| Muscleblind-like 1 (MBNL1) is a splicing factor whose improper cellular localization is a central component of myotonic dystrophy (DM). In DM, the lack of properly localized MBNL1 leads to mis-splicing of many pre-mRNAs. The mechanism by which MBNL1 regulates it pre-mRNA targets is not well understood. In order to determine the mechanism by which MBNL1 regulates alternative splicing, a consensus RNA binding motif for Mbl (the Drosophila ortholog of MBNL1) and MBNL1 were determined using SELEX (Systematic Evolution of Ligands by Exponential Enrichment). These consensus motifs allowed for the identification of high affinity endogenous sites within pre-mRNAs that are regulated by MBNL1. In vitro binding studies showed that MBNL1 bound to RNAs that contained the consensus motif surrounded by pyrimidines.;Some of these sites were identified upstream of exon 5 within the MBNL1 pre-mRNA, and we have shown that MBNL1 auto-regulates the exclusion of exon 5 in HeLa cells. The region of the MBNL1 gene that includes exon 5 and flanking intronic sequence is highly conserved in vertebrate genomes. The 3' end of intron 4 is non-canonical in that it contains an AAG 3' splice site and a predicted branchpoint that is 141 nucleotides from the 3' splice site. Using a mini-gene that includes exon 4, intron 4, exon 5, intron 5 and exon 6 of MBNL1, we show that MBNL1 regulates inclusion of exon 5. Mapping of the intron 4 branchpoint confirms that branching occurs primarily at the predicted distant branchpoint. Structure probing and footprinting reveal that the highly conserved region between the branchpoint and the 3' splice site is primarily unstructured, and MBNL1 binds within this region of the pre-mRNA, which we have termed the MBNL1 response element. Deletion of the response element eliminates MBNL1 splicing regulation and leads to complete inclusion of exon 5, which is consistent with the suppressive effect of MBNL1 on splicing.;This dissertation includes previously published co-authored material as well as my recent co-authored material that has been submitted for publication. |
