FIG. 6.

RBM4 acts antagonistically to PTB in alternative α-TM pre-mRNA splicing. The pαTM-8/9a/9b (A) or pαTM-1/2b/3 (B) minigene was cotransfected into HEK 293 cells along with the expression vector encoding FLAG-tagged PTB alone (lanes 2) or with increasing amounts of the vector encoding FLAG-RBM4 (lanes 3 through 5). Lanes 1 show cotransfection of the reporter with the empty effector vectors. Splicing of the α-TM transcripts was assayed by RT-PCR as in Fig. 4. GAPDH was used as a control for pαTM-8/9a/9b. (C) Recombinant mouse RBM4 (lanes 2 and 4) or mock eluate (lanes 1 and 3) was incubated with ³²P-labeled α-TM intron 9a RNA probe (wild type or CU1 mutant) followed by UV cross-linking. The reactions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography. The RNA probe contained a fragment corresponding to nucleotides 9 through 34 of α-TM intron 9a (Materials and Methods). (D) FLAG-PTB-containing cell lysate was prepared from transiently transfected HEK 293 cells and subsequently incubated with wild-type (lanes 1 and 2) or mutant (lanes 3 and 4) ³²P-labeled α-TM intron 9a probe. For competition, the experiment was performed with the wild-type RNA probe using cell lysate prepared from mock-transfected HEK 293 cells (lane 5) or cells that transiently expressed either FLAG-PTB (lane 6) or FLAG-RBM4 (lane 7) or both proteins (lane 8). UV cross-linking and analysis were performed as in panel C. Western blotting using anti-FLAG is shown in the lower panel. The asterisk represents an unidentified protein of ∼70 kDa.