OTTR-seq Profiling Reveals Dynamic tRNA Modification Landscapes Across Diverse Archaeal Species

Here, we apply Ordered Two-Template Relay sequencing (OTTR-seq) to survey more than a dozen common tRNA modification types across nine archaeal species spanning diverse ecological niches. We uncover coordinated, 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 as tRNA modification anti-determinants.

Comparative analyses also reveal lineage-specific divergence in the domain architectures of tRNA methyltransferases, including Trm1, Trm10, Trm11, and Trm14. We further refine known identity elements such as the G10oU25 pairing and highlight novel structural contexts that may 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.