CONTRIBUTION OF MUSCLEBLIND-LIKE1 (MBNL1) BINDING EVENTS TO DOSE-RESPONSE BEHAVIOR OF MBNL1
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Mis-regulation of the alternative splicing factor muscleblind-like I (MBNL 1) plays a significant role in the disease myotonic dystrophy (OM). MBNL 1 regulates alternative splicing of genes involved in development of skeletal muscle, heart and the central nervous system. In myotonic dystrophy the lack of properly localized MBNL 1 leads to mis-splicing of many pre-mRNAs. One of the mis-spliced pre-mRNAs is the MBNLJ pre-mRNA that codes for the MBNL 1 protein (an auto-regulated event). Specifically, the mis-splicing is aberrant inclusion of exon 5 in the MBNLJ pre-mRNA. Previous work has shown that intron 4 of the MBNLJ gene is highly conserved and contains multiple MBNLI binding sites. It has been shown that a 90-nucleotide region within intron 4 is necessary for regulation by MBNL 1. This study investigates the sensitivity of the auto-regulated MBNLJ splicing event by generating MBNL 1 dose-response curves using HEK293 cells with an inducible MBNL 1 expression system. Coupling this expression system with MBNLJ constructs containing deletions of various MBNLI binding sites within the important 90 nucleotide regulation region, it was shown that a single central MBNL1 site was fundamentally more important that other sites for splicing regulation.