The extensive m⁵C epitranscriptome of Thermococcus kodakarensis is generated by a suite of RNA methyltransferases...

RNAs are often modified to invoke new activities. 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, but the synthesis and impacts of m⁵C have yet to be fully investigated. Here, we map m⁵C in the model hyperthermophile, Thermococcus kodakarensis. We demonstrate that m⁵C is ~25x more abundant in T. kodakarensis than human cells, and the m⁵C epitranscriptome includes ~10% of unique transcripts. T. kodakarensis rRNAs harbor tenfold more m⁵C compared to Eukarya or Bacteria. We identify at least five RNA m⁵C methyltransferases (R5CMTs), and strains deleted for individual R5CMTs lack site-specific m⁵C modifications that limit hyperthermophilic growth. We show that m⁵C is likely generated through partial redundancy in target sites among R5CMTs. The complexity of the m⁵C epitranscriptome in T. kodakarensis argues that m⁵C supports life in the extremes.