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

Abstract

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.

We are very grateful to Andrzej Malkiewicz (Lodz, Poland) for providing nucleoside standards for mcm⁵U and mcm⁵s²U. We thank Alan Diamond (University of Illinois at Chicago) for providing pBPLUGA-Gpx1. pSport1-SECISBP2 was a generous gift from Laura V. Papp (Queensland Institute of Medical Research, Australia). We are grateful to Anders Bekkelund and Tine W. Abrahamsen for their help with plasmid construction and to Cecilie G. Castellanos, Guro F. Lien, and Hege Wiksen for excellent technical assistance and animal care. We thank GenOway, Lyon, France; The Norwegian Transgenic Center (NTS); and the Center for Comparative studies at Oslo University Hospital HF for the excellent service they provided.

This work was supported by grants from the FRIBIO and FUGE programs of the Research Council of Norway.