Mammalian ALKBH8 Possesses tRNA Methyltransferase Activity Required for the Biogenesis of Multiple Wobble Uridine Modifications Implicated in Translational Decoding

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 (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.tRNAs are frequently modified at the wobble uridine, a feature that is believed to either promote or restrict wobbling depending on the type of modification. In the case of eukaryotes, the functions of wobble uridine modifications have been studied in the greatest detail in Saccharomyces cerevisiae. Here, the modifications 5-methoxycarbonylmethyluridine (mcm⁵U), 5-methoxycarbonylmethyl-2-thiouridine (mcm⁵s²U), and 5-carbamoylmethyluridine (ncm⁵U) or its 2′-O-ribose-methylated form, ncm⁵Um, are found in 11 out of 13 wobble uridine-containing tRNAs (22). mcm⁵U and mcm⁵s²U are mostly found in “split” codon boxes, where the pyrimidine- and purine-ending codons encode different amino acids, while ncm⁵U is found in “family” codon boxes, where all four codons encode a single amino acid. Early reports based on in vitro experiments suggested that wobble nucleosides, such as mcm⁵U, ncm⁵U, and their derivatives, may restrict wobbling (17, 37, 45), but the results of a recent comprehensive study performed in vivo in S. cerevisiae show that such modifications can improve the reading both of the cognate, A-ending codons and of the wobble, G-ending codons (22). This may suggest that the primary role of these modified nucleosides is to improve translational efficiency rather than to restrict wobbling.The characterization of wobble uridine modifications in higher eukaryotes is very limited, and little is known about the enzymes that introduce them. In mammals, mcm⁵s²U has been found in the wobble position of tRNA^Glu(UUC), tRNA^Lys(UUU), and tRNA^Arg(UCU) (40). Unlike yeast, mammals possess a specialized tRNA that is responsible for recoding the UGA stop codon to insert the 21st amino acid, selenocysteine (Sec). The mammalian tRNA^Sec population consists of two subpopulations containing either mcm⁵U or the ribose-methylated derivative mcm⁵Um in the wobble position. Interestingly, ribose methylation of mcm⁵U in tRNA^Sec appears to have a role in regulating selenoprotein synthesis, as the expression of some selenoproteins, such as glutathione peroxidase 1 (Gpx1), appears to be promoted by mcm⁵Um-containing tRNA^Sec (5, 7, 9, 32).Some years ago, the Escherichia coli AlkB protein was found to be a 2-oxoglutarate- and iron-dependent dioxygenase capable of demethylating the lesions 1-methyladenosine and 3-methylcytosine in DNA (13, 42). Multicellular organisms generally possess several different AlkB homologues (ALKBH), and bioinformatics analysis has identified eight different mammalian ALKBH proteins, denoted ALKBH1 to ALKBH8 in humans and Alkbh1 to Alkbh8 in mice, as well as the somewhat-less-related, obesity-associated FTO protein (2, 16, 30). Among the ALKBH proteins of unknown function, ALKBH8 is the only one containing additional annotated protein domains. Here, the AlkB domain is localized between an N-terminal RNA recognition motif (RRM) and a C-terminal methyltransferase (MT) domain. Interestingly, the MT domain has sequence homology to the S. cerevisiae tRNA methyltransferase Trm9, which has been shown to catalyze the methyl esterification of modified wobble uridine (U34) residues of tRNA^Arg and tRNA^Glu, resulting in the formation of mcm⁵U and mcm⁵s²U, respectively (23, 43). Until recently, human ALKBH8 was incorrectly annotated in the protein sequence database, and another human protein, KIAA1456, has been designated the human Trm9 homologue (3, 23).We have generated for this study Alkbh8-targeted mice that lack exons critical for both the MT and AlkB activities of Alkbh8. The mice did not display any overt phenotype, but tRNA from these mice was completely devoid of mcm⁵U, mcm⁵s²U, and mcm⁵Um, and the relevant tRNA isoacceptors instead contained the acid form 5-carboxymethyluridine (cm⁵U) and/or the amide forms ncm⁵U/ncm⁵s²U. Furthermore, we show that recombinant ALKBH8 and TRM112 form a heterodimeric complex capable of catalyzing the methyl esterification of cm⁵U and cm⁵s²U to mcm⁵U and mcm⁵s²U, respectively. In agreement with the involvement of mcm⁵Um in selenoprotein synthesis, we observed a reduced level of Gpx1 in the Alkbh8^−/− mice, and tRNA^Sec from these mice showed a reduced ability to decode the UGA stop codon to Sec.