Modulation of alternative splicing of trafficking genes by genome editing reveals functional consequences in muscle biology

Abstract

Alternative splicing is a regulatory mechanism by which multiple mRNA isoforms are generated from single genes. Numerous genes that encode membrane trafficking proteins are alternatively spliced. However, there is limited information about the functional consequences that result from these splicing transitions. Here, we developed appropriate tools to study the functional impact of alternative splicing in development within the most in vivo context. Secondly, we provided evidence of the physiological implications of splicing regulation during muscle development. Our previous work in mouse heart development identified three trafficking genes that are regulated by alternative splicing between birth and adulthood: the clathrin heavy chain, the clathrin light chain-a, and the trafficking kinesin binding protein-1. Here, we demonstrated that alternative splicing regulation of these three genes is tissue- and developmental stage-specific. To identify the functional consequences of splicing regulation in vivo, we used genome editing to block the neonatal-to-adult splicing transitions. We characterized the phenotype of one of these mouse lines and demonstrated that when splicing regulation of the clathrin heavy chain gene is prevented mice exhibit an increase in body and muscle weights which is due to an enlargement in myofiber size. The significance of this work has two components. First, we revealed novel roles of the clathrin heavy chain in muscle growth and showed that its regulation by alternative splicing contributes to muscle development. Second, the new mouse lines will provide a useful tool to study how splicing regulation of three trafficking genes affects tissue identity acquisition and maturation in vivo.