Pervasive non-triplet alternative splicing drives functional isoform diversity

Abstract Alternative mRNA splicing is an important mechanism for regulating gene expression and generating transcriptomic diversity. Most cases of alternative splicing studied to date are triplet, meaning that both isoforms retain the same translational reading frame. Indeed, non-triplet alternative splicing is sometimes considered evidence of splicing errors or noise. Nevertheless, some examples of functionally important non-triplet alternative splicing exist. We set out to determine the global prevalence, regulation, and function of non-triplet alternative splicing in vivo in C. elegans . Here we use RNA-Seq and bioinformatic analysis of wild-type and NMD-deficient mutants to categorize the molecular consequences of non-triplet alternative splicing into three classes: NMD-sensitive isoforms, alternative C-terminal length isoforms, and dual-coding isoforms. We identify hundreds of non-triplet alternative splicing events across these three categories. Genetic and molecular analyses reveal cases of developmental regulation, splicing factor autoregulation, cell-specific splicing, and physiologically important isoform-specific function. Analysis of human transcriptomes reveals broadly similar patterns and distributions of non-triplet alternative splicing. Together these experiments demonstrate the importance of non-triplets, a large but underappreciated class of alternative splicing, for regulating gene expression and generating protein-coding diversity.