Combinatorial Mutagenesis of MBNL1 Zinc Fingers Elucidates Distinct Classes of Regulatory Events

Abstract

The RNA binding protein and alternative splicing factor Muscleblind-like 1 (MBNL1) has been a topic of intense study due to its role in myotonic dystrophy (DM) pathogenesis. MBNL1 contains four zinc finger (ZF) RNA binding domains arranged in two pairs. Through combinatorial mutagenesis of the ZF domains, we demonstrate that the pairs of ZFs have differential affinity for RNA and subsequently differential splicing activities. We evaluated splicing and binding activity for six MBNL1-mediated splicing events and found that the splicing activity profiles for the ZF mutants vary among transcripts. Clustering analysis of splicing profiles revealed that two distinct classes of MBNL1 pre-mRNA substrates exist. For some of the RNA transcripts tested, binding and splicing activity of the ZF mutants correlated. However, for some transcripts it appears that MBNL1 exerts robust splicing activity in the absence of RNA binding. We demonstrate that functionally distinct classes of MBNL1-mediated splicing events exist as defined by requirements for ZF-RNA interactions.