Dual-site specificity of the archaeal tRNA m ² G methyltransferase Trm14

<jats:title>Abstract</jats:title> <jats:p> <jats:italic>N</jats:italic> <jats:sup>2</jats:sup> -methylguanosine (m <jats:sup>2</jats:sup> G) is widely found at multiple positions in tRNAs across the three domains of life. Tryptophan tRNA from <jats:italic>Thermococcus kodakarensis</jats:italic> contains m <jats:sup>2</jats:sup> G at position 67. We previously proposed that the tRNA m <jats:sup>2</jats:sup> G methyltransferase Trm14 is responsible for m <jats:sup>2</jats:sup> G67 formation in tRNA <jats:sup>Trp</jats:sup> from <jats:italic>T. kodakarensis</jats:italic> , although Trm14 was originally identified as the enzyme catalyzing m <jats:sup>2</jats:sup> G6 formation in tRNA <jats:sup>Cys</jats:sup> in <jats:italic>Methanocaldococcus jannaschii</jats:italic> . Thus, it remained unclear whether Trm14 could also methylate G67. Here, we characterized archaeal Trm14. Biochemical analyses using recombinant <jats:italic>T. kodakarensis</jats:italic> Trm14 revealed that the enzyme catalyzes m <jats:sup>2</jats:sup> G formation at positions 6 and 67 in <jats:italic>T. kodakarensis</jats:italic> tRNA <jats:sup>Cys</jats:sup> and tRNA <jats:sup>Trp</jats:sup> transcripts, respectively. Mass spectrometric analyses demonstrated the loss of m <jats:sup>2</jats:sup> G6 and m <jats:sup>2</jats:sup> G67 in native tRNA <jats:sup>Cys</jats:sup> and tRNA <jats:sup>Trp</jats:sup> , respectively, from a <jats:italic>T. kodakarensis trm14</jats:italic> gene disruptant strain, providing direct evidence for the dual-site specificity of <jats:italic>T. kodakarensis</jats:italic> Trm14. The growth phenotype of the <jats:italic>trm14</jats:italic> gene disruptant strain was comparable to that of the wild-type strain. In contrast, a <jats:italic>trm14</jats:italic> / <jats:italic>trm11</jats:italic> double disruptant, in which <jats:italic>trm11</jats:italic> encodes the tRNA m <jats:sup>2</jats:sup> G10/m <jats:sup>2</jats:sup> <jats:sub>2</jats:sub> G10 methyltransferase, exhibited severe growth retardation at 95 °C. This suggests that m <jats:sup>2</jats:sup> G6/m <jats:sup>2</jats:sup> G67 and m <jats:sup>2</jats:sup> G10/m <jats:sup>2</jats:sup> <jats:sub>2</jats:sub> G10 cooperatively contribute to cellular fitness at high temperatures. Biochemical analyses revealed that Trm14 methylates all 46 <jats:italic>T. kodakarensis</jats:italic> tRNA transcripts. Furthermore, we found that recombinant <jats:italic>M. jannaschii</jats:italic> Trm14 methylated both positions. In contrast, the bacterial ortholog TrmN modified only position 6 in tRNA. Overall, this study expands our understanding of archaeal Trm14 by demonstrating its broader substrate specificity and the physiological significance of these modifications under hyperthermophilic conditions. </jats:p>