Myotonic dystrophy (DM) is the most common form of adult onset muscular dystrophy. DM is an autosomal dominant neuromuscular disorder that is caused by a (CTG)n repeat expansion in the 3' UTR of the DM protein kinase (DMPK) gene. The long term objective of the proposed research is to elucidate how a triplet repeat expansion in the 3' UTR of a gene leads to a dominantly inherited disease. Current evidence suggests that DM pathogenesis is associated with the accumulation of DMPK mutant allele transcripts within the nucleus. Our working 'sequestration' hypothesis is that DM is an RNA dominant disease in which the (CUG)n expansion forms an exceptionally stable double stranded RNA (dsRNA) hairpin structure. This unusual RNA hairpin acts as a high affinity binding site for triplet repeat expansion dsRNA binding proteins that possibly play important roles in nucleocytoplasmic RNA export. Large repeat expansions associated with severe disease lead to sequestration of these proteins on DMPK mutant allele transcripts and a dominant negative effect on the export of other RNAs. This proposal is focused on testing this RNA dominance model using several different experimental approaches. First, the hypothesis that (CUG)n expansion RNAs have a dominant negative effect on mRNA export will be directly examined using RNA microinjection into frog oocyte and mammalian fibroblast nuclei. Second, the sequestration hypothesis predicts that expansion binding proteins should accumulate in nuclear foci together with DMPK mutant transcripts. Therefore, we will complete the characterization of several proteins that preferentially recognize large (CUG)n expansions, and determine the subcellular distribution of these proteins in normal and DM patient cells. Third, preferred RNA binding sites for these expansion binding proteins will be characterized by in vitro and in vivo analyses with particular emphasis on identifying RNAs that normally associate with these proteins. Fourth, we will determine if expansion binding proteins are involved in mRNA export by combining the use of monoclonal antibodies and recombinant proteins with the microinjection system developed in the first aim. Fifth, the relevance of RNA dominance to other neuromuscular and neurological diseases will be investigated. These studies have important implications for elucidating molecular mechanisms involved in DM pathogenesis and cellular strategies which facilitate the exchange of genetic information between the nucleus and cytoplasm.
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