Fig 1: The regulation of the alternative splicing of the U2AF2 exon 2 by IWS1 and IWS1 phosphorylation, depends on the p52 isoform of the H3K36me3 reader LEDGF and its splicing partner SRSF1.a (Upper panel) Lysates of the indicated cells were probed with antibodies, as shown. RT-PCR addressing exon 2 U2AF2 splicing in the same cells. (Middle and Lower Panels) U2AF2 E2/E3 ±SD, was determined by quantification of the RT-PCR results and by qRT-PCR. b (Upper Panel) Lysates of NCI-H522 cells transfected with the indicated siRNAs, probed with antibodies, as shown. RT-PCR addressing exon 2 U2AF2 splicing. (Middle and Lower Panels) U2AF2 E2/E3 ±SD, was determined by quantification of the RT-PCR results and by qRT-PCR. c (Upper panel) Domains of the p52 and p75 LEDGF isoforms (see Ferris et al., 201050). (Middle panel) Western blot of the indicated NCI-H522 cells, probed for LEDGF expression and RT-PCR, addressing U2AF2 exon 2 splicing in the same cells. (Lower panels) U2AF2 E2/E3 ratio ±SD, was determined by quantification of the RT-PCR results and by qRT-PCR. d ChIP of p52/LEDGF in NCI-H522 cells, transduced with V5-p52/LEDGF. Mean fold enrichment in p52/LEDGF binding (anti-V5 IP, vs IgG control IP) to the indicated U2AF2 regions±SD. V5-p52/LEDGF expression, in figure S5G. e (Upper panel) Immunoblotting, showing SRSF1 expression in the indicated NCI-H522 cells. RT-PCR addressing U2AF2 exon 2 splicing. (Lower panels) U2AF2 E2/E3 ratio ±SD, was determined by quantification of the RT-PCR results and by qRT-PCR. f SRSF1 ChIP in the indicated V5-SRSF1-transduced NCI-H522 cells. Mean fold enrichment in SRSF1 binding (anti-V5 IP, vs IgG control IP) to the indicated U2AF2 regions±SD. V5-SRSF1 expression in Supplementary figure S6b. g RIP, addressing SRSF1 binding to U2AF2 RNA in panel f cells. Mean fold enrichment in SRSF1 binding (anti-V5 IP, vs IgG control IP) in the indicated U2AF2 regions ±SD. All assays were done, using three biological replicates, in triplicate. n.s non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (one-sided unpaired t-test). h Phosphorylation of IWS1 recruits SETD2 in CTD of RNA Pol II. SetD2 trimethylates histone H3K36 co-transcriptionally. p52/LEDGF and its partner SRSF1, bind histone H3K36me3 promoting exon 2 inclusion in the mature U2AF2 transcript.