Figure 2.

Identification of RNA–RNA interactions in double affinity-purified spliceosomal complexes. Gradient fractions 7–15 containing double affinity-purified complexes or 0.5% of the unfractionated tobramycin eluate (E) were UV irradiated in the presence (+; lanes 1 and 3–12) or absence (−; lane 2) of psoralen (AMT); note that the input of the gradient fractionated material, that is, the anti-SF3a66 eluate, is not shown. Recovered RNA was separated by denaturing PAGE and transferred to a nylon membrane that was hybridized sequentially with ³²P-labeled probes against the U2 snRNA (C), U1 (B) or pre-mRNA (A). A low level of ³²P-labeled pre-mRNA (present in the purified complexes) is detected in panels B and C, but crosslinked pre-mRNA species were not observed before Northern blotting. The identity of the crosslinked species is indicated on the right, where ‘PRE' indicates pre-mRNA and ‘intPRE' internally crosslinked pre-mRNA. Asterisks: the identity of this double band is unknown.