During spliceosome activation, a large structural rearrangement occurs that involves the release of two small nuclear RNAs, U1 and U4, and the addition of a protein complex associated with Prp19p. We show here that the Prp19p-associated complex is required for stable association of U5 and U6 with the spliceosome after U4 is dissociated. Ultraviolet crosslinking analysis revealed the existence of two modes of base pairing between U6 and the 5' splice site, as well as a switch of such base pairing from one to the other that required the Prp19p-associated complex during spliceosome activation. Moreover, a Prp19p-dependent structural change in U6 small nuclear ribonucleoprotein particles was detected that involves destabilization of Sm-like (Lsm) proteins to bring about interactions between the Lsm binding site of U6 and the intron sequence near the 5' splice site, indicating dynamic association of Lsm with U6 and a direct role of Lsm proteins in activation of the spliceosome.

The Prp19p protein of the budding yeast Saccharomyces cerevisiae is an essential splicing factor and is associated with the spliceosome during the splicing reaction. We have previously shown that Prp19p is not tightly associated with small nuclear ribonucleoprotein particles but is associated with a protein complex consisting of at least eight protein components. By sequencing components of the affinity-purified complex, we have identified Cef1p as a component of the Prp19p-associated complex, Ntc85p. Cef1p could directly interact with Prp19p and was required for pre-mRNA splicing both in vivo and in vitro. The c-Myb DNA binding motif at the amino terminus of Cef1p was required for cellular growth but not for interaction of Cef1p with Prp19p or Cef1p self-interaction. We have identified a small region of 30 amino acid residues near the carboxyl terminus required for both cell viability and proteinprotein interactions. Cef1p was associated with the spliceosome in the same manner as Prp19p, i.e. concomitant with or immediately after dissociation of U4. The antiCef1p antibody inhibited binding to the spliceosome of Cef1p, Prp19p, and at least three other components of the Prp19p-associated complex, suggesting that the Prp19p-associated complex is likely associated with the spliceosome and functions as an integral complex.The eukaryotic spliceosome is a multicomponent ribonucleoprotein particle composed of five small nuclear RNAs, U1, U2, U4/U6, and U5, and a number of protein factors (for reviews, see Refs. 1-6). Spliceosome assembly is a stepwise process involving sequential binding of small nuclear RNAs and protein factors (7-13). During spliceosome assembly, U1 first binds to the 5Ј splice site followed by binding of U2 to the branch site through base pair interactions between the small nuclear RNAs and the intron sequences. U4/U6 and U5 are then added to the spliceosome as a preformed three-small nuclear RNP particle. This triggers a conformational rearrangement of the spliceosome in which base pairing of U1 with the 5Ј splice site is replaced by U6, and base paired U4/U6 unwinds to form new base pairings between U6 and U2 (14 -17). U1 and U4 thus become only loosely associated with the spliceosome, which is now activated and ready for catalytic reactions. It is believed that such structural rearrangements of the spliceosome are mediated by protein factors. Although several proteins containing the DEX(D/H) box motif have been shown RNA unwindase activity (6, 18 -21), no substrate specificity could be demonstrated. It remains a question what dictates the substrate specificity and mediates conformational rearrangement of the spliceosome during spliceosome assembly.We have previously shown that the yeast Saccharomyces cerevisiae Prp19p protein is essential for pre-mRNA splicing and is required before the first step of the splicing reaction. Prp19p is not tightly associated with small nuclear RNAs but is associated with the spliceosome immediately after or concomitant with dissociation of U4 from the spliceosome, s...

The Prp19p-associated complex is essential for the yeast pre-mRNA splicing reaction. The complex consists of at least eight protein components, but is not tightly associated with spliceosomal snRNAs. By a combination of genetic and biochemical methods we previously identified four components of this complex, Ntc25p, Ntc85p, Ntc30p and Ntc20p, all of them being novel splicing factors. We have now identified three other components of the complex, Ntc90p, Ntc77p and Ntc31p. These three proteins were also associated with the spliceosome during the splicing reaction in the same manner as Prp19p, concurrently with or immediately after dissociation of U4 snRNA. Two-hybrid analysis revealed that none of these proteins interacted with Prp19p or Ntc25p, but all interacted with Ntc85p. An interaction network between the identified components of the Prp19p-associated complex is demonstrated. Biochemical analysis revealed that Ntc90p, Ntc31p, Ntc30p and Ntc20p form a subcomplex, which, through interacting with Ntc85p and Ntc77p, can associate with Prp19p and Ntc25p to form the Prp19p-associated complex. Genetic analysis suggests that Ntc31p, Ntc30p and Ntc20p may play roles in modulating the function of Ntc90p.

The yeast Saccharomyces cerevisiae Prp19p protein is an essential splicing factor and a spliceosomal component. It is not tightly associated with small nuclear RNAs (snRNAs) but is associated with a protein complex consisting of at least eight proteins. We have identified two novel components of the Prp19p-associated complex, Ntc30p and Ntc20p. Like other identified components of the complex, both Ntc30p and Ntc20p are associated with the spliceosome in the same manner as Prp19p immediately after or concurrently with dissociation of U4, indicating that the entire complex may bind to the spliceosome as an intact form. Neither Ntc30p nor Ntc20p directly interacts with Prp19p, but both interact with another component of the complex, Ntc85p. Immunoprecipitation analysis revealed an ordered interactions of these components in formation of the Prp19p-associated complex. Although null mutants of NTC30 or NTC20 showed no obvious growth phenotype, deletion of both genes impaired yeast growth resulting in accumulation of precursor mRNA. Extracts prepared from such a strain were defective in pre-mRNA splicing in vitro, but the splicing activity could be restored upon addition of the purified Prp19p-associated complex. These results indicate that Ntc30p and Ntc20p are auxiliary splicing factors the functions of which may be modulating the function of the Prp19p-associated complex.Splicing of pre-mRNA requires five small nuclear RNAs (snRNAs) 1 and a large number of protein factors, which assemble into a large ribonucleoprotein complex called the spliceosome (for reviews, see Refs. 1-6). Spliceosome assembly is a multistep process that involves sequential binding of snRNAs to the pre-mRNA in an order of U1, U2, then U4/U6 and U5 as a preformed tri-snRNP particle. A subsequent conformational rearrangement results in dissociation of U1 and U4, accompanied by new base pair formation between U2 and U6 and between U6 and the 5Ј splice site, leading to the formation of the active spliceosome on which the catalytic reactions take place.Functional studies of snRNAs have revealed their important roles in recognition and alignment of splice sites mediated through base pair interactions between snRNAs and the intron sequences during spliceosome assembly. Although numerous protein splicing factors have been identified, their functional roles are not well understood. The DEx(D/H) box proteins are among the best characterized protein factors and have been shown RNA unwindase activity (6 -10). It is generally believed that these proteins play essential roles in modulating structural change of the spliceosome during spliceosome assembly by either unwinding RNA base pairing or by hydrolyzing ATP to provide energy required for conformational rearrangement (6). In addition, a U5 protein with strong sequence similarity to ribosomal translocase EF-2 was demonstrated to have GTP binding activity and implicated in the structural rearrangement of RNA (11).We have previously shown that the yeast Saccharomyces cerevisiae Prp19p protein is an essentia...