(A) Schematic of the exon/intron structure of Srsf10. Usage of the downstream (dn) major splice site in Srsf10 exon 2 (GT.AG, U2-type) leads to exon 3 inclusion and a non-protein coding isoform, while usage of the upstream minor splice site (AT.AC, U12-type) results in exon 4 inclusion. A minor 5’ splice site in exon 3 is present but not used in the endogenous context (dotted lines). * indicate stop codons. (B) Srsf10 minigene splicing upon siRNA-mediated knockdown of endogenous Srsf10. Top: exon/intron structure of the Srsf10 minigene reporter containing mouse exons 2 to 4 (and complete intervening introns) with indicated primer binding sites (arrows). HeLa cells were transfected with control siRNA (siCtrl) or against human Srsf10 (siSrsf10), incubated for 24 hr followed by minigene transfection. After 48 hr splicing was analyzed with the indicated primers. Bottom: exemplary gel and quantification of the dn-E3 isoform (n = 5, mean ± SD). (C) Knockdown and rescue of SRSF10. Top: Western Blot of SRSF10 after siRNA-mediated knockdown and transfection with overexpression vectors for the different GFP-tagged Srsf10 isoforms. VINCULIN was used as loading control. Middle: Exemplary gel of Srsf10 minigene splicing upon knockdown and rescue. Bottom: Quantification of the dn-E3 isoform (n ≥ 3, mean ± SD). (D) Exon/intron structure of the Srsf10 minigenes used for mutational analysis. Exon and intron sequences were replaced by sequences containing a minor intron from glia maturation factor beta (Gmfb exons 4 to 5 including the minor intron 4, marked in red). Below the sequence of the identified ESE is shown. (E) Quantification of Srsf10 minigene splicing upon knockdown and rescue. HeLa cells were transfected with the mutated minigenes (D) and analyzed as in (B). Splicing of mutants is shown relative to the wt from (B) and for each mutant relative to the Ctrl siRNA (n = 5, mean ± SD). Student’s t test-derived p values *p<0.05, **p<0.01, ***p<0.001.