Thermococcus kodakarensis bisulfite-sequenced RNA

We carried out global analysis of methyl-5 cytidine in RNA from Thermococcus kodakarensis, a hyperthermophilic archaeon with an extensive collection of RNA modifying enzymes. In triplicate and across two biological conditions, we took an ultra-deep sequenced approach of bisulfite treated RNA to comprehensively map m5C sites with single nucleotide resolution. In duplicate, we additionally bisulfite-sequenced RNA from 13 individual strains, each deleted for a unique, non-essential putative methyltransferase. We found 232 m5C sites in a diverse RNAs and identified at least four bona fide RNA m5C methyltransferases responsible for installing individual m5C modifications. Shifts in the m5C-epitranscriptome profile were observed across biological conditions. Cells deleted for individual R5CMTs exhibited impaired growth at increasing temperatures, indicating a role for m5C residues to support life at high temperatures. RNA methyltransferases identified in this work exhibited in vitro methyltransferase activity with apparent specificity for substrate RNAs identified from bisulfite sequencing. Lastly, we demonstrated that some sites are targeted for modification by multiple methyltransferases, indicating a partially redundant network of enzymes that maintain the epitranscriptome. Taken together, we identified a relatively large collection of RNA methyltransferases with demonstrated methylation activity that maintain an extensive and dynamic m5C-epitranscriptome in T. kodakarensis that supports life in the extremes.