A COMPREHENSIVE BIOCHEMICAL AND GENETIC-ANALYSIS OF THE YEAST U1 SNRNP REVEALS 5 NOVEL PROTEINS

The U1 snRNP is essential for recognition of the pre-mRNA 5'-splice si te and the subsequent assembly of the spliceosome. Yeast U1 snRNP is c onsiderably more complex than its metazoan counterpart, which suggests possible differences between yeast and metazoa in early splicing even ts. We have comprehensively analyzed the composition of yeast U1 snRNP s using a combination of biochemical, mass spectrometric, and genetic methods. We demonstrate the specific association of four novel U1 snRN P proteins, Snu71p, Snu65p, Nam8p, and Snu56p, that have no known meta zoan homologues. A fifth protein, Npl3p, is an abundant cellular compo nent that reproducibly co-purifies with the U1 snRNP, but its associat ion is salt-sensitive. Therefore, we are unable to establish conclusiv ely whether it binds specifically to the U1 snRNP. Interestingly, Nam8 p and Npl3p were previously assigned functions in (pre-m)RNA-metabolis m; however, so far, no association with U1 snRNP has been demonstrated or proposed. We also show that the yeast SmB protein is a U1 snRNP co mponent. Yeast U1 snRNP therefore contains 16 different proteins, incl uding seven snRNP cove proteins, three homologues of the metazoan U1 s nRNP-specific proteins, and six yeast-specific U1 snRNP proteins. We h ave simultaneously continued the characterization of additional mutant s isolated in a synthetic lethal (MUD) screen for genes that functiona lly cooperate with U1 snRNA. Consistent with the biochemical results, mud10, mud15, and mud16 are alleles of SNU56, NAM8, and SNU65, respect ively. mud10 and mud15 affect the in vivo splicing efficiency of nonca nonical introns. Moreover, mud10p strongly affects the in vitro format ion of splicing complexes, and extracts from the mud15 strain contain a U1 snRNP that migrates aberrantly on native gels. Finally, we show t hat Nam8p/Mud15p contributes to the stability of U1 snRNP.