Regulatory interplay between SR proteins governs<i>CLK1</i>kinase splice variants production

The CLK1 kinase phosphorylates SR proteins to modulate their splicing regulatory activity. Skipping of alternative exon 4 on the <i>CLK1</i> pre-mRNA produces a CLK1 variant lacking the catalytic site. Here, we aimed to understand how various SR proteins integrate into the regulatory program that controls <i>CLK1</i> exon 4 splicing. Previously, we observed that the depletion of SRSF10 promoted the inclusion of <i>CLK1</i> exon 4. Using the expression of tagged proteins and CRISPR/Cas9-mediated knockouts in HCT116 cells, we now identify TRA2β, TRA2α, SRSF4, SRSF5, SRSF7, SRSF8, and SRSF9 as activators of exon 4 inclusion. In contrast, SRSF3, SRSF10, and SRSF12 elicit exon 4 skipping. Using CRISPR/dCas13Rx and RNA immunoprecipitation assays, we map an enhancer in exon 4 interacting with TRA2β. Notably, CLK1 kinase inhibitors antagonized the repressor activity of HA-SRSF10, HA-SRSF12, and HA-SRSF3. Our results suggest that <i>CLK1</i> exon 4 inclusion is determined primarily by a balance between the activities of TRA2 proteins and CLK-phosphorylated SRSF3. CLK-phosphorylated SRSF10 and SRSF12 would interact with TRA2 proteins to prevent their enhancer activity, allowing SRSF3 to enforce exon 4 skipping more efficiently. Our study provides insight into the complex regulatory network controlling the alternative splicing of <i>CLK1</i>, which uses CLK1-mediated phosphorylation of SR proteins to regulate the inclusion of catalytic exon 4 in <i>CLK1</i> transcripts.