Suppression of the AG mutation by increasing the distance between AGs and by stronger Py-tracts. (A) AG-AG insertion mutants. Analysis of the indicated mutants were as in Figure 1. The first ag indicates the position of the AG created by the ALPS mutation; the second corresponds to the natural intron 5 3′ splice site. Underlined ags are those used when exon 6 is included (determined by sequencing of the spliced products). (B) The 3′ss sequence of WT, AG, and Py-tract mutants. Mutated/inserted nucleotides are in bold. (/) Intron/exon boundary. The box summarizes the conclusion from the results of parts C and D that at least six uridines are required to promote exon 6 inclusion in the AG mutant. (C) Analysis of the effects of uridine insertions in the AG mutant, analyzed as in A. (D) Analysis of mutants showing different arrangements of uridine-rich regions in the Py-tract, analyzed as in A. (E) Spliceosome assembly analysis of AG-AG insertion and Py-tract mutants, analyzed as in Figure 2D at the indicated times of incubation. (F) Psoralen-mediated crosslinking analysis for detection of U1/Fas pre-mRNA and U2/Fas pre-mRNA base-pairing interactions, performed as in Figure 2E with wild-type or the indicated mutant RNAs. (G) U2AF65 crosslinking-immunoprecipitation assays were carried out as in Figure 3A using wild-type or the indicated mutant RNAs.
