Department of Molecular Biosciences, University of Oslo, PO Box 1041 Blindern, Oslo 0316, Norway.
Nat Commun. 2011 Feb 1;2:172. doi: 10.1038/ncomms1173.
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.
哺乳动物有 9 种不同的同源物（ALKBH1-9）与大肠杆菌 DNA 修复脱甲基酶 AlkB 同源。ALKBH2 是一种真正的 DNA 修复酶，但其他 ALKBH 蛋白的体内功能仍然难以捉摸。最近的研究表明，ALKBH8 含有一个额外的转移 RNA(tRNA)甲基转移酶结构域，它可以将其前体 5-羧甲基尿嘧啶（cm(5)U）转化为 wobble 核苷 5-甲氧基羰基甲基尿嘧啶（mcm(5)U）。在这项研究中，我们报告说（R）-和 5-甲氧基羰基羟甲基尿嘧啶（mchm(5)U）是哺乳动物 tRNA-Arg(UCG)和 tRNA-Gly(UCC)中的存在物，分别代表修饰 RNA 核苷的第一个非对映异构体对。通过体外和体内研究，我们表明 mchm(5)U 的两种非对映异构体均由 mcm(5)U 生成，并且 ALKBH8 的 AlkB 结构域特异性地将 mcm(5)U 羟化为 tRNA-Gly(UCC)中的（S）-mchm(5)U。这些发现扩展了 ALKBH 加氧酶的功能超越了核酸修复，并增加了对哺乳动物 wobble 尿嘧啶修饰及其生物发生的现有知识。
