FIG. 4.

Competition of Tra2 binding restores M1 splicing. In vitro splicing assays performed on ³²P-labeled M1/ftz 3′ RNA in the presence (Tra2) or absence (Mock) of repressive amounts (3 pmol) of unlabeled recombinant Tra2 protein and RNA competitors are shown. Splicing precursors, intermediates, and products were resolved on denaturing polyacrylamide-urea gels, and their mobilities are indicated. The lariat intron product is not resolved from the pre-mRNA. (A) Competitions with the dsx ESE RNA in increasing amounts (0, 0.5, 2.5, and 12.5 pmol; triangles). Splicing is restored at moderate competitor concentrations (lanes 3 and 4), but the highest concentration of competitor RNA (lanes 5 and 9) caused nonspecific repression of splicing (compare control lanes 6 to 9). Lane 1 shows a reaction in which no ATP, Tra2, or competitors were added. (B) A similar set of reactions were carried out with RNA 5 (Fig. 3A) from within the M1 intron. Restoration of splicing is evident at the two highest concentrations of competitor (compare lane 1 to lanes 3 and 4). (C) A similar set of reactions carried out with RNA 2, which spans part of exon 3 and the 5′ end of the intron. Restoration of splicing can be seen by comparing lane 1 to lanes 2 to 4. Again, a nonspecific inhibition of splicing by high concentrations of the competitor was observed in lane 4 compared to lanes 5 to 8. (D) Competition with the same molar amounts of the unlabeled 207-nt negative-control RNA derived from sequences in dsx RNA outside of the enhancer (see Materials and Methods). No restoration of M1 splicing was observed (lanes 2 to 4).