Stages in the second reaction of pre-mRNA splicing: the final step is ATP independent

We have analyzed pre-mRNA splicing in yeast extracts immunodepleted of the PRP18 protein. We find that while the first step of splicing (cleavage at the 5' splice site, and generation of the exon 1 and lariat intermediates) is unaffected by the absence of PRP18, the second step of splicing (excision of the lariat intron and formation of mRNA) is substantially slower in the absence of PRP18. The splicing intermediates that are formed in the absence of PRP18 can be rapidly chased into products by the addition of purified PRP18 protein. This chasing is not dependent on ATP, implying that ATP is not required during the second cleavage-and-ligation reaction. This result suggests that there are ordered stages within the second step of splicing and that PRP18 acts late in the second step, perhaps during the catalytic step. The ATP independence also supports the idea that this reaction proceeds by a transesterification mechanism.