Comprehensive nucleoside analysis of archaeal RNA modification profiles reveals an m⁷G in the conserved P loop of 23S...

Extremophilic Archaea employ diverse RNA modifications for survival. Our understanding of the modified nucleosides and their functions in Archaea is far from complete. Here, we establish an extensive profile of nucleoside modifications in thermophilic and mesophilic Archaea. Through liquid chromatography-tandem mass spectrometry (LC-MS/MS) and rigorous non-coding RNA depletion, we identify four previously unannotated modifications in archaeal mRNA. Nucleoside analysis conducted on total, large, small, and mRNA-enriched subfractions of hyperthermophile Thermococcus kodakarensis reveals modifications whose relative abundance is dynamically responsive to growth temperatures. To predict archaeal RNA-modifying enzymes, we leverage open-access databases to compare putative functional domains with previously annotated enzymes. Our approach leads to the discovery of a methyltransferase responsible for the installation of m⁷G in the P loop of 23S rRNA peptidyl transferase center in T. kodakarensis. The methyltransferase activity is confirmed in vitro with synthetic substrates and in vivo by assessing the presence of the m⁷G modification in a genetic deletion strain.