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

ABSTRACT: Over 170 distinct chemical modifications expand the RNA alphabet, yet all known natural modifications occur on nucleobases or ribose sugars; none have been identified on the phosphate backbone. In contrast, phosphate backbone modifications, and particularly phosphorothioates (PS), a replacement of a non-bridging oxygen with a sulfur group, are central to RNA therapeutics, enhancing stability and bioavailability. While PS modifications are known on DNA, they were never reliably detected on natural RNA. Here, we establish sequencing- and mass spectrometry–based workflows that quantitatively map RNA PS at single-nucleotide resolution. Across diverse archaeal species, we identify highly specific PS at conserved positions in ribosomal RNAs (rRNAs) and transfer RNA (tRNA) hotspots, including in proximity to the peptidyltransferase center. Across most targets PS deposition was highly dynamic with sulfur availability, and in a subset of targets PS deposition was temperature-dependent. Guided by evolutionary distribution, we uncover a novel enzyme family, broadly divided into two subclasses, that installs PS on distinct tRNA or rRNA substrates. Loss of these enzymes in diverse species leads to phenotypes ranging from complete inability to grow to temperature-dependent growth lag, and biophysical follow-ups reveal a role for phosphorothioates in thermostabilization. Our findings reveal the first natural RNA backbone modification, its enzymatic machinery, and initial functional roles, supplying both the tools and the map for further mechanistic dissection and potential therapeutic exploitation, and raise the possibility that additional natural backbone modifications may remain to be discovered.

ORGANISM(S): Pyrococcus furiosus Methanomassiliicoccus luminyensis synthetic construct Methanococcus maripaludis Pyrodictium abyssi Haloferax volcanii Thermococcus kodakarensis Methanobrevibacter smithii Methanosarcina mazei Methanosphaera stadtmanae

PROVIDER: GSE315103 | GEO | 2026/09/17

REPOSITORIES: GEO