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 (mcm 5 U) 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 mcm 5 U. 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 (mcm 5 s 2 U) and 5-methoxycarbonylmethyl-2-O-methyluridine (mcm 5 Um), respectively. Despite the complete loss of all of these uridine modifications, Alkbh8 ؊/؊ mice appear normal. However, the selenocysteine-specific tRNA (tRNA Sec ) is aberrantly modified in the Alkbh8 ؊/؊ mice, and for the selenoprotein Gpx1, we indeed observed reduced recoding of the UGA stop codon to selenocysteine.

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 (S)-5-methoxycarbonylhydroxymethyluridine (mchm 5 U), hydroxylated forms of mcm 5 U, are present in mammalian tRNA UCG Arg , and tRNA UCC Gly , 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 UCC Gly . 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.

Bacterial and mammalian AlkB proteins are iron(II)- and 2-oxoglutarate-dependent dioxygenases that reverse methylation damage, such as 1-methyladenine and 3-methylcytosine, in RNA and DNA. An AlkB-domain is encoded by the genome of numerous single-stranded, plant-infecting RNA viruses, the majority of which belong to the Flexiviridae family. Our phylogenetic analysis of AlkB sequences suggests that a single plant virus might have acquired AlkB relatively recently, followed by horizontal dissemination among other viruses via recombination. Here, we describe the first functional characterization of AlkB proteins from three plant viruses. The viral AlkB proteins efficiently reactivated methylated bacteriophage genomes when expressed in Escherichia coli, and also displayed robust, iron(II)- and 2-oxoglutarate-dependent demethylase activity in vitro. Viral AlkB proteins preferred RNA over DNA substrates, and thus represent the first AlkBs with such substrate specificity. Our results suggest a role for viral AlkBs in maintaining the integrity of the viral RNA genome through repair of deleterious methylation damage, and support the notion that AlkB-mediated RNA repair is biologically relevant.