The goal of this project is to determine the molecular basis for myotonic dystrophy (DM), a dominantly inherited disease caused by an expanded CTG trinucleotide repeat in the 3' untranslated region of the DMPK. DM is a multi-systemic disorder characterized by progressive skeletal muscle weakness, cardiomyopathy and arrhythmias, cataracts, and abnormalities in brain and endocrine function. Despite identification of the genetic defect six years ago, the molecular basis of the disease is unknown. DMPK transcripts containing the expanded repeat accumulate in nuclear foci. One hypothesis is that the expanded repeat creates a grain-of-function in the DMPK RNA which has a trans-dominant effect on the expression of other genes. A proposed mediator of the nucleus of DM cells. Our preliminary results establish the previously hypothesized link between the nuclear accumulation of expanded-CUG transcripts, nuclear accumulation of CUG-BP, and alteration of tissue-specific RNA processing in DM cells. We demonstrate that CUG-BP positively regulates splicing of the cardiac troponin T (cTNT) alternative exon 5 by binding to a previously characterize muscle-specific splicing element. cTNT exon 5 is aberrantly included in DM striated muscle, consistent with nuclear accumulation of a positive splicing regulator. The trans-dominant effect on cTNT splicing was reproduced in normal muscle cells by co-transfection of cTNT minigenes with expanded DMPK minigenes containing up to 1440 CTG repeats. Wild-type cTNT minigenes but not minigenes with a mutated CUG-BP binding site expressed enhanced levels of exon 5 inclusion demonstrating the role of CUG-BP in the trans-dominant effect. The goals of this proposal are to: (i) characterize expression of a novel protein closely related to CUG-BP (etr-3) recently identified in an EST library (ii) determine the mechanism of etr-3 and CUG-BP nuclear accumulation in DM cells; (iii) identify genes that are regulated post-transcriptionally by etr-3 and CUG-BP which are likely to directly mediate DM pathogenesis; (iv) establish stable fibroblast and muscle cell lines that inducible express expanded-CUG RNA; (v) establish lines of transgenic mice that express expanded-CUG RNA. This proposal will directly test the hypothesis that the expanded trinucleotide creates a gain-of-function mutation in the RNA. It will also establish experimental systems to investigate the basis of a novel pathogenic mechanism and ultimately, test means of therapeutic intervention.
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