title: Editing and methylation at a single site by functionally interdependent activities.
abstract: Nucleic acids undergo naturally occurring chemical modifications. Over 100 different modifications have been described and every position in the purine and pyrimidine bases can be modified; often the sugar is also modified. Despite recent progress, the mechanism for the biosynthesis of most modifications is not fully understood, owing, in part, to the difficulty associated with reconstituting enzyme activity in vitro. Whereas some modifications can be efficiently formed with purified components, others may require more intricate pathways. A model for modification interdependence, in which one modification is a prerequisite for another, potentially explains a major hindrance in reconstituting enzymatic activity in vitro. This model was prompted by the earlier discovery of tRNA cytosine-to-uridine editing in eukaryotes, a reaction that has not been recapitulated in vitro and the mechanism of which remains unknown. Here we show that cytosine 32 in the anticodon loop of Trypanosoma brucei tRNAThris methylated to 3-methylcytosine (m3C) as a pre-requisite for C-to-U deamination. Formation of m3C in vitro requires the presence of both the T. brucei m3C methyltransferase TRM140 and the deaminase ADAT2/3. Once formed, m3C is deaminated to 3-methyluridine (m3U) by the same set of enzymes. ADAT2/3 is a highly mutagenic enzyme, but we also show that when co-expressed with the methyltransferase its mutagenicity is kept in check. This helps to explain how T. brucei escapes 'wholesale deamination' of its genome while harbouring both enzymes in the nucleus. This observation has implications for the control of another mutagenic deaminase, human AID, and provides a rationale for its regulation.
authors.affiliation: Department of Microbiology, Ohio State Biochemistry Program and The Center for RNA Biology, The Ohio State University, Columbus, Ohio 43210, USA.
authors.forename: Mary Anne T
authors.initials: MA
authors.lastname: Rubio
authors.affiliation: Department of Microbiology, Ohio State Biochemistry Program and The Center for RNA Biology, The Ohio State University, Columbus, Ohio 43210, USA.|Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati, Cincinnati, Ohio 45221, USA.
authors.forename: Kirk W
authors.initials: KW
authors.lastname: Gaston
authors.affiliation: Department of Microbiology, Ohio State Biochemistry Program and The Center for RNA Biology, The Ohio State University, Columbus, Ohio 43210, USA.
authors.forename: Katherine M
authors.initials: KM
authors.lastname: McKenney
authors.affiliation: Department of Microbiology, Ohio State Biochemistry Program and The Center for RNA Biology, The Ohio State University, Columbus, Ohio 43210, USA.
authors.forename: Ian M C
authors.initials: IM
authors.lastname: Fleming
authors.affiliation: Department of Microbiology, Ohio State Biochemistry Program and The Center for RNA Biology, The Ohio State University, Columbus, Ohio 43210, USA.|Institute of Parasitology, Biology Centre and Faculty of Sciences, University of South Bohemia, 37005 České Budějovice (Budweis), Czech Republic.
authors.forename: Zdeněk
authors.initials: Z
authors.lastname: Paris
authors.affiliation: Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati, Cincinnati, Ohio 45221, USA.
authors.forename: Patrick A
authors.initials: PA
authors.lastname: Limbach
authors.affiliation: Department of Microbiology, Ohio State Biochemistry Program and The Center for RNA Biology, The Ohio State University, Columbus, Ohio 43210, USA.
authors.forename: Juan D
authors.initials: JD
authors.lastname: Alfonzo
mesh_terms: D000926:Anticodon / Q000378:metabolism; D001483:Base Sequence; D003596:Cytosine / Q000031:analogs & derivatives / Q000378:metabolism; D003641:Deamination; D008745:Methylation; D008780:Methyltransferases / Q000378:metabolism*; D009700:Nucleoside Deaminases / Q000378:metabolism*; D017393:RNA Editing; D012363:RNA, Transfer, Thr / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics*; D014529:Uridine / Q000378:metabolism
chemical_list: D000926:Anticodon; D012363:RNA, Transfer, Thr; C036386:3-methylcytosine; D003596:Cytosine; D008780:Methyltransferases; D009700:Nucleoside Deaminases; D014529:Uridine