thiouridine and 5-methoxycarbonylmethyluridine

Transfer RNAs are synthesized as a primary transcript that is processed to produce a mature tRNA. As part of the maturation process, a subset of the nucleosides are modified. Modifications in the anticodon region often modulate the decoding ability of the tRNA. At position 34, the majority of yeast cytosolic tRNA species that have a uridine are modified to 5-carbamoylmethyluridine (ncm(5)U), 5-carbamoylmethyl-2'-O-methyluridine (ncm(5)Um), 5-methoxycarbonylmethyl-uridine (mcm(5)U) or 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U). The formation of mcm(5) and ncm(5) side chains involves a complex pathway, where the last step in formation of mcm(5) is a methyl esterification of cm(5) dependent on the Trm9 and Trm112 proteins.. Both Trm9 and Trm112 are required for the last step in formation of mcm(5) side chains at wobble uridines. By co-expressing a histidine-tagged Trm9p together with a native Trm112p in E. coli, these two proteins purified as a complex. The presence of Trm112p dramatically improves the methyltransferase activity of Trm9p in vitro. Single tRNA species that normally contain mcm(5)U or mcm(5)s(2)U nucleosides were isolated from trm9Δ or trm112Δ mutants and the presence of modified nucleosides was analyzed by HPLC. In both mutants, mcm(5)U and mcm(5)s(2)U nucleosides are absent in tRNAs and the major intermediates accumulating were ncm(5)U and ncm(5)s(2)U, not the expected cm(5)U and cm(5)s(2)U.. Trm9p and Trm112p function together at the final step in formation of mcm(5)U in tRNA by using the intermediate cm(5)U as a substrate. In tRNA isolated from trm9Δ and trm112Δ strains, ncm(5)U and ncm(5)s(2)U nucleosides accumulate, questioning the order of nucleoside intermediate formation of the mcm(5) side chain. We propose two alternative explanations for this observation. One is that the intermediate cm(5)U is generated from ncm(5)U by a yet unknown mechanism and the other is that cm(5)U is formed before ncm(5)U and mcm(5)U.

Topics: Esterification; Mutation; RNA, Transfer; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Thiouridine; tRNA Methyltransferases; Uridine

Uridines in the wobble position of tRNA are almost invariably modified. Modifications can increase the efficiency of codon reading, but they also prevent mistranslation by limiting wobbling. In mammals, several tRNAs have 5-methoxycarbonylmethyluridine (mcm5U) or derivatives thereof in the wobble position. Through analysis of tRNA from Alkbh8-/- mice, we show here that ALKBH8 is a tRNA methyltransferase required for the final step in the biogenesis of mcm5U. We also demonstrate that the interaction of ALKBH8 with a small accessory protein, TRM112, is required to form a functional tRNA methyltransferase. Furthermore, prior ALKBH8-mediated methylation is a prerequisite for the thiolation and 2'-O-ribose methylation that form 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) and 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm5Um), respectively. Despite the complete loss of all of these uridine modifications, Alkbh8-/- mice appear normal. However, the selenocysteine-specific tRNA (tRNASec) is aberrantly modified in the Alkbh8-/- mice, and for the selenoprotein Gpx1, we indeed observed reduced recoding of the UGA stop codon to selenocysteine.

Topics: AlkB Homolog 8, tRNA Methyltransferase; Amino Acid Sequence; Animals; Dioxygenases; Gene Targeting; Humans; Mice; Mice, Knockout; Molecular Sequence Data; Molecular Structure; Nucleic Acid Conformation; Protein Biosynthesis; RNA, Transfer; Sequence Alignment; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Thiouridine; tRNA Methyltransferases; Uridine