Electrophoresis of ribonucleoproteins reveals an ordered assembly pathway of yeast splicing complexes

Abstract

Three splicing complexes formed with a yeast pre-messenger RNA during in vitro splicing can be resolved by non-denaturing gel electrophoresis after incubation in the presence of non-specific competitor RNA. The time course of the appearance of these complexes and their composition suggest that they represent an ordered pathway of splicing complex assembly.

The biochemical defects ofprp4-1andprp6-1yeast splicing mutants reveal that the PRP6 protein is required for the accumulation of the [U4/U6.U5] tri-snRNP

“…The U4/U6 snRNA association is normally favored leading to undetectable amounts of the free U4 snRNP. The U4/U6 snRNA dissociation occurs after spliceosome formation takes place, as a critical step in the splicing pathway (4,5,7,12). In the prp4-1 mutant, this step could occur aberrantly: the PRP4 ts protein may dissociate from the U4/U6 snRNP particle, leading to the degradation of the U6 snRNA, or, alternatively, the PRP4 ts protein may allow the degradation of the U6 snRNA in the particle, leading to the dissociation of the PRP4 protein from the U4 snRNA.…”

“…The formation of the spliceosome has been mostly analyzed in vitro and is an ordered process that involves the stepwise binding of the different snRNPs to the pre-mRNA (4)(5)(6)(7). The Ul snRNP interacts first to form the commitment complex (8)(9)(10)(11); the U2 snRNP then binds to this complex to form a prespliceosome.…”

Identification and purification of a 62,000-dalton protein that binds specifically to the polypyrimidine tract of introns.

“…Pre-mRNAs have been shown to interact with snRNPs and proteins in nuclear extracts to form splicing com plexes (Brody and Abelson 1985;Grabowski et al 1985) that can be separated by gel electrophoresis Sharp 1986, 1987;Pikielny et al 1986;Lamond et al 1988). Three complexes (A,B,C) are discernible using Tris-glycine native gels (Fig.…”

Schizosaccharomyces pombe U4 small nuclear RNA closely resembles vertebrate U4 and is required for growth

“…The high evolutionary conservation of U6 and the release (or less-tight association) of U4 from the spliceosome prior to intron cleavage (Pikielny et al, 1986;Cheng & Abelson, 1987;Lamond et al, 1988) has led to the further suggestion that U6 is the enzymically active component, and that the role of V4 is to act as a carrier for U6 and to block its activity until the appropriate moment. However, it is notable that only a part of the region of highest primary sequence conservation between V4 from Schizo pombe and vertebrates corresponds to the V4jU6 interaction domain.…”

“…Major events in spliceosome assembly include the association of UI and U2 small nuclear ribonucleoproteins (snRNPs) with the 5' splice junction and site of branch formation, respectively. U4 and U6 snRNA are associated by hydrogen-bonding and are found in a single snRNP particle (Bringmann et al, 1984;Hashimoto & Steitz, 1984) and may associate with the spliceosome as a U4/U5/U6 complex; U4 is released from the spliceosome before intron cleavage (Pikielny et al, 1986;Cheng & Abelson, 1987;Lamond et al, 1988). The site and mechanism of action of U4 snRNP remains obscure but it is essential for splicing activity in vitro.…”