Figure 3.

RBM4 mediates the exclusion of the MEF2Cγ region by recruiting the U2AF65 subunit in a CU element-dependent manner. (A) The diagram presents the wild-type and mutant MEF2C minigenes in an overexpressing plasmid. (B) C3H10T1/2 cells were transfected with the MEF2C minigene vector and cultured in growth (GM) or differentiating medium (DM). (C) C3H10T1/2 cells were cotransfected with the MEF2C minigene vector and a mock vector, or the FLAG-RBM4-expressing vector, or the FLAG-RBM4 Iso2-expressing vector. Transfected cells were cultured in GM for 24 h. (D) The wild-type (CU) or mutant (CUgg) MEF2C minigene vector was cotransfected with the mock vector, or the FLAG-RBM4-expressing vector into C3H10T1/2 cells. RT-PCR and immunoblotting assays were performed as described in a previous panel. The number below the gel indicates the relative ratio of MEF2Cγ- over the total MEF2C transcripts using TotalLab Quant Software. (E) The schematic shows the probe sequence flanking (italics) and within the MEF2Cγ region, with the putative polypyrimidine tract and splice site underlined. (F) The mock eluate (E) from the Ni2+ agarose resin or recombinant His-tagged RBM4 protein (1, 2, or 4 μg) was incubated with 10 nM DIG-labeled wild-type (CU) or mutant (CUgg) probes. The mixtures were fractionated in an 8% native acrylamide gel and transferred to a Nylon membrane. The membrane was probed using an HRP-conjugated anti-DIG Fab fragment. (G) Nuclear extracts prepared from C3H10T1/2 cells were incubated with the DIG-labeled CU or CUgg probes and recombinant RBM4 protein, followed by UV crosslinking. Reactions were analyzed using an immunoblotting assay with the indicated antibody. The bar graph shows the relative ratio of MEF2Cγ- over total MEF2C transcripts and the relative level of UV-crosslinked proteins using TotalLab Quant Software (ND, no difference; * p < 0.05; ** p < 0.01; *** p < 0.005).