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. 2016 Jun 13;7:ncomms11840. doi: 10.1038/ncomms11840

Figure 1. Identification of an alternative splicing isoform of TEAD4 regulated by RBM4.

Figure 1

(a) Schematics of human TEAD4 pre-mRNA and protein. The full-length TEAD4 contains 1–12 exons, including the DNA-binding domain in the N terminal, whereas exon 3 of TEAD4 can be skipped to generate a new isoform whose translation is started at exon 6 to produce a truncated TEAD4-S protein. TEAD4-S lacks an N-terminal DNA-binding domain, but still contains an intact C-terminal YAP-binding motif. (b) The localizations of TEAD4-FL and TEAD4-S were determined by immunofluorescence assay. Flag-tagged TEAD4-FL or TEAD4-S was transfected into cells. The immunofluorescence assay was performed with anti-Flag antibody to show the localizations of Flag-TEAD4. Scale bar, 25 μm. (c) Interaction of two TEAD4 isoforms with YAP protein as judged by co-IP experiment. The Flag-tagged TEAD4-FL or TEAD-S was co-transfected with HA-tagged YAP into 293T cells, and the binding of YAP was detected. (d) 293 cells expressing RBM4 on tetracycline induction were collected at different time points after induction to determine the expression levels of TEAD4-FL and TEAD4-S. (e) RBM4 regulates the splicing of TEAD4 in various cancer cells, including PANC1, HT1080, MDA-MB-231 and HepG2. The cells were stably transfected with RBM4 or vector control, and the splicing of TEAD4 was examined by RT–PCR. The representative gels were shown. (f) The schematic of TEAD4 pre-mRNA where the potential RBM4-binding site (CGGCCGG) in red. TEAD4 splicing reporters with the indicated mutations (mut1: CTTATA and mut2: GTAACG) were generated (upper panel). TEAD4 splicing reporters containing wild-type RBM4-binding site (lower-left panel) or mutations (lower-right panel) were co-expressed with RBM4 or vector control in 293T cells to assay for the splicing change of TEAD4. Representative gels from triplicate experiments were shown, with the mean±s.d. of TEAD4-S% plotted below representative gels. (g) Binding of TEAD4 pre-mRNAs with RBM4 was examined by RNA-immunoprecipitation assay in cells exogenously expression FLAG-RBM4 or vector control. (h) 293 cells were co-transfected with Flag-RBM4 or vector control and the indicated mutant or wild-type (WT) TEAD4 reporters, and then immunoprecipitated with anti-Flag antibody. The co-precipitated RNAs were detected by RT–PCR.