FIG. 6.

Effects of the 5′ cap and splice site sequence on snRNA interactions with pre-mRNA during time courses of splicing reactions. (A) Ad1WW pre-mRNA, capped with m⁷GpppG or ApppG, was incubated under splicing conditions in the presence of psoralen at 30°C. After the times shown above the lanes, reaction mixtures were removed and irradiated for 1 min each at ambient temperature. The results were analyzed by gel electrophoresis. The bands at the foot of the panel are intramolecular cross-links. Parallel reactions included (i) duplicates that contained an exogenous protein but showed the same features on quantitative analysis, (ii) reactions in the absence of MgCl₂ and ATP, and (iii) reactions that were incubated for 40 and 90 min but not irradiated to allow splicing efficiency to be measured. (B) Ad1WW, Ad1WM, and Ad1MW pre-mRNAs were incubated under splicing conditions as in panel A, but for longer periods; irradiated; and analyzed as in panel A. The unlabelled lanes contained RNA from parallel reactions for 2.7 h that had not been irradiated so that the efficiency of splicing could be measured. The band shown as comprising U6 snRNA cross-linked to the downstream site, which is most obvious in the reaction of Ad1MW (lanes 21 to 24), has not been demonstrated directly to be this molecule, but its substrate specificity, kinetics, and mobility are consistent with this identification. (C) Identical to panel A in all respects, including the parallel reactions, but with Ad1CC pre-mRNA.