Alternative pre-mRNA splicing has a major impact on cellular functions and development with the potential to fine-tune cellular localization, posttranslational modification, interaction properties, and expression levels of cognate proteins. The plasticity of regulation sets the stage for cells to adjust the relative levels of spliced mRNA isoforms in response to stress or stimulation. As part of an exon profiling analysis of mouse cortical neurons stimulated with high KCl to induce membrane depolarization, we detected a previously unrecognized exon (E24a) of the CASK gene, which encodes for a conserved peptide insertion in the guanylate kinase interaction domain. Comparative sequence analysis shows that E24a appeared selectively in mammalian CASK genes as part of a >3,000 base pair intron insertion. We demonstrate that a combination of a naturally defective 5 splice site and negative regulation by several splicing factors, including SC35 (SRSF2) and ASF/SF2 (SRSF1), drives E24a skipping in most cell types. However, this negative regulation is countered with an observed increase in E24a inclusion after neuronal stimulation and NMDA receptor signaling. Taken together, E24a is typically a skipped exon, which awakens during neuronal stimulation with the potential to diversify the protein interaction properties of the CASK polypeptide. 1. Introduction Alternative pre-mRNA splicing generates protein diversity throughout the transcriptome, whereas mistakes in its regulation underlie a variety of human diseases [1, 2]. Mechanisms of exon skipping and inclusion, as well as 5′ and 3′ splice site selection, are commonly used to produce multiple mRNA isoforms from a single gene. Regulation occurs during the dynamic stages of spliceosome assembly and involves the interactions of small nuclear ribonucleoprotein complexes (snRNPs) and numerous protein factors, such as SR, hnRNP, and KH-type splicing factors, with the pre-mRNA [3, 4]. Internal cassette exons are recognized through interactions at the 5′ splice site with U1 followed by U6 snRNP, together with interactions at the branch site/3′ splice site region with U2 snRNP and U2 auxiliary factor (U2AF). Members of the SR protein family contribute essential roles as enhancers of exon recognition when the splice sites are less than ideal, which is typically the case for mammals. Their modular protein structures allow for the simultaneous recognition of exonic RNA sequence motifs (via the N-terminal RNA-binding domain) and components of U1 snRNP and/or U2AF bound to their respective splice sites (via the C-terminal
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