Alternative Splicing is Coordinated by RNA-binding Proteins and Impacts Transcript Stability During Myogenesis

Alternative splicing of pre-mRNA is a co- and post-transcriptional process that occurs in more than 90% of human genes. By selectively including or excluding specific regions, alternative splicing expands the proteome from a limited number of genes. Tissue-specific RNA-binding proteins (RBPs) bind sequences surrounding an alternative exon to control splicing decisions. Interestingly, the stability of an alternative splice form can affect whether the mRNA is translated into a functional protein. However, it is not known how RBPs and transcript stability might contribute to isoform-specific expression during skeletal muscle development and differentiation (myogenesis). Here, we hypothesized that alternative splicing is governed in part by specific RBPs during myogenesis and that the stability of splice isoforms contributes to how these isoforms function in the regulation of muscle maturation. To investigate the regulation and expression of alternative splice forms in muscle, we utilized the mouse C2C12 muscle cell line that undergoes myogenesis and mimics developmental splicing transitions observed in skeletal muscle in vivo. We found that the RBPs muscle-blind (MBNL) 1, MBNL2, polypyrimidine tract binding protein (PTBP) 1, and PTBP2 were downregulated during myogenesis while CUGBP elav-like family member 2 (CELF2) was upregulated, as measured by western blot. Expression of the CELF1 and quaking (OKI) proteins were unchanged during myogenesis. Several splicing transitions were altered during C2C12 cell differentiation when specific RBPs were depleted. We further demonstrated that alternatively spliced exons from several genes contribute to transcript stability by either promoting or inhibiting mRNA degradation. Alternative exons may alter microRNA binding sites, and we have identified potential binding sites in transcripts with different stabilities. Together, these data implicate a complex model of specific temporal regulation of alternatively spliced gene expression in muscle, which could be used in the future as a potential therapeutic mechanism to understand and combat disease progression.Bachelor of Scienc