A novel N4,N4-dimethylcytidine in the archaeal ribosome enhances hyperthermophily

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 an RNA modification, N4,N4-dimethylcytidine (m⁴₂C), at the universally conserved C918 in the 16S rRNA helix 31 loop. Here, we characterize and structurally resolve a class of RNA methyltransferase that generates m⁴₂C whose function is critical for hyperthermophilic growth. m⁴₂C is synthesized by the activity of a unique family of RNA methyltransferase containing a Rossman-fold that targets only intact ribosomes. The phylogenetic distribution of the newly identified m⁴₂C synthase family implies that m⁴₂C is biologically relevant in each domain. Resistance of m⁴₂C to bisulfite-driven deamination suggests that efforts to capture m⁵C profiles via bisulfite sequencing are also capturing m⁴₂C.