The Evolutionary Landscape of tRNA Modifications in Archaea: Insights from High-Throughput Sequencing.

Transfer RNA (tRNA) modifications play essential roles in structure, stability and decoding accuracy, yet the evolutionary dynamics and diversity of these modifications remain incompletely understood. Recent advances in high-throughput sequencing, including Ordered Two-Template Relay sequencing (OTTR-seq), now facilitate systematic, transcriptome-wide detection of tRNA modifications at single-base resolution. Here we employed OTTR-seq to comprehensively profile tRNA modifications across nine archaeal species spanning diverse ecological niches, including thermophiles, methanogens, acidophiles, and halophiles. We identified coordinated and mutually exclusive methylation patterns at acceptor stem position 6 and 67 in hyperthermophiles, as well as unexpected clade-specific co-modifications at core positions 10 and 26, demonstrating tolerance of canonical anti-determinants in Thermoprotei. Comparative genomic analyses revealed clear associations between modifications and evolutionary changes in enzyme domain architectures, including divergence in homologs of Trm14, Trm10, Trm11, and Trm1. We also expanded upon previously characterized identity elements in the D-stem, such as the conserved G10oU25 pair critical for Trm11 specificity, providing additional insight into determinants that likely govern enzyme-substrate interactions. Together, these findings offer valuable insights into archaeal tRNA modification biology and evolutionary dynamics, and provide a foundation for guiding targeted biochemical experiments and mechanistic studies.