Abstract 531: Mechanistic and Functional Differences in Rna Binding and Processing Activities of the Muscle- and Non-muscle Rbfox2 Isoforms

Abstract

Myotonic Dystrophy type 1 (DM1), the most prevalent form of adult onset muscular dystrophy, is caused by CTG trinucleotide repeat expansion in the 3’-UTR of DMPK gene. Over 80% of DM1 patients exhibit heart dysfunctions, which are the second leading cause for DM1-related deaths. Recently, we demonstrated that aberrant expression of a non-muscle splice isoform of RNA-binding protein RBFOX2 triggers cardiac conduction delay, atrioventricular heart blocks, and spontaneous arrhythmogenesis in DM1 heart. RBFOX2 is a master regulator of tissue-specific alternative splicing and a pair of mutually exclusive 43-nucleotide(nt) and 40-nt exons in its C-terminal domain encode the muscle (RBFOX2 43 ) and non-muscle (RBFOX2 40 ) isoforms. The RBFOX2 40 isoform is predominantly expressed in the fetal heart, and is replaced by the RBFOX2 43 isoform in development, specifically within the cardiomyocytes of adult hearts. To deconstruct the splicing regulatory networks of RBFOX2 43 and RBFOX2 40 isoforms, characterize their respective RNA binding landscapes, and determine the RBFOX2 40 -driven transcriptome alterations in DM1 heart tissue, we performed eCLIP and high-resolution RNA-sequencing studies on cardiomyocytes isolated from wild type (expressing the normal muscle-specific RBFOX2 43 isoform), Rbfox2 Δ43/Δ43 (expressing the non-muscle RBFOX2 40 isoform), and RBFOX2 40 overexpressing (OE) mice. By integrating genome-wide RNA binding and processing activities for the two RBFOX2 isoforms, we found that a switch from the muscle-specific (RBFOX2 43 ) to non-muscle (RBFOX2 40 ) isoform provokes DM1-like cardiac pathology by altering the mRNA abundance and splicing of genes encoding components of the conduction system and/or contractile apparatus. Further, through subnuclear fractionation and protein-protein interaction studies, we demonstrate that the higher-order assembly of LASR (large assembly of splicing regulators) complexes formed by the RBFOX2 40 isoform boost its splicing activity and promote the generation of pathogenic splice variants of voltage-gated ion channels and other components of the cardiac conduction system.