title: Box H/ACA snoRNAs are preferred substrates for the trimethylguanosine synthase in the divergent unicellular eukaryote Trichomonas vaginalis.
abstract: The 2,2,7-trimethylguanosine caps of eukaryal snRNAs and snoRNA are formed by the enzyme Tgs1, which catalyzes sequential guanine-N2 methylations of m(7)G caps. Atypically, in the divergent unicellular eukaryote Trichomonas vaginalis, spliceosomal snRNAs lack a guanosine cap and the recombinant T. vaginalis trimethylguanosine synthase (TvTgs) produces only m(2,7)G in vitro. Here, we show by direct metabolic labeling that endogenous T. vaginalis RNAs contain m(7)G, m(2,7)G, and m(2,2,7)G caps. Immunodepletion of TvTgs from cell extracts and TvTgs add-back experiments demonstrate that TvTgs produces m(2,7)G and m(2,2,7)G caps. Expression of TvTgs in yeast tgs1Δ cells leads to the formation of m(2,7)G and m(2,2,7)G caps and complementation of the lethality of a tgs1Δ mud2Δ strain. Whereas TvTgs is present in the nucleus and cytosol of T. vaginalis cells, TMG-containing RNAs are localized primarily in the nucleolus. Molecular cloning of anti-TMG affinity-purified T. vaginalis RNAs identified 16 box H/ACA snoRNAs, which are implicated in guiding RNA pseudouridylation. The ensemble of new T. vaginalis H/ACA snoRNAs allowed us to predict and partially validate an extensive map of pseudouridines in T. vaginalis rRNA.
authors: Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095-1489, USA.
authors: Augusto
authors: A
authors: Simoes-Barbosa
authors: Kausik
authors: K
authors: Chakrabarti
authors: Michael
authors: M
authors: Pearson
authors: Delphine
authors: D
authors: Benarroch
authors: Stewart
authors: S
authors: Shuman
authors: Patricia J
authors: PJ
authors: Johnson
mesh_terms: D001483:Base Sequence; D001692:Biological Transport; D006151:Guanosine / Q000031:analogs & derivatives / Q000378:metabolism; D008745:Methylation; D008780:Methyltransferases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012315:RNA Caps / Q000378:metabolism; D020537:RNA, Small Nucleolar / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D014246:Trichomonas vaginalis / Q000201:enzymology* / Q000235:genetics
chemical_list: D012315:RNA Caps; D020537:RNA, Small Nucleolar; D006151:Guanosine; C017778:N(2),N(2),7-trimethylguanosine; D008780:Methyltransferases; C456271:trimethylguanosine synthase