The PRP18 gene, which had been identified in a screen for pre-mRNA splicing mutants in Saccharomyces cerevisiae, has been cloned and sequenced. Yeast strains bearing only a disrupted copy of PRP18 are temperature sensitive for growth; even at a low temperature, they grow extremely slowly and do not splice pre-mRNA efficiently. This unusual temperature sensitivity can be reproduced in vitro; extracts immunodepleted of PRP18 are temperature sensitive for the second step of splicing. The PRP18 protein has been overexpressed in active form in Escherichia coli and has been purified to near homogeneity. Antibodies directed against PRP18 precipitate the U4/U5/U6 small nuclear ribonucleoprotein particle (snRNP) from yeast extracts.From extracts depleted of the U6 small nuclear RNA (snRNA), the U4 and U5 snRNAs can be immunoprecipitated, while no snRNAs can be precipitated from extracts depleted of the U5 snRNA. PRP18 therefore appears to be primarily associated with the U5 snRNP. The antibodies against PRP18 inhibit the second step of pre-mRNA splicing in vitro. Together, these results imply that the U5 snRNP plays a role in the second step of splicing and suggest a model for the action of PRP18.The splicing of pre-mRNA takes place in two sequential cleavage-and-ligation reactions (reviewed in references 26, 27, and 59). In the first step, the pre-mRNA is cut at the 5' splice site, releasing the first exon; concomitantly, the 5' phosphoryl group at the end of the intron is ligated to the 2' hydroxyl of an internal adenosine, yielding a branched RNA termed a lariat. In the second step, cleavage at the 3' splice site is accompanied by ligation of the two exons, yielding the product mRNA and releasing the intron as a lariat. Each cleavage and ligation step is thought to be a concerted, transesterification reaction in which the number of phosphodiester bonds is conserved. The identical reactions occur during group II self-splicing, leading to the hypothesis that RNA is also the catalyst in pre-mRNA splicing (36). The splicing of pre-mRNA occurs on a large ribonucleoprotein particle (RNP) called the spliceosome (13, 24), which consists of five small nuclear RNAs (snRNAs), Ul, U2, U4, U5, and U6, and a large but undetermined number of proteins (17,25,39,53).pre-mRNA splicing occurs in all eukaryotes, and its mechanism appears to be conserved from yeasts to metazoans. Although much has been learned about the functions of the snRNAs and the splicing proteins, a complete accounting of the role of any of the splicing factors cannot presently be given. Each snRNA is part of a small nuclear RNP (snRNP) (reviewed in reference 44). The Ul and U2 snRNPs are involved in recognition of splice sites; Ul base pairs to the 5' splice site, and U2 base pairs to the branchpoint sequence (see reference 57 and previously cited reviews). The U4 snRNA base pairs to the U6 snRNA, and together they form a single snRNP (12, 29). The U5 snRNP binds to the U4/U6 snRNP, forming a stable complex (9,17,39 reaction. U4 is apparently needed only for assemb...