The purpose of this study was to investigate the role of the mutant CUGn RNA in the induction of stress in type 1 myotonic dystrophy (DM1) cells and in the stress-mediated inhibition of protein translation in DM1. To achieve our goals, we performed HPLC-based purification of stress granules (SGs), immunoanalysis of SGs with stress markers TIA-1, CUGBP1, and ph-eIF2, site-specific mutagenesis, and examinations of RNA-protein and protein-protein interactions in myoblasts from control and DM1 patients. The cause-and-effect relationships were addressed in stable cells expressing mutant CUG repeats. We found that the mutant CUGn RNA induces formation of SGs through the increase of the double-stranded RNA-dependent protein kinase (PKR) and following inactivation of eIF2α, one of the substrates of PKR. We show that SGs trap mRNA coding for the DNA repair and remodeling factor MRG15 (MORF4L1), translation of which is regulated by CUGBP1. As the result of the trapping, the levels of MRG15 are reduced in DM1 cells and in CUG-expressing cells. These data show that CUG repeats cause stress in DM1 through the PKR-ph-eIF2α pathway inhibiting translation of certain mRNAs, such as MRG15 mRNA. The repression of protein translation by stress might contribute to the progressive muscle loss in DM1.
"olved in the splicing of several genes directly implicated in the multisystem phenotype of DM patients , such as cardiac Troponin ( cTNT ) , IR and chloride channel 1 ( CLCN1 ; Osborne et al . , 2009 ) . Overactivation of PKR also inactivates its substrate eIF2α , inhibiting the translation of specific mRNAs , such as the DNA repair factor MRG15 ( Huichalaf et al . , 2010 ) ."
[Show abstract][Hide abstract] ABSTRACT: Myotonic dystrophy type I (DM1 or Steinert’s disease) and type II (DM2) are multisystem disorders of genetic origin. Progressive muscular weakness, atrophy and myotonia are the most prominent neuromuscular features of these diseases, and other clinical manifestations such as cardiomyopathy, insulin-resistance and cataracts are also common. From a clinical perspective, most DM symptoms are interpreted as a result of an accelerated aging (cataracts, muscular weakness and atrophy, cognitive decline, metabolic dysfunction, etc.), including an increased risk of developing tumors. From this point of view, DM1 could be described as a progeroid syndrome since a notable age-dependent dysfunction of all systems occurs. The underlying molecular disorder in DM1 consists of the existence of a pathological (CTG)n triplet expansion in the 3’ untranslated region of the DMPK gene, whereas (CCTG)n repeats in the first intron of the CNBP/ZNF9 gene cause DM2. The expansions are transcribed into (CUG)n and (CCUG)n-containing RNA, respectively, which form secondary structures and sequester RNA-binding proteins, such as the splicing factor muscleblind-like protein (MBNL), forming nuclear aggregates known as foci. Other splicing factors, such as CUGBP, are also disrupted, leading to a spliceopathy of a large number of downstream genes linked to the clinical features of these diseases. Skeletal muscle regeneration relies on muscle progenitor cells, known as satellite cells, which are activated after muscle damage, and which proliferate and differentiate to muscle cells, thus regenerating the damaged tissue. Satellite cell dysfunction seems to be a common feature of both age-dependent muscle degeneration (sarcopenia) and muscle wasting in DM and other muscle degenerative diseases. This review aims to describe the cellular, molecular and macrostructural processes involved in the muscular degeneration seen in DM patients, highlighting the similarities found with muscle aging.
"Among the mis-splicing events that have been documented , splicing reversions occurring in the muscle chloride channel CLCN1 , , the insulin receptor INSR  and BIN1  contribute respectively to myotonia, insulin resistance and muscle weakness. Since MBNL1 has also been implicated in transcription and other aspects of RNA biogenesis –, and since CUGBP1 can regulate translation , , other defects in gene expression are expected. Moreover, the CUG repeat expansion may have other effects on gene expression, as suggested by a study in a CTG repeat-expressing mouse that identified changes in the abundance of many extracellular matrix mRNAs . "
[Show abstract][Hide abstract] ABSTRACT: With the goal of identifying splicing alterations in myotonic dystrophy 1 (DM1) tissues that may yield insights into targets or mechanisms, we have surveyed mis-splicing events in three systems using a RT-PCR screening and validation platform. First, a transgenic mouse model expressing CUG-repeats identified splicing alterations shared with other mouse models of DM1. Second, using cell cultures from human embryonic muscle, we noted that DM1-associated splicing alterations were significantly enriched in cytoskeleton (e.g. SORBS1, TACC2, TTN, ACTN1 and DMD) and channel (e.g. KCND3 and TRPM4) genes. Third, of the splicing alterations occurring in adult DM1 tissues, one produced a dominant negative variant of the splicing regulator RBFOX1. Notably, half of the splicing events controlled by MBNL1 were co-regulated by RBFOX1, and several events in this category were mis-spliced in DM1 tissues. Our results suggest that reduced RBFOX1 activity in DM1 tissues may amplify several of the splicing alterations caused by the deficiency in MBNL1.
PLoS ONE 09/2014; 9(9):e107324. DOI:10.1371/journal.pone.0107324 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In recent years, histochemistry at light and electron microscopy has increasingly been applied to investigate basic mechanisms of skeletal muscle diseases; in particular, the study in situ of skeletal muscle cell nuclei proved to be crucial for elucidating some pathogenetic mechanisms of skeletal muscle wasting in myotonic dystrophy (DM) and sarcopenia. DM is an autosomal dominant disorder whose multisystemic features originate form nucleotide expansions: (CTG)n in the dystrophy myotonic protein kinase (DMPK) gene on chromosome 19q13 in DM type 1 (DM1), or (CCTG)n in intron 1 of the CNBP gene (previously know as zinc finger 9 gene, ZNF9) on chromosome 3q21 in DM type 2 (DM2). Sarcopenia is an age-related condition characterized by the decline of muscle mass, strength and function, whose causes are still poorly known and probably manifold (e.g., altered levels of anabolic hormones and inflammatory mediators, impairment of proteolytic and autophagic pathways, mitochondrial or neuromuscular dysfunction, loss of satellite cells). Interestingly, skeletal muscles in both DM and sarcopenia show myofibre atrophy, fibre size variability and centrally located nuclei, as well as a reduced satellite cells' effectiveness. Based on ex vivo and in vitro studies, we have demonstrated that both myofibres and satellite cells of DM and sarcopenic muscles exhibit a massive nuclear rearrangement of the structural and molecular factors responsible for pre-mRNA transcription and maturation: the impairment in the pre-mRNA post transcriptional pathways would thus account for the aging-reminiscent muscle phenotype of DM patients suggesting that the skeletal muscle wasting observed in DM and sarcopenia may result from similar cellular mechanisms.
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