Fig. 7. Effect of RBM4 on in vitro splicing of the adenovirus major late (AdML) pre-mRNA. The top diagram shows the AdML pre-mRNA and splicing products. Splicing was carried out in HeLa cell nuclear (lane 1) or S100 extract (lanes 2 and 11), or S100 extract supplemented with SR proteins (lanes 3 and 12, 0.15 µg; lane 4, 0.3 µg; lanes 5–10 and 13–17, 0.6 µg). In addition, the reactions contained increasing amounts of purified wild-type RBM4 (lanes 7–10), 50 ng wild-type (lane 15), 100 ng RRM mutant RBM4 (lane 16) or 100 ng MBP–CAD (lane 17). The mock fraction containing no recombinant protein was added as control (lanes 6 and 14). Size markers are given on the left. Pre-mRNA and splicing intermediates and products are depicted in the middle. Note that mRNA (D) generated by the use of the distal 5′ splice site is susceptible for degradation in extract as described elsewhere (Tarn and Steitz, 1994; a degraded fragment denoted by the asterisk). However, the levels of the intron (triangles) excised by splicing at the distal site obviously elevated with increasing amounts of RBM4 (lanes 7–10).