^GT4 allele was maintained on a mixed C57Bl6/129 background. Reduction of Mbnl2 mRNA in skeletal muscle of Mbnl2^GT4/GT4 homozygotes, when compared with WT littermates, was confirmed by Northern blot using 1.5 µg of polyadenylated RNA probed with a 500 bp fragment of the Mbnl2 3' UTR (nt 2292–2793, accession no. NM_175341 [GenBank] ).

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-GAPDH mouse monoclonal (Biogenesis, Brentwood, NH, USA), anti-hnRNP H rabbit polyclonal (J Wilusz, Ft. Collins, CO, USA), anti-hnRNP I (M Garcia-Blanco, Durham, NC, USA), and secondary antibodies conjugated with Alexa 488, Alexa 680, or horse radish peroxidase (HRP) (Invitrogen Molecular Probes, Carlsbad, CA, USA).

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 hind limb 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 Supplementary Material, Table S1. 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 Supplementary Material, Table S2. 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 <10%), the initial analysis to identify splicing differences was carried out on muscle RNA from individual mice in each of the following categories (ages): WT FVB (day 2 and 6 months), HSA^LR (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 HSA^LR and WT mice (n=6 per group, age 6 months).

FISH (fluorescence in situ hybridization) and 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.

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 30 min. 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, USA). 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 (areaxintensity) in nuclear foci was determined using Metaview software (Universal Imaging Corp., Downington, PA, USA). We also used Metaview software to determine the average MBNL1 signal intensity in the nucleoplasm, exclusive of nuclear foci. Multiple nuclei (42–65) 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 (10 mM Tris pH 7.6, 2% SDS, 2 mM DTT, 1 mM PMSF, 2 mM Benzamidine and 1xProtease Arrest). For human biopsy muscles, 10 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 polyclonal A2764 (1:10 000), anti-CUG-BP1 mouse monoclonal 3B1 (1:500) or anti-GAPDH 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, 15 µl 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 175 V/cm in eight 20 ms square wave pulses at 1 s 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 5–7 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, USA). Cells were observed 1 or 2 days following transfection.

	SUPPLEMENTARY MATERIAL

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 
Supplementary material is available at HMG online.

	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).

Conflict of Interest statement. No conflict of interest to report.

	FOOTNOTES

 
Present address: Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.

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				S.-i. Hino, S. Kondo, H. Sekiya, A. Saito, S. Kanemoto, T. Murakami, K. Chihara, Y. Aoki, M. Nakamori, M. P. Takahashi, et al. Molecular mechanisms responsible for aberrant splicing of SERCA1 in myotonic dystrophy type 1 Hum. Mol. Genet., December 1, 2007; 16(23): 2834 - 2843. [Abstract] [Full Text] [PDF]

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				K. P. Smith, M. Byron, C. Johnson, Y. Xing, and J. B. Lawrence Defining early steps in mRNA transport: mutant mRNA in myotonic dystrophy type I is blocked at entry into SC-35 domains J. Cell Biol., September 7, 2007; 178(6): 951 - 964. [Abstract] [Full Text] [PDF]

				Y. Yuan, S. A. Compton, K. Sobczak, M. G. Stenberg, C. A. Thornton, J. D. Griffith, and M. S. Swanson Muscleblind-like 1 interacts with RNA hairpins in splicing target and pathogenic RNAs Nucleic Acids Res., August 15, 2007; (2007) gkm601v1. [Abstract] [Full Text] [PDF]

				T. Moroy and F. Heyd The impact of alternative splicing in vivo: Mouse models show the way RNA, August 1, 2007; 13(8): 1155 - 1171. [Abstract] [Full Text] [PDF]

				J. D. Lueck, C. Lungu, A. Mankodi, R. J. Osborne, S. L. Welle, R. T. Dirksen, and C. A. Thornton Chloride channelopathy in myotonic dystrophy resulting from loss of posttranscriptional regulation for CLCN1 Am J Physiol Cell Physiol, April 1, 2007; 292(4): C1291 - C1297. [Abstract] [Full Text] [PDF]

				T. A. Cooper Regulation of chloride ion conductance during skeletal muscle development and in disease. Focus on "Chloride channelopathy in myotonic dystrophy resulting from loss of posttranscriptional regulation for CLCN1" Am J Physiol Cell Physiol, April 1, 2007; 292(4): C1245 - C1247. [Full Text] [PDF]

				T. A. Cooper A Reversal of Misfortune for Myotonic Dystrophy? N. Engl. J. Med., October 26, 2006; 355(17): 1825 - 1827. [Full Text] [PDF]

				R. J. Osborne and C. A. Thornton RNA-dominant diseases Hum. Mol. Genet., October 15, 2006; 15(suppl_2): R162 - R169. [Abstract] [Full Text] [PDF]

				R. N. Kanadia, J. Shin, Y. Yuan, S. G. Beattie, T. M. Wheeler, C. A. Thornton, and M. S. Swanson Reversal of RNA missplicing and myotonia after muscleblind overexpression in a mouse poly(CUG) model for myotonic dystrophy PNAS, August 1, 2006; 103(31): 11748 - 11753. [Abstract] [Full Text] [PDF]

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