UV cross-linking analysis of proteins that bind to the exonic splicing enhancer sequences. (A) UV cross-linking of wt, ex-1, ex-2, and ex-3 to HeLa nuclear extracts. 32P-labeled pre-mRNA consisting of exon 16 and its flanking upstream (67 nt's) and downstream (36 nt's) intronic sequences were subjected to UV cross-linking using HeLa nuclear extracts. Either tRNA or WT sense transcripts were added to compete with WT substrates. tRNAs were added to ex-1, ex-2, and ex-3 substrates as competitor. t indicates tRNA; and wt, wt sense transcript. (Top) The full-length gel. (Bottom) The enlargement of the approximately 30- to 40-kDa region of the gel. (B) Purified SR proteins. (C) UV cross-linking of wt or ex-3 using purified SR proteins in the presence of tRNA. The arrow indicates approximately 33 kDa cross-linked band. (D) Identification by immunoprecipitation of SR proteins cross-linked to wt and ex-3. 32P-labeled RNA substrates from wt or ex-3 were subjected to UV cross-linking using HeLa nuclear extracts. The cross-linked protein-RNA mixtures were digested with RNAse A, precleared with protein A–Sepharose beads, and immunoprecipitated overnight at 4°C using anti-SF2, anti-9G8, or anti-SC35 antibodies. The immunoprecipitates were washed, subjected to SDS-PAGE electrophoresis on a 12% gel, and autoradiographed.
