Loss of the Muscle-Specific Chloride Channel in Type 1 Myotonic Dystrophy Due to Misregulated Alternative Splicing

Charlet-B., Nicolas; Savkur, Rajesh S.; Singh, Gopal K.; Philips, Anne V.; Grice, Elizabeth A.; Cooper, Thomas A.

doi:10.1016/s1097-2765(02)00572-5

Cited by 549 publications

(493 citation statements)

References 55 publications

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“…Furthermore, we performed RT-PCR for Clcn1 and Tnnt3 in our mice and uncovered splicing abnormalities (Fig. 2c) similar to those in transgenic mice overexpressing CUG repeats 15 , in the Mbnl1ΔE3 knockout mouse 16 and in individuals with myotonic dystrophy [14][15][16] . Skeletal muscle histology also showed induction of central nuclei, nuclear clumping and fiber size variation as seen in individuals with DM1 (Fig.…”

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confidence: 62%

“…The cardiac conduction abnormalities observed in these mice are exactly like those seen in up to 70% of individuals with DM1: namely, heart block and atrioventricular node dysfunction 13 . This is the first transgenic mouse model with cardiac conduction Myotonia in DM1 and DM2 is associated with reduced chloride channel (Clcn1, also known as Clc-1) expression and splicing abnormalities of Clcn1 mRNA 14,15 . We found ClC-1 protein significantly reduced or absent from the muscle membranes in mice in which transgene expression was induced (Fig.…”

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confidence: 99%

“…Myotonia in DM1 and DM2 is associated with reduced chloride channel (Clcn1, also known as Clc-1) expression and splicing abnormalities of Clcn1 mRNA 14,15 . We found ClC-1 protein significantly reduced or absent from the muscle membranes in mice in which transgene expression was induced (Fig.…”

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Reversible model of RNA toxicity and cardiac conduction defects in myotonic dystrophy

et al. 2006

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Myotonic dystrophy (DM1), the most common muscular dystrophy in adults, is caused by an expanded (CTG) n tract in the 3′ UTR of the gene encoding myotonic dystrophy protein kinase (DMPK) 1 , which results in nuclear entrapment of the 'toxic' mutant RNA and interacting RNAbinding proteins (such as MBNL1) in ribonuclear inclusions 2 . It is unclear if therapy aimed at eliminating the toxin would be beneficial. To address this, we generated transgenic mice expressing the DMPK 3′ UTR as part of an inducible RNA transcript encoding green fluorescent protein (GFP). We were surprised to find that mice overexpressing a normal DMPK 3′ UTR mRNA reproduced cardinal features of myotonic dystrophy, including myotonia, cardiac conduction abnormalities, histopathology and RNA splicing defects in the absence of detectable nuclear inclusions. However, we observed increased levels of CUG-binding protein (CUG-BP1) in skeletal muscle, as seen in individuals with DM1. Notably, these effects were reversible in both mature skeletal and cardiac muscles by silencing transgene expression. These results represent the first in vivo proof of principle for a therapeutic strategy for treatment of myotonic dystrophy by ablating or silencing expression of the toxic RNA molecules.Common features of adult-onset DM1 include myotonia, progressive skeletal muscle loss, cardiac conduction defects, smooth muscle dysfunction, cataracts and insulin resistance 2 . The normal number of CTG repeats (n = 5 to ~30) is higher (n = 50 to >3,000) in individuals with DM1 (ref. 1 ). Unlike the wild-type transcript, mutant DMPK mRNA forms nuclear aggregates 3,4 and is thought to trigger dominant effects by aberrant interactions with or altered activity of RNA splicing factors, principally members of the muscleblind-like (MBNL) family (such as MBNL1) and the CUG-BP and ETR3-like factor (CELF) family (such as CUG-BP1), leading to abnormal splicing of specific RNAs such as chloride channel (Clcn1), insulin Correspondence should be addressed to M.S.M. (mahadevan@virginia.edu). 4 These authors contributed equally to this work. AUTHOR CONTRIBUTIONSM.S.M., R.S.Y., Q.Y., C.D.F.-M., T.D.B. and L.H.P. performed experimental work and data analysis. S.B. generated the transgene constructs. M.S.M. was responsible for conceptual design and execution. COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests. One potential therapeutic approach in DM1 is to get rid of the toxic RNA from cells. However, it is unclear if this will alleviate the effects of the disease. We used the tetracycline (Tet) inducible system with the reverse tetracycline transactivator (rtTA) to generate double transgenic mice harboring (i) a Tet-responsive, DMPK promoter 10,11 -driven transgene (named GFP-DMPK 3′ UTR) expressing the DMPK 3′ UTR mRNA as part of a GFP transcript, and (ii) a constitutively expressed rtTA transgene (Fig. 1a) Fig. 1). Notably, RNA blots of skeletal muscle RNA showed two major species due to alternative use of polyadenylation signal...

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Reversible model of RNA toxicity and cardiac conduction defects in myotonic dystrophy

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“…Aberrant processing of mRNAs in tumor cells has been described for the MDM2, RasGRP4, SLP-65, TLE1, TLE4, MTA1 and CD44 genes and is emerging as an important characteristic of tumor cells (Bartel et al, 2002;Kumar et al, 2002;Reuther et al, 2002;Yang et al, 2002;Jumaa et al, 2003;Venables, 2004;Watermann et al, 2006) and is observed in other diseases, such as myotonic dystrophy (Charlet-B et al, 2002). In aberrant splicing, portions of exons, portions of introns or both are retained within transcripts that fail to be purged by the cellular pathways designed to scavenge abnormal mRNAs, such as nonsense-mediated decay (Culbertson, 1999;Wilkinson and Shyu, 2002).…”

Section: Discussionmentioning

confidence: 99%

Cancer cells express aberrant DNMT3B transcripts encoding truncated proteins

et al. 2007

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Cancer cells display an altered distribution of DNA methylation relative to normal cells. Certain tumor suppressor gene promoters are hypermethylated and transcriptionally inactivated, whereas repetitive DNA is hypomethylated and transcriptionally active. Little is understood about how the abnormal DNA methylation patterns of cancer cells are established and maintained. Here, we identify over 20 DNMT3B transcripts from many cancer cell lines and primary acute leukemia cells that contain aberrant splicing at the 5 0 end of the gene, encoding truncated proteins lacking the C-terminal catalytic domain. Many of these aberrant transcripts retain intron sequences. Although the aberrant transcripts represent a minority of the DNMT3B transcripts present, Western blot analysis demonstrates truncated DNMT3B isoforms in the nuclear protein extracts of cancer cells. To test if expression of a truncated DNMT3B protein could alter the DNA methylation patterns within cells, we expressed DNMT3B7, the most frequently expressed aberrant transcript, in 293 cells. DNMT3B7-expressing 293 cells have altered gene expression as identified by microarray analysis. Some of these changes in gene expression correlate with altered DNA methylation of corresponding CpG islands. These results suggest that truncated DNMT3B proteins could play a role in the abnormal distribution of DNA methylation found in cancer cells.

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“…A multi-step model for DM1 pathogenesis has been proposed [reviewed in reference 2]: (1) the mutant gene is transcribed, giving rise to transcripts that contain an expanded CUG repeat (CUG exp ) [3]; (2) the CUG exp transcripts accumulate in RNA nuclear (ribonuclear) foci [4]; (3) RNA binding proteins, including muscleblind 1 (MBNL1), are sequestered in the ribonuclear foci [5,6]; (4) altered activity of splicing factors, such as MBNL1 and CUG binding protein 1, leads to abnormal alternative splicing for a sub-group of pre-mRNAs [7,8]; and (5) expression of inappropriate splice products leads to symptoms of DM1. For example, CUG exp RNA triggers abnormal alternative splicing of the ClC-1 chloride channel, and the predominant ClC-1 splice products expressed in DM1 muscle are devoid of ion channel activity [9][10][11]. Deficiency of ClC-1 channels contributes to myotonia in DM1, and can be reversed in a transgenic mouse model by overexpressing MBNL1 to levels that exceed the capacity of CUG exp RNA to sequester proteins [12].…”

Section: Introductionmentioning

confidence: 99%

Ribonuclear foci at the neuromuscular junction in myotonic dystrophy type 1

Wheeler

Krym

Thornton

2007

Neuromuscular Disorders

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In myotonic dystrophy type 1 (DM1) the muscle fibers express RNA containing an expanded CUG repeat (CUG exp ). The CUG exp RNA is retained in the nucleus, forming ribonuclear foci. Splicing factors in the muscleblind (MBNL) family are sequestered in ribonuclear foci, resulting in abnormal regulation of alternative splicing. In extrajunctional nuclei, these effects on splicing regulation lead to reduced chloride conductance and altered insulin receptor signaling. Here we show that CUG exp RNA is also expressed in subsynaptic nuclei of muscle fibers and in motor neurons in DM1, causing sequestration of MBNL1 protein in both locations. In a transgenic mouse model, expression of CUG exp RNA at high levels in extrajunctional nuclei replicates many features of DM1, but the toxic RNA is poorly expressed in subsynaptic nuclei and the mice fail to develop denervation-like features of DM1 myopathology. Our findings indicate that subsynaptic nuclei and motor neurons are at risk for DM1-induced spliceopathy, which may affect function or stability of the neuromuscular junction.

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Loss of the Muscle-Specific Chloride Channel in Type 1 Myotonic Dystrophy Due to Misregulated Alternative Splicing

Cited by 549 publications

References 55 publications

Reversible model of RNA toxicity and cardiac conduction defects in myotonic dystrophy

Reversible model of RNA toxicity and cardiac conduction defects in myotonic dystrophy

Cancer cells express aberrant DNMT3B transcripts encoding truncated proteins

Ribonuclear foci at the neuromuscular junction in myotonic dystrophy type 1

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