Ancestral intronic splicing regulatory elements in the SCNα gene family

SCNα genes encode components of voltage-gated sodium channels that are crucial for generating electrical signals. Humans have ten paralogous SCNα genes, some of which contain duplicated mutually exclusive exons 5a and 5b. In reconstructing their evolutionary history, we found multiple unannotated copies of exon 5 in distant species and showed that exon 5 duplication goes back to a common ancestor of the SCNα gene family. We char-acterized splicing patterns of exons 5a and 5b across tissues, tumors, and developmental stages, and demonstrated that the nonsense mediated decay (NMD) system is not the ma-jor factor contributing to their mutually exclusive choice. Comparison of SCN2A , SCN3A , SCN5A , and SCN9A intronic nucleotide sequences revealed multiple Rbfox2 binding sites and two highly conserved intronic splicing regulatory elements (ISRE) that are shared be-tween paralogs. Minigene mutagenesis and blockage by antisense oligonucleotides showed that the formation of RNA structure between ISRE promotes exon 5b skipping in SCN9A . The inclusion of exon 5b is also suppressed in siRNA-mediated knockdown of Rbfox2, which makes the collective action of RNA structure and Rbfox2 compatible with the model of a structural RNA bridge. ISRE sequences are conserved from human to elephant shark and may represent an ancient, evolutionarily conserved regulatory mechanism. Our results demonstrate the power of comparative sequences analysis in application to paralogs for elucidating splicing regulatory programs.