Snt309p modulates interactions of Prp19p with its associated components to stabilize the Prp19p-associated complex essential for pre-mRNA splicing

Abstract

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 SNT309-deleted strain (⌬SNT309) were defective in splicing but could be complemented by addition of the purified Prp19p-associated complex. Isolation of the Prp19p-associated complex from ⌬SNT309 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 ⌬SNT309 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 Prp19p-associated 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)(13)(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.

Identification and Characterization of Two Novel Components of The Prp19p-associated Complex, Ntc30p and Ntc20p

“…In the absence of Snt309p, Prp19p becomes only loosely associated with other components. In addition, the association of Ntc85p with Ntc30p and Ntc20p was also weakened, but association between Ntc30p and Ntc20p was not affected (16). This indicates that Snt309p, although not interacting directly with Ntc85p, affects interactions between Ntc85p and Ntc30p and between Ntc85p and Ntc20p.…”

“…This finding suggests that formation of the Prp19p-associated complex may involve sequential interactions of the components in the following order: Snt309p binds to Prp19p first, followed by binding of Ntc85p, then Ntc30p and Ntc20p. The fact that Ntc30p and Ntc20p do not interact with each other but remain associated even after dissociation from Ntc85p (16) suggests that they may interact with another factor(s) to form a subcomplex regardless of the presence of Ntc85p. A scheme of interactions between the known components of the Prp19p-associated complex is shown in Fig.…”

“…We have previously demonstrated that Snt309p plays a role in modulating interaction of Prp19p with other associated components (16). In the absence of Snt309p, Prp19p becomes only loosely associated with other components.…”

“…Snt309p was identified by screening synthetic lethal mutants to the prp19 mutation (Snt stands for synthetic lethal to PRP nineteen) (15). Although the SNT309 gene is not required for yeast growth, Snt309p, through interaction with Prp19p, plays an important role in modulating interactions of Prp19p with other associated components to stabilize the Prp19p-associated complex (16). Both proteins are associated with the spliceosome immediately after or simultaneously with dissociation of U4.…”

Inorganic Phosphate Deprivation Causes tRNA Nuclear Accumulation via Retrograde Transport inSaccharomyces cerevisiae

“…Our studies uncovered a single synthetic-enhanced growth interaction between snt309D and los1D. Since Snt309 is a component of spliceosomes (Chen et al 1999), it is unclear why snt309D and los1D would have enhanced genetic interactions.…”