[Tokyo Metropolitan University] Original source (Japanese) https://kyodonewsprwire.jp/release/202609246495
Points
We discovered that the linkers (connection points) between ribosomal RNA (rRNA) and transfer RNA (tRNA) in archaea, microorganisms that inhabit high-temperature environments, are modified with sulfur-containing chemical modifications called “thiophosphate modifications.” We then elucidated the molecular-level mechanism, including the function of enzymes, of how these modifications are produced.
This modification suggests that it plays an essential role in the survival of archaea in high-temperature environments.
Thiophosphate modification has been widely used as an artificial technique to make RNA drugs less susceptible to degradation, but this study is the first in the world to prove that the same modification also exists in naturally occurring RNA. By elucidating the mechanism by which this modification is created, it is expected that this will lead to new technologies for efficiently synthesizing RNA drugs in the desired shape (three-dimensional structure).
Overview
Yuko Nobu, a specially appointed researcher, and Mango Taoka, an associate professor, at the Graduate School of Science, Tokyo Metropolitan University, have discovered that in archaeal rRNA and tRNA, there is a sulfur modification (thiophosphate-modified linker) with a specific three-dimensional structure in the “phosphate linker” region that connects adjacent nucleosides. This research involved the Weizmann Institute (Israel), Colorado State University (USA), Christian Albrecht University (Germany), Virginia Tech (USA), Tel Aviv University (Israel), University of Regensburg (Germany), University of Strasbourg (France), Jagiellonian University (Poland), Veterinary and Medical University Vienna (Austria), National Cancer Institute (USA), and Medical University Vienna (Austria).
The thiophosphate-modified linkers of rRNA were concentrated in the region that reads the genetic code of messenger RNA (mRNA) and in the site where the central reaction for protein synthesis (peptide transfer reaction, Note 1) occurs, and the amount of modification varied depending on the temperature at which the archaea grew. Furthermore, it was found that the thiophosphate-modified linkers of tRNA are necessary to maintain the stability of tRNA.
Furthermore, the research group identified two new enzymes (Esti-A and Esti-B) that perform this stereoselective thiophosphate modification, using information from proteins thought to have evolutionarily similar functions as a clue. They found that the loss of function of these enzymes leads to various effects, such as archaea being unable to survive at all or their growth slowing down at high temperatures.
The results of this research were published in the academic journal “Cell,” published by Cell Press, on September 18, 2026 (Japan time). This research was supported by the Japan Society for the Promotion of Science (JSPS) Grants-in-Aid for Scientific Research 25K15441 and 24K01517, and the TMU GAP Fund (Entrepreneurial Activity Support Program).
Background of the Research
RNA has a chain-like structure in which four types of nucleosides—adenosine, guanosine, cytidine, and uridine—are linked together via phosphate-containing “junctions” (phosphate linkers) (Figure 1). To date, more than 150 types of natural chemical modifications have been reported to this basic structure, including methylation, acetylation, and amino acid addition. All of these known modifications occur on the base or ribose portion, and none have been found to occur on the phosphate linker. In 2020, Wu et al. reported the discovery of thiophosphate-modified linkers in prokaryotes and eukaryotes (Note 2), but this report was later refuted by Kaiser et al. (Note 3), and many RNA researchers came to believe that “thiophosphate-modified linkers do not exist in RNA.”
Chemical structure of RNA with thiophosphate-modified linker
RNA is composed of nucleosides, which are basically ribose molecules with one base (A, U, G, or C) attached to the 1′ position. RNA is a macromolecule formed when these nucleosides are linked together between the 3′ and 5′ positions by linkers (usually phosphate groups).
On the other hand, thiophosphate-modified linkers are a central chemical modification in RNA drugs that enhances stability and efficacy in the body, and are an important modification used in almost all RNA drugs except mRNA drugs.
Research Details
In this study, we identified sulfur on the phosphate linkers of archaeal rRNA and tRNA, proving the existence of thiophosphate-modified linkers in naturally occurring RNA (Figure 2). Thiophosphate-modified linkers have a three-dimensional structure centered on the phosphorus atom (“optical isomers” that take on two mirror-image forms), but all naturally occurring modified linkers were R-isomers. We also established a method for easily and rapidly identifying and quantifying thiophosphate-modified linkers from a large number of samples with the accuracy to distinguish a single linker. When this method was applied to various archaeal species, we found that (1) thiophosphate-modified linkers are present near the active site of the rRNA peptide transfer reaction, (2) their amount fluctuates depending on the presence of sulfur in the growth medium, and (3) some thiophosphate-modified linkers increase with increasing growth temperature. Next, using the evolutionary distribution of structurally similar proteins as a clue, we identified two novel enzymes, Esti-A and Esti-B, essential for the synthesis of thiophosphate linkers in archaea. Deficiencies in these enzymes in various archaeal species resulted in a range of effects, from slow growth at high temperatures to other effects. In particular, it has become clear that thiophosphate-modified linkers of tRNA are necessary for tRNA stability.
This study is the first to discover RNA with a thiophosphate linker in which one of the four oxygen atoms bound to the phosphorus of the linker is replaced by sulfur. Furthermore, it was found that enzymes called Esti-A or Esti-B are utilized for this oxygen-sulfur substitution in the linker.
Significance and ripple effects of the research
This discovery is the first to reveal the thiophosphate modification that occurs in the linker region of natural RNA and the enzyme system that produces it. Furthermore, the functional characteristics of this modification were analyzed. This provides a foundation for further investigation into how thiophosphate modification functions in vivo and offers an important set of enzymes for the application of RNA medicine in the treatment and prevention of diseases. Thiophosphate-modified linkers, which play a crucial role in controlling drug efficacy, are indispensable components of RNA medicines. Therefore, “stereoselective synthesis,” which selects and produces only the desired shape (three-dimensional structure), is the goal in RNA drug synthesis. However, there is currently no convenient method, making commercial synthesis difficult. In this respect, the synthetic enzymes Esti-A and Esti-B discovered in this study are expected to be useful in future RNA drug synthesis.
Glossary
Note 1: The peptide transfer reaction active site is located in the ribosomal large subunit and is the site that catalyzes the reaction of joining the carboxyl group and amino group of an amino acid. It is composed of rRNA and attaches a peptide chain bound to tRNA to an amino acid on another tRNA. As a result, the peptide chain is elongated and a protein is synthesized.
Note 2: In a paper published in 2020 by Wu et al. (DOI: 10.1021/acschembio.0c00163), they discovered thiophosphate modifications in the linker region of rRNA in prokaryotes and eukaryotes, and reported that the gene group involved in thiophosphate modification of DNA may also be involved in RNA modification.
Note 3: In a paper published in 2021 by Kaiser et al. (DOI: 10.1002/anie.202106215), they pointed out common pitfalls in RNA modification analysis using mass spectrometry and questioned the existence of thiophosphate modifications in RNA as reported by Wu et al.
Publication Information Publication: Cell
Title: Discovery of Natural RNA Phosphorothioates and their Writer Machinery
Authors: Alexander Maman, Yuko Nobe*, Kristin A. Fluke*, Katrin Weidenbach, Alexandra N. Harte, Brett W. Burkhart, Jakub Nowak, Priyadarshini Mukherjee, Deepak Kumar Choudhary, Anatoly Kustanovich1, Katharina Vogl, Kiall Suazo, Danijela Radovanović, Donna Matzov, Walter Rossmanith, Jordan L. Meier, Moran Shalev-Benami, Dina Grohmann, Kylie D. Allen, Eric Westhof, Sebastian Glatt, Ruth A. Schmitz, Thomas J. Santangelo#, Masato Taoka#, Schraga Schwartz1# (*etc. contributions, #corresponding author)
DOI:10.1016/j.cell.2026.08.034
Phosphate backbone epitranscriptomics: Discovery of natural RNA phosphorothioates and their writer machinery, Cell via PuBMed
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.
Astrobiology, Genomics, evolution,
