Fig. 2

SRSF10 controls alternative splicing of pre-CDC25A through exon 6 skipping. A Pie graph depicting the alternative splicing events after splicing factor SRSF10 knockdown in Hep3B cells. The exon skipping accounted for large part of the AS events. B GO enrichment for potential targets of SRSF10. The top 5 upregulated and 5 downregulated pathways after SRSF10 knockdown are shown, which converged the AS events toward G2/M pathways. C Heatmap of cell-cycle related genes encompassing significantly changed exon skipping events under the SRSF10 silencing condition. CDC25A☆ ranked the highest and was selected as a potential target. All experiments were conducted in triplicate, and the results are presented as the log value (base 2) of the sh-SRSF10/control cell ratio. D Preliminary comparison of exon 6 skipping events between sh-SRSF10 and the control group. E Exon skipping in CDC25A measured in Hep3B cell line with stable knockdown of SRSF10 and the control. Left panel, mean ± SD of the percent-splice-in (PSI) from three experiments; the q value was calculated by paired Student’s t test. Right panel, diagram of exon 6 inclusive (Long, L) and exclusive (Exon 6 deleted, △E6) isoforms in the fate of pre-CDC25A splicing. F Schematic of three designed primers for the following semi-RT‒PCR. Primer set 1 was utilized to amplify all exon 6 inclusive and exclusive variants, while primer sets 2 and 3 were specific for only the L and △E6 isoforms individually, respectively. G Alternative splicing of CDC25A in exon 6 was examined by RT‒PCR (primer set 1) and western blot in two HCC cell lines. The enhanced CDC25A exon 6 missing isoform was verified under SRSF10 overexpression, while deleting SRSF10 resulted in long isoform retention. H Direct binding between the indicated SRSF10 protein and endogenous CDC25A RNA fragments was verified by CLIP-qPCR assay and Flag-western blotting detection. Flag-tagged SRSF10 wild type (WT) and motif RRM1 (11-84aa) deletion mutants (△RRM) were constructed, and the upper panel demonstrates that SRSF10 RRM1 deletion strikingly decreased its binding to exon 6. In the lower panel, immunoblotting with Flag-SRSF10 showed the ideal input of foreign SRSF10 protein, and displayed similar binding ability of SRSF10(WT) and SRSF10(△RRM) to endogenous CDC25A mRNA. I SRSF10 (△RRM) was unable to restore CDC25A exon 6 skipping. CDC25A exon 6 inclusive and exclusive isoforms and their corresponding proteins were detected under SRSF10 silencing in two HCC cell lines. The SRSF10(WT) substantially restored the endogenous SRSF10 pattern, whereas SRSF10 (△RRM) displayed no effects in exon 6 exclusion and remained responsive to SRSF10 deprivation