title: Genetic and biochemical analysis of yeast and human cap trimethylguanosine synthase: functional overlap of 2,2,7-trimethylguanosine caps, small nuclear ribonucleoprotein components, pre-mRNA splicing factors, and RNA decay pathways.
abstract: Trimethylguanosine synthase (Tgs1) is the enzyme that converts standard m(7)G caps to the 2,2,7-trimethylguanosine (TMG) caps characteristic of spliceosomal small nuclear RNAs. Fungi and mammalian somatic cells are able to grow in the absence of Tgs1 and TMG caps, suggesting that an essential function of the TMG cap might be obscured by functional redundancy. A systematic screen in budding yeast identified nonessential genes that, when deleted, caused synthetic growth defects with tgs1Delta. The Tgs1 interaction network embraced proteins implicated in small nuclear ribonucleoprotein function and spliceosome assembly, including Mud2, Nam8, Brr1, Lea1, Ist3, Isy1, Cwc21, and Bud13. Complementation of the synthetic lethality of mud2Delta tgs1Delta and nam8Delta tgs1Delta strains by wild-type TGS1, but not by catalytically defective mutants, indicated that the TMG cap is essential for mitotic growth when redundant splicing factors are missing. Our genetic analysis also highlighted synthetic interactions of Tgs1 with proteins implicated in RNA end processing and decay (Pat1, Lsm1, and Trf4) and regulation of polymerase II transcription (Rpn4, Spt3, Srb2, Soh1, Swr1, and Htz1). We find that the C-terminal domain of human Tgs1 can function in lieu of the yeast protein in vivo. We present a biochemical characterization of the human Tgs1 guanine-N2 methyltransferase reaction and identify individual amino acids required for methyltransferase activity in vitro and in vivo.
authors.affiliation: Department of Microbiology and Molecular Medicine, University of Geneva, CH1211 Geneva, Switzerland. Stephane.Hausmann@medecine.unige.ch
authors.forename: Stéphane
authors.initials: S
authors.lastname: Hausmann
authors.forename: Sushuang
authors.initials: S
authors.lastname: Zheng
authors.forename: Michael
authors.initials: M
authors.lastname: Costanzo
authors.forename: Renee L
authors.initials: RL
authors.lastname: Brost
authors.forename: Dominique
authors.initials: D
authors.lastname: Garcin
authors.forename: Charles
authors.initials: C
authors.lastname: Boone
authors.forename: Stewart
authors.initials: S
authors.lastname: Shuman
authors.forename: Beate
authors.initials: B
authors.lastname: Schwer
mesh_terms: D000595:Amino Acid Sequence; D001669:Biochemical Phenomena; D020134:Catalytic Domain; D017353:Gene Deletion; D016681:Genome, Fungal / Q000235:genetics; D006151:Guanosine / Q000031:analogs & derivatives* / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006801:Humans; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D017434:Protein Structure, Tertiary; D012313:RNA / Q000235:genetics / Q000378:metabolism*; D012326:RNA Splicing / Q000235:genetics*; D017411:Ribonucleoproteins, Small Nuclear / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D017411:Ribonucleoproteins, Small Nuclear; D006151:Guanosine; D006153:Guanosine Diphosphate; C017778:N(2),N(2),7-trimethylguanosine; D012313:RNA; D008780:Methyltransferases; C456271:trimethylguanosine synthase
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