Prp16p is a DEAH-box ATPase that transiently associates with the spliceosome to promote the structural transition required for the second chemical step. Yeast strains carrying the cold-sensitive allele prp16-302 stall the release of Prp16p at low temperatures, yet splice precursors with aberrant branchpoints at increased frequency. To identify new factors involved in the regulation of splicing fidelity, we sought suppressors of the prp16-302 growth phenotype. Deletion of the nonessential ISY1 gene (1) improves growth of prp16-302 strains, (2) alleviates stalling, and (3) restores fidelity of branchpoint usage to wild-type levels. Isy1p is a subunit of the NineTeen Complex containing Prp19p, an essential E3 ubiquitin ligase homolog required for splicing. Notably, ⌬isy1 PRP16 strains display reduced fidelity of 3-splice site selection. Consistent with a recent two-state model of the spliceosome, our genetic and biochemical results suggest that Isy1p acts together with U6 snRNA to promote a spliceosomal conformation favorable for first-step chemistry. We propose that deletion of ISY1 favors the premature release of Prp16p, thus promoting second-step chemistry of precursors with inappropriate 3-splice sites.[Keywords: Isy1p; Prp16p; pre-mRNA splicing; fidelity] Supplemental material is available at http://www.genesdev.org. Nuclear pre-mRNA splicing, the process that removes introns from primary transcripts in eukaryotic cells, is an essential step in gene expression. Splicing is catalyzed by the spliceosome, an extraordinarily complex ribonucleoprotein (RNP) machine composed of five small nuclear RNAs (snRNAs)-U1, U2, U4, U5, and U6-and about a hundred proteins (Burge et al. 1998;Jurica and Moore 2003). The pre-mRNA splicing reaction consists of two sequential transesterifications involving the intron-defining sequences at the 5Ј-splice site, 3Ј-splice site, and branchpoint. In the first chemical step, the conserved adenosine at the branch site attacks the 5Ј-splice site, producing a branched lariat intermediate and a free 5Ј-exon. In the second step, the 5Ј-exon attacks the 3Ј-splice site, resulting in ligation of 5Ј-and 3Ј-exons and release of the intron lariat. Because single-nucleotide precision is required, cells must have mechanisms to control the fidelity of pre-mRNA splicing.Enzymes belonging to the DExH/D-box superfamily of RNA-dependent ATPases remodel contacts within RNPs by promoting the exchange of RNA-RNA or RNA-protein pairing partners (Staley and Guthrie 1998;Tanner and Linder 2001). Prp16p is an essential DEAHbox ATPase that associates transiently with the spliceosome to activate the second catalytic step of splicing (Schwer and Guthrie 1991). ATP hydrolysis by Prp16p is required for release of the enzyme from the spliceosome and subsequent second-step catalysis (Schwer and Guthrie 1991), and promotes a conformational rearrangement of the spliceosome (Schwer and Guthrie 1992). Prp16p was originally identified in a screen for factors that allow splicing of pre-mRNAs containing branchpoint mutations (...
Cryptic unstable transcripts (CUTs) are short, 300-600-nucleotide (nt) RNA polymerase II transcripts that are rapidly degraded by the nuclear RNA exosome in yeast. CUTs are widespread and probably represent the largest share of hidden transcription in the yeast genome. Similarly to small nucleolar and small nuclear RNAs, transcription of CUT-encoding genes is terminated by the Nrd1 complex pathway. We show here that this termination mode and ensuing CUTs degradation crucially depend on the position of RNA polymerase II relative to the transcription start site. Notably, position sensing correlates with the phosphorylation status of the polymerase C-terminal domain (CTD). The Nrd1 complex is recruited to chromatin via interactions with both the nascent RNA and the CTD, but a permissive phosphorylation status of the latter is absolutely required for efficient transcription termination. We discuss the mechanism underlying the regulation of coexisting cryptic and mRNA-productive transcription.
The spliceosome is a complex small nuclear (sn)RNA-protein machine that removes introns from pre-mRNAs via two successive phosphoryl transfer reactions. The chemical steps are isoenergetic, yet splicing requires at least eight RNA-dependent ATPases responsible for substantial conformational rearrangements. To comprehensively monitor pre-mRNA conformational dynamics, we developed a strategy for single molecule FRET (smFRET) that utilizes a small, efficiently spliced yeast pre-mRNA, Ubc4, in which donor and acceptor fluorophores are placed in the exons adjacent to the 5′ and 3′ splice sites. During splicing in vitro we observe a multitude of generally reversible, time-and ATP-dependent conformational transitions of individual pre-mRNAs. The conformational dynamics of branchpoint and 3′ splice site mutants differ from one another and from wild-type. Because all transitions are reversible, spliceosome assembly appears to be occurring close to thermal equilibrium.
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