Fig. 4: 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. | Nature Communications

Fig. 4: 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.

From: RETRACTED ARTICLE: AKT3-mediated IWS1 phosphorylation promotes the proliferation of EGFR-mutant lung adenocarcinomas through cell cycle-regulated U2AF2 RNA splicing

Fig. 4

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.

Back to article page