The AG mutant inhibits U2 snRNP recruitment. (A) Inhibition of pre-mRNA splicing in vitro by the AG mutant. Uniformly α32P-UTP labeled RNAs corresponding to the wild type (WT) or AG mutant Fas genomic region between exon 5 and position +25 of intron 6 were incubated with HeLa cell nuclear extracts under splicing conditions (see Materials and Methods) in the absence or presence of ATP for 15 and 30 min. RNA species were subsequently purified and analyzed by electrophoresis in 13% denaturing polyacrylamide gels. The positions of pre-mRNA, splicing intermediates, and products are indicated. (B) Quantification of lariat intermediates and products for WT and AG mutant transcripts from three independent experiments as in A. Average relative values, determined by PhosphorImager analyses, and SDs are shown. (C) Spliceosome assembly assays corresponding to in vitro splicing mixes as in A incubated for the indicated times and treated with 5 mg/mL of heparin for 10 min at room temperature prior to complex analysis by native agarose-polyacrylamide gel electrophoresis. The positions of heterogeneous nuclear RNP (H), A, B, and C complexes are shown. (D) Spliceosome assembly analysis of WT or AG RNAs corresponding to exon 6, the 3′ 68 nt of intron 5 and the 5′ 25 nt of intron 6. Analyses were as in B except that electrophoresis was carried out in 1.5% low-melting point agarose gels. The identities of the complexes are indicated. (E) Psoralen-mediated crosslinking to detect base pairing interactions between U1 and U2 snRNAs with WT or AG exon 6 splice sites. Splicing mixes as in C in the presence of psoralen (22.2 mg/mL), using mock-treated HeLa cell nuclear extracts or extracts in which U1, U2, U4, or U5 snRNAs were inactivated by RNase H–mediated digestion. The reactions were incubated for 20 min at 30°C and irradiated with long-wave UV light. RNA was isolated and analyzed by electrophoresis in denaturing 6% polyacrylamide gels. The positions of the free RNA and the U1/pre-mRNA and U2/pre-RNA crosslinked species are indicated.
