The regulation of alternative splicing associated with Myotonic Dystrophy
Warf, Michael Bryan
Myotonic Dystrophy (DM) is a genetic disorder with multisystemic symptoms that is caused by expression (as RNA) of expanded repeats of CTG or CCTG in the genome. It is hypothesized that the protein MBNL1 (M[barbelow]uscleb[barbelow]lin[barbelow]d-l[barbelow]ike-1) is sequestered to the expanded CUG or CCUG RNAs. MBNL1 regulates the alternative splicing of a variety of pre-mRNAs and its mis-localization results in mis-splicing of a subset of pre-mRNAs that are linked to the symptoms found in DM patients. I initially demonstrated that MBNL1 can bind short structured CUG and CCUG repeats with high affinity and specificity in vitro . Next, I was able to determine and articulate the first structure of a binding site of MBNL1 in an endogenous pre-mRNA that it regulates. I found that MBNL1 binds a stem-loop in the cardiac troponin T (cTNT) pre-mRNA. The stem-loop contains two mismatches and resembles both CUG and CCUG repeats. I determined that MBNL1 regulated exon 5 by directly competing with the essential splicing factor U2AF65 for binding upstream of exon 5. When U2AF65 is prevented from binding, factors in the spliceosome can no longer be recruited and the following exon is skipped. Furthermore, I found that MBNL1 and U2AF65 compete by binding mutually exclusive RNA structures. I also characterized a potential therapeutic approach for DM. Current data suggest that if MBNL1 is released from sequestration, disease symptoms may be alleviated. Using a targeted screen of small molecules known to bind structured nucleic acids, I identified the small molecule pentamidine as a compound that disrupted MBNL1 binding to CUG repeats in vitro . I showed in cell culture that pentamidine was able to reverse the mis-splicing of two pre-mRNAs affected in DM. Pentamidine also significantly reduced the formation of RNA foci in tissue culture cells, which are characteristic of DM. MBNL1 was released from the foci in the treated cells. Furthermore, pentamidine partially rescued splicing defects of two pre-mRNAs in mice expressing expanded CUG repeats. This dissertation includes three previously published co-authored publications.