FIG. 4.

Cross-linking of p65 occurs at the 5′ splice site. (A) MB cross-linking with different GC+DX RNAs truncated from the 3′ end by linearization at the restriction sites indicated in the top panel. In the upper panel, the RNA is shown schematically with exons as empty boxes, the intron as a line, the pyrimidine tract as the solid rectangle, and the branch point as the solid circle. Full-length BamHI RNA undergoes both steps of splicing, AccI lacks the 3′ splice site YAG and exon and goes through step 1 only, and XhoI contains only the 5′ exon and 5′ splice site and is blocked before A complex assembly, while StyI RNA contains only the 5′ exon. The RNAs were incubated with HeLa nuclear extracts under normal splicing conditions for 15 min before MB cross-linking. Note that cross-linking of p65 occurs with all RNAs except GC+DX/StyI, which lacks the 5′ splice site. (B) Time course of MB cross-linking with GC+DX/XhoI. Note that p65 cross-linking appears as normal but does not decline. (C) p65 cross-linking to RNA with a 5′ splice site-specific label. GC+DX RNAs containing a single ³²P label at the 5′ exon-intron junction were incubated with 40% HeLa nuclear extracts under normal splicing conditions for 15 min before being subjected to cross-linking. Lane 1, MB cross-linking; lane 2, UV cross-linking. p65 MB cross-linked efficiently to RNA with the 5′ splice site label, but was not detected by UV cross-linking. Lane M, protein size markers (kilodaltons).