Thermococcus kodakarensis bisulfite-sequenced RNA. Thermococcus kodakarensis

1. While many modifications are limited in frequency, restricted to non-coding RNAs, or present only in select organisms, 5-methylcytidine (m5C) is abundant across diverse RNAs and fitness-relevant across Domains of life. Here, we mapped m5C in the model hyperthermophile, Thermococcus kodakarensis. We demonstrate that the m5C epitranscriptome includes ~10% of unique and sufficiently expressed transcripts. T. kodakarensis rRNAs harbor tenfold more m5C compared to most Eukarya or Bacteria, and m5C levels dynamically respond to environmental cues. We identified at least five RNA m5C methyltransferases (R5CMTs) that modify RNAs in vivo and in vitro. 2. Ribosome structure and activity are challenged at high temperatures, often demanding modifications to ribosomal RNAs (rRNAs) to retain translation fidelity. LC-MS/MS, bisulfite-sequencing, and high-resolution cryo-EM structures of the archaeal ribosome identified a new RNA modification, N4,N4-dimethylcytidine (m4,2C), at the universally conserved C918 in the 16S rRNA helix 31 loop.