Fig 1: HnRNP F induces YAP 3'UTR splicing and the effect of hnRNP F on YAP 3'UTR-mediated mRNA stability is alternative splicing dependent. (A). PC-3 cells were transfected with siNon, sihnRNPU, sihnRNPF (40 nM each) (left panel), or were transfected with empty vector, pFRT-TO-HIS-FLAG-HA-hnRNPU or p3X-FLAG-hnRNPF at 0.5 µg/mL (right panel). Twenty-four h after incubation, total RNA was isolated and the semi-quantitative RT-PCR assay was performed (n = 3). PCR fragments were amplified by RT-PCR using primers as described in the Figure 1C, to specifically amplify the full-length YAP 3'UTR and alternatively spliced YAP 3'UTR. ß-actin gene was used as an internal control for RNA quality and loading. Significance was tested using one-way analysis of variance (ANOVA) with Holm–Sidak method post hoc test, where * p < 0.05 compared to the control. (B). Schematic diagram of the luciferase reporter used in assay. Another G-tract located in full-length YAP 3'UTR at 3219 to 3230 was not shown in this diagram. (C). PC-3 cells were transfected with 0.5 µg of pGL3-YAP(AS)-3'UTR-Luc, pGL3-YAP-3'UTR-Luc or pGL3-YAP-3'UTR-Mut-Luc along with 40 nM siRNA against non-silencing control (siNon) or hnRNP F (sihnRNPF) for 24 h (n = 3). The cells were also cotransfected with a pSV-ß-galactosidase vector, and the ß-galactosidase activity was used to normalize the luciferase activity. Luciferase activity is presented as the fold induction relative to the control. Data are means ± s.d from triplicate analysis. Significance is tested using ONE-WAY ANOVA with Holm-Sidak method post hoc test, where * denotes p < 0.05 compared to the siNon transfected cells; where # denotes p < 0.05 compared to the cells cotransfected with pGL3-YAP(AS)-3'UTR-Luc and same siRNA.