Overexpression of CUG Triplet Repeat-binding Protein, CUGBP1, in Mice Inhibits Myogenesis

Timchenko, Nikolai A.; Patel, Roma H.; Iakova, Polina; Cai, Zong-Jin; Quan, Ling; Timchenko, Lubov

doi:10.1074/jbc.m312923200

Cited by 185 publications

(216 citation statements)

References 35 publications

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“…This is analogous to results from the CUG-BP1 transgenic mouse 28 and the Mbnl1ΔE3 knockout mouse 16 , in which perturbations of only one (CUG-BP1) or the other (MBNL-1) protein results in DM1-like splicing defects and DM1 pathology. Therapeutic strategies aimed at addressing only one side of the balance (that is, correction of MBNL1 sequestration or decreasing CUG-BP1) may not be sufficient, as both these proteins may have other functions in addition to their roles as splicing factors 17 .…”

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confidence: 99%

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

et al. 2006

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“…The association of CUGBP1 with its mRNA targets influences their alternative splicing, translation and turnover [17]. CUGBP1 is ubiquitously expressed and plays a critical role in various physiological and pathological cellular processes, including skeletal muscle differentiation and atrophy [18][19][20][21], cell proliferation [22], heart development and disease [23][24][25], and tumour growth [26]. Recently, several studies indicate that CUGBP1 may be involved in metabolic disorders.…”

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confidence: 99%

RNA-binding protein CUGBP1 regulates insulin secretion via activation of phosphodiesterase 3B in mice

Zhai

Yang

et al. 2016

Diabetologia

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Aims/hypothesis CUG-binding protein 1 (CUGBP1) is a multifunctional RNA-binding protein that regulates RNA processing at several stages including translation, deadenylation and alternative splicing, as well as RNA stability. Recent studies indicate that CUGBP1 may play a role in metabolic disorders. Our objective was to examine its role in endocrine pancreas function through gain-and loss-of-function experiments and to further decipher the underlying molecular mechanisms. Methods A mouse model in which type 2 diabetes was induced by a high-fat diet (HFD; 60% energy from fat) and mice on a standard chow diet (10% energy from fat) were compared. Pancreas-specific CUGBP1 overexpression and knockdown mice were generated. Different lengths of the phosphodiesterase subtype 3B (PDE3B) 3′ untranslated region (UTR) were cloned for luciferase reporter analysis. Purified CUGBP1 protein was used for gel shift experiments.Results CUGBP1 is present in rodent islets and in beta cell lines; it is overexpressed in the islets of diabetic mice. Compared with control mice, the plasma insulin level after a glucose load was significantly lower and glucose clearance was greatly delayed in mice with pancreas-specific CUGBP1 overexpression; the opposite results were obtained upon pancreas-specific CUGBP1 knockdown. Glucose-and glucagon-like peptide1 (GLP-1)-stimulated insulin secretion was significantly attenuated in mouse islets upon CUGBP1 overexpression. This was associated with a strong decrease in intracellular cAMP levels, pointing to a potential role for cAMP PDEs. CUGBP1 overexpression had no effect on the mRNA levels of PDE1A, 1C, 2A, 3A, 4A, 4B, 4D, 7A and 8B subtypes, but resulted in increased PDE3B expression. CUGBP1 was found to directly bind to a specific ATTTGTT sequence residing in the 3′ UTR of PDE3B and stabilised PDE3B mRNA. In the presence of the PDE3 inhibitor cilostamide, glucose-and GLP-1-stimulated insulin secretion was no longer reduced by CUGBP1 overexpression. Similar to CUGBP1, PDE3B was overexpressed in the islets of diabetic mice. Conclusions/interpretation We conclude that CUGBP1 is a critical regulator of insulin secretion via activating PDE3B. Repressing this protein might provide a potential strategy for treating type 2 diabetes.

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“…16 The most commonly accepted theory is that (CTG) n expansion could affect differentiation of DM myoblasts by interfering with the signals leading to the withdrawal of cell cycle and the shift toward the differentiation program. 26,32,33 However, it should be noted that the majority of the data on in vitro differentiation of DM muscle cells derive from studies on DM1 fibroblasts converted into skeletal muscle, 18,19 from primary muscle cell cultures obtained from cDM1 fetuses 31 or from mouse muscle cells. 14,15,17,33,34 No studies focusing on muscle differentiation of human primary DM1 myoblasts cultures obtained from adult muscle DM1 biopsies are available, underlining the need to work with a model as close as possible to human regenerating skeletal muscle.…”

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Normal myogenesis and increased apoptosis in myotonic dystrophy type-1 muscle cells

et al. 2010

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Myotonic dystrophy (DM) is caused by a (CTG) n expansion in the 3 0 -untranslated region of DMPK gene. Mutant transcripts are retained in nuclear RNA foci, which sequester RNA binding proteins thereby misregulating the alternative splicing. Controversy still surrounds the pathogenesis of the DM1 muscle distress, characterized by myotonia, weakness and wasting with distal muscle atrophy. Eight primary human cell lines from adult-onset (DM1) and congenital (cDM1) patients, (CTG) n range 90-1800, were successfully differentiated into aneural-immature and contracting-innervated-mature myotubes. Morphological, immunohistochemical, RT-PCR and western blotting analyses of several markers of myogenesis indicated that in vitro differentiation-maturation of DM1 myotubes was comparable to age-matched controls. In all pathological muscle cells, (CTG) n expansions were confirmed by long PCR and RNA fluorescence in situ hybridization. Moreover, the DM1 myotubes showed the splicing alteration of insulin receptor and muscleblind-like 1 (MBNL1) genes associated with the DM1 phenotype. Considerable myotube loss and atrophy of 15-day-differentiated DM1 myotubes indicated activated catabolic pathways, as confirmed by the presence of apoptotic (caspase-3 activation, cytochrome c release, chromatin fragmentation) and autophagic (P62/LC3) markers. Z-VAD treatment significantly reduced the decrease in myonuclei number and in average width in 15-day-differentiated DM1 myotubes. We thus propose that the muscle wasting typical in DM1 is due to impairment of muscle mass maintenanceregeneration, through premature apoptotic-autophagic activation, rather than altered myogenesis. Myotonic dystrophy (DM) is a multi-systemic disorder caused by two different microsatellite expansions in non-coding regions. Together, these two mutations affect 1 out of 8000 individuals and represent the most common form of muscular dystrophy in adults. DM1 and DM2 have common symptoms such as myotonia, muscle weakness and early cataract development. 1,2 Although DM1 and DM2 initially affect different muscles (distal versus proximal), histological analysis of the muscular tissues shows common aspects such as central nucleation. The classic form of DM1 is characterized by muscle distress with myotonia, progressive muscle weakness and wasting. Atrophy has also been reported, occurring preferentially in type-1 fibers in DM1 and in type-2 in DM2. 3 DM1 but not DM2 also presents a congenital form (cDM1), characterized by a high neonatal mortality and symptoms such as hypotonia, mental retardation and respiratory distress. 4,5 DM1 is associated with an unstable (CTG) n trinucleotide expansion located in the 3 0 -untranslated (3 0 -UTR) region of the DM protein kinase (DMPK) gene on chromosome 19q13.3. The mutant DMPK transcript, containing the expanded (CTG) n sequence, accumulates in discrete nuclear foci able to sequester various nuclear factors such as RNAbinding proteins or splicing regulators, causing different and highly variable downstream deleterious effects. 2,6...

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Overexpression of CUG Triplet Repeat-binding Protein, CUGBP1, in Mice Inhibits Myogenesis

Cited by 185 publications

References 35 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

RNA-binding protein CUGBP1 regulates insulin secretion via activation of phosphodiesterase 3B in mice

Normal myogenesis and increased apoptosis in myotonic dystrophy type-1 muscle cells

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