Strict 3′ splice site sequence requirements for U2 snRNP recruitment after U2AF binding underlie a genetic defect leading to autoimmune disease

The AG mutation does not inhibit initial recognition of the 3′splice site by the U2AF heterodimer. (A) Crosslinking-immunoprecipitation of U2AF65 to assess binding to wild-type (WT) and AG mutant RNAs. ³²P-uridine–labeled WT and AG RNAs comprising the 3′ 68 nucleotides of intron 5, exon 6, and 5′ 25 nucleotides of intron 6 were incubated with nuclear extracts under splicing conditions for the indicated times. After ultraviolet irradiation and RNase treatment, U2AF65 was immunoprecipitated using the MC3 monoclonal antibody (Gama-Carvalho et al. 1997). Immunoprecipitated materials were fractionated by sodium-dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis. The position of RNA-crosslinked U2AF65 is indicated. The ratios of U2AF65 crosslinking to WT versus AG probes (WT/AG) at 5, 15, and 30 min and the SD corresponding to four independent experiments are indicated below the corresponding lanes. An aliquot of the products of crosslinking before immunoprecipitation was analyzed by electrophoresis and serves as loading control. (B) U2AF35 crosslinking-immunoprecipitation to WT and mutant AG. Assays were carried out as in A for the indicated times points using α-³²P-guanosine–labeled RNAs. U2AF35 was immunoprecipitated using a rabbit polyclonal antibody (Zuo and Maniatis 1996). The positions of the U2AF heterodimer components crosslinked to the RNAs are indicated. The ratio of U2AF35 crosslinking to WT versus AG RNAs at 5 and 15 min and the SD for three independent experiments are indicated below the corresponding lanes. (C) U2AF65 crosslinking-immunoprecipitation to various 3′ splice site mutants. Assays were carried out as in A with the indicated mutant RNAs. Ratios and SDs correspond to three independent experiments. (D) Improving exon definition does not relieve the effect of the AG mutation. The indicated mutant minigenes were transfected and RNAs analyzed as in Figure 1. U1comp indicates a mutant minigene with enhanced complementarity between U1 snRNA and the intron 6 5′ splice site (positions −2 and −3 from the exon 6–intron 6 boundary were mutated to A and C, and positions +7 and +8 in intron 6 were mutated to A and T). The m0 mutant replaces the uridine-rich PTB-binding silencer in exon 6 (UUUGUCUUCUUCUUUU) by a random sequence (AAUGCACACUCACCAG) (Izquierdo et al. 2005).