Snt 309 p modulates interactions of Prp 19 p with its associated components to stabilize the Prp 19 p-associated complex essential for pre-mRNA splicing

The SNT309 gene was identified via a mutation that causes lethality of cells in combination with a prp19 mutation. We showed previously that Snt309p is a component of the Prp19p-associated complex and that Snt309p, like Prp19p, is associated with the spliceosome immediately after or concomitantly with dissociation of U4 from the spliceosome. We show here that extracts prepared from the SNT309deleted strain (DSNT309) were defective in splicing but could be complemented by addition of the purified Prp19passociated complex. Isolation of the Prp19p-associated complex from DSNT309 extracts indicated that the complex was destabilized in the absence of Snt309p and dissociated on affinity chromatography, suggesting a role of Snt309p in stabilization of the Prp19p-associated complex. Addition of the affinity-purified Prp19p–Snt309p binary complex to DSNT309 extracts could reconstitute the Prp19p-associated complex. Genetic analysis further suggests that Snt309p plays a role in modulating interactions of Prp19p with other associated components to facilitate formation of the Prp19passociated complex. A model for how Snt309p modulates such interactions is proposed. Splicing of pre-mRNA takes place on a multicomponent ribonucleoprotein particle called the spliceosome, which consists of five small nuclear RNAs (snRNAs) and a number of protein factors (see refs. 1–8 for reviews). Numerous protein factors involved in the splicing reaction have been identified in mammals and yeast. Some are intrinsic components of the spliceosome (9–11), whereas others associate with the spliceosome only transiently (12–14). Many of the protein factors are components of the sn ribonucleoprotein particles (see refs. 1 and 15–18 for reviews). Yeast genetics provides a powerful tool for identification of protein factors involved in the splicing reaction, as well as for studying interactions between these components. A large number of PRP (precursor RNA processing) genes that encode protein splicing factors have been identified by screening temperature-sensitive mutants defective in pre-mRNA splicing (19, 20). Other genes were identified through genetic interactions with introns, PRP genes, or snRNA genes (21–27). Over 40 genes that encode protein factors of the splicing machinery have been identified, but the precise functions of these proteins are not well understood. The yeast PRP19 gene was among the genes identified in a screen for temperature-sensitive mutants defective in splicing (20). Biochemical characterizations indicated that the Prp19p protein is essential for the pre-mRNA splicing reaction in vitro. The protein is not tightly associated with snRNAs, but is associated with the spliceosome during the splicing reaction (11, 28). Prp19p seems to associate with the spliceosome concomitantly with or just after dissociation of U4 from the spliceosome (14, 28), suggesting a possible role for mediating conformational rearrangement at this step of the spliceosome assembly process. Attempts to purify the Prp19p protein for functional studies led to the identification of a protein complex consisting of at least seven proteins associated with Prp19p (29). Among them, at least three can directly interact with Prp19p as analyzed by Far Western blotting (29). The SNT309 gene, which encodes a component of the Prp19p-associated complex, was identified in a genetic screen for mutations that exacerbate the phenotype of prp19 mutations (27). Cells disrupted in the SNT309 gene, although viable, are temperature-sensitive and accumulated pre-mRNA at the nonpermissive temperature. Snt309p can directly interact with Prp19p but not with any other component in the complex. It was shown further that Snt309p is associated with the spliceosome at the same time as Prp19p (27). In this report, we show that in the absence of Snt309p, the Prp19p-associated complex is destabilized and cannot function properly in the splicing reaction. Snt309p seems to play a role in modulating interactions of Prp19p with other components of the Prp19passociated complex. A model for how Snt309p modulates such interactions is proposed. MATERIALS AND METHODS Strains and Plasmids. The following strains were used: BJ2168 (MATa prc1 prb1 pep4 leu2 trp1 ura3), YSCC1 (MATa prc1 prb1 pep4 leu2 trp1 ura3 PRP19-HA), YSCC20 (MATa prc1 prb1 pep4 leu2 trp1 ura3 NTC20-HA), YHR13 (MATa prc1 prb1 pep4 leu2 trp1 ura3 PRP19-HA DSNT309::LEU2), and YHR203 (MATa prc1 prb1 pep4 leu2 trp1 ura3 NTC20-HA DSNT309:: LEU2). The following plasmids were used: pBS.SNT309 (4.0-kb fragment of the SNT309 gene in pBS), pG1.SNT309 [a 824-bp BamHI fragment isolated from pGEM.SNT309 (27) was inserted into the BamHI site of pG1], and pET.SNT309 (a 824-bp BamHI fragment isolated from pGEM.SNT309 was inserted into pET15b). Construction of the SNT309-Deleted, PRP19 or NTC20 Epitope-Tagged Strain. The SNT309-deleted, PRP19 or NTC20 epitope-tagged strain was constructed by one-step gene displacement of strains YSCC1 and YSCC20, which are derivatives of strain BJ2168 in that the Prp19p or Ntc20p The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. PNAS is available online at www.pnas. org. This paper was submitted directly (Track II) to the Proceedings office. Abbreviations: snRNA, small nuclear RNA; 40P, 40% saturated ammonium sulfate fraction of the splicing extract; CEN, centromere. ‡Present address: Department of Biomolecular Chemistry, University of Wisconsin Medical School, Madison, WI 53706. §Present address: Department of Forestry, University of Aberdeen, Aberdeen AB24 5UA, U.K. ¶To whom reprint requests should be addressed.