Phosphate backbone epitranscriptomics: Discovery of natural RNA phosphorothioates and their writer machinery

Over 150 modifications expand the RNA alphabet, yet all known natural modifications occur on nucleobases or ribose sugars, with none identified on the phosphate backbone. In contrast, phosphorothioates (PSs), in which a non-bridging phosphate oxygen is replaced with sulfur, are central to RNA therapeutics but have never been reliably detected in natural RNAs. Here, we develop sequencing- and mass spectrometry-based approaches to quantitatively map RNA PSs at single-nucleotide resolution. Across diverse archaeal species, we identify stereospecific PS modifications at rRNA and tRNA hotspots, which are dynamically regulated by sulfur availability and temperature. We uncover a diverse enzyme family that selectively modifies tRNA/rRNA substrates and whose evolutionary presence/absence matches the distribution of PSs. Enzyme loss causes inviability or temperature sensitivity, and functional analyses reveal that tRNA PSs enhance tRNA stability. These findings establish the first natural RNA phosphate-backbone modification and its enzymatic machinery, providing a foundation for mechanistic and functional exploration.

National Cancer Institute
St. Jude Children's Research Hospital
Jagiellonian University
Universität Regensburg
Université de Strasbourg
Christian-Albrechts-Universität zu Kiel
Colorado State University
Virginia State University
University of Chinese Academy of Sciences
Tel Aviv University
Medizinische Universität Wien - AKH
Tokyo Metropolitan University
Weizmann Institute of Science