Myotonic dystrophy (DM) is an autosomal dominant disorder affecting 1 in 8500 people worldwide. Individuals with DM exhibit progressive muscular dystrophy, arrhythmias and CNS damage. Mutations are expanded CTG or CCTG repeats in untranslated regions of DMPK and ZNF9 genes respectively. The expanded repeats are expressed as CUG (DM1) or CCUG (DM2) repeat RNA, which accumulate as discrete foci in nuclei. The pathogenesis of DM1 involves misregulation of alternative splicing resulting in expression of inappropriate protein isoforms causing specific symptoms. However, the mechanism by which expanded repeats alter pre-mRNA alternative splicing is unclear. Two RNA binding proteins have been implicated in DM1 pathogenesis: MBNL1 and CUG-BP1. CUG-BP1 regulates alternative splicing of cardiac troponin T (cTNT), insulin receptor (IR) and chloride channel (CIC-1) pre-mRNAs that are mis-spliced in DM1 striated muscle. The functional consequences of mis-splicing of CIC-1 and IR directly correlate with myotonia and insulin resistance, respectively, observed in DM1. The splicing patterns observed for these pre-mRNAs are consistent with increased CUG-BP1 activity observed in DM1 striated muscle. MBNL proteins bind and colocalize with the foci of expanded CUG repeat RNA in DM1 cells. MBNL proteins directly regulate splicing of cTNT and IR alternative exons. Therefore, MBNL1 and CUG-BP1 proteins are the major regulators of alternative splicing of pre-mRNAs that are misregulated in DM1. The goal of this proposal is to understand how expanded CUG repeat RNA disrupts the regulation by CUG-BP1 and MBNL1. In this study I will: i) establish and characterize stable cell lines which inducibly express CUG repeat RNA, ii) characterize the effects of CUG repeat RNA on steady state levels and nuclearcytoplasmic distribution of CUG-BP1 and MBNL1, iii) determine whether CUG repeat RNA affects post-translational modifications of CUG-BP1 and MBNL1 and characterize the significance of these modifications on alternative splicing.
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