The Landscape of tRNA Modifications in Archaea.

Transfer RNA (tRNA) modifications are essential for structural integrity, decoding fidelity, and stress adaptation, yet their evolutionary dynamics remain poorly understood. Here, we apply Ordered Two-Template Relay sequencing (OTTR-seq) to comprehensively profile tRNA modifications across nine archaeal species spanning diverse ecological niches. We uncover coordinated and mutually exclusive methylation at acceptor stem positions 6 and 67 in hyperthermophiles, as well as clade-specific co-modification at positions 10 and 26, which are typically known for their importance as tRNA modification anti-determinants. Comparative analyses also reveal lineage-specific divergence in the domain architectures of tRNA methyltransferases, including Trm14, Trm10, Trm11, and Trm1. We further refine known identity elements such as the G10oU25 pairing, and highlight novel structural contexts that facilitate or prevent modification. These findings exemplify the co-evolution of tRNAs and their modifying enzymes, providing new insights into how archaea may fine-tune translation in extreme environments. The scope of these data and comparative analyses establish a multispecies framework for future biochemical, mechanistic, and predictive modeling efforts.