title: Rearrangement of competing U2 RNA helices within the spliceosome promotes multiple steps in splicing.
abstract: Nuclear pre-messenger RNA (pre-mRNA) splicing requires multiple spliceosomal small nuclear RNA (snRNA) and pre-mRNA rearrangements. Here we reveal a new snRNA conformational switch in which successive roles for two competing U2 helices, stem IIa and stem IIc, promote distinct splicing steps. When stem IIa is stabilized by loss of stem IIc, rapid ATP-independent and Cus2p-insensitive prespliceosome formation occurs. In contrast, hyperstabilized stem IIc improves the first splicing step on aberrant branchpoint pre-mRNAs and rescues temperature-sensitive U6-U57C, a U6 mutation that also suppresses first-step splicing defects of branchpoint mutations. A second, later role for stem IIa is revealed by its suppression of a cold-sensitive allele of the second-step splicing factor PRP16. Our data expose a spliceosomal progression cycle of U2 stem IIa formation, disruption by stem IIc, and then reformation of stem IIa before the second catalytic step. We propose that the competing stem IIa and stem IIc helices are key spliceosomal RNA elements that optimize juxtaposition of the proper reactive sites during splicing.
authors.affiliation: Center for Molecular Biology of RNA Department of Molecular, Cell, and Developmental Biology, University of California at Santa Cruz, Santa Cruz, California 95064, USA. perriman@biology.ucsc.edu
authors.forename: Rhonda J
authors.initials: RJ
authors.lastname: Perriman
authors.forename: Manuel
authors.initials: M
authors.lastname: Ares
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D053487:DEAD-box RNA Helicases / Q000378:metabolism; D008954:Models, Biological; D009154:Mutation; D009690:Nucleic Acid Conformation; D010641:Phenotype; D020365:RNA Helicases; D012322:RNA Precursors / Q000378:metabolism; D012326:RNA Splicing; D000072260:RNA Splicing Factors; D012342:RNA, Small Nuclear / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D017381:Spliceosomes / Q000378:metabolism*; D013696:Temperature; D015534:Trans-Activators / Q000378:metabolism
chemical_list: C114067:CUS2 protein, S cerevisiae; D012322:RNA Precursors; D000072260:RNA Splicing Factors; D012342:RNA, Small Nuclear; D016601:RNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; C405484:U2 small nuclear RNA; D000251:Adenosine Triphosphatases; C075669:PRP16 protein, S cerevisiae; C064447:PRP5 protein, S cerevisiae; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases