Fig. 4: Srsf1-mediated alternative splicing promotes EDB exon inclusion.

a Representation of the FN pre-mRNA containing two conserved GA-rich exonic splicing enhancers (ESEs) in extra exon 25 coding for the EDB domain and multiple sequence alignment from 9 different vertebrate species. Letter size indicates conservation and asterisks show identical nucleic acids. b Left: Molecular docking of the Srsf1 RRM2 (orange) with the ESE1 RNA motif 5’- AGGAGAAG-3’ (green and yellow) showing hydrogen bond interactions. Right: Comparison between the published structure of human RRM2 with 5’-UGAAGGAC-3’ (grey)²⁶ and the docking results for 5’-AGGAGAAG-3’ (green) showing two major differences in positioning (U → A and C → G). c Semi-quantiative PCR analysis of Srsf1 expression in early passage cultured FAPs and freshly isolated cultured MuSC-derived myoblasts. d PCR analysis and PSI quantification of endogenous EDB splicing of the FN mRNA in MuSC-derived myoblasts treated with siSrsf1 compared to the siSCR control. Two different siRNAs targeting Srsf1 with the suffix _a and _b were designed. e Schematic of the minigene construct containing the extra EDB exon 25 and flanking regions including exon 24 and 26 and graphical representation of the experimental workflow to assess the effects of Srsf1 knockdown. Arrows indicate primers used to assess mingene specific transcripts independent from endogenous FN. f PCR analysis and PSI quantifcation of EDB splicing of the FN minigene mRNA in MuSC-derived myoblasts after transfection with increasing amounts of siSrsf1 compared to the siSCR control. g PCR analysis and PSI quantification of EDB splicing of the FN minigene mRNA in mouse fibroblasts overexpressing Srsf1 or the empty vector (EV) control. h Schematic of the proposed working model for EDB exon splicing. In MuSCs, Srsf1 enhances EDB exon 25 inclusion through RRM binding to GA-rich motifs in the FN pre-mRNA. In FAPs, Srsf1 levels are low and the EDB exon 25 is spliced. Bars represent means ± SEM from myoblasts isolated from n = 3 biological replicates per condition, each from separate young mice. P-values were calculated using a one-tailed Student’s t-test (c,g) or one-way ANOVA with Dunnett’s (d, f) post-hoc test. ***p < 0.001, **p < 0.01, *p < 0.05. Source data are provided as a Source Data file.