Comprehensive Nucleoside Analysis of Archaeal RNA Modification Profiles Reveals a m7G in the Conserved P-loop of 23S rRNA

ABSTRACT: Extremophilic archaea employ diverse chemical RNA modifications, providing a rich source of new enzymes for biotechnologically valuable RNA manipulations. Our understanding of the modified nucleoside profiles in Archaea, as well as the functions and dynamic regulation of specific RNA modifications is far from complete. Here, we established an extensive profile of nucleoside modifications in thermophilic and mesophilic Archaea through highly sensitive LC-MS/MS analysis and rigorous non-coding RNA depletion, identifying - with high confidence - at least four previously unannotated modifications in archaeal mRNAs. Nucleoside quantification analysis conducted on total, large, small, and mRNA-enriched subfractions of the model hyperthermophilic archaeon Thermococcus kodakarensis revealed a series of modifications whose abundance is dynamically responsive to growth temperatures, implying that specific RNA modifications are fitness relevant under specific growth conditions. To predict the RNA-modifying enzymes most likely to generate the new and dynamic RNA modifications, we leveraged a bioinformatics analysis of open-access databases to annotate likely functional domains of archaeal proteins. Putative enzyme activities were confirmed in vitro and in vivo by assessing the presence of the target RNA modification in genetic deletion strains of T. kodakarensis. Our approach led to the discovery of a methyltransferase-encoded gene responsible for m7G modification in the P-loop of 23S rRNA peptidyl transferase center. This novel finding validates an effective platform for discovering RNA-modifying enzymes through LC-MS/MS analysis that will accelerate efforts of the community towards uncovering the complex and dynamic roles of RNA modifications.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Thermococcus Kodakarensis

SUBMITTER: Yueh-Lin Tsai  

LAB HEAD: Ivan R.

PROVIDER: PXD060644 | Pride | 2025-02-12

REPOSITORIES: pride