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 (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.

ALKBH8-mediated formation of a novel diastereomeric pair of wobble nucleosides in mammalian tRNA

“…S10), possibly due to non-enzymatic hydrolysis of the labile ester bond in (S)-mchm 5 U, as previously reported 23 . In agreement with previous findings 22 (Figs 1d and 2g). Moreover, tRNA UCC Gly from the gene-targeted mice gave rise to alternative additional peaks, agreeing with the altered modification pattern observed by LC-MS/MS.…”

“…tRNA isolated from KI(MT + ) mice displayed a strong accumulation of mcm 5 U, consistent with mcm 5 U being a direct substrate for the ALKBH8 oxygenase. In agreement with previous results 22 , we detected substantial amounts of 5-carboxymethyluridine (cm 5 U) and elevated levels of ncm 5 U in tRNA from the MTdeficient Alkbh8 − / − and KI(Ox + ) mice. Notably, we also detected 5-carbamoylhydroxymethyluridine (nchm 5 U) and trace amounts of 5-carboxyhydroxymethyluridine (chm 5 U), the hydroxylated forms of ncm 5 U and cm 5 U, in tRNA from the MT-deficient mice (note that the R and S diastereomers of these nucleosides could not be separated chromatographically).…”

“…We and others recently showed that ALKBH8 contains a MT domain, which is the functional mammalian Trm9 homologue, and requires a small accessory protein, TRM112, for activity (Fig. 1b) 21,22 .…”

NSUN 3 and ABH 1 modify the wobble position of mt‐t RNA Met to expand codon recognition in mitochondrial translation

“…While the Ten‐Eleven Translocation (TET) protein family of dioxygenases primarily mediates oxidation of m 5 C in nuclear DNA and has also been implicated in histone modification, most of the members of the AlkB‐like Fe(II)/alpha‐ketoglutarate‐dependent dioxygenases (ALKBH) have been shown to act on RNA (reviewed in Shen et al , 2014; Fedeles et al , 2015; Li et al , 2015; Ougland et al , 2015). These include FTO (ALKBH9) that is implicated together with ALKBH5 in the oxidative removal of several modifications including 6‐methyladenosine (m 6 A) from RNA and ALKBH8 that is involved in the generation of 5‐methoxycarbonylmethyluridine (mcm 5 U) in cytoplasmic tRNAs (Fu et al , 2010a,b; Songe‐Møller et al , 2010; Jia et al , 2011; Thalhammer et al , 2011; Berulava et al , 2013; Zheng et al , 2013). So far, only ALKBH7, which was suggested to act on protein substrates during necrosis (Fu et al , 2013; Solberg et al , 2013; Wang et al , 2014), and ALKBH1/ABH1 have been reported to localise to mitochondria; however, the cellular localisation of ABH1 has been a matter of debate (Pan et al , 2008; Westbye et al , 2008; Ougland et al , 2012).…”

Lysine methylation of VCP by a member of a novel human protein methyltransferase family

“…Site-directed mutagenesis was performed using Quikchange II site-directed mutagenesis kit (Stratagene). Proteins were produced using standard methods 28,29 , variations are described in the Supplementary Methods.…”