Fig. 5: IWS1 phosphorylation controls the CDCA5/ERK phosphorylation feedback loop, through U2AF2 alternative RNA splicing. | Nature Communications

Fig. 5: IWS1 phosphorylation controls the CDCA5/ERK phosphorylation feedback loop, through U2AF2 alternative RNA splicing.

From: RETRACTED ARTICLE: AKT3-mediated IWS1 phosphorylation promotes the proliferation of EGFR-mutant lung adenocarcinomas through cell cycle-regulated U2AF2 RNA splicing

Fig. 5

a Functional domain composition of U2AF65. Numbers mark the domain boundaries. Exon 2 encodes the RS domain. b HEK-293T cells were transduced with the V5-tagged splice variants of U2AF2, encoding V5-U2AF65α and V5-U2AF65β. Anti V5-U2AF65 immunoprecipitates and input lysates were probed with the indicated antibodies. c Anti-U2AF65, anti-IgG mouse isotype control immunoprecipitates and input lysates of NCI-H522 cells transduced with the indicated constructs were probed with antibodies, as shown. d (Upper panel) The ratio of spliced to unspliced CDCA5 and GUSB (control) RNAs was measured in the indicated NCI-H522 cells by qRT-PCR. (Middle and Lower panels) RIP assays of U2AF65 and Prp19 in the same cells. Anti-U2AF65 or anti-Prp19 IP, vs IgG control IP were used to calculate the mean fold binding enrichment in the indicated RNA regions ±SD. Primers listed in Supplementary Table 2. Primer location in Supplementary Fig. 7b. e (Upper panel) shIWS1 rescue by U2AF65α and U2AF65β. Spliced/unspliced RNA ratios of CDCA5 and GUSB, measured by qRT-PCR, as in Fig. 5d. (Middle and Lower panels) RIP assays of U2AF65 and Prp19 in the same cells. Data presented as in figure 5d. f Cytosolic CDCA5 mRNA levels in the indicated shIWS1-transduced cells were rescued by IWS1 and U2AF65α, as determined by q-RT-PCR. GAPDH-normalized CDCA5 mRNA ±SD. Validation of fractionation in Supplementary Fig. 7e. g Lysates of NCI-H522 cells, transduced with the indicated constructs, were probed with antibodies, as shown. DM-A and DM-E are the S79/S209AA and S79/S209EE Sororin mutants, respectively. h (Upper panels) Lysates of KRAS and EGFR mutant cell lines, transduced with the indicated constructs, were probed with antibodies as shown. RT-PCR, using U2AF2 exon 1 and 3 primers. (Lower Left panel) Bars show the qRT-PCR-determined U2AF2 E2/E3 ratios in NCI-H522 and NCI-H1299 cells, and in the KRAS and EGFR mutant cell lines, relative to the shControl±SD. (Lower Right panel) Bars show the shIWS1-induced percent reduction of ERK-phosphorylation in the same cell lines, normalized to tubulin ± SD. EGFR mutant and non-mutant cells were compared, using a one-way ANOVA. All assays were in triplicate, on three biological replicates. n.s non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (one-sided unpaired t-test).

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