title: ALKBH8-mediated formation of a novel diastereomeric pair of wobble nucleosides in mammalian tRNA.
abstract: Mammals have nine different homologues (ALKBH1-9) of the Escherichia coli DNA repair demethylase AlkB. ALKBH2 is a genuine DNA repair enzyme, but the in vivo function of the other ALKBH proteins has remained elusive. It was recently shown that ALKBH8 contains an additional transfer RNA (tRNA) methyltransferase domain, which generates the wobble nucleoside 5-methoxycarbonylmethyluridine (mcm(5)U) from its precursor 5-carboxymethyluridine (cm(5)U). In this study, we report that (R)- and 5-methoxycarbonylhydroxymethyluridine (mchm(5)U), hydroxylated forms of mcm(5)U, are present in mammalian tRNA-Arg(UCG), and tRNA-Gly(UCC), respectively, representing the first example of a diastereomeric pair of modified RNA nucleosides. Through in vitro and in vivo studies, we show that both diastereomers of mchm(5)U are generated from mcm(5)U, and that the AlkB domain of ALKBH8 specifically hydroxylates mcm(5)U into (S)-mchm(5)U in tRNA-Gly(UCC). These findings expand the function of the ALKBH oxygenases beyond nucleic acid repair and increase the current knowledge on mammalian wobble uridine modifications and their biogenesis.
authors.affiliation: Department of Molecular Biosciences, University of Oslo, PO Box 1041 Blindern, Oslo 0316, Norway.
authors.forename: Erwin
authors.initials: E
authors.lastname: van den Born
authors.forename: Cathrine B
authors.initials: CB
authors.lastname: Vågbø
authors.forename: Lene
authors.initials: L
authors.lastname: Songe-Møller
authors.forename: Vibeke
authors.initials: V
authors.lastname: Leihne
authors.forename: Guro F
authors.initials: GF
authors.lastname: Lien
authors.forename: Grazyna
authors.initials: G
authors.lastname: Leszczynska
authors.forename: Andrzej
authors.initials: A
authors.lastname: Malkiewicz
authors.forename: Hans E
authors.initials: HE
authors.lastname: Krokan
authors.forename: Finn
authors.initials: F
authors.lastname: Kirpekar
authors.forename: Arne
authors.initials: A
authors.lastname: Klungland
authors.forename: Pål Ø
authors.initials: PØ
authors.lastname: Falnes
mesh_terms: D000071503:AlkB Homolog 8, tRNA Methyltransferase; D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid; D003062:Codon / Q000235:genetics*; D019295:Computational Biology; D051379:Mice; D015394:Molecular Structure; D014176:Protein Biosynthesis / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism*; D012353:RNA, Transfer, Gly / Q000737:chemistry*; D013045:Species Specificity; D053719:Tandem Mass Spectrometry; D014529:Uridine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D012359:tRNA Methyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D003062:Codon; D012333:RNA, Messenger; D012353:RNA, Transfer, Gly; C030689:5-methoxycarbonylmethyluridine; C548496:ALKBH8 protein, mouse; D000071503:AlkB Homolog 8, tRNA Methyltransferase; D012359:tRNA Methyltransferases; D014529:Uridine