title: Analysis of synthetic lethality reveals genetic interactions between the GTPase Snu114p and snRNAs in the catalytic core of the Saccharomyces cerevisiae spliceosome.
abstract: Conformational changes of snRNAs in the spliceosome required for pre-mRNA splicing are regulated by eight ATPases and one GTPase Snu114p. The Snu114p guanine state regulates U4/U6 unwinding during spliceosome activation and U2/U6 unwinding during spliceosome disassembly through the ATPase Brr2p. We investigated 618 genetic interactions to identify an extensive genetic interaction network between SNU114 and snRNAs. Snu114p G domain alleles were exacerbated by mutations that stabilize U4/U6 base pairing. G domain alleles were made worse by U2 and U6 mutations that stabilize or destabilize U2/U6 base pairing in helix I. Compensatory mutations that restored U2/U6 base pairing in helix I relieved synthetic lethality. Snu114p G domain alleles were also worsened by mutations in U6 predicted to increase 5' splice site base pairing. Both N-terminal and G domain alleles were exacerbated by U5 loop 1 mutations at positions involved in aligning exons while C-terminus alleles were synthetically lethal with U5 internal loop 1 mutations. This suggests a spatial orientation for Snu114p with U5. We propose that the RNA base pairing state is directly or indirectly sensed by the Snu114p G domain allowing the Snu114p C-terminal domain to regulate Brr2p or other proteins to bring about RNA/RNA rearrangements required for splicing.
authors.affiliation: The University of Manchester, Faculty of Life Sciences, Manchester M13 9PT, United Kingdom.
authors.forename: Lily Novak
authors.initials: LN
authors.lastname: Frazer
authors.forename: Simon C
authors.initials: SC
authors.lastname: Lovell
authors.forename: Raymond T
authors.initials: RT
authors.lastname: O'Keefe
mesh_terms: D020029:Base Pairing / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005804:Genes, Lethal; D016297:Mutagenesis, Site-Directed / Q000379:methods; D009154:Mutation; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012342:RNA, Small Nuclear / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017415:Ribonucleoprotein, U5 Small Nuclear / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017381:Spliceosomes / Q000378:metabolism*; D013489:Suppression, Genetic
chemical_list: D012342:RNA, Small Nuclear; D017415:Ribonucleoprotein, U5 Small Nuclear; C472240:SNU114 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C405484:U2 small nuclear RNA; C405482:U4 small nuclear RNA; C405486:U6 small nuclear RNA; D020558:GTP Phosphohydrolases