MBNL1 binds GC motifs embedded in pyrimidines to regulate alternative splicing

Muscleblind-like 1 (MBNL1) regulates alternative splicing and is a key player in the disease mechanism of myotonic dystrophy (DM). In DM, MBNL1 becomes sequestered to expanded CUG/CCUG repeat RNAs resulting in splicing defects, which lead to disease symptoms. In order to understand MBNL1’s role in both the disease mechanism of DM and alternative splicing regulation, we sought to identify its RNA-binding motif. A doped SELEX was performed on a known MBNL1-binding site. After five rounds of SELEX, MBNL1 selected pyrimidine-rich RNAs containing YGCY motifs. Insertion of multiple YGCY motifs into a normally MBNL1-independent splicing reporter was sufficient to promote regulation by MBNL1. MBNL1 was also shown to regulate the splicing of exon 22 in the ATP2A1 pre-mRNA, an exon mis-spliced in DM, via YGCY motifs. A search for YGCY motifs in 24 pre-mRNA transcripts that are mis-spliced in DM1 patients revealed an interesting pattern relative to the regulated exon. The intronic regions upstream of exons that are excluded in normal tissues relative to DM1, are enriched in YGCY motifs. Meanwhile, the intronic regions downstream of exons that are included in normal tissues relative to DM1, are enriched in YGCY motifs

Auto-Regulation of the MBNL1 Pre-mRNA

xiv, 59 p. : ill. (some col.)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.Committee in charge: Kenneth Prehoda, Chair; J. Andrew Berglund, Advisor; Victoria J. de Rose, Member; Alice Barkan, Member; Karen Guillemin, Outside Membe