(A) Schematic of the PM5-10 pre-mRNA. (B) RNA composition of MS2 affinity-purified spliceosomal complexes assembled on PM5-10 pre-mRNA. RNA was recovered from the indicated complexes, separated by denaturing PAGE and detected by silver staining. RNA identities are indicated on the left. (C) Affinity-purified B, B028 and Bact complexes were UV-irradiated +/− psoralen (Ps) as indicated. RNA-RNA crosslinks were identified by Northern blot analyses, incubating sequentially with 32P-labelled probes against U1, U2, U4, and U6 snRNAs. Several bands were observed solely in the presence of psoralen, and were detected by two or more probes, indicating crosslinking of two (or more) of these RNAs. The blot was stripped of each 32P-probe prior to incubation with a subsequent probe. 32P-labelled PM5-10 pre-mRNA, on which the B, B028 and Bact complexes were formed, is visible in all panels. The positions of crosslinked RNA species are indicated. The less efficient formation of the U2/U6 crosslink (U2/U6 helix II) in both B and B028 compared to Bact, suggests this region is shielded in B/B028 by proteins and thus less accessible to psoralen.