Prp16p, Slu7p, and Prp8p interact with the 3′ splice site in two distinct stages during the second catalytic step of pre-mRNA splicing

For the second catalytic step of pre-mRNA splicing to occur, a 3′ splice site must be selected and juxtaposed with the 5′ exon. Four proteins, Prp16p, Slu7p, Prp17p, Prp18p, and an integral spliceosomal protein, Prp8p, are known to be required for the second catalytic step. prp8-101, an allele of PRP8 defective in 3′ splice site recognition, exhibits specific genetic interactions with mutant alleles of the other second step splicing factors. The prp8-101 mutation also results in decreased crosslinking of Prp8p to the 3′ splice site. To determine the role of the step-two-specific proteins in 3′ splice site recognition and in binding of Prp8p to the 3′ splice site, we performed crosslinking studies in mutant and immunodepleted extracts. Our results suggest an ordered pathway in which, after the first catalytic step, Prp16p crosslinks strongly to the 3′ splice site and Prp8p and Slu7p crosslink weakly. ATP hydrolysis by Prp16p affects a conformational change that reduces the crosslinking of Prp16p with the 3′ splice site and allows stronger crosslinking of Prp8p and Slu7p. Thus, the 3′ splice site appears to be recognized in two stages during the second step of splicing. Strong 3′ splice site crosslinking of Prp8p and Slu7p also requires the functions of Prp17p and Prp18p. Therefore, Prp8p and Slu7p interact with the 3′ splice site at the latest stage of splicing prior to the second catalytic step that can currently be defined, and may be at the active site.