Abstract
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.
Highlights
Alternative pre-mRNA splicing generates protein diversity throughout the transcriptome, whereas mistakes in its regulation underlie a variety of human diseases [1, 2]
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
Internal cassette exons are recognized through interactions at the 5 splice site with U1 followed by U6 small nuclear ribonucleoprotein complexes (snRNPs), together with interactions at the branch site/3 splice site region with U2 snRNP and U2 auxiliary factor (U2AF)
Summary
Alternative pre-mRNA splicing generates protein diversity throughout the transcriptome, whereas mistakes in its regulation underlie a variety of human diseases [1, 2]. Their silencing and enhancing roles depend upon the RNA code or map of the relative positions of their binding sites in the pre-mRNA and whether or not their functions antagonize or promote those of the core snRNPs. Tissue and developmental stage-specific expression and modification of these factors modulate splicing changes in a spatial and temporal manner. Roles for UAGG motifs, splicing factor hnRNP A1, and functional NMDA receptors have been implicated in the inducible skipping of E19 of the NMDA R1 transcript in cortical neurons stimulated with high KCl [18] While these studies highlight insights into mechanisms of inducible exon skipping, comparatively less is known about inducible exon inclusion. These results suggest a model in which E24a is in the midst of an evolutionary transition from that of a pseudoexon harbored in an intron sequence and that of a functional coding exon emerging with the potential to diversify the properties of the CASK polypeptide when activated by cellular stimulation
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