Biology subjects

Yamagami, R.

Publications and source records attributed to Yamagami, R..

4 recordsLinked to original sources

bioRxiv2026

Dual-site specificity of the archaeal tRNA m2G methyltransferase Trm14

N2-methylguanosine (m2G) is widely found at multiple positions in tRNAs across the three domains of life. Tryptophan tRNA from Thermococcus kodakarensis contains m2G at position 67. We previously proposed that the tRNA m2G methyltransferase Trm14 is responsible for m2G67 formation in tRNATrp from T. kodakarensis, although Trm14 was originally identified as the enzyme catalyzing m2G6 formation in tRNACys in Methanocaldococcus jannaschii. Thus, it remained unclear whether Trm14 could also methylate G67. Here, we characterized archaeal Trm14. Biochemical analyses using recombinant T. kodakarensis Trm14 revealed that the enzyme catalyzes m2G formation at positions 6 and 67 in T. kodakarensis tRNACys and tRNATrp transcripts, respectively. Mass spectrometric analyses demonstrated the loss of m2G6 and m2G67 in native tRNACys and tRNATrp, respectively, from a T. kodakarensis trm14 gene disruptant strain, providing direct evidence for the dual-site specificity of T. kodakarensis Trm14. The growth phenotype of the trm14 gene disruptant strain was comparable to that of the wild-type strain. In contrast, a trm14/trm11 double disruptant, in which trm11 encodes the tRNA m2G10/m22G10 methyltransferase, exhibited severe growth retardation at 95 {degrees}C. This suggests that m2G6/m2G67 and m2G10/m22G10 cooperatively contribute to cellular fitness at high temperatures. Biochemical analyses revealed that Trm14 methylates all 46 T. kodakarensis tRNA transcripts. Furthermore, we found that recombinant M. jannaschii 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.

molecular biology↗

bioRxiv2026

Optimized tRNA Structure-seq reveals robust tRNA secondary structures in S. cerevisiae under mild stress conditions

RNA structure plays a crucial role in diverse biological processes beyond the translation of genetic information. Therefore, the development of reliable methods for RNA structure prediction is essential for understanding RNA structure-related functions, however accurate and comprehensive RNA structure prediction remains challenging. Here, we focus on secondary structure prediction of transfer RNA (tRNA) using structure probing coupled with next-generation sequencing (tRNA Structure-seq). In silico prediction of Saccharomyces cerevisiae tRNA secondary structures achieves only 56.9% accuracy on average. Incorporation of dimethyl sulfate (DMS) probing data improve prediction accuracy to 87.4%, which is still not sufficient for practical tRNA structure prediction. To overcome this, we optimized the tRNA Structure-seq analysis pipeline by explicitly incorporating natural tRNA modifications detected in tRNA sequencing data and by refining pseudo-free energy parameters specifically optimized for tRNA structure prediction. Using this optimized pipeline, the average prediction accuracy is remarkably improved to 94%. Furthermore, analysis of multiple structural conformations predicted from DMS probing data indicates that S. cerevisiae tRNAs predominantly adopt the canonical cloverleaf secondary structure under in vivo conditions. Finally, we examined tRNA structures under mild stress conditions, including heat stress, osmotic stress, and antibiotic stress. These perturbations had minimal effects on in vivo tRNA secondary structure, demonstrating that S. cerevisiae tRNAs maintain structural stability under physiologically relevant stress conditions. In summary, our results establish an optimized tRNA Structure-seq analysis that enables highly accurate tRNA secondary structure prediction and reveals the intrinsic robustness of tRNA structures in living cells.

molecular biology↗

bioRxiv2025

A transfer RNA methyltransferase with an unusual domain composition catalyzes 2'-O-methylation at position 6 in tRNA.

Thermococcus kodakarensis tRNATrp contains 2'-O-methylcytidine at position 6 (Cm6). However, the tRNA methyltransferase responsible for the modification has not been identified. Using comparative genomics we predicted TK1257 as a candidate for the modification. Biochemical and mass spectrometry studies of purified recombinant TK1257 gene product show it to possess a tRNA methyltransferase activity for Cm6 formation. This protein has a highly unusual composition of domains, containing N-terminal ferredoxin-like, SPOUT catalytic and THUMP domains. Previous to this study, all known THUMP-related tRNA methyltransferases were shown to contain a Rossmann fold catalytic domain and the nucleosides they produced were N2-methylguanosine and/or N2, N2-dimethylguanosine. Therefore, our findings extend the knowledge of architecture of tRNA methyltransferases. We named the TK1257 gene product TrmTS and showed it can synthesize Am6 and Um6 as well as Cm6. A trmTS gene deletion strain showed slight growth retardation at high temperatures. Site-directed mutagenesis studies based on structural model revealed catalytically and structurally important amino acid residues in TrmTS and identified a TrmTS-specific linker is structurally essential. We showed that TrmTS recognizes the 3'-CCA terminal region and a stretch loop connected with at least two stems in RNA. Finally, we constructed a model of the binding between TrmTS and tRNA.

biochemistry↗

bioRxiv2023

RNA folding studies inside peptide-rich droplets reveal roles of modified nucleosides at the origin of life

Compartmentalization of RNA in biopolymer-rich membraneless organelles is now understood to be pervasive and critical for the function of extant biology and has been proposed as a prebiotically-plausible way to accumulate RNA. However, compartment-RNA interactions that drive encapsulation have the potential to influence RNA structure and function in compartment- and RNA sequence-dependent ways. Herein, we detail Next-Generation Sequencing (NGS) experiments performed for the first time in membraneless compartments called complex coacervates to characterize the fold of many different transfer RNAs (tRNAs) simultaneously under the potentially denaturing conditions of these compartments. Strikingly, we find that natural modifications favor the native fold of tRNAs in these compartments. This suggests that covalent RNA modifications could have played a critical role in metabolic processes at the origin of life. One Sentence SummaryWe demonstrate that RNA folds into native secondary and tertiary structures in protocell models and that this is favored by covalent modifications, which is critical for the origins of life.

biochemistry↗