Translational regulation by oxidative desulfuration of tRNA modifications

Domain: biology
Published: 2026-03-10
Editorial outcome: major_revision

Abstract
Modifications in the anticodon region of transfer RNA (tRNA) are essential for accurate and efficient protein synthesis. 5-Methyl-2-thiouridine derivatives (xm5s2U) are major modifications at the wobble position of tRNA anticodons decoding purine-ending two-codon sets. Although the thiocarbonyl group of xm5s2U enhances decoding efficiency, it is chemically susceptible to oxidative desulfuration, yielding 4-pyrimidinone derivatives (xm5h2U). Here, we identify xm5h2U derivatives in human cells and mouse tissues and confirm their cellular formation by spike-in experiments. Desulfurized tRNAs carrying 5-methoxycarbonylmethyl-4-pyrimidinone (mcm5h2U) show impaired codon recognition in a human reconstituted in vitro translation system. The mcm5h2U modification reduces aminoacylation of tRNAs for lysine, glutamate, and glutamine, but not arginine. Cryogenic electron microscopy reveals the structural basis of altered AAA/AAG decoding by mcm5h2U at the ribosomal A-site. These findings reveal a mechanism by which oxidative desulfuration of tRNA modifications dynamically regulates codon recognition and protein synthesis under oxidative stress conditions in human and mammalian cells.

Introduction
Higher-order biological processes emerge from sophisticated regulation of gene expression at multiple stages at both transcriptional and post-transcriptional levels, including translational control. In protein synthesis, transfer RNA (tRNA) anticodons recognize and decode messenger RNA (mRNA) codons on the ribosome. tRNAs undergo the most extensive and diverse range of post-transcriptional modifications, particularly within the anticodon region. To date, ~150 types of RNA modifications have been identified in various RNA molecules across all domains of life, with tRNAs comprising over 80% of these modifications1. Recent research indicates that tRNA modifications respond dynamically to cellular metabolite levels and environmental stresses, temporally and spatially influencing translational regulation significantly2–7.

The anticodon of tRNA, located at positions 34, 35, and 36, recognizes a specific codon on the mRNA through base pairing at the A-site of the ribosome. In this interaction, the first and second nucleotides of the codon form Watson–Crick base pairs with the third (position 36) and second (position 35) nucleotides of the anticodon, respectively. These two Watson–Crick base pairs in the codon–anticodon helix are strictly monitored through A-minor interactions8 with conserved residues in the decoding center of the small ribosomal subunit (corresponding to A1492, A1493, and G530 of the bacterial 16S rRNA)9. These interactions trigger a large conformational rearrangement of the small subunit and induce GTP hydrolysis by the elongation factor bound to the ribosome, thereby ensuring accurate decoding during protein synthesis. By contrast, base pairing between the third nucleotide of the codon and the first nucleotide (position 34) of the anticodon does not necessarily follow the Watson–Crick pairing rules; instead, wobble base pairing is permitted10. This mechanism allows 61 sense codons to be efficiently decoded by a limited number of tRNAs. The first position of the anticodon often contains diverse modifications that promote or modulate codon–anticodon interactions, thereby enabling accurate and efficient decoding of the genetic code11–13. Defects in tRNA modifications frequently impair multiple stages of protein synthesis—including aminoacylation, decoding, and translocation—and can lead to pathological consequences2,14–16.

2-Thiouridine (s2U) and its derivatives (xm5s2U; Fig. 1a, b) are highly conserved wobble modifications found at the first position of the anticodon in all domains of life17–19. In bacteria and archaea, 5-methylaminomethyl-2-thiouridine (mnm5s2U) and 5-carboxymethylaminomethyl-2-thiouridine (cmnm5s2U) occur in total tRNA for Glu, Gln, and Lys. In eukaryotes, 5-methoxycarbonylmethyl-2-thiouridine (5-methylcarboxymethyl-2-thiouridine, mcm5s2U)20 and 5-taurinomethyl-2-thiouridine (τm5s2U)21 are present in cytoplasmic and mitochondrial tRNAs for Lys, Glu and Gln, respectively22,23. Mammalian cytosolic tRNAArg responsible for AGR codons also has mcm5s2U24. Recent studies identified 2′-O-methyl-2-thiouridine (s2Um) and 5-cyanomethyl-2-thiouridine (cnm5s2U) in archaeal tRNAs25–27. The s2U derivatives are also present at position 54 of tRNAs. 5-Methyl-2-thiouridine (m5s2U) and 1-methyl-4-thiopseudouridine (m1s4Ψ) are present in thermophilic bacteria and archaea28–30.

The xm5s2U modification performs pleiotropic roles in translation, including efficient aminoacylation of tRNAs31–33, accurate and efficient decoding of purine-ending codons (NNR)17,34, and rapid translocation on the ribosome35. The xm5s2U modification favors a C3′-endo sugar pucker conformation due to steric repulsion between 2-thiocarbonyl and 2′-hydroxyl groups36,37, so that xm5s2U prevents misdecoding of pyrimidine-ending codons (NNY). In addition, structural analyses revealed that mnm5s2U34 base pairs with the third guanosine of NNG codons at the A-site of the ribosome via an anionic U • G wobble pairing38,39. In this base pairing geometry, the uracil base of xm5s2U34 moves toward the minor groove39, rather than the major groove as occurs in canonical U • G wobble pairing10. In this geometry, 2-thio group of xm5s2U contributes to stacking interaction with the second base pair of the codon–anticodon helix38. To make this wobble pair possible, N3 of the uracil base should be deprotonated. In fact, s2U modification promotes acidity of the N3 atom of the uracil base by lowering its pKa value40. This is the molecular basis by which xm5s2U facilitates the decoding of G-ending codons. However, according to structural study of the anticodon-stem loop bound to 30S ribosomal subunit41, the mcm5s2U–G base pair adopts a Watson–Crick-like geometry, and it was proposed that mcm5s2U may undergo a tautomeric shift of the uracil base from keto- to enol form, allowing it to form non-canonical hydrogen bonds with G.

Biogenesis of 2-thio modification of xm5s2U34 has been extensively studied in both bacteria and eukaryotes17,18,30. Cysteine desulfurase (bacterial IscS and eukaryotic Nfs1) initiates s2U formation by extracting a sulfur atom from cysteine as a persulfide, which is then relayed by several sulfur mediators and transferred to U34 of tRNA by thiouridylases (bacterial MnmA and the eukaryotic CTU1/CTU2 complex)42–46. In bacteria, the sulfur is relayed by multiple sulfur mediators through persulfide chemistry45, whereas in eukaryotes, sulfur transfer is facilitated by a ubiquitin-activating E1-like enzyme and ubiquitin-like proteins43,47. In bacteria, loss of s2U modification due to deficiency of mnmA or sulfur mediators causes a slow growth phenotype46, highlighting the physiological importance of this modification. Disruption of the CTU1/CTU2 complex impairs cytoplasmic s2U modification in yeast48, worms42, and plants49. Intriguingly, loss of cytoplasmic s2U showed a synthetic lethality together with deficiency of 5-methoxycarbonylmethyl modification of mcm5s2U48. On the other hand, loss of mitochondrial s2U modification due to mutation of the yeast mitochondrial tRNA-specific 2-thiouridylase gene mtu1 resulted in deficiency of mitochondrial protein synthesis and respiratory function50. It is known that pathogenic mutations in the human MTU1 gene cause reversible infantile liver failure (RILF), a heritable and acute liver failure followed by spontaneous recovery at an early stage of life51,52. Loss-of-function mutations in the CTU2 gene cause severe primary microcephaly, ambiguous male genitalia, dysmorphic facies, polydactyly, and renal agenesis (DREAM-PL)53,54.

Due to the chemical properties of the thiocarbonyl group, xm5s2U modifications are chemically unstable, and readily oxidized and desulfurized under in vitro oxidative conditions, forming uridine or 4-pyrimidinone derivatives (xm5h2U)(Fig. 1b)55,56. h2U predominantly adopts the C2′-endo form57, suggesting less stable base pairing with A or G than s2U. Synthetic RNA duplexes containing an h2U • A pair exhibited significantly lower melting temperatures than those with an s2U • A pair, while duplexes with either an s2U • G or h2U • G pair displayed similar melting temperatures58. If desulfuration of xm5s2U occurs in cells, xm5h2U34 is likely to negatively regulate protein synthesis. In fact, mcm5h2U and cm5h2U, the desulfurized forms of mcm5s2U and cm5s2U, have been found in tRNAs from yeast and various human cultured cells regardless of the presence or absence of oxidative stress, reaching up to 11% depending on the efficiency of the antioxidant defense mechanisms59. However, their mechanistic roles in translational regulation have not been elucidated.

Through a spike-in experiment, we confirmed that h2U derivatives are naturally occurring in cellular processes, rather than artifacts produced during tRNA extraction and purification. The mcm5h2U34 modification significantly impairs the aminoacylation efficiency of tRNAs for Lys, Glu, and Gln, eliminates AAA decoding, and substantially decreases AAG decoding at the A-site of the ribosome. This alteration affects protein synthesis in a human reconstituted in vitro translation system. Cryogenic electron microscopy (cryo-EM) structural analyses of ribosomes complexed with desulfurized tRNAs and AAR codons showed that the mcm5h2U • G pair has a minor groove-shifted U • G wobble geometry, whereas the mcm5h2U • A pair adopts a unique geometry with a single hydrogen bond (H-bond). These findings indicate that oxidative desulfuration of tRNA modifications influences protein synthesis under oxidative stress conditions.

Results
Detection of h2U derivatives in mammalian and bacterial tRNAs
We reviewed our previous liquid chromatography-mass spectrometry (LC-MS) data on cytoplasmic and mitochondrial tRNAs in mammalian tissues and human culture cells22, and detected unassigned nucleosides with a molecular mass 32 Da lower than that of mcm5s2U (m/z 333) and τm5s2U (m/z 398), implying that h2U derivatives are indeed present in cellular tRNAs. In LC-MS nucleoside analyses of total RNA from mouse livers, we clearly detected unassigned nucleosides with m/z 301 (Fig. 1c). In addition, we also detected nucleosides with m/z 366 in mitochondrial tRNALys isolated from pig liver (Fig. 1d). Collision-induced dissociation (CID) of these nucleosides generated base-related ions (BH+) with loss of unmodified ribose (Fig. 1e, f), suggesting that the 32 Da loss originates from their bases.

To confirm that these nucleosides are h2U derivatives, we prepared authentic h2U derivatives by in vitro oxidation of mouse tRNA fraction and pig mitochondrial tRNALys, and found massive accumulation of mcm5h2U (Fig. 1c) and τm5h2U (Fig. 1d). Next, we conducted LC-MS co-injection analyses of nucleosides from total RNA and its in vitro oxidized form, and demonstrated that the unassigned nucleosides co-eluted with the authentic h2U derivatives (Fig. 1c, d), confirming the presence of mcm5h2U and τm5h2U in mammalian tissues.

We also detected an unassigned nucleoside (m/z 272) with molecular mass 32 Da lower than that of mnm5s2U (m/z 304) in Escherichia coli tRNAs (Supplementary Fig. 1a, b). LC-MS co-injection with in vitro oxidized total tRNA confirmed the identity of this nucleoside as mnm5h2U (Supplementary Fig. 1a). To test whether mnm5h2U arises from desulfuration of mnm5s2U, we analyzed nucleosides from the E. coli ΔmnmA strain, which lacks 2-thiolation. In this strain, neither mnm5s2U nor mnm5h2U was detected (Supplementary Fig. 1c), supporting that mnm5h2U is derived from mnm5s2U. Similarly, we analyzed cytoplasmic tRNAGln from wild-type Saccharomyces cerevisiae and a ctu1Δ strain lacking 2-thiolation of mcm5s2U (Supplementary Fig. 1d). In wild-type tRNAGln, mcm5s2U was detected as the major modification, whereas the ctu1Δ strain showed only mcm5U and no detectable mcm5h2U (Supplementary Fig. 1d). These results confirm that mcm5h2U, like mnm5h2U, is generated from the corresponding 2-thiouridine derivative.

Confirmation of h2U derivatives produced in cells
Given the chemical nature of xm5s2U modifications, we initially hypothesized that their h2U derivatives might result from artificial oxidation during preparation of tRNAs in vitro. To confirm that h2U derivatives, namely, mcm5h2U, τm5h2U, and h2U, are indeed generated in mammalian cells, we conducted a spike-in experiment using E. coli tRNA as a tracer. This approach allowed us to estimate the extent of artificial oxidation of xm5s2U during in vitro handling of tRNAs. As a tracer, we isolated E. coli tRNAGln1, which bears cmnm5s2U at position 34 of the anticodon (Fig. 2a). This tRNA was chosen because cmnm5s2U and its desulfurized derivative, cmnm5h2U, are distinct from xm5s2U and xm5h2U found in mammalian tRNAs. In our experiment, E. coli tRNAGln1 was added to a mouse liver homogenate as a spike-in tracer, followed by extraction of total RNA and purification of individual tRNAs, including the tracer, using reciprocal circulating chromatography (RCC)60 (Fig. 2a).

To monitor artificial desulfuration of s2U derivatives during RNA extraction and tRNA purification, we measured the rate of conversion of cmnm5s2U to cmnm5h2U in the tracer tRNA (Fig. 2a). We isolated cytoplasmic tRNALys and tRNAGln, as well as mitochondrial tRNALys and tRNAGlu, along with E. coli tRNAGln1 as a tracer from mouse liver lysates. The tracer tRNA was then digested with RNase T1, and its anticodon-containing fragments were analyzed by capillary LC nano-electrospray ionization (ESI)-MS, revealing the presence of cmnm5s2U, cmnm5h2U, and cmnm5U (Fig. 2b). We detected cmnm5h2U in ~2% of the tracer tRNA prior to the spike-in experiment (Fig. 2b and Supplementary Fig. 2a). CID analysis confirmed the presence of cmnm5h2U at position 34 (Fig. 2c). After isolation with mouse tRNAs, the level of cmnm5h2U remained virtually unchanged (Fig. 2b, d, and Supplementary Fig. 2b, c), indicating that artificial formation of h2U derivatives during handling of tRNAs was negligible under our experimental conditions.

Using this approach, we identified mcm5h2U in 9.9% and 7.2% of mouse cytoplasmic tRNALys and tRNAGln, respectively (Fig. 3a–d). CID analyses confirmed the presence of mcm5h2U at position 34 in both tRNAs (Fig. 3e, f). Similarly, τm5h2U was detected in 2.3% and 2.9% of mitochondrial tRNALys and tRNAGlu, respectively (Fig. 3g–j). h2U was also identified in 0.4% and 1.3% of these tRNAs, respectively (Fig. 3g–j). The presence of τm5h2U at position 34 in both mitochondrial tRNAs was further validated by CID analyses (Fig. 3k, l).

To expand on these findings, we performed similar analyses in various cultured cell lines (Supplementary Fig. 3). We observed that mcm5h2U was present at varying levels across different cell lines. Notably, ~15% of tRNALys in HeLa cells contained mcm5h2U, while it was scarcely detected in A549 and T24 cells.

Translation efficiency decreases upon desulfuration of tRNAs
Given that 2-thiouridine derivatives play crucial roles in accurate and efficient translation, we wondered whether h2U derivatives affect translation in a codon-specific manner. We examined the function of desulfurized tRNAs using a human cell-free translation system. To this end, we prepared desulfurized tRNAs by in vitro oxidation with H2O256 and confirmed efficient conversion of mcm5s2U to mcm5h2U (Supplementary Fig. 4a). As a reporter mRNA, Renilla luciferase (Rluc) with an N-terminal hemagglutinin (HA) was used (Fig. 4a), and translation initiation was mediated by the Hepatitis C virus (HCV) internal ribosome entry site (IRES) (Fig. 4a). As a control mRNA, we employed a mutant HiBiT peptide that is unaffected by tRNA desulfuration (Fig. 4a). Since mcm5s2U is present in four mammalian cytoplasmic tRNAs for Lys, Glu, Gln, and Arg (encoded by AGR codons), two Lys residues in the HiBiT peptide (VSGWRLFKKIS) were substituted with Arg encoded by CGN codons to generate a mutant HiBiT construct (SGWRLFRRIS) which forms a complex with Large BiT, reconstituting functional nanoluciferase activity.

The Rluc reporter mRNA and either desulfurized or untreated total tRNA were added to a reconstituted human cell-free protein synthesis system consisting of human 80S ribosomes, translation factors, and aminoacyl-tRNA synthetases61. The activity of Rluc was measured by luciferase assay. As expected, significant Rluc activity was observed when untreated tRNA was used, whereas it was markedly reduced when desulfurized tRNA was used (Fig. 4b). This result was corroborated by western blotting using an anti-HA antibody (Fig. 4c).

To demonstrate that this effect is specific to certain codons, the mutant HiBiT mRNA was translated and nanoluciferase activity was measured. We observed similar levels of activity regardless of whether desulfurized or untreated tRNA was used (Fig. 4b). Among 378 amino acid residues in the Rluc reporter sequence, 77 codons are read by the four tRNA species containing mcm5s2U, accounting for 22% of the total (Supplementary Data 1). Specifically, it consists of 62 A-ending codons and 15 G-ending codons. The G-ending codons are redundantly decoded by isodecoders having either mcm5s2U34 or C34, whereas the A-ending codons are decoded solely by mcm5s2U34-tRNAs. These findings suggest that the translational activity of mcm5s2U-modified tRNAs is impaired by desulfuration.

Since desulfurized tRNAs had less activity in an in vitro translation, we next examined whether desulfurized tRNAs participate in protein synthesis in cells by detecting mcm5h2U in polysome-associated tRNAs. HEK293T cell lysate was first separated into the supernatant (free RNA) and crude ribosome fractions using a sucrose cushion (Fig. 4d), followed by sucrose density gradient (SDG) centrifugation to obtain the polysome fraction (Fig. 4e). Small RNA were then obtained from the free RNA and polysomal RNA, and subjected to the LC-MS nucleoside analysis, showing that mcm5h2U is present in the polysomal RNA (Fig. 4f, g). As expected, the signal intensity of mcm5h2U in the polysomal RNA was lower than that in the total RNA and free RNA fractions. These results suggest that a portion of desulfurized tRNAs participates in translation, whereas others might be excluded.

Effect of tRNA desulfuration on aminoacylation
It has been revealed that the desulfuration of mcm5s2U impairs protein synthesis. We next investigated which step of protein synthesis is affected by mcm5h2U. Although 2-thiomodification of xm5s2U is known to contribute to efficient aminoacylation of tRNAs in bacteria31–33, the role of the 2-thiogroup of mcm5s2U in tRNA aminoacylation remains poorly understood in mammals. To examine the effect of desulfuration of mcm5s2U on aminoacylation, we performed in vitro aminoacylation reactions using recombinant human aminoacyl-tRNA synthetases and total tRNAs with or without desulfuration.

As a control, we used tRNAs Thr, which do not have mcm5s2U. Little change in Thr incorporation into total tRNAs was observed regardless of desulfuration treatment (Fig. 5a). Next, we compared the incorporation activities of four amino acids (Arg, Gln, Glu, and Lys) into total tRNAs with or without desulfuration. For Arg, no difference in the incorporation efficiency was observed between desulfurized and untreated total tRNA (Fig. 5b), indicating that arginyl-tRNA synthetase (RARS1) does not recognize mcm5s2U34 in tRNAArg (AGR) for its aminoacylation. By contrast, significant reductions in aminoacylation activity were observed for tRNAs for Gln, Glu, and Lys upon desulfuration (Fig. 5c–e). Considering that tRNAs charged with these amino acids are a mixture of isoacceptors including isodecoders (Supplementary Data 2)62, some parts of which carry mcm5s2U34, it became evident that desulfuration of mcm5s2U34 significantly impairs aminoacylation of these tRNAs. Thus, we found that the three aminoacyl-tRNA synthetases, glutaminyl-tRNA synthetase (QARS1), glutamyl-prolyl-tRNA synthetase 1 (EPRS1), and lysyl-tRNA synthetase (KARS1), specifically recognize the chemical structure of the 2-thiocarbonyl group in mcm5s2U to achieve efficient aminoacylation. These findings indicate that desulfuration of mcm5s2U negatively regulates cytoplasmic translation by lowering the aminoacylation efficiency of tRNAs for Gln, Glu, and Lys.

Effect of tRNA desulfuration on codon recognition
Next, we investigated the effect of mcm5s2U desulfuration on tRNA codon recognition on the ribosome. The xm5s2U34 modification is crucial for accurate and efficient recognition of purine nucleosides at the third position of NNR codons17,34. We measured the ability of human tRNALys with or without desulfuration to bind both AAA and AAG codons at the A-site on the ribosome. As a positive control, untreated tRNALys efficiently bound to both codons (Fig. 6a, b). Desulfurized tRNALys showed almost no binding to the AAA codon (Fig. 6a) and a ~60% reduction in binding to the AAG codon (Fig. 6b). These results reveal that mcm5h2U34 significantly impairs codon recognition at the A-site on the ribosome.

Structural insights into codon recognition mediated by h2U derivatives
To obtain structural insights into the molecular mechanism by which mcm5h2U34 reduces codon recognition ability, we conducted cryo-EM single-particle analyses of the ribosome complex with human cytoplasmic tRNALys bearing mcm5s2U34 or mcm5h2U34, P-site tRNA, and mRNA with AAA or AAG at the A-site. To this end, human cytoplasmic tRNALys was desulfurized by treating twice with H2O2, confirming the high frequency of mcm5h2U34 (Supplementary Fig. 4b). After several classification and focused classification targeting A-site tRNA, we determined cryo-EM structures of four ribosome complexes with A- and P-site tRNAs at 2.26–3.18 Å resolution (Fig. 7a, Supplementary Fig. 5a–d, Supplementary Fig. 6, and Supplementary Data 3). Ribosomes adopt the closed conformation in all four structures (Fig. 7b), with the codon-anticodon helix at the A-site strictly recognized by A-minor interactions with A1492 and A1493 in helix 44 and G530 in helix 18 of 16S ribosomal RNA (rRNA). In particular, we clearly observed cryo-EM densities for 5-methoxycarbonylmethyl- and 2-thiocarbonyl groups of mcm5s2U34 (Fig. 7c). On the other hand, no density was observed for the 2-thiocarbonyl group in mcm5h2U34 (Fig. 7d). Additionally, the 5-methoxycarbonylmethyl groups of mcm5s2U34 and mcm5h2U34 interact with C1054 of 16S rRNA in all four structures (Supplementary Fig. 7a–d).

The mcm5s2U34 modification pairs with A3 of the AAA codon via a canonical Watson-Wick geometry (Fig. 7c), whereas mcm5s2U34 pairs with G3 of the AAG codon via a non-canonical base pairing involving a minor groove-shifted U (Fig. 7c). The geometry of the mcm5s2U • G3 base pair is discussed in more detail in the Supplementary Discussion. Meanwhile, mcm5h2U34 pairs with A3 via a unique geometry involving a single H-bond between O4 of mcm5h2U34 and N6 of A3 (Fig. 7d, e). The N3 atom of mcm5h2U no longer acts as a proton donor due to its chemical nature, so that it is not involved in any additional H-bonding (Fig. 7e). The observed geometry with a single H-bond could explain why s2U-to-h2U conversion results in loss of the ability to recognize the AAA codon (Fig. 6a).

The mcm5h2U • G pair adopts a similar geometry to the non-canonical wobble base pair mnm5s2U • G, with U* shifted into the minor groove (Fig. 7d, e). Given that the 2-thiocarbonyl group of mcm5s2U stabilizes C3′ endo ribose puckering36,37, mcm5s2U • G pairing should be more stable than mcm5h2U • G pairing. In addition, the 2-thiocarbonyl group of mcm5s2U lowers the pKa of N3 atom40, and the 5-methylcarboxymethyl group also contributes to the structural rearrangement to form the minor groove-shifted mcm5s2U • G pairing38. This structural observation is consistent with our biochemical evidence showing that s2U-to-h2U conversion decreases the ability to decode the AAG codon (Fig. 6b).

Methods
Biological resources
E. coli strain BW25113 was obtained from National BioResource Project E. coli (NBRP-E. coli) at the National Institute of Genetics (Mishima, Japan). The ΔmnmA strain was obtained from the E. coli KEIO collection resource (Tokyo, Japan)76. S. cerevisiae BY4742 (WT) strain (Matα; his3-1; leu2-0; ura3-0) and ctu1Δ strain (BY4742 background) were obtained from EUROSCARF (Germany). Mouse and pig livers were purchased from Funakoshi (Tokyo, Japan). MKN45 (RCB1001), HeLa (RCB0007), HEK293T (RCB2202) and HuH-7 (RCB1366) were obtained from RIKEN BRC (Tsukuba, Japan). A549 (CCL-185), Hep G2 (HB-8065), MCF7 (HTB-22), T24 (HTB-4), and RAW264.7 (TIB-71) were obtained from ATCC. MOLM-13 (C0003003) was obtained from Addexbio (San Diego).

Cell culture
E. coli cells were cultivated in Luria-Bertani (LB) medium with vigorous shaking at 37°C. S. cerevisiae cells were cultivated in Yeast Peptone Dextrose (YPD) medium (2% peptone, 1% yeast extract, and 2% glucose) at 30°C. HeLa, HuH-7, hep G2, MCF7, and A549 cells were cultured at 37°C and 5% CO2 in Dulbecco’s modified Eagle’s medium (high-glucose; Sigma) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (Fujifilm Wako Pure Chemical Corporation). MOLM-13, MKN45, and RAW264.7 cells were cultured at 37°C and 5% CO2 in Roswell Park Memorial Institute (RPMI) 1640 medium (Nacalai Tesque) supplemented with 10% FBS. T24 cells were cultured at 37°C and 5% CO2 in McCoy’s 5A (modified) Medium (Gibco) supplemented with 10% FBS.

For oxidative stress conditions, HeLa cells were cultured until they reached 70–80% confluency, then treated for 1 h with oxidative stress-inducing agents: 2 mM H2O2 (Nacalai Tesque), 20 μM menadione (Sigma-Aldrich), or 0.25–1 mM rotenone (Tokyo Chemical Industry). Although 2 mM H2O2 is far above the IC50 value for HeLa cells (50 μM), we did not observe any cell detachment nor apoptosis in our condition. Following treatment, cells were washed with PBS. Total RNA was extracted as shown below.

RNA extraction and isolation of individual tRNAs
Total RNA was extracted from E. coli as described77. Briefly, E. coli cells were suspended in a 1:1 mixture of water-saturated phenol and RNA extraction buffer A [50 mM NaOAc (pH 5.2) and 10 mM Mg(OAc)2], followed by two cycles of freeze-thawing with liquid nitrogen, and vigorous stirring for 1 h at room temperature. After centrifugation, the aqueous phase was subjected to chloroform extraction and 2-propanol precipitation.

Total RNA from yeast cells were prepared as follows. Yeast cells were resuspended in 5–10 weights of RNA extraction buffer B [50 mM NaOAc (pH 5.2), 10 mM EDTA, 1% SDS, and 14.3 mM β-mercaptoethanol], and an equal volume of water-saturated phenol was added. The mixture was shaken for 1 h, followed by centrifugation at 8000 × g for 30 min at 4°C. The supernatants were collected, treated with an equal volume of chloroform, followed by isopropanol precipitation. The pellets were dissolved in water, and added with 2% (v/v) 1 M MES-NaOH (pH 6.0), 2.5% (v/v) 5 M NaCl, and 40% (v/v) 2-butoxyethanol. The mixture was then placed on ice for 30 min, followed by centrifugation at 8000 × g for 30 min at 4°C to remove precipitates. The supernatants were subjected to ethanol precipitation to obtain the total RNA. To obtain small RNA fraction, total RNA was subjected to an anion-exchange resin (DEAE Sepharose Fast Flow; Cytiva) in low salt buffer A [20 mM HEPES-KOH (pH 7.5), 250 mM NaCl, and 2 mM dithiothreitol (DTT)]. After washing the resin with the same buffer, small RNA fractions were eluted with high salt buffer A [20 mM HEPES-KOH (pH 7.5), 1 M NaCl, and 2 mM DTT].

Total RNA from cultured cells or frozen tissue were prepared using an isolation reagent comprising 40% (w/v) phenol, 1% (v/v) ethyleneglycol, 2 M guanidine thiocyanate, 20 mM tri-sodium citrate, 0.1 M NaOAc (pH 4.5), 0.2% (w/v) N-lauroylsarcosine sodium salt, 50 mM salicylic acid, 10 μM aluminon, 6.9 mM 8-hydroxyquinoline, 0.0001 w/v% BPB. For highly reducing conditions, the same isolation reagent was used with the addition of 50 mM DTT. Alternatively, total RNA were extracted from cultured cells or frozen tissue using TriPure Isolation Reagent (Roche). Frozen tissues were cryogenically ground in a GRINDOMIX GM200 knife mill (Roche) prior to extraction. For further purification, total RNA were subjected to an anion-exchange resin (DEAE Sepharose Fast Flow; Cytiva) in low salt buffer B [10 mM HEPES-KOH (pH 7.5), 250 mM NaCl, and 2 mM DTT], and small RNA fractions were eluted with high salt buffer B [10 mM HEPES-KOH (pH 7.5), 1 M NaCl and 2 mM DTT]. To obtain total tRNA fractions or class I tRNA fractions, total RNA was separated by on 7 M urea denaturing polyacrylamide gel electrophoresis (PAGE) and stained with SYBR Gold (Invitrogen), then purified by excising the corresponding gel bands.

Individual tRNAs were isolated by RCC60,78. Sequences of 5′-terminal ethylcarbamate (EC) amino-modified DNA probes (Sigma Aldrich) complementary to target tRNAs are listed in Supplementary Data 6. Each probe was covalently immobilized on NHS-activated Sepharose 4 Fast Flow resin (Cytiva), packed in a custom-made tip, and attached to a custom-made multichannel head on the RCC device. Small RNA fractions obtained by DEAE chromatography were dissolved in 6 × NHE buffer [1.2 M NaCl, 30 mM HEPES-KOH (pH 7.5), 15 mM EDTA-NaOH pH 8.0 and 1 mM DTT], and passed through the tip by auto-pipetting at 65°C. After washing the tip columns with 0.1 × NHE at 40°C, bound tRNAs were eluted with 0.1 × NHE at 68°C. The purity of isolated tRNAs was confirmed by 10% PAGE with 7 M urea.

RNA mass spectrometry
Isolated tRNAs were digested with RNase T1 and analyzed by capillary LC/nano-ESI-MS as described78–80. One picomole of isolated tRNA was digested with 20 U of RNase T1 (Thermo Fisher Scientific) in 20 mM NH4OAc (pH 5.3) at 37°C for 45 min. A linear ion trap-orbitrap hybrid MS instrument (LTQ Orbitrap XL; Thermo Fisher Scientific) was equipped with a splitless nano HPLC system (DiNa; Techno Alpha) using a nano-LC trap column (L-column2 ODS; 0.3 × 5 mm; CERI) and a capillary column (HiQ Sil C18W-3; 0.1 × 100 mm; Techno Alpha). Digested fragments were separated for 35 min at a flow rate of 300 nL/min with a linear gradient from 2 to 100% solvent B. The solvent system consisted of 0.4 M 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP; pH 7.0, solvent A) and 0.4 M HFIP (pH 7.0) in 50% methanol (solvent B). The eluent was ionized using an ESI source in negative mode, with a scan range of m/z 600–2000. Xcalibur 2.0.7 (Thermo Fisher Scientific) was used for system operation.

LC-MS shotgun analyses of tRNA modification were conducted as described81 with slight modification. Five picomoles of class I tRNA fraction was digested with 50 units of RNase T1 (Thermo Fisher Scientific) in 20 mM NH4OAc (pH 5.3) at 37°C for 45 min. Digested fragments were analyzed by a DiNa nano-flow HPLC system (Techno Alpha) coupled with an Orbitrap Eclipse Tribrid MS instrument (Thermo Fisher Scientific). The percentage intensity of each modification was calculated from the ratio of mass chromatogram peak areas of RNA fragments with and without the target modification using QualBrowser in Xcalibur 4.2.28.14 software (Thermo Fisher Scientific).

Nucleoside analyses of tRNAs were performed as described79,82. Four micrograms of total RNA were digested into nucleosides with 0.1 U of nuclease P1 (Wako Pure Chemical Industries) in 10 mM NH4OAc (pH 5.3) for 1 h at 37°C, followed sequentially by 0.1 U of phosphodiesterase I (PDase I; Worthington Biochemical Corporation) in 100 mM ammonium bicarbonate, and 0.16 U of bacterial alkaline phosphatase (BAP; E. coli C75; Takara). For hydrophilic interaction chromatography (HILIC)/ESI-MS, digests were dissolved in 90% acetonitrile/10% water and applied to a ZIC-cHILIC column (3 μm particle size, 2.1 × 150 mm; Merck Millipore) connected to a Q Exactive Hybrid Quadrupole-Orbitrap MS instrument (Thermo Fisher Scientific) and an Ultimate 3000 LC system (Thermo Fisher Scientific). Nucleosides were separated at a flow rate of 100 μL/min in a multistep gradient comprising 90-85% solvent B from 0 to 10 min, 85–30% solvent B from 10 to 30 min with gradient curve 7, 30% solvent B for 10 min, then a return to 90% solvent B. Proton adducts of nucleosides were scanned in positive ion mode across an m/z range of 110–950. Xcalibur 4.4 (Thermo Fisher Scientific) was used for system operation.

Preparation of h2U-modified tRNAs by in vitro desulfuration
In vitro oxidation/desulfuration of s2U-modified tRNAs was conducted according to the literature56 with slight modification. Human or mouse tRNA mixtures (400 μg) were dissolved in 10 mM HEPES-KOH (pH 7.4) and 100 mM H2O2 and incubated for 24 h at room temperature, followed by performing ethanol precipitation twice. For individual tRNAs, the isolated tRNALys (40 μg) was dissolved in 10 mM HEPES-KOH buffer (pH 7.4) and 50 mM H2O2 and incubated for 24 h at room temperature, followed by ethanol precipitation twice. For complete desulfuration, the reaction was conducted twice to achieve the highest s2U-to-h2U conversion rate.

Preparation of mRNAs for cell-free translation
The pUC-T7-HCV-HA-Rluc plasmid was used for Rluc mRNA transcription. For the control, we designed pUC-T7-HCV-mHiBiT plasmid encoding a mutant HiBiT peptide with 27 amino acids (MSTNPNTLSTTGGSSGWRLFRRISGGS) that does not contain any Lys, Glu, Gln, or Arg residues (encoded by AGR codons). For plasmid construction, a DNA segment spanning T7 promotor, HCV-IRES, and mutant HiBiT sequences was PCR-amplified from the pUC-T7-HCV-HA-Rluc plasmid as a template, followed by a second round of PCR to add an XbaI restriction enzyme site to the 3′ end of the product. The amplified product was inserted into the HindIII and XbaI sites of the same vector. Finally, according to the literature83,84, the beginning of the HCV-IRES sequence was mutated from MSTNPKPQRKT to MSTNPNTLSTT to generate the pUC-T7-HCV-mHiBiT plasmid by site-directed mutagenesis to avoid codons read by h2U-containing tRNAs. All primers used for plasmid construction are listed in Supplementary Data 6.

Plasmids were linearized by XbaI (Takara) digestion, and mRNAs were synthesized by in vitro transcription with T7 RNA polymerase using a HiScribe™ T7 High Yield RNA Synthesis Kit (NEB).

Reconstituted human cell-free translation
Reconstituted cell-free translation was carried out as described61 with modifications. Briefly, 1.26 mg/mL eEF1 complex, 100 μg/mL eEF2, 5 μg/mL eRF1/3, 0.48 μM 40S ribosomal subunit, 0.48 μM 60S ribosomal subunit, 150 μg/mL aminoacyl-tRNA synthetase mixture, and 100 μM amino acid mixture (Promega) were mixed in a test tube in reaction buffer containing 50 mM HEPES-KOH (pH 7.5), 55.2 mM potassium acetate, 6 mM magnesium acetate, 200 μM spermidine trihydrochloride, 60 μg/mL creatine kinase, 20.6 mM creatine phosphate, 1 mM DTT, 1.36 mM ATP, and 840 μM each of GTP, UTP, and CTP. This reaction mixture was then mixed with mRNA (1.56 μM Rluc mRNA, or 1.66 μM mHiBiT mRNA) and total tRNAs with or without desulfuration (1 mg/mL) in a final volume of 10 μL, followed by incubation at 32°C for 3 h.

Luciferase assay
One microliter of cell-free translation product of Rluc or mHiBiT mRNAs was used for measuring luciferase activity using a Renilla Luciferase Assay System (Promega) or a Nano-Glo HiBiT Lytic Detection System (Promega), respectively. Relative light units (RLU) of luminescence were measured by a GloMax 96 Microplate Luminometer (Promega).

Western blotting and antibodies
Cell-free translation products of Rluc mRNAs were separated by 15% SDS-PAGE and electroblotted onto an Amersham Hybond P PVDF membrane (Cytiva) using a Transblot Turbo apparatus (Bio-Rad). To detect the HA epitope of Rluc products, anti-HA-tag mAb (M180-3; MBL) was used as primary antibody at a 1:7000 dilution, and horseradish peroxidase (HRP)-conjugated donkey anti-mouse IgG (715-035-150; Jackson ImmunoResearch) was used as secondary antibody at a dilution of 1:20,000. To detect recombinant eEF-1, anti-His-tag mAb-HRP-DirecT (D291-7; MBL) was used at a dilution of 1:10,000. Target proteins were detected using Pierce ECL Plus Western Blotting Substrate (Thermo Fisher Sciences) and visualized with an ImageQuant LAS4000 mini instrument (Cytiva). Band intensities were analyzed by ImageJ 1.53t.

Polysomal RNA preparation
HEK293T cells (~80% confluency) cultured in 25 dishes with 15-cm diameter were lysed with a lysis buffer comprising 20 mM Tris-HCl (pH 7.5), 150 mM KCl, 3 mM MgCl2, 0.5% Triton X-100, 1 mM DTT, 50 μg/mL cycloheximide. The lysate was clarified by centrifugation at 13,000 × g for 10 min at 4°C. The cell lysate was loaded onto a cushion buffer [20 mM Tris-HCl (pH 7.5), 150 mM KCl, 3 mM MgCl2, 1 mM DTT, 50 μg/ml cycloheximide, and 20% sucrose] and ultracentrifuged at 35,000 rpm and 4°C for 4 h by a Optima L-90K ultracentrifuge (Beckman) with a 70 Ti rotor (Beckman). Free RNA fraction was extracted from the supernatant using TriPure Isolation Reagent (Roche). The crude ribosome pellet was re-suspended in a ribosome binding buffer [20 mM Tris-HCl (pH 7.5), 150 mM KCl, 3 mM MgCl2, 1 mM DTT, 50 μg/ml cycloheximide], and then loaded onto a 10-50% sucrose gradient in an SDG buffer [20 mM Tris-HCl (pH 7.5), 150 mM KCl, 3 mM MgCl2, 1 mM DTT, 50 μg/ml cycloheximide] and ultracentrifuged at 25,000 rpm and 4°C for 4.5 h by a Optima L-90K ultracentrifuge (Beckman) with a SW28 rotor (Beckman). The gradients were fractionated with continuous measurement of absorbance at 260 nm using a TRIAX flow cell (BioComp) and a fraction collector (Gilson, Fisher Scientific). The polysomal fraction was collected, followed by RNA precipitation by adding 3 volume of ethanol and incubating overnight at –80°C85. Then, polysomal RNA was extracted from the pellet using TriPure Isolation Reagent (Roche). To concentrate small RNA fraction, polysomal RNA, free RNA, and total RNA were subjected to an anion-exchange resin (DEAE Sepharose Fast Flow; GE Healthcare) with low salt buffer [10 mM HEPES-KOH (pH 7.5), 250 mM NaCl, and 2 mM DTT]. After washing the resin with the same buffer, small RNA fractions were eluted with high salt buffer [10 mM HEPES-KOH (pH 7.5), 1 M NaCl and 2 mM DTT], followed by ethanol precipitation.

Aminoacylation assay
Aminoacylation of each tRNA was performed in a 20 μL reaction mixture containing 100 mM HEPES-KOH (pH 7.5), 50 mM KCl, 6 mM MgCl2, 2 mM DTT, 2 mM ATP, 15–30 μM 14C-labeled amino acids, 20 μM total tRNA with or without desulfuration and 750 ng aminoacyl-tRNA synthetase mixture61 at 37°C. Before aminoacylation reaction, total tRNA was deacylated with 100 mM CHES-NaOH (pH 9.0) at 37°C for 45 min. The concentration and specific activity of 14C-labeled amino acids used in this study were as follows: 15 μM L-[14C(U)]-Threonine (6.5 GBq/mmol; American Radiolabeled Chemicals), 15 μM L-[14C(U)]-Arginine (11.1 GBq/mmol; American Radiolabeled Chemicals), 30 μM L-[14C(U)]-Glutamine (9.0 GBq/mmol; PerkinElmer), 30 μM L-[14C(U)]-Glutamic acid (8.1 GBq/mmol; American Radiolabeled Chemicals), and 15 μM L-[14C(U)]-Lysine (12.1 GBq/mmol; PerkinElmer). At each time point, the reaction was quenched by spotting 4 μL aliquots of reaction mixture onto a Whatman 3MM Chr filter paper (Cytiva). The filter paper was pre-soaked in a 5% trichloroacetic acid solution before use. The spotted filter paper was washed three times for 15 min each in ice-cold 5% trichloroacetic acid, washed with ethanol and dried under a heat lamp. Acid-insoluble radioactivity of aminoacyl-tRNAs was measured by a liquid scintillation analyzer (PerkinElmer).

Ribosomal A-site tRNA binding
tRNA binding to the A-site on the ribosome was carried out as described86,87 with modifications. mRNAs containing the Shine-Dalgarno sequence and an AAR codon (R = A or G) codons or a UUC codon (negative control) at the A-site (Supplementary Data 6) were chemically synthesized. Human cytoplasmic tRNALys bearing mcm5s2U or mcm5h2U was dephosphorylated in buffer containing 10 mM HEPES-KOH (pH 7.5) and 0.008 units of bacterial alkaline phosphatase (E. coli C75; Takara) at 55°C for 30 min, followed by gel purification of the intact tRNAs. The dephosphorylated tRNAs were 5′ phosphorylated with [γ−32P]ATP (PerkinElmer) by T4 polynucleotide kinase (NEB), followed by gel purification of the 5′−32P-labeled tRNAs. Radioactivity of labeled tRNAs was measured by Cherenkov counting using a liquid scintillation analyzer (PerkinElmer). The specific activities of labeled tRNAs were adjusted to 20,000 cpm/pmol by adding respective cold tRNALys with or without desulfuration. First, E. coli 70S ribosomes (1.6 pmol) were incubated with E. coli tRNAfMet (25 pmol) and mRNA (20 pmol) at 37°C for 40 min in binding buffer containing 50 mM HEPES-KOH (pH 7.5), 120 mM KCl, 6.5 mM Mg(OAc)2, 1 mM DTT, and 0.5 mM spermine to occupy the P-site of the ribosome. Next, 0.1, 0.25, or 0.5 pmol of the 32P-labeled tRNALys was added to the mixture and incubated at 37°C for 15 min, then at 0°C for 30 min. The mixture was spotted onto double-layered Amersham Protran nitrocellulose membrane (Cytiva) and Hybond-N+ nylon membrane (Cytiva) using a 96-well dot-blot vacuum apparatus (SANPLATEC, Japan). The membranes were washed twice with 200 μL of binding buffer, dried, exposed to an imaging plate, and visualized on an FLA-7000 image analyzer (Fujifilm). The amount of bound tRNA was quantified using Multi Gauge V3.0 (Fujifilm).

Single-particle cryo-EM analysis of ribosomes with human tRNAs
The cryo-EM single-particle analysis of the ribosome–tRNA complexes was basically performed in accordance with our previous studies5,78,86,88. E. coli 70S ribosomes were prepared from the MRE600 strain as previously described5, resuspended in RBS buffer containing 20 mM HEPES-KOH (pH 7.6), 10 mM Mg(OAc)2, 30 mM NH4Cl, and 6 mM β-mercaptoethanol. Methionylation and formylation of tRNAiMet were carried out as described previously89. To form a ribosome complex, E. coli 70S ribosomes were mixed with mRNA and P-site tRNA in a mixture containing 20 mM HEPES-KOH (pH 7.6), 10 mM Mg(OAc)2, 30 mM NH4Cl, 6 mM β-mercaptoethanol, 100 nM 70S ribosomes, 500 nM mRNA, and 500 nM E. coli fMet-tRNAiMet, followed by incubation at 37°C for 15 min. Next, 500 nM of human cytoplasmic tRNALys was added as A-site tRNA and the mixture was incubated at 37°C for 30 min, followed by further incubation on ice for 1 h. Grid preparation was carried out as previously described5.

Ribosome particle images were collected using a Titan Krios transmission electron microscope (Thermo Fisher Scientific) operated at an accelerating voltage of 300 kV and equipped with a K3 camera (Gatan). Automated image acquisition was performed at a nominal magnification of ×105,000 with a defocus range from 0.5 to 2.5 mm using a K3 direct electron detector (Gatan) in CDS-counting mode (0.8285 Å/pixel; Supplementary Data 3). The total exposure of electrons at the specimen level was ~50 e−/Å2, and dose was fractionated into 48 frames (Supplementary Data 3).

Images were recoded as movie micrographs and image processing was performed using RELION-3.1.390. Details of image processing for each ribosome complex are described in Supplementary Fig. 5. Movies were motion-corrected with the program implemented in RELION, and contrast transfer function (CTF) parameters were estimated with CTFFIND-4.191. Ribosome particles were automatically picked by crYOLO-1.8.292. The obtained particles were initially subjected to 2D classification to discard ice contamination, and the remaining particles were then auto-refined to reconstruct a consensus map. Subsequently, based on the alignment of consensus reconstruction, 3D classification was performed. The subgroup of 70S ribosome particles was auto-refined and further classified using a mask around A-site tRNA to discard ribosomes with a vacant A-site. Subgroups of A-site tRNA-bound ribosomes were further auto-refined followed by CTF refinement, Bayesian polishing and another round of 3D refinement and postprocessing. The final resolution reached 2.26–3.18 Å. The angular distribution of particle projections was calculated by RELION-3.1.390. The local resolution of each map was estimated by RELION-3.1.390 (Supplementary Fig. 6a–d).

The high-resolution structure of the E. coli 70S ribosome complexed with P-site tRNAiMet and A-site tRNA (PDB: 7y7e)5 was used as a starting model and ridged body-fitted to the obtained cryo-EM maps. Models of mRNA and A-site tRNA were manually constructed using COOT-0.9.593, and tRNA modifications were manually inserted into the model. The chemical structure of mcm5h2U was modeled using eLBOW94. These initial models were real space-refined to cryo-EM maps by the phenix.real space refine routine95. These models were corrected using UCSF Chimera-1.1496 and COOT-0.9.593, followed by a final phenix.real space refine routine using Phenix-1.1895. Refined final models were validated using MOLPROBITY97. Map vs. model FSC values were calculated using Phenix-1.1895 (Supplementary Fig. 6e, f). Graphical figures were prepared using UCSF Chimera-1.1496 or UCSF ChimeraX 1.398.

Synonymous codon usage bias analysis
Gene lists associated with the Gene Ontology (GO) term “response to oxidative stress” (GO:0006979) were retrieved using AmiGO 2 (http://amigo.geneontology.org). Gene names were mapped to corresponding NCBI RefSeq accessions using the UniProt ID mapping tool (https://www.uniprot.org/id-mapping). The coding sequences (CDS) corresponding to the RefSeq identifiers were then downloaded from NCBI in FASTA format. For each sequence, valid open reading frames (ORFs)—defined as regions starting with a canonical ATG initiation codon and terminating at the first in-frame stop codon (TAA, TAG, or TGA)—were extracted from the FASTA files.

Relative synonymous codon usage (RSCU) values were calculated using a custom Python script with Biopython v1.81, based on the observed versus expected frequencies of each synonymous codon.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In their manuscript “Translational Regulation by Desulfurated tRNA Modification: Implications for Oxidative Stress Responses” the authors identify h2U derivatives in tRNA samples prepared form several organisms and cells. The desulfurated tRNAs are described to be less efficient in the recognition of their cognate codons at ribosomes and show reduced aminoacylation efficiency.\n\nThe authors suggest that oxidative desulfuration of tRNA modulates protein synthesis under oxidative stress conditions. Nevertheless, they do not provide any evidence that those events happen in vivo and that the presence of h2U derivatives is modulated under stress conditions. The formation of these derivatives through a cellular process is indirectly demonstrated and not fully convincing, which questions the significance of the data provided regarding aminoacylation efficiency and codon-anticodon recognition.\n\nIn addition, xm5s2U modifications have been demonstrated to be important in several organisms and conditions for efficient protein translation upon stress (see for example PMID: 26052047). The formation of 2-deoxouridine derivatives can well be an unwanted damage of the tRNA, resulting in molecules that are toxic, should not participate to protein translation and are probably been degraded.\n\nTherefore, I support the publication in Nature Communications only upon more convincing demonstration of the presence and the function of 2-deoxouridine derivatives in vivo.\n\nIn addition, I have also the following major points:\n1. The oxidated form is convincingly demonstrated to be derived from mnm5s2U in bacteria, thanks to the use of E. coli ΔmnmA strain. The authors should use a similar approach in eukaryotes. Knockout of the enzymes for the 5-Methyl-2-thiouridine derivatives will strengthen the results.\n2. The spike-in experiments demonstrate that h2U derivative formation during handling of tRNAs was negligible, but not that these derivatives are effectively present in the cells. These h2U derivatives could be produced, for example, during lysis when the cellular components including tRNAs get in contact with H2O2 or other cellular radicals in the mix due to the lipid membrane dissolution. I suggest to use controlled conditions in the initial lysis and also to carefully compare several lysis methods.\n3. Are these h2U derivatives effectively involved in protein translation in vivo? Could these derivatives be, for example, identified and quantified in tRNAs extracted from polysomal fractions? In cells with high fractions of these derivatives ( ~15% of tRNALys in HeLa cells contained mcm5h2U), is the translation of the cognate codons effectively affected?\n4. Is there is a purification step of the tRNA after H2O2 treatment? In the Methods section it is not mentioned if the left over H2O2 in the tRNA preparation was removed, as this can interfere with the subsequent reactions, for example, aminoacylation or in vitro translation.\n5. The author state in the discussion „we have quantified h2U derivatives under various oxidative stress conditions, but are yet to find conditions that reproducibly increase h2U levels”. The fat that the authors did not find conditions that reproducibly increase h2U levels is a major concern to doubt the biological function of these derivatives. In any case it would be useful to show also these negative results. Which conditions and stresses have been used so far without observing any modulation?\n6. In figure 4 Western of the HiBit construct is missing as control.\n\nMinor points:\nThe author state “We reviewed our previous liquid chromatography-mass spectrometry (LC-MS) data”. Please add a reference. Which previous data were reviewed?\nWhat is the peak indicated by an asterisk in fig S1A? Please clarify in the legend.", "reviewer_2": "Review of the manuscript entitled \"Translational Regulation by Desulfurated tRNA Modification: Implications for Oxidative Stress Responses\" by Yufeng Mo, Kensuke Ishiguro, Kenjyo Miyauchi, Yuriko Sakaguchi, Naho Akiyama, Ayaka Murayama, Kodai Machida, Hiroaki Imataka, Akio Yamashita, Mikako Shirouzu, and Tsutomu Suzuki submitted to Nature Comminications.\n\nThe authors describe the identification of h2U derivatives in human and bacterial cells as well as in mouse liver tissue. These h2U derivatives are products of sulfur removal from natural 2-thiouridine derivatives present in four transfer RNAs specific for lysine, glutamine, glutamic acid, and asparagine. The authors claim that the h2U derivatives are the products of natural oxidative desulfuration of intact tRNAs isolated from cytoplasmic and mitochondrial fractions. In a series of mass spectrometry experiments, they confirm the presence of x5h2U at position 34 of the tRNA, which was originally the position of the xms2U derivatives. Since this is the first position of the anticodon, they examine the characteristics of the oxidized tRNA. They noted the decreased efficiency of translation in a human reconstituted in vitro system with free cells and the decreased efficiency of h2U-tRNALys, Glu and Gln aminoacylation. Finally, they concluded that desulfuration of native tRNA specific for lysine (with mcm5s2U) affects codon recognition when the damaged tRNA is bound to either a programmed AAA or AAG ribosome, by a loss of AAA codon recognition followed by to some degree decreased AAG codon recognition. To confirm the molecular structures of the mcm5h2U and A and mcm5h2U and G base pairs, they used a cryo-EM approach and discovered the “anionic type” mcm5h2U-G base pair. They also show the weak mcm5h2U-A pair (with only one hydrogen bond) as well as the two original base pairs mcms2U-A and mcm5s2U-G. In their general conclusions, they state that desulfuration of the tRNA thio-modification modulates protein synthesis under oxidative stress.\n\nAlthough the data presented are convincing and to some extent original, the manuscript needs to be thoroughly revised to be considered for publication.\n\nMajor corrections:\n- First of all, the presence of mcm5h2U has already been detected in yeast, several human cancer cell lines and also in modified 293 cells deprived of oxidative system gene expression and cultured under normal and oxidative stress conditions (doi: 10.3390/ijms252212336). Therefore, such statements as on page 4 and in some other fragments of the manuscript such as: “However, the occurrence of oxidative desulfuration of xm5s2U within cells has not yet been investigated.” is incorrect. The manuscript should refer to the published data and correct the text in the manuscript.\n- The authors dissect the oxidative desulfuration of 2-thiouridine to a 4-pyrimidinone derivative and omit the other product, the uridine derivative. As previously reported, the composition of the products of the desulfuration reaction depends on the pH of the reaction environment and on the nature of the substituent at position 5 of the 2-thiouridine moiety (doi: 10.1016/j.bmcl.2013.08.114, doi: 10.1016/j.bmc.2015.07.030). The authors completely ignored such a xm5s2U property, did not explain how they assessed the influence of xm5h2U-tRNA and excluded the influence of the presence of xm5U-tRNA products in the investigated processes (translation/aminoacylation), which were performed under physiological conditions (pH 7.4). Of particular interest in this context is the result of binding of the oxidized mcm5S2U-tRNA to the mRNA ribosome, assuming that part of the thio-tRNA is converted to its oxo derivative, which facilitates U-A binding. Moreover, the efficiency of aminoacylation also depends on the presence of the thio modification in the wobble position (identity element for AARS). Therefore, the observed effect could be due to the presence of x5h2U and x5U-tRNA in the whole tRNA, which reduce the activity of AARS. This question needs clarification/discussion.\n- The oxidative desulfuration reaction scheme shown in Figure 1b, in which only one desulfuration product xm5s2U is shown, may be misleading to the readers and is inconsistent with the previous studies conducted in vitro and in the cellular systems as discussed above. The authors are advised to correct the schematic and possibly note under the figure that only the xm5h2U type desulfuration product was monitored.\n- The interpretation and discussion of the molecular structures of the base pairs of mcm5s2U/mcm5h2U with A and with G requires some clarification. The base pair mcm5s2U-A is (as expected) in a typical Watson-Crick mode, while the desulfurized base pair mcm5h2U-A has only one possible hydrogen bond. The authors claim a weak bond in this base pair. The authors can address the confirmation of their result using the previously shown data of thermodynamic measurements performed for the 4-pyrimidinone type nucleoside analogs incorporated into the RNA duplex. These data show that h2U-type units form weak base pairs with A and more stable ones with G (data are given for h2U and geranylated 2-thiouridine-containing RNA duplexes). In addition, the authors are advised to point out that in such a measurement, the addition of a silver anion (sealing properties) increases the stability of the h2U-A duplex, which is further evidence for the existence of a weak h2U-A base pair.\n- Importantly, the mcms2U-G base pair is not an anionic base pair (Fig. 7c) and is incorrectly interpreted as a base pair with the hydrogen bond between N2H of G and N3H of U (hydrogen bonds marked in red). The authors should check the interpretation of the team of P. Agris, who assigned such a base pair to the C-G type, where 2-thiouridine is pre-structured to its keto-enol form and provides three hydrogen bonds between G and mcm5s2U (doi:10.1016/j.jmb.2011.12.048). Fig. 7c and the interpretation in the text should be improved.\n- The sentence : “In addition, structural analyses revealed that xm5s2U34 base-pairs with the third guanosine of NNG codons at the A site of the ribosome via an anionic U • G wobble pairing 33,34” refers to only one of two different cases, in one of which the xm5s2U-G base pair can adopt “a new wobble base pair” for mnm5s2U-G (Rozow et al 2016) and in the other the C-G like mode is found for mcm5s2U-G (Vendeix et al 2012). In the following text, the description refers to the new wobble base pair mnm5s2U-G, which is not related to the discussed mcm5s2U-G case, because the electronic properties of mcm5 and mnm5 are quite different and have different effects on the dissociation of the N3H proton. This fragment of the introduction requires a more detailed comment. Ref. 34 should be placed before Ref. 33.\n- Authors are advised to pay more attention to correct citation, naming of compounds and interpretation of published data: e.g. page 9: mcm5h2U-G is not an anionic base pair. Similarly, mcm5s2U-G is not an anionic base pair. Only mnm5s2U-G has been shown to form a new wobble base pair in which the ionized mnm5s2U is bound to G. The discussed h2U-G and ges2U-G form base pairs of the 4-pyrimidinone-G type, and the pair mnm5s2U-G can also be categorized in the same group of modes. The total charge of this U-unit is 0, although the charge is distributed between the nucleobase and the mnm5. Do you think that “the anionic” base pair in this case is a correct description? Please correct the text accordingly.\n- It is not clear how xm5s2U prevents misdecoding of the pyrimidine ending codons NNY due to the 3’-endo sugar pucker. The topic needs reference / discussion.\n- The description on page 9 is misleading: “Given that the 2-thiocarbonyl group of mcm5s2U stabilizes C3’-endo ribose puckering 31,32, mcm5s2U-G pairing should be more stable than mcm5h2U-G pairing. In addition, the 2-thiocarbonyl group of mcm5s2U promotes the acidity of the N3 atom (Ref. 37), thereby contributing to the anionic mcm5s2U-G pairing 34.” The authors provide no evidence to support the conclusion of the first statement. Also, the acidity of N3 in mcm5s2U is similar to that of the 5-non-substituted 2-thiouridine (see Ref. 37) , and the mcm5s2U-G base pairing is not anionic. Therefore, the conclusions drawn here must be properly proved and descriptions corrected.\n- The discussion of the discovered/confirmed data should be deepened and completed.\n\nMinor corrections:\n- please do not introduce “2-deoxouridine” as another colloquial name for 4-pyrimidinone riboside. This name is misleading compared to usually used 2’-deoxyuridine.\n- as a flag laboratory for modified nucleosides, the authors are advised to consider using a proper name for modified nucleoside, abbreviated as mcm5s2u. The proper name (used by other authors) is “5-methylcarboxymethyl-2-thiouridine” and NOT “5-methoxycarbonylmethyl-2-thiouridine”. This proper name is logical in relation to the abbreviation used: methyl – m and carboxy -c. So it is rather confusing that mcm5 is read here as “methoxy” (for which a correct abbreviation/symbol should be “mo”), “carbonyl” (it has no abbreviation, and “c” is typically used for “carboxy(l)\"). The use of confusing names leads to confusion in the nomenclature of modified nucleosides.\n- The references to 35 and 36, which refer to the wobble base pair U-G occurring in the major groove, are misleading. The classical pairs U-G and G-U adopt the wobble mode, but these two base pairs are NOT isosteric, the feature which is important for the ribosomal context. While the G34-U3 base pair can be accommodated on the ribosome, the U34-G3 base pair is not allowed in this spatial architecture (see the work of Westhof). “Anionic” G-U in ribosomal RNA are interesting newly discovered modes, but their discussion is not relevant here (Ref. 33).\n- The description of the results – the sentence about the experiments with bacterial knock-out S2U synthetase should be corrected. You write that ”the E. coli ΔmnmA strain in which mnm5s2U is converted to mnm5U (Figure S1c)”. This is an incorrect description, as in this bacterial strain the thiolation of the nucleoside mnm5U in the tRNA is abandoned.\n- On page 3 (Introduction) the authors list the x5s2U derivatives present in tRNA in different organisms (prokaryotes and eukaryotes), and for cytoplasmic tRNA in eukaryotes, but they cite the work of Johansson et al 2008 (Ref 16) concerning tRNA from yeast S. cerevisiae. Since tRNA modifications in eukaryotes vary by organism and the manuscript focuses primarily on human tRNA, a more detailed description of human tRNAs and their modified thio-uridines is recommended. The situation of human mitochondrial tRNA is quite clear and frequently cited in the literature, but precise information on specific human cytoplasmic tRNA is only cursorily given, so a detailed description would be greatly appreciated.\n- In the chapter \"Confirmation of h2U derivatives produced in cells\", the authors give the h2U content in cytoplasmic and mitochondrial tRNAs from different cells with an accuracy of tenths of a percent. They should explain how these values were calculated and which value was taken as 100%. Do the authors believe that the h2U content in cells does not depend on individual cell characteristics, cell culture and environmental conditions? The h2U content in animal tissues is presented in a similar way. How do the authors explain the differences? No comments are given on the formation of uridine derivatives.\n- Did the authors take into account that the total tRNA fraction used in the assay for cell-free translation contains tRNAs that read codons with G regardless of desulfuration, e.g. tRNACUULys, tRNACUCGlu or tRNACUGGln? Please comment on this question in the discussion.", "reviewer_3": "This work describes the identification and mechanistic studies of a tRNA modification, mcm5h2U34, present naturally in cells and tissues. In contrast to most RNA modifications studied in the past, mcm5h2U34 seems to be derived from cellular oxidation from mcm5s2U, possibly under oxidative stress and not synthesized by a cellular enzyme. Cells contain many chemically reactive metabolic products such as reactive oxygen species (ROS) that can react non-enzymatically with RNA, however, the functional consequence of such tRNA product on translation have not been studied much. This work starts with a methodology to detect and quantify mcm5h2U34, and identification in mouse tissues. This is followed by in vitro studies of mcm5h2U34 showing its deleterious effect on translation and on tRNA charging, ending with cryo-EM structures with an atomic rationale of how mcm5h2U34 affects decoding on the ribosome. The results are of high quality and clearly presented. This exciting work provides a streamlined methodology to study the functional consequences of many potential RNA damages and provides in-depth functional analysis of this frequently occurring RNA modification.\n\nComment:\n1. A major issue is that no cellular data is shown that accompanies the biological regulation model in Fig. 8. On p. 10, the authors state: “Indeed, we have quantified h2U derivatives under various oxidative stress conditions, but are yet to find conditions that reproducibly increase h2U levels.”? What are the conditions tested? What is the charging level of the mcm5h2U34 modified tRNAs compared to their mcm5s2U34 counterpart in cells?\n2. The functional model would expect to find lower fractions of mcm5h2U34 tRNAs on the polysome compared to the total RNA input. Have the authors tested this?\n3. Would mcm5h2U34 tRNA specifically affect translation of stress response proteins? This may be addressed through computational codon usage analysis.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Response to Reviewer comments:\nFirst, we thank the reviewers for their thoughtful and helpful review of our manuscript, and for providing valuable and productive suggestions for its improvement. Our point-by-point responses to each of the reviewers’ comments are shown below. Changes to the main text are marked in yellow.\nReviewer #1 (Remarks to the Author):\nIn their manuscript “Translational Regulation by Desulfurated tRNA Modification: Implications for Oxidative Stress Responses” the authors identify h2U derivatives in tRNA samples prepared form several organisms and cells. The desulfurated tRNAs are described to be less efficient in the recognition of their cognate codons at ribosomes and show reduced aminoacylation efficiency.\nThe authors suggest that oxidative desulfuration of tRNA modulates protein synthesis under oxidative stress conditions. Nevertheless, they do not provide any evidence that those events happen in vivo and that the presence of h2U derivatives is modulated under stress conditions. The formation of these derivatives through a cellular process is indirectly demonstrated and not fully convincing, which questions the significance of the data provided regarding aminoacylation efficiency and codon-anticodon recognition.\nIn addition, xm5s2U modifications have been demonstrated to be important in several organisms and conditions for efficient protein translation upon stress (see for example PMID: 26052047). The formation of 2-deoxouridine derivatives can well be an unwanted damage of the tRNA, resulting in molecules that are toxic, should not participate to protein translation and are probably been degraded.\nTherefore, I support the publication in Nature Communications only upon more convincing demonstration of the presence and the function of 2-deoxouridine derivatives in vivo.\nWe sincerely appreciate the reviewer’s thoughtful and constructive comments. We fully agree that demonstrating the in vivo relevance of h2U derivatives is essential to support our conclusions. In our revised manuscript, we have made several key improvements to address these important concerns.\nIn addition, I have also the following major points:\n1. The oxidated form is convincingly demonstrated to be derived from mnm5s2U in bacteria, thanks to the use of E. coli ΔmnmA strain. The authors should use a similar approach in eukaryotes. Knockout of the enzymes for the 5-Methyl-2-thiouridine derivatives will strengthen the results.\nWe totally agree with reviewer #1 that knockout of the enzymes formcm5s2U in eukaryotes will strengthen the result. We have accordingly performed LC/MS analysis with yeast ctu1Δ strain in which 2-thiolation of mcm5s2U is missing. In the MS chromatograms (Figure S1d), mcm5s2U as well as mcm5h2U were absent, confirming that mcm5h2U is derived from mcm5s2U.\n2. The spike-in experiments demonstrate that h2U derivative formation during handling of tRNAs was negligible, but not that these derivatives are effectively present in the cells. These h2U derivatives could be produced, for example, during lysis when the cellular components including tRNAs get in contact with H2O2 or other cellular radicals in the mix due to the lipid membrane dissolution. I suggest to use controlled conditions in the initial lysis and also to carefully compare several lysis methods.\nThank you for pointing this out. To minimize oxidation by radicals that may arise during cell lysis, we used a highly reducing conditions by adding 50 mM DTT in the initial lysis buffer. We also monitored the formation of cmnm5h2U by introducing tracer tRNA at the onset of lysis (Figure S2c). The results showed that the initial lysis conditions had little effect on the frequency of h²U derivative formation.Very recently, Prof. Sierant and Nawrot group, who first demonstrated in vitro desulfuration of s2U derivatives, reported the presence of mcm5h2U and its derivative in yeast and human cells (PMID: 39596401). Although they did not perform spike-in experiments as we did, their findings serve as an independent confirmation of the existence of h2U derivatives in cells.\n3. Are these h2U derivatives effectively involved in protein translation in vivo? Could these derivatives be, for example, identified and quantified in tRNAs extracted from polysomal fractions? In cells with high fractions of these derivatives ( ~15% of tRNALys in HeLa cells contained mcm5h2U), is the translation of the cognate codons effectively affected?\nThank you for the productive suggestion. In this study, we first separated human culture cell lysate into the supernatant (free RNA) and crude ribosome fractions using a sucrose cushion (Figure 4d), followed by sucrose density gradient (SDG) centrifugation to obtain the polysome fraction (Figure 4e). Small RNAs were then obtained from the free RNA and polysomal RNA, and subjected to the LC/MS nucleoside analysis, showing that mcm5h2U is present in the polysomal RNA (Figure 4f, g). As expected, the signal intensity of mcm5h2U in the polysomal RNA was lower than that in the total RNA and free RNA fractions. These results suggest that a portion of desulfurated tRNAs participates in translation, whereas others may fail to be aminoacylated or be excluded during the codon recognition step.\n4. Is there is a purification step of the tRNA after H2O2 treatment? In the Methods section it is not mentioned if the left over H2O2 in the tRNA preparation was removed, as this can interfere with the subsequent reactions, for example, aminoacylation or in vitro translation.\nWe apologize for the oversight in not providing details about the steps following H O treatment. After H O treatment, the tRNA was purified by performing ethanol precipitation twice. We have now added this information to the Methods section.\n5. The author state in the discussion „we have quantified h2U derivatives under various oxidative stress conditions, but are yet to find conditions that reproducibly increase h2U levels”. The fat that the authors did not find conditions that reproducibly increase h2U levels is a major concern to doubt the biological function of these derivatives. In any case it would be useful to show also these negative results. Which conditions and stresses have been used so far without observing any modulation?\nThank you for pointing this out. We have listed the negative results in Table S4. We repeatedly cultured HeLa cells under various concentrations of H O , menadione, and rotenone, and analyzed changes in mcm5h U levels. However, under none of these conditions did we observe a significant increase in mcm5h U levels. Although it remains unclear under which specific conditions h U derivatives are upregulated, desulfurated tRNAs actually have a biological function to down-regulate protein synthesis in the cell.\n6. In figure 4 Western of the HiBit construct is missing as control.\nWe failed to detect the mutant HiBiT by western blotting due toits small size (<2 kDa). Instead, as a control, we compared its luminescence activities between untreated and desulfurated tRNAs.\nMinor points\nThe author state “We reviewed our previous liquid chromatography-mass spectrometry (LC-MS) data”. Please add a reference. Which previous data were reviewed?\nThank you for the comment. We have added the following reference.\nSuzuki, T. et al., Complete chemical structures of human mitochondrial tRNAs. Nature Commun., 11, 4269 (2020)\nWhat is the peak indicated by an asterisk in fig S1A? Please clarify in the legend.\nThe peak indicated by an asterisk is a non-specific peak. It has been added to the legend.\nReviewer #2 (Remarks to the Author):\nReview of the manuscript entitled \"Translational Regulation by Desulfurated tRNA Modification: Implications for Oxidative Stress Responses\" by Yufeng Mo, Kensuke Ishiguro, Kenjyo Miyauchi, Yuriko Sakaguchi, Naho Akiyama, Ayaka Murayama, Kodai Machida, Hiroaki Imataka, Akio Yamashita, Mikako Shirouzu, and Tsutomu Suzuki submitted to Nature Comminications.\nThe authors describe the identification of h2U derivatives in human and bacterial cells as well as in mouse liver tissue. These h2U derivatives are products of sulfur removal from natural 2-thiouridine derivatives present in four transfer RNAs specific for lysine, glutamine, glutamic acid, and asparagine. The authors claim that the h2U derivatives are the products of natural oxidative desulfuration of intact tRNAs isolated from cytoplasmic and mitochondrial fractions. In a series of mass spectrometry experiments, they confirm the presence of x5h2U at position 34 of the tRNA, which was originally the position of the xms2U derivatives. Since this is the first position of the anticodon, they examine the characteristics of the oxidized tRNA. They noted the decreased efficiency of translation in a human reconstituted in vitro system with free cells and the decreased efficiency of h2U-tRNALys, Glu and Gln aminoacylation. Finally, they concluded that desulfuration of native tRNA specific for lysine (with mcm5s2U) affects codon recognition when the damaged tRNA is bound to either a programmed AAA or AAG ribosome, by a loss of AAA codon recognition followed by to some degree decreased AAG codon recognition. To confirm the molecular structures of the mcm5h2U and A and mcm5h2U and G base pairs, they used a cryo-EM approach and discovered the “anionic type” mcm5h2U-G base pair. They also show the weak mcm5h2U-A pair (with only one hydrogen bond) as well as the two original base pairs mcms2U-A and mcm5s2U-G. In their general conclusions, they state that desulfuration of the tRNA thio-modification modulates protein synthesis under oxidative stress.\nAlthough the data presented are convincing and to some extent original, the manuscript needs to be thoroughly revised to be considered for publication.\nWe sincerely thank the reviewer for the careful reading of our manuscript and for the thoughtful and constructive summary of our work. We appreciate the acknowledgment that the data are convincing and contain original aspects. At the same time, we fully recognize the need for a thorough revision, as pointed out, to further improve the clarity, accuracy, and scientific rigor of the manuscript.\nMajor corrections:\n- First of all, the presence of mcm5h2U has already been detected in yeast, several human cancer cell lines and also in modified 293 cells deprived of oxidative system gene expression and cultured under normal and oxidative stress conditions (doi: 10.3390/ijms252212336). Therefore, such statements as on page 4 and in some other fragments of the manuscript such as: “However, the occurrence of oxidative desulfuration of xm5s2U within cells has not yet been investigated.” is incorrect. The manuscript should refer to the published data and correct the text in the manuscript.\nWe thank the reviewer for this important and constructive comment. The publication by Sierant et al. (Int. J. Mol. Sci. 2024, 25, 12336; PMID: 39596401), which clearly demonstrated the presence of desulfurated tRNA modifications such as mcm5h2U in yeast and human cells under oxidative stress conditions, became available after we finalized the manuscript. We acknowledge that our original statement was outdated and misleading in light of their findings. Accordingly, we have revised the relevant sentences throughout the manuscript to accurately reflect the prior work by Sierant et al. and have cited their study at appropriate locations (e.g., Introduction, and Discussion sections). These revisions clarify the novelty and scope of our own findings in relation to this recent publication.\n- The authors dissect the oxidative desulfuration of 2-thiouridine to a 4-pyrimidinone derivative and omit the other product, the uridine derivative. As previously reported, the composition of the products of the desulfuration reaction depends on the pH of the reaction environment and on the nature of the substituent at position 5 of the 2-thiouridine moiety (doi: 10.1016/j.bmcl.2013.08.114, doi: 10.1016/j.bmc.2015.07.030). The authors completely ignored such a xm5s2U property, did not explain how they assessed the influence of xm5h2U-tRNA and excluded the influence of the of the presence of xm5U-tRNA products in the investigated processes (translation/aminoacylation), which were performed under physiological conditions (pH 7.4). Of particular interest in this context is the result of binding of the oxidized mcm5S2U-tRNA to the mRNA ribosome, assuming that part of the thio-tRNA is converted to its oxo derivative, which facilitates U-A binding. Moreover, the efficiency of aminoacylation also depends on the presence of the thio modification in the wobble position (identity element for AARS). Therefore, the observed effect could be due to the presence of x5h2U and x5U-tRNA in the whole tRNA, which reduce the activity of AARS. This question needs clarification/discussion.\nThank you for this valuable comment. As the reviewer rightly pointed out, previous studies have shown that oxidation of s2U derivatives can yield both 4-pyrimidinone and uridine derivatives, and that the composition of the resulting products depends on the pH of the reaction environment and the nature of the C5 substituent. Based on this finding, we have revised Figure 1b to indicate that uridine derivatives (xm5U) can also be generated by oxidation and desulfuration of xm5s2U.\nBased on careful evaluation of oxidation conditions, we treated human class I tRNA fractions and isolated human tRNALys with 100 mM or 50 mM H O at pH 7.4 for 24 hours at room temperature. We confirmed that under these conditions, mcm5s2U was almost entirely converted to mcm5h2U, with only minimal formation of mcm5U (Figure S4a,b). We confirmed the reproducibility of this reaction. From these results, we cautiously conclude that the contribution of mcm5U formation in our experimental system is limited.Therefore, all relevant experiments we performed—including in vitro translation, aminoacylation, A-site tRNA binding assays, and cryo-EM—were conducted using tRNAs containing mcm5h2U as a major modification, allowing us to exclude the influence of mcm5U from our interpretation. However, since the exact mechanism of desulfuration in cells remains unclear, it is possible that mcm5U is generated alongside h U derivatives to some extent. We have added a relevant description addressing this point in the Discussion.\n- The oxidative desulfuration reaction scheme shown in Figure 1b, in which only one desulfuration product xm5s2U is shown, may be misleading to the readers and is inconsistent with the previous studies conducted in vitro and in the cellular systems as discussed above. The authors are advised to correct the schematic and possibly note under the figure that only the xm5h2U type desulfuration product was monitored.\nWe have revised Figure 1b to indicate that uridine derivatives (xm5U) can also be generated by oxidation and desulfuration of xm5s2U. We have added a note to Figure 1b to clarify that our analysis focused exclusively on xm5h2U.\n- The interpretation and discussion of the molecular structures of the base pairs of mcm5s2U/mcm5h2U with A and with G requires some clarification. The base pair mcm5s2U-A is (as expected) in a typical Watson-Crick mode, while the desulfurized base pair mcm5h2U-A has only one possible hydrogen bond. The authors claim a weak bond in this base pair. The authors can address the confirmation of their result using the previously shown data of thermodynamic measurements performed for the 4-pyrimidinone type nucleoside analogs incorporated into the RNA duplex. These data show that h2U-type units form weak base pairs with A and more stable ones with G (data are given for h2U and geranylated 2-thiouridine-containing RNA duplexes). In addition, the authors are advised to point out that in such a measurement, the addition of a silver anion (sealing properties) increases the stability of the h2U-A duplex, which is further evidence for the existence of a weak h2U-A base pair.\nThanks for the valuable suggestion. We have made revisions in discussion part accordingly by mentioning and citing the previous data of thermodynamic measurements performed for the 4-pyrimidinone type nucleoside analogs incorporated into the RNA duplex (Elzbieta Sochacka et al. 2015).\n- Importantly, the mcms2U-G base pair is not an anionic base pair (Fig. 7c) and is incorrectly interpreted as a base pair with the hydrogen bond between N2H of G and N3H of U (hydrogen bonds marked in red). The authors should check the interpretation of the team of P. Agris, who assigned such a base pair to the C-G type, where 2-thiouridine is pre-structured to its keto-enol form and provides three hydrogen bonds between G and mcm5s2U (doi:10.1016/j.jmb.2011.12.048). Fig. 7c and the interpretation in the text should be improved.\nThank you for this insightful comment. As correctly noted, according to Vendeix et al., 2012 (PMID: 22227389), the mcm5s2U–G base pair adopts a Watson–Crick-like geometry, and they proposed that mcm5s2U may undergo a tautmeric shift of the uracil base from keto-to-enol form, allowing it to form non-canonical hydrogen bonds with G. It is important to note, however, that there are several critical differences between the conditions used in their crystallographic analysis and those in our cryo-EM study. They solved a crystal structure of a ribosome complex with a chemically-synthesized anticodon stem-loop (ASL) and the Thermus thermophilus 30S subunit (PMID: 22227389). The crystallization was conducted under conditions involving high concentrations of precipitants and at pH 6.5. Moreover, the resolution of their electron density map was not sufficient to unambiguously determine the geometry of the base pair. In contrast, we solved a cryo-EM structure of a ribosome complex with native tRNALys purified from human culture cells and the E. coli 70S ribosome. The cryo-EM grids were prepared at pH 7.6, which is much closer to physiological conditions, compared to the crystallization conditions. As shown in Figure 7c, both the mcm5s2U34 and G3 bases are clearly visible in the cryo-EM density, which supports a wobble geometry shifted toward the minor groove.\nOn the other hand, structural study of E. coli tRNALys bound to Thermus thermophilus 70S ribosome performed by X-ray crystallography revealed that mnm5s2U34 forms a wobble base pair with G3 that is shifted toward the minor groove (PMID: 26791911). To enable this geometry, mnm5s2U34 must adopt a tautomeric form in which the N3 position acts as a proton acceptor; accordingly, the mnm5s2U–G3 pair has been referred to as an anionic U–G base pair (PMID: 37068913). The pKa values of N3 in various uridine derivatives have been experimentally determined (PMID: 28088758): the pKa of N3 is 9.15 for unmodified uridine, 8.05 for mcm5s2U, and 7.28 for mnm5s2U. According to the Henderson–Hasselbalch equation, at pH 7.6 the proportion of deprotonated N3 is approximately 2.8% for uridine, 26% for mcm5s2U, and 68% for mnm5s2U. Since these calculations assume an ideal aqueous environment, it remains unclear how these values may be affected within the ribosomal context. Nevertheless, both mcm5s2U and mnm5s2U are statistically more prone to exist in an anionic form compared to uridine. Therefore, the minor-groove-shifted geometry of the mcm5s2U34–G3 base pair observed in this study is reasonably interpreted as an anionic U–G base pair.\nNotably, in the study by Vendeix et al. (PMID: 22227389), crystallization was carried out at pH 6.5, where only about 2.7% of mcm5s2U would be deprotonated at N3 according to the Henderson–Hasselbalch equation. This suggests that mcm5s2U was predominantly in its protonated form, which may have contributed to differences in base-pair geometry observed in their crystallographic structure.\nThe above discussion has been included as a Supplementary Discussion.\n- The sentence : “In addition, structural analyses revealed that xm5s2U34 base-pairs with the third guanosine of NNG codons at the A site of the ribosome via an anionic U • G wobble pairing 33,34” refers to only one of two different cases, in one of which the xm5s2U-G base pair can adopt “a new wobble base pair” for mnm5s2U-G (Rozow et al 2016) and in the other the C-G like mode is found for mcm5s2U-G (Vendeix et al 2012). In the following text, the description refers to the new wobble base pair mnm5s2U-G, which is not related to the discussed mcm5s2U-G case, because the electronic properties of mcm5 and mnm5 are quite different and have different effects on the dissociation of the N3H proton. This fragment of the introduction requires a more detailed comment. Ref. 34 should be placed before Ref. 33.\nAs pointed out, we have clarified in the Supplementary Discussion that the geometries of the mnm5s2U–G base pair reported by Rozov et al. (2016) and the mcm5s2U–G base pair described by Vendeix et al. (2012) are different, and that the electronic properties of mcm5 and mnm5 differ, including their pKa values of N3.\nWe also noted that the crystallization by Vendeix et al. was performed at pH 6.5, whereas our cryo-EM analysis was carried out at pH 7.6, which may have contributed to the observed differences in geometry.\nAs described above, the pKa of N3 in mcm5s2U is 8.05, and according to the Henderson–Hasselbalch equation, at pH 7.6 approximately 26% of mcm5s2U would be deprotonated at N3, while at pH 6.5 only about 2.7% would be deprotonated. Therefore, it is reasonable to assume that mcm5s2U was mostly in the protonated state under the crystallization conditions, which may have led to differences in base-pair geometry.\nAs suggested, Rozov et al. has been placed before Westhof et al.\n- Authors are advised to pay more attention to correct citation, naming of compounds and interpretation of published data: e.g. page 9: mcm5h2U-G is not an anionic base pair. Similarly, mcm5s2U-G is not an anionic base pair. Only mnm5s2U-G has been shown to form a new wobble base pair in which the ionized mnm5s2U is bound to G. The discussed h2U-G and ges2U-G form base pairs of the 4-pyrimidinone-G type, and the pair mnm5s2U-G can also be categorized in the same group of modes. The total charge of this U-unit is 0, although the charge is distributed between the nucleobase and the mnm5. Do you think that “the anionic” base pair in this case is a correct description? Please correct the text accordingly.\nThank you for the comment. As suggested, we have revised the text to refer to the mcm5s2U–G and mcm5h2U–G base pair as a “minor-groove-shifted base pair” rather than definitively calling them an “anionic base pair.”\n- It is not clear how xm5s2U prevents misdecoding of the pyrimidine ending codons NNY due to the 3’-endo sugar pucker. The topic needs reference / discussion.\nThank you for the comment. We have cited the relevant studies (Refs. 32 and 33) reporting that the 2-thiocarbonyl group and the 2'-hydroxyl group are in van der Waals proximity and contribute to the stabilization of the C3'-endo sugar pucker in the introduction.\n- The description on page 9 is misleading: “Given that the 2-thiocarbonyl group of mcm5s2U stabilizes C3’-endo ribose puckering 31,32, mcm5s2U-G pairing should be more stable than mcm5h2U-G pairing. In addition, the 2-thiocarbonyl group of mcm5s2U promotes the acidity of the N3 atom (Ref. 37), thereby contributing to the anionic mcm5s2U-G pairing 34.” The authors provide no evidence to support the conclusion of the first statement. Also, the acidity of N3 in mcm5s2U is similar to that of the 5-non-substituted 2-thiouridine (see Ref. 37) , and the mcm5s2U-G base pairing is not anionic. Therefore, the conclusions drawn here must be properly proved and descriptions corrected.\nAs the reviewer correctly pointed out, the acidity of N3 in mcm5s2U is similar to that of s2U without a C5-substituent. However, in this context, we are comparing it with unmodified uridine to emphasize that the N3 proton in mcm5s2U is shifted toward greater acidity. As described above, our structural model places the mcm5s2U base pair in a wobble geometry shifted toward the minor groove, and it is not feasible to model it in a Watson–Crick geometry. We have already discussed the difference in geometry between Vendeix et al. (2012, PMID: 22227389) and our study in Supplementary Discussion. In our observed geometry, deprotonation of the N3 atom in mcm5s2U is necessary. As previously noted, at pH 7.6, only 2.8% of uridine exists in the deprotonated form, whereas approximately 26% of mcm5s2U is deprotonated. Therefore, mcm5s2U is statistically more prone to deprotonation, and the mcm5s2U–G base pair can reasonably be interpreted as adopting an anionic G–U geometry.\n- The discussion of the discovered/confirmed data should be deepened and completed.\nTaking into account the many valuable comments we received, we have revised and expanded the Discussion to provide a more in-depth analysis. Supplementary Discussion has also been provided.\nMinor corrections:\n- please do not introduce “2-deoxouridine” as another colloquial name for 4-pyrimidinone riboside. This name is misleading compared to usually used 2’-deoxyuridine.\nWe agree that “2-deoxouridine” is potentially confusing, so we have adopted an original nomenclature to clarify the distinction.\n- as a flag laboratory for modified nucleosides, the authors are advised to consider using a proper name for modified nucleoside, abbreviated as mcm5s2u. The proper name (used by other authors) is “5-methylcarboxymethyl-2-thiouridine” and NOT “5-methoxycarbonylmethyl-2-thiouridine”. This proper name is logical in relation to the abbreviation used: methyl – m and carboxy -c. So it is rather confusing that mcm5 is read here as “methoxy” (for which a correct abbreviation/symbol should be “mo”), “carbonyl” (it has no abbreviation, and “c” is typically used for “carboxy(l)\"). The use of confusing names leads to confusion in the nomenclature of modified nucleosides.\nIn this study, we have followed the nomenclature established by the RNA modification database MODOMICS. As the names used in MODOMICS are widely accepted in the field, we believe our terminology adheres to a standard and commonly used convention. That said, as you correctly pointed out, “5-methylcarboxymethyl-2-thiouridine” is also a valid systematic name, and we have now included it alongside the MODOMICS nomenclature in the revised manuscript.\n- The references to 35 and 36, which refer to the wobble base pair U-G occurring in the major groove, are misleading. The classical pairs U-G and G-U adopt the wobble mode, but these two base pairs are NOT isosteric, the feature which is important for the ribosomal context. While the G34-U3 base pair can be accommodated on the ribosome, the U34-G3 base pair is not allowed in this spatial architecture (see the work of Westhof). “Anionic” G-U in ribosomal RNA are interesting newly discovered modes, but their discussion is not relevant here (Ref. 33).\nThank you for your comment regarding the spatial distinction between U–G and G–U base pairs. We have removed Ref 36 citation in the relevant section and revised the description accordingly. Ref. 34 (ref 33 in previous version) was replaced by Westhof, E., et al. (2019). \"The multiple flavors of GoU pairs in RNA.\" J Mol Recognit 32(8): e2782.\n- The description of the results – the sentence about the experiments with bacterial knock-out S2U synthetase should be corrected. You write that ”the E. coli ΔmnmA strain in which mnm5s2U is converted to mnm5U (Figure S1c)”. This is an incorrect description, as in this bacterial strain the thiolation of the nucleoside mnm5U in the tRNA is abandoned.\nThank you for your comment. We have revised the wording accordingly to ensure clarity and accuracy.\n- On page 3 (Introduction) the authors list the x5s2U derivatives present in tRNA in different organisms (prokaryotes and eukaryotes), and for cytoplasmic tRNA in eukaryotes, but they cite the work of Johansson et al 2008 (Ref 16) concerning tRNA from yeast S. cerevisiae. Since tRNA modifications in eukaryotes vary by organism and the manuscript focuses primarily on human tRNA, a more detailed description of human tRNAs and their modified thio-uridines is recommended. The situation of human mitochondrial tRNA is quite clear and frequently cited in the literature, but precise information on specific human cytoplasmic tRNA is only cursorily given, so a detailed description would be greatly appreciated.\nThank you for your comment. We have now cited a recent review article on 2-thiouridines in human cytoplasmic tRNAs (PMID: 28218716) and revised the corresponding section to strengthen the description.\n- In the chapter \"Confirmation of h2U derivatives produced in cells\", the authors give the h2U content in cytoplasmic and mitochondrial tRNAs from different cells with an accuracy of tenths of a percent. They should explain how these values were calculated and which value was taken as 100%. Do the authors believe that the h2U content in cells does not depend on individual cell characteristics, cell culture and environmental conditions? The h2U content in animal tissues is presented in a similar way. How do the authors explain the differences? No comments are given on the formation of uridine derivatives.\nIt is well known that the signal intensity of molecules detected by mass spectrometry is significantly influenced by differences in ionization efficiency among analytes. In this study, since RNA fragments are detected as negative ions derived from their phosphate groups, we calculated the modification ratios based on the intensity ratio between each modified fragment and its corresponding unmodified counterpart in the mass chromatogram. Our previous studies have shown that differences in ionization efficiency due to base composition are minimal (PMID:29760464). Therefore, we calculated the modification levels by taking the total signal intensity of all RNA fragments detected in the mass chromatogram as 100% and determining the proportion of each fragment. Additionally, we have added a carefully worded discussion addressing the possibility that variations in h2U levels may be influenced by cellular oxidative stress response, redox balance, and the degradation of h²U-modified tRNAs\n- Did the authors take into account that the total tRNA fraction used in the assay for cell-free translation contains tRNAs that read codons with G regardless of desulfuration, e.g. tRNACUULys, tRNACUCGlu or tRNACUGGln? Please comment on this question in the discussion.\nThank you for your comment. We have added a description about the presence of isoacceptors with C34 that redundantly contribute to decoding of G-ending codons in the result section.\nReviewer #3 (Remarks to the Author):\nThis work describes the identification and mechanistic studies of a tRNA modification, mcm5h2U34, present naturally in cells and tissues. In contrast to most RNA modifications studied in the past, mcm5h2U34 seems to be derived from cellular oxidation from mcm5s2U, possibly under oxidative stress and not synthesized by a cellular enzyme. Cells contain many chemically reactive metabolic products such as reactive oxygen species (ROS) that can react non-enzymatically with RNA, however, the functional consequence of such tRNA product on translation have not been studied much. This work starts with a methodology to detect and quantify mcm5h2U34, and identification in mouse tissues. This is followed by in vitro studies of mcm5h2U34 showing its deleterious effect on translation and on tRNA charging, ending with cryo-EM structures with an atomic rationale of how mcm5h2U34 affects decoding on the ribosome. The results are of high quality and clearly presented. This exciting work provides a streamlined methodology to study the functional consequences of many potential RNA damages and provides in-depth functional analysis of this frequently occurring RNA modification.\nWe sincerely thank the reviewer for the thoughtful and encouraging summary of our work. We greatly appreciate the recognition of both the novelty and the technical rigor of our study. As the reviewer pointed out, mcm5h2U34 represents a unique class of RNA modification—likely generated through non-enzymatic oxidative processes—and we are pleased that our efforts to develop a robust detection methodology and to explore its functional consequences on translation have been well received. We hope that this study will help pave the way for further investigations into the biological significance of oxidatively derived RNA modifications.\nComment:\n1. A major issue is that no cellular data is shown that accompanies the biological regulation model in Fig. 8. On p. 10, the authors state: “Indeed, we have quantified h2U derivatives under various oxidative stress conditions, but are yet to find conditions that reproducibly increase h2U levels.”? What are the conditions tested?\nThank you for the comment. We repeatedly cultured HeLa cells under various concentrations of H O , menadione, and rotenone, and analyzed changes in mcm5h U levels. However, under none of these conditions did we observe a significant increase in mcm5h U levels. We suspect this may be due to the rapid activation of antioxidant responses, which effectively eliminate intracellular ROS. We have now included the quantification results of h²U derivatives under the oxidative stress conditions tested so far in Table S4.\nWhat is the charging level of the mcm5h2U34 modified tRNAs compared to their mcm5s2U34 counterpart in cells?\nRegarding in vitro aminoacylation efficiency, we have confirmed that, with the exception of tRNAArg, tRNAs carrying the mcm5h2U modification (tRNALys, tRNAGln, and tRNAGlu) exhibit reduced aminoacylation efficiency compared to their mcm5s2U-modified counterparts. However, since mcm5h2U34-modified tRNAs constitute only a few percent of the total tRNA population in cells, it is technically challenging to distinguish them from mcm5s2U34-modified tRNAs and accurately determine their aminoacylation levels in vivo.\n2. The functional model would expect to find lower fractions of mcm5h2U34 tRNAs on the polysome compared to the total RNA input. Have the authors tested this?\nThank you for the valuable suggestion. We received a similar recommendation from Reviewer #1, and accordingly, we fractionated human culture cell lysates using sucrose density gradient (SDG) centrifugation to collect the supernatant and polysome fractions. We then extracted tRNAs from each fraction and performed nucleoside analysis by LC/MS. The results showed that the signal intensity of mcm5h2U in the polysome fraction was lower compared to that in the total RNA and supernatant fractions (Figure 4f). These findings suggest that while a portion of desulfurated tRNAs may participate in translation, others are likely excluded either due to failure in aminoacylation or rejection during codon recognition.\n3. Would mcm5h2U34 tRNA specifically affect translation of stress response proteins? This may be addressed through computational codon usage analysis.\nThank you for the valuable suggestion. We performed Relative Synonymous Codon Usage (RSCU) analysis on a set of genes associated with the Gene Ontology (GO) term \"response to oxidative stress\" (GO:0006979). Compared with the RSCU values calculated from all human protein coding genes (PMID: 35859338), we observed no significant differences in codon usage for Lys, Gln, or Glu. However, for Arg, the usage of CGN codons was decreased, while that of AGR codons was relatively increased. Desulfuration of mcm5s2U led to reduced aminoacylation capacity in tRNAs for Lys, Gln, and Glu, whereas tRNAArg retained full aminoacylation activity. Although the codon recognition properties of tRNAArg bearing mcm5s2U remain to be investigated, its resistance to desulfuration may contribute to the preferential usage of AGR codons in oxidative stress-responsive genes. This additional analysis has been included in discussion part with Table S5.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nWe sincerely thank you for your time and effort in co-reviewing our manuscript as part of the Nature Communications Early Career Researcher initiative. We truly appreciate your thoughtful feedback and the opportunity to benefit from your insights.\nResponse to the reviewer #2ʼ comments\nThe authors have addressed some of the concerns raised in the previous round of review.\nThey clarified that xm5h2U is not a novel modificaon but rather a cellular response to oxidave stress derived from xm5s2U. They also expanded their invesgaon to include the presence of xm5h2U in the polysomal fracon and atempted to interpret structures using Cryo-EM. Despite these improvements, the manuscript sll falls short of the standards required for publicaon and needs substanal revision in four crical areas.\nWe sincerely thank the reviewer for reading our revised manuscript so carefully and for providing numerous critical and constructive comments. We fully appreciate the considerable time and effort you devoted to this detailed evaluation. We are also grateful for your positive assessment of our additional analyses of polysomal RNAs and the structural interpretation.\nAt the same time, as you pointed out in the four areas of concern, we fully recognize that substantial revisions are required. Below, we provide our detailed responses and rebuttals to each point. We sincerely hope that our revisions adequately address your concerns.\n1. Incomplete acknowledgment of prior work on h2U detecon in cells\nIn response to the suggeson that the authors should cite the first report of desulfurated 5-substuted 2-thiouridine forms observed in cellular systems, they did include a reference to the relevant study. However, they failed to emphasize a crucial finding: h2U had already been idenfied in cells - both under oxidave stress and in condions without stress, including in cells lacking anoxidant systems.\nSpecifically, the earlier work demonstrated for the first me that mcm5h2U, a desulfurated derivave of mcm5s2U, as well as desulfurated immature forms (e.g., cm5s2U) were present in both yeast and human cells not submited to oxidave stress. The study included a range of human cell lines, including seven cancer types (HeLa, A375, MCF7, U87, MOLT-4, K562, A431) and the non-cancerous HEK293 line. Notably, the levels of h2U varied between cell types, depending on the efficiency of their anoxidant defense, reaching up to 11%.\nThis essenal context (even in short) is missing from the current manuscript.\nWe apologize for the oversight in not citing it in a comprehensive way. In this revised version, we have added the fact that h2U has been identified in cells, detected regardless of the presence or absence of oxidative stress, and even observed in cells lacking antioxidant systems. In particular, we now revised this section as follows,\n“In fact, mcm5h2U and cm5h2U, the desulfurized forms of mcm5s2U and cm5s2U, have been found in tRNAs from yeast and various human cultured cells regardless of the presence or absence of oxidative stress, reaching up to 11% depending on the efficiency of the antioxidant defense mechanisms (Sierant et al, 2024).”\nFor instance:\nLine 128: “Although desulfurated derivaves such as mcm5h2U have been detected in yeast and human cells under oxidave condions (Ref 56), their mechanisc roles in translaonal regulaon have not been fully elucidated.”\nIn the Discussion, the authors briefly state that the cited publicaon used a different detecon method but failed to elaborate on what that method entailed. It should be clearly stated that the referenced study esmated modificaon levels using calibraon curves derived from synthec standards, rather than desulfurated tRNA prepared in-house. This disncon is important for understanding the accuracy and reproducibility of the measurements.\nThe section being pointed out here is probably the following description starting from line 363.\n“Although the recent publication by Sierant et al. (2024) also detected mcm5h2U in yeast and human cultured cells and analyzed its behavior under oxidative stress conditions, their detection method differs from ours, and therefore further validation is required.”\nWe have rewritten this part as shown below.\n“Sierant et al. (2024) likewise detected mcm5h2U in yeast and human cultured cells and analyzed its behavior under oxidative stress using calibration curves derived from synthetic standards”\nMoreover, the authors use the phrase ‘clear evidence’ mulple mes:\nLine 131: “In this study, we present ‘clear evidence’ of the presence h2U derivaves in human cells and mouse ssues.”\nLine 215: “These results provide ‘clear evidence’ that oxidave desulfuraon of 2-thiouridine derivaves naturally occurs in human cultured cells.”\nThis phrasing implicitly undermines the clarity of evidence presented in prior studies (including Ref 56), which had already provided compelling data on the presence of mcm5h2U and related compounds. Such wording could be perceived as dismissive of foundaonal work in the field. It is essenal that the authors discuss their findings in the context of exisng literature, presenng facts objecvely and respecully acknowledging prior contribuons.\nThank you for your comment regarding the wording. All of the data in this manuscript were obtained before the publication of Sierant et al. (2024), and therefore it was necessary for us to rule out the possibility of oxidation during the extraction process through spike-in experiments. We used the term “clear evidence” to reinforce this conclusion, and it was never intended to imply any criticism of the clarity of evidence presented in prior studies. To avoid any misunderstanding, we have removed these two sentences.\n2. Concerns Regarding Oxidave Stress Condions and Data Consistency\nA significant issue in the current manuscript is the choice of hydrogen peroxide (H₂O₂) concentraon used to induce oxidave stress in HeLa cells. The authors applied a concentraon of 2 mM H₂O₂, which appears to be inappropriately high given that the reported IC₅₀ for H₂O₂ in HeLa cells is 50 μM (as stated in Sierant et al., Ref. 56). This discrepancy raises concerns about the physiological relevance of the experiment and the integrity of the results.\nThe use of 2 mM H₂O₂ likely exceeds the oxidave stress threshold compable with cell viability. Such a high concentraon may induce oxidaon in already dead or dying cells, which then become an in vitro system, compromising the biological interpretaon of the results. Importantly, this could explain the inconsistencies observed in the data. For example, the increase in h2U levels was minimal—only a 2% rise—which contradicts the expectaon of significant desulfuraon under oxidave stress. This low increase raises the possibility of an improperly conducted experiment or issues with the H₂O₂ reagent itself. If the later is the case, it would cast doubt on the reliability of other experiments using the same oxidave treatment.\nThe use of 2 mM H O to treat human cultured cells for several hours has been reported in multiple studies (PMID: 19212855, 37309898, 33326779, 12023051, etc.), and therefore our experiment cannot be considered extreme or unconventional. As the reviewer correctly pointed out, though, 2 mM H O is far above the IC50 value for HeLa cells (50 μM) and indeed strongly inhibits cell growth. Under our experimental conditions, we did not observe any detachment of HeLa cells from the culture dish. The reviewer’s concern that oxidative reactions may have occurred in cells that were already dead or dying is certainly valid. Nevertheless, treatment of HeLa cells with 2 mM H O for 1 hour resulted in almost no change in the proportion of h2U derivatives. If this were essentially an in vitro “dead-cell” model, the ratio of h2U species should have increased clearly and substantially. Sierant et al. (2024) also noted that treatment of yeast with 100 mM H O likely produced h2U species through a direct oxidation process occurring in vitro. Consistent with these findings, the oxidative stress conditions we employed did not lead to a marked increase in h2U derivatives. This suggests, again in agreement with Sierant et al. (2024), that the cellular antioxidant system remained functional and effectively minimized tRNA damage.\nFurthermore, the authors should consider the specific sensivity of HeLa and other tested cell lines to H₂O₂. It is well-established that the cellular response to oxidave agents varies depending on the anoxidant system of the cell, including glutathione levels and catalase acvity. The authors should reference studies such as PMCID: PMC6527626 to support a more nuanced discussion of cellular suscepbility and anoxidant defense variability.\nThank you very much. We have long been searching for conditions in which h2U derivatives increase, using various oxidative stress inducers. However, as the reviewer correctly pointed out, we believe that multiple antioxidant systems robustly protect the cells and suppress tRNA oxidation. That said, we suspect that there may be specific biological contexts—such as particular growth conditions or stages of cellular differentiation—in which h2U derivatives do increase, and we hope to capture such phenomena in our future studies.\nWe have added a discussion on the diversity of cellular sensitivity and antioxidant defenses, citing the papers the reviewer kindly pointed out.\nIn the Discussion secon, the authors state: “Approximately 15% of tRNALys in HeLa cells contained mcm5h2U, whereas litle was detected in A549 and T24 cells (Figure S3).”\nHowever, this observaon is not consistent with the findings of Sierant et al. (2024), who demonstrated that A549 cells exhibit a sensivity to H₂O₂ similar to that of HeLa cells. Based on this, one would expect a comparable degree of mcm5s2U damage and corresponding levels of mcm5h2U in both cell types. The divergence in outcomes suggests either methodological differences that were not disclosed or inconsistencies in experimental execuon. This contradicontradicon must be addressed directly by the authors.\nAs you correctly pointed out, according to the results of Sierant et al. (2024), HeLa and A549 cells show comparable sensitivity to H O , with IC50 values of 50 µM and 70 µM, respectively. In our experiments, under untreated conditions, the level of mcm5h2U in tRNALys was approximately 15% in HeLa cells, whereas it was almost undetectable in A549 cells. This difference may be attributable to cell line–specific variations in the regulatory mechanisms of antioxidant systems.\nMoreover, since mcm5h2U levels do not change even when cells are treated with H O , we believe it is not appropriate to directly link H O sensitivity with the basal mcm5h2U levels observed under untreated conditions.\nA further point of concern is the apparently redundant experimental design shown in Supplementary Figures S4A and S4B. The data appear nearly idencal despite being described as the result of separate oxidaons (\"double\" in vitro oxidaon of tRNALys). It is unclear why a second oxidaon step was necessary if the outcome did not differ from the first one.\nThe experiment shown in Figure S4A represents the result of a single round of H O treatment applied to the class I tRNA fraction. Although 14.3% of mcm5s2U remained, we judged that this level was still acceptable for use in our in vitro translation assays.\nIn contrast, the experiment in Figure S4B was conducted using purified tRNALys.\nAfter the first round of H O treatment, 5.3% of mcm5s2U remained. However, because this sample was intended for cryo-EM analysis, it was necessary to reduce the residual mcm5s2U level as much as possible. Therefore, we performed a second round of H O treatment, which ultimately reduced the mcm5s2U level to 0.7%.\nDuring this process, the level of mcm5U increased slightly (0.7% → 2.5% → 2.7%), but we determined that this increase was not significant enough to affect the downstream experiments. Indeed, in the cryo-EM analysis, the density corresponding to mcm5h2U was clearly visible.\n3. Structural interpretaon of the mcm5s2U-G base pair\nThe structural analysis and discussion related to the mcm⁵s²U-G base pair presented in Figure 7c and its corresponding Supplementary Discussion are both conceptually confusing and technically flawed. Instead of clarifying the structural basis for wobble pairing involving modified uridines, the authors provide a misleading and overly speculave interpretaon, lacking consistency with established structural and mechanisc knowledge in the field.\nThe authors atempt to address the reviewer’s earlier comments by providing a detailed discussion of the base pairing behavior of mcm⁵s²U-G within the tRNA–mRNA–ribosome complex. Unfortunately, the current explanaon reflects a misunderstanding of the role of the wobble posion and the behavior of modified uridines during decoding. Hence, the authors' esmaon of the “ionized” state of mcm⁵s²U is both unnecessary and misleading. Unlike mnm⁵s²U, which can exist in a zwiterionic form (U), mcm⁵s²U does not ionize under physiological condions in the same way. Therefore, the concept of an “anionic base pair” is incorrect and should be avoided. As noted in Rozov et al. (Ref. 34), there is no menon of “anionic U-G wobble pairing”; this is a misrepresentaon of prior work. If needed, the more accurate terminology is “zwiter ionic U” pairing with G (see Ref. 37), though even this does not apply in the case of mcm⁵s²U.\nIt is actually not clear why the discussion is so extensive, since the structure of this base pairing has already been proposed and a careful comparison shows that this new cryo-TEM structure obtained by the authors is very similar, if not idencal, to the structure obtained by X-ray crystallography.\nA reliable mechanisc explanaon was first provided by Vendeix et al. (2012), who described a tautomeric enol form (E4) of mcm⁵s²U that forms a C–G-like base pair via three hydrogen bonds, which is very similar to the Watson-Crick geometry. Despite possible discrepancies between their and your experimental condions, this geometry was consistently observed in both structural studies. Therefore, it is reasonable to consider this structure as the most likely conformaon under the current condions and to recognize it as such in this context.\nIndeed, as shown by Vendeix et al. (2012), the enol form of mcm5s2U can form three hydrogen bonds with G3, adopting a Watson–Crick–like GC base-pairing geometry. We examined this possibility in detail. When we measured the distances between the corresponding atoms using our cryo-EM structural model (Figure IA), the values obtained for the Watson–Crick–like geometry proposed by Vendeix et al. were as follows:\n• U4(OH) – G6(O): 3.8 Å\n• U3(N) – G1(NH): 3.8 Å\n• U2(S) – G2(NH₂): 3.6 Å\nDistances in the range of 3.6–3.8 Å are too long to support hydrogen bonding interactions.\nBy contrast, in the geometry proposed by Rosov & Westhof, the distances are:\n• U4(O) – G1(NH): 3.1 Å\n• U3(N) – G2(NH₂): 2.9 Å\nTherefore, based on our cryo-EM results, it is difficult to support a Watson–Crick–like geometry, and the minor groove–shifted geometry proposed by Rozov and Westhof is a more plausible interpretation (Figure IA). In addition, the resolution of the ASL + AAG codon + 30S subunit complex reported by Vendeix et al. (PDB: 3T1Y) is 2.8 Å, whereas the resolution of our native human tRNALys + AAG codon + 70S ribosome complex is 2.26 Å, indicating that our structure provides a more reliable model.\nFigure IB shows the mcm5s2U–G base-pairing model from Vendeix et al. (3T1Y). In this model, a Watson–Crick–like geometry is made possible by assuming an enol tautomer of mcm5s2U, and the interatomic distances fall within 2.7–3.1 Å, which at first glance appears reasonable. However, when this structure is visualized in a CPK representation (Figure IC), three steric clashes become evident, indicating that the model is structurally strained.\nMoreover, when the mcm5s2U–G pair from 3T1Y is superimposed onto our cryo-EM–derived model, a clear misalignment is observed (Figure ID). Finally, we compared the two models using the Q-score (PMID: 32042190), which evaluates the fitting between a model and the density. When the mcm5s2U–G model obtained in this study was overlaid onto the cryo-EM density and the average Q-score of the uracil base was calculated to be 0.82 (Figure IE), corresponding to a resolution of 1.28 Å. In contrast, when the 3T1Y mcm5s2U–G coordinates (Vendeix et al., 2012) were fitted into the cryo-EM density, the average Q-score of the uracil base was 0.57 (Figure IF), equivalent to a resolution of 3.17 Å. These results clearly demonstrate that our model represents a more reliable base-pairing geometry.\nIn the Supplementary Discussion, we outline several important differences between the two structures. Specifically, in the crystallographic analysis by Vendeix et al. (2012), the ribosome complex was formed by soaking chemically synthesized ASL, mRNA, and paromomycin into crystals of the T. thermophilus 30S subunit under conditions with high concentrations of precipitants and at pH 6.5. In contrast, our cryo-EM structure was determined using native human tRNALys and the E. coli 70S ribosome, with grids prepared at pH 7.6, a condition much closer to physiological pH. As noted above, our cryo-EM reconstruction also provides a higher-resolution and therefore more reliable structural model.\nIndeed, as the reviewer pointed out, unlike mnm5s2U, mcm5s2U does not form a zwitterion, and therefore whether it can adopt an anionic U–G base-pairing geometry is open to debate. In fact, considering that the pKa values of the N3 atom in U, mcm5s2U, and mnm5s2U are 9.15, 8.05, and 7.28, respectively, the reviewer’s comment is well taken. However, according to the Henderson–Hasselbalch equation, the proportion of deprotonated N3 at pH 7.6 is 2.8% for U, 26% for mcm5s2U, and 68% for mnm5s2U, indicating that mcm5s2U is statistically more likely to adopt an anionic form than uridine. While it remains to be further examined whether the mcm5s2U–G base pair indeed forms an anionic U–G interaction, our high-resolution cryo-EM structure clearly rules out the Watson–Crick–like geometry proposed by Vendeix et al. that requires the enol tautomeric shift of mcm5s2U.\nIn this revised version, we therefore describe the mcm5s2U34–G3 interaction as a minor groove–shifted GU wobble geometry, and we mention the possibility of an anionic U–G base pair only as one of several potential interpretations in the Supplementary Discussion.\nA few words of discussion: it must be emphasized that at the ribosome, all codon units are rigidly fixed in the A-site. Two first base pairs, that is N1-N36 and N2-N35 form the codon - ancodon minihelix. For the formaon of the third base pair binding, the first ancodon nucleoside (N34) exhibits a degree of spaal flexibility to allow forming the base pair with N3. This flexibility is a defining feature of the nucleosides in wobble posion. Modified uridines such as mcm⁵s²U at posion 34 are specifically tailored to accommodate this flexibility, enabling them to pair either with A3 (via standard Watson–Crick geometry) or G3 (via alternave, non-canonical arrangements).\nWe have added a brief explanation of the relevant background to the Introduction.\nClassical Crick wobble pairs, such as U34-G, in which U binds to G in major groove, are not spaally compable with the ribosomal decoding center (as demonstrated in the works of Westhof team), prompng the evoluon of modified uridines capable of forming base pairs of minor-groove shied U with G. For G3 decoding, this involves U* adopng an altered structure/tautomer to interact with G - not a shi of the enre base pair, as is mistakenly stated mulple mes in the manuscript.\nWe have added relevant explanations in Supplementary Discussion describing the minor groove–shifted GU wobble base pair.\nThus, several misleading or technically incorrect statements should be revised:\nAbstract: “The mcm5h2U • G pair shows a minor-groove shied U • G wobble geometry”\nIncorrect: it is not the pair that shis, but rather U that adopts a minor-groove shied tautomeric structure. Suggested revision: “The mcm5h2U • G interacon involves “a minor-groove-shied U” base pairing with G in a non-standard geometry.” (here it is analogy to a new wobble base pair).\nAs suggested, we have revised it.\nLine 319: “mcm5s2U34 pairs with G3 of the AAG codon via a minor-groove shied G-U wobble pairing” Suggested revision: “mcm5s2U34 pairs with G3 of the AAG codon via a non-canonical base pairing involving a minor-groove-shied U.”\nAs suggested, we have revised it.\nLine 328: “The mcm5h2U-G pair adopts a similar geometry to the minor-groove-shied U-G wobble geometry.” Suggested revision: “The mcm5h2U-G pair adopts a similar geometry to the new wobble base pair mnm5s2U-G, with U shied into the minor groove.”\n⁎\nAs suggested, we rephrased it as follows.\n“The mcm5h2U–G pair adopts a similar geometry to the non-canonical wobble base pair mnm5s2U–G, with U* shifted into the minor groove.”\nLine 330: “The 2-thiocarbonyl group of mcm5s2U promotes the acidity of the N3 atom (Ref 37), thereby contribung to the minor-groove-shied mcm5s2U-G pairing.” This is an oversimplified and parally misleading biochemical interpretaon. Suggested revision: “The presence of the 2-thiocarbonyl group lowers the pKa of N3 by approximately 1 unit, increasing its proton-dissociaon potenal; however, minor-groove shied geometry arises from structural rearrangements primarily due to C5 substuons, not ionizaon alone.”\nAs suggested, we rephrased it as follows.\n“The 2-thiocarbonyl group of mcm5s2U lowers the pKa of N3 atom, and the 5-methylcarboxymethyl group also contributes structural rearrangement to form the minor-groove-shifted mcm5s2U-G pairing.”\nHydrogen bonding mis-assignments and structural clarificaons The hydrogen bond patern proposed by the authors is not chemically plausible:\n“U4(O)-G1(NH) and U3(NH)-G2(NH2)” This arrangement in the second base pair includes two hydrogen donors with no acceptor, which is not in line with the state of the art.\nCorrect interpretaon based on structural precedents:\no Classical wobble (Crick):\nU2(O) – G1(NH)\nU3(NH) – G6(O)\no New wobble (Rozov & Westhof):\nU4(O) – G1(NH)\nU3(N) – G2(NH₂)\no Alternave non-canonical geometry (suggested to be used here):\nU4(OH) – G6(O)\nU3(N) – G1(NH)\nU2(O) – G2(NH₂)\nThe authors should re-consider the hydrogen bonding network shown in Fig. 7c, ideally providing distance measurements between donors and acceptors to validate the geometry. This would significantly improve the credibility and clarity of the structural data.\nWe calculated the hydrogen-bond distances for the proposed enol-form base pairing and have added these values to Figure 7c.\n4. Implausible Selecvity of Oxidaon Products A parcularly quesonable result reported in the manuscript is the observaon that under cellular condions, s²U undergoes only desulfuraon to yield h²U, without any formaon of the oxidized product U. This finding contradicts all earlier literature concerning the chemical behavior of 2-thiouridine under oxidave condions (e.g. Rao et al., 1974; Watanabe et al., 1980; Sochacka et al., 2011), which consistently demonstrated that U is a prominent oxidaon product of s²U, both in chemical models and in biological systems.\nThe absence of U in the authors' data is not explained or discussed, and given the well-documented oxidave pathways of s²U, it is difficult to accept this result without an in-depth mechanisc raonale or addional experimental validaon. As it stands, this contradicts known biochemistry and casts doubt on the specificity or sensivity of the detecon methods used.\nAs the reviewer pointed out, in our in vitro oxidation experiments using H O , the mcm5s2U derivatives in tRNA underwent desulfuration exclusively to mcm5h2U, with little formation of mcm5U. This was an unexpected result, because the previous study by Sochacka et al. (2011) reported that oxidation of s2U-containing RNA produces both h2U species and U species.\nBased on that report, we had anticipated that mcm5U-containing tRNA would also be generated alongside mcm5h2U-containing tRNA, and thus assumed that the subsequent challenge would be how to separate tRNAs containing mcm5h2U from those containing mcm5U.\nHowever, under our experimental conditions, as shown in Figure S4a,b, even after performing two rounds of oxidation, the increase in mcm5U was limited to only about 2%. In the study by Sochacka et al. (2011), chemically synthesized ASL RNA was oxidized with 100 mM H O in 10 mM Tris buffer (pH 7.4). In contrast, we oxidized human or mouse tRNA fractions with 100 mM H O in 10 mM HEPES–KOH (pH 7.4), and purified human tRNALys with 50 mM H O in the same HEPES–KOH buffer. The reason for this discrepancy is unclear, but the RNA samples themselves differ, and the different buffer composition might also contribute.\nFor product identification, we digested tRNA with RNase T , analyzed the resulting 12-mer RNA fragment in negative-ion mode, and quantified the species based on peak intensities in the mass chromatogram. This is a highly reliable method, and it is known that the measurement is not influenced by different chemical structures of modified nucleosides (PMID: 29760464, 27348091). Therefore, there is no doubt that mcm5s2U is converted to mcm5h2U with high yield under our conditions. Indeed, in our cryo-EM structural analysis, clear density corresponding to the mcm5h2U base was visualized.\nRegarding the papers the reviewer kindly introduced, in Rao et al. (1974), tRNAs were oxidized under various conditions, but oxidation products were not analyzed. Watanabe et al. (1980) oxidized the s²T nucleoside with 116 mM H O in 50 mM sodium phosphate (pH 8.0), but because the substrate is chemically different (s2T instead of mcm5s2U), is a nucleoside rather than an RNA, and the buffer and pH conditions differ, the results are not directly comparable to ours.\n5. Minor Correcons\nSeveral editorial issues and ambiguous phrasing remain in the manuscript:\nLine 96:\n“they proposed”\nThis phrase lacks clarity. Please specify who proposed the stated idea — e.g., provide the author(s) or reference directly in the sentence.\nLine 308:\n“resuluon”\nThis is a spelling error. Please correct to “resoluon.”\nThank you very much. They were revised accordingly."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have adequately addressed my issues and those of the other reviewers, and have improved the manuscript. I do not have additional comments.", "reviewer_2": "Review file is attached", "reviewer_3": "The authors adequately addressed my comments.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Response to Reviewer comments:\nFirst, we thank the reviewers for their thoughtful and helpful review of our manuscript, and for providing valuable and critical comments. Our point-by-point responses to each of the reviewersʼ comments are shown below.\nReviewer #2 (Remarks to the Author):\nI accept the explanations provided for points 1 and 3–5, as well as the corresponding corrections introduced in the manuscript.\nHowever, regarding point 2, I consider that the experiments involving oxidation of tRNA in a cellular system using 2 mM hydrogen peroxide are somewhat debatable. I recommend that the authors indicate in the text that, under their experimental conditions, such a high concentration of H2O2 did not cause cell detachment or apoptosis. Therefore, it would be advisable to include in the manuscript the sentence from the rebuttal: “Although 2 mM H2O2 is far above the IC50 value for HeLa cells (50 μM) and indeed strongly inhibits cell growth, under our experimental conditions, we did not observe any detachment of HeLa cells from the culture dish.”\nUnder this condition, I accept the authorsʼ response and approve the manuscript for publication in Nature Communications.\nWe thank Reviewer #2 for the constructive evaluation and for accepting our explanations for points 1 and 3–5. Regarding point 2, as suggested, we have revised the manuscript to explicitly state that under our experimental conditions, treatment with 2 mM H O did not cause detectable cell detachment or apoptosis.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nReviewer #5 (Remarks to the Author):\nI concur with Reviewer #2ʼs assessment that the manuscript does not convincingly demonstrate the physiological relevance of desulfurated tRNA modifications. The experimental data presented do not adequately support the proposed mechanistic model in which desulfuration of tRNA modifications is posited to modulate protein synthesis under oxidative stress. A significant limitation is that most of the experiments were conducted in vitro, and notably, the authors report that inducing oxidative stress in cells does not lead to increased tRNA desulfuration. The rationale provided by the authors for this observation is insufficient and lacks substantive mechanistic explanation.\nFurthermore, I share Reviewer #2ʼs concern regarding the concentrations of H2O2 used in the experiments; these levels exceed the standard range typically used to model oxidative stress and may instead induce DNA damage, thereby confounding the interpretation of results. If the authors contend that variations in mcm5h2U levels among different cell lines are correlated with intrinsic antioxidant capacity and susceptibility to oxidative stress, this claim must be directly substantiated with experimental evidence. For example, measuring reactive ROS levels in cell lines is a feasible approach and should be incorporated to strengthen their argument.\nAt present, the manuscript does not provide physiologically relevant data to support its conclusions, and further targeted experiments and clearer mechanistic explanations are necessary to address these fundamental concerns. The authorsʼ response and revisions do not adequately address any of these valid concerns. Their replies are mostly argumentative, lacking substantial and credible evidence supported by experimental data. The statement that “the cellular antioxidant system remained functional and effectively minimized tRNA damage” is inaccurate; there is ample evidence in the literature demonstrating that induced oxidative stress can modulate tRNA modification and induce tRNA damage, including the formation of tRNA-derived fragments. Additionally, the manuscript contains discrepancies in the mechanistic explanation of tRNA desulfuration effects on aminoacylation and decoding properties, as uncharged tRNA are largely excluded from the ribosomal A site. Presenting a polysome profile in which only a negligible amount of mcm5h2U is detected in the polysomal fractions does not convincingly demonstrate its involvement in translation. The mere co-sedimentation of mcm5h2U with polysomes does not confirm its specific association with these complexes; rather, it simply indicates that mcm5h2U is present in particles of similar size. Therefore, additional evidence is needed to substantiate a functional role in translation beyond co-sedimentation alone.\nIn my assessment, Reviewer #2ʼs requests for greater physiological relevance and mechanistic clarity are both justified and essential for maintaining the integrity and novelty of the study. This expectation is further reinforced by the precedent established in prior work, particularly by Sierant et al. (2024). The authorsʼ initial omission of this related research and their lack of direct citation points to a concerning gap in scholarly diligence. While the authors have now addressed this oversight by referencing Sierant et al. (2024) in their revised discussion, this addition alone does not resolve the underlying issues. The authors contend that Sierant et al. (2024) did not elucidate a mechanistic role for desulfurated tRNA modifications in translation and argue that their own work does offer new mechanistic insights. However, this manuscript stops short of providing substantive mechanistic or physiological evidence necessary to support meaningful advances in understanding. Without robust experimental substantiation and clear mechanistic explanation, the present manuscript fails to deliver genuine conceptual progress or novelty and does not meet the standards expected for publication in Nature Communications.\nWe thank Reviewer #5 for the careful and critical evaluation of our manuscript. As noted, this study was not designed to comprehensively establish the in vivo physiological relevance of desulfurated tRNA modifications, and we acknowledge the experimental limitations of the current work.\nThe primary aim of this study was to demonstrate that h2U derivatives, previously considered mainly as in vitro oxidation products, are present in cells and tissues, and to define their molecular effects on translation, including impacts on aminoacylation, codon recognition, and tRNA–ribosome interactions.\nAccordingly, the model proposed here addresses potential mechanistic roles of desulfurated tRNA modifications rather than their functions under specific physiological conditions.\nRegarding the use of high H₂O₂ concentrations, we have revised the Discussion to clarify that the apparent protection observed under millimolar H₂O₂ conditions is more plausibly explained by the chemical buffering capacity of the intracellular environment, rather than by biologically specific antioxidant defense mechanisms, and we have revised the text to avoid overinterpretation.\nWe agree that in vivo analyses will be essential to establish physiological significance. In particular, identifying physiological conditions that reproducibly increase tRNA desulfuration will enable future in vivo studies directly addressing the biological relevance of these modifications.\nReviewer #6 (Remarks to the Author):\n*Do you consider the points raised by Reviewer #2 to be technically important and necessary for the integrity of the study?\nI agree with Reviewer #2 that the authors had not adequately provided proper attribution for the prior studies of the desulfuration products of mcm5s2U and cm5s2U. The authors have now responded by noting the key paper by Sierant, M. et al. and discussing the implications of the paper for the present results. The authors have responded adequately to other seemingly less important points raised by Reviewer #2.\n*Do you feel the authorsʼ responses and revisions adequately address these concerns?\nYes, the authors have adequately addressed the concerns of Reviewer #2.\n*Are any of Reviewer #2ʼs requests unreasonable, beyond the scope of the study, or unlikely to add significant value?\nReviewer #2 was aggressively rigorous about important issues, such as proper attribution of prior studies, and about some relatively less important issues, such as quantitative comparisons of modification levels in the same cell line studied in two different laboratories. However, the authorsʼ responses to all of Reviewer #2ʼs comments have strengthened the manuscript by improving scholarship and improving accessibility to the field for the general reader.\n*Does the revised manuscript maintain a clear conceptual advance and novelty?\nOverall, the authors have provided both novelty and conceptual advance in the studies of desulfuration of tRNA modifications. While the observation of oxidative desulfuration of xs2U modifications is not novel, the authorsʼ quantitative and mechanistic analysis of the translational consequences of the desulfurated ribonucleotides is new and important. Using the Suzuki Labʼs unique reciprocal circulating chromatography method for purifying individual tRNAs, the rigorous site-specific quantification of the xs2U modifications and their desulfuration products in individual tRNAs is an important step forward. The most novel features of the work are the effects of loss of xs2U modifications on aminoacylation and codon recognition by specific tRNAs and the structural evidence of the effects of the loss of xs2U modifications on tRNA binding in the context of a ribosome. These are very important results.\nThe exchange between reviewer and authors about the high levels of hydrogen peroxide used in the studies reveals a lack of understanding of the chemical biology of reactive oxygen species and a poorly worded explanation for the observed lack of effect of H2O2 on the xs2U levels. The authors argue that the lack of change in xs2U levels even at H2O2 concentrations three order-of-magnitude higher than the LD50 is due to “antioxidant defense mechanisms”. This is not accurate. The cells died at micromolar concentrations of H2O2 (LD50) when the antioxidant defense mechanisms (e.g., catalase, SOD, etc.) were exceeded. The protective effects of the cellular environment against millimolar H2O2 concentrations is nothing more than the buffering effect of high concentrations of carbohydrates, proteins, lipids, and metabolites, not the result of biologically-relevant specific antioxidant defense mechanisms.\nWe thank Reviewer #6 for the careful and thoughtful assessment of our revised manuscript and for the positive evaluation of its conceptual advance and novelty. We also appreciate the reviewerʼs comments on the interpretation of experiments using high concentrations of H O .\nIn response, we have revised the Discussion to clarify that the lack of detectable changes in h2U levels under high H O conditions should not be attributed to biologically specific antioxidant defense mechanisms. Instead, we now emphasize that, at millimolar H₂O₂ concentrations, enzymatic antioxidant systems are rapidly overwhelmed and that the observed effects are more plausibly explained by the chemical buffering capacity of the intracellular environment, arising from abundant proteins, metabolites, lipids, and other redox-active components. These revisions align the Discussion with the established chemical biology of ROS.\nWe thank Reviewer #6 for highlighting this point, which has helped improve the accuracy and clarity of the manuscript."}, {"round": 3, "reviewer_comments": {"reviewer_2": "I accept the explanations provided for points 1 and 3–5, as well as the corresponding corrections introduced in the manuscript.\nHowever, regarding point 2, I consider that the experiments involving oxidation of tRNA in a cellular system using 2 mM hydrogen peroxide are somewhat debatable. I recommend that the authors indicate in the text that, under their experimental conditions, such a high concentration of H2O2 did not cause cell detachment or apoptosis. Therefore, it would be advisable to include in the manuscript the sentence from the rebuttal: “Although 2 mM H2O2 is far above the IC50 value for HeLa cells (50 μM) and indeed strongly inhibits cell growth, under our experimental conditions, we did not observe any detachment of HeLa cells from the culture dish.”\nUnder this condition, I accept the authorsʼ response and approve the manuscript for publication in Nature Communications.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_5": "I concur with Reviewer #2ʼs assessment that the manuscript does not convincingly demonstrate the physiological relevance of desulfurated tRNA modifications. The experimental data presented do not adequately support the proposed mechanistic model in which desulfuration of tRNA modifications is posited to modulate protein synthesis under oxidative stress. A significant limitation is that most of the experiments were conducted in vitro, and notably, the authors report that inducing oxidative stress in cells does not lead to increased tRNA desulfuration. The rationale provided by the authors for this observation is insufficient and lacks substantive mechanistic explanation.\nFurthermore, I share Reviewer #2ʼs concern regarding the concentrations of H2O2 used in the experiments; these levels exceed the standard range typically used to model oxidative stress and may instead induce DNA damage, thereby confounding the interpretation of results. If the authors contend that variations in mcm5h2U levels among different cell lines are correlated with intrinsic antioxidant capacity and susceptibility to oxidative stress, this claim must be directly substantiated with experimental evidence. For example, measuring reactive ROS levels in cell lines is a feasible approach and should be incorporated to strengthen their argument.\nAt present, the manuscript does not provide physiologically relevant data to support its conclusions, and further targeted experiments and clearer mechanistic explanations are necessary to address these fundamental concerns. The authorsʼ response and revisions do not adequately address any of these valid concerns. Their replies are mostly argumentative, lacking substantial and credible evidence supported by experimental data. The statement that “the cellular antioxidant system remained functional and effectively minimized tRNA damage” is inaccurate; there is ample evidence in the literature demonstrating that induced oxidative stress can modulate tRNA modification and induce tRNA damage, including the formation of tRNA-derived fragments. Additionally, the manuscript contains discrepancies in the mechanistic explanation of tRNA desulfuration effects on aminoacylation and decoding properties, as uncharged tRNA are largely excluded from the ribosomal A site. Presenting a polysome profile in which only a negligible amount of mcm5h2U is detected in the polysomal fractions does not convincingly demonstrate its involvement in translation. The mere co-sedimentation of mcm5h2U with polysomes does not confirm its specific association with these complexes; rather, it simply indicates that mcm5h2U is present in particles of similar size. Therefore, additional evidence is needed to substantiate a functional role in translation beyond co-sedimentation alone.\nIn my assessment, Reviewer #2ʼs requests for greater physiological relevance and mechanistic clarity are both justified and essential for maintaining the integrity and novelty of the study. This expectation is further reinforced by the precedent established in prior work, particularly by Sierant et al. (2024). The authorsʼ initial omission of this related research and their lack of direct citation points to a concerning gap in scholarly diligence. While the authors have now addressed this oversight by referencing Sierant et al. (2024) in their revised discussion, this addition alone does not resolve the underlying issues. The authors contend that Sierant et al. (2024) did not elucidate a mechanistic role for desulfurated tRNA modifications in translation and argue that their own work does offer new mechanistic insights. However, this manuscript stops short of providing substantive mechanistic or physiological evidence necessary to support meaningful advances in understanding. Without robust experimental substantiation and clear mechanistic explanation, the present manuscript fails to deliver genuine conceptual progress or novelty and does not meet the standards expected for publication in Nature Communications.", "reviewer_6": "*Do you consider the points raised by Reviewer #2 to be technically important and necessary for the integrity of the study?\nI agree with Reviewer #2 that the authors had not adequately provided proper attribution for the prior studies of the desulfuration products of mcm5s2U and cm5s2U. The authors have now responded by noting the key paper by Sierant, M. et al. and discussing the implications of the paper for the present results. The authors have responded adequately to other seemingly less important points raised by Reviewer #2.\n*Do you feel the authorsʼ responses and revisions adequately address these concerns?\nYes, the authors have adequately addressed the concerns of Reviewer #2.\n*Are any of Reviewer #2ʼs requests unreasonable, beyond the scope of the study, or unlikely to add significant value?\nReviewer #2 was aggressively rigorous about important issues, such as proper attribution of prior studies, and about some relatively less important issues, such as quantitative comparisons of modification levels in the same cell line studied in two different laboratories. However, the authorsʼ responses to all of Reviewer #2ʼs comments have strengthened the manuscript by improving scholarship and improving accessibility to the field for the general reader.\n*Does the revised manuscript maintain a clear conceptual advance and novelty?\nOverall, the authors have provided both novelty and conceptual advance in the studies of desulfuration of tRNA modifications. While the observation of oxidative desulfuration of xs2U modifications is not novel, the authorsʼ quantitative and mechanistic analysis of the translational consequences of the desulfurated ribonucleotides is new and important. Using the Suzuki Labʼs unique reciprocal circulating chromatography method for purifying individual tRNAs, the rigorous site-specific quantification of the xs2U modifications and their desulfuration products in individual tRNAs is an important step forward. The most novel features of the work are the effects of loss of xs2U modifications on aminoacylation and codon recognition by specific tRNAs and the structural evidence of the effects of the loss of xs2U modifications on tRNA binding in the context of a ribosome. These are very important results.\nThe exchange between reviewer and authors about the high levels of hydrogen peroxide used in the studies reveals a lack of understanding of the chemical biology of reactive oxygen species and a poorly worded explanation for the observed lack of effect of H2O2 on the xs2U levels. The authors argue that the lack of change in xs2U levels even at H2O2 concentrations three order-of-magnitude higher than the LD50 is due to “antioxidant defense mechanisms”. This is not accurate. The cells died at micromolar concentrations of H2O2 (LD50) when the antioxidant defense mechanisms (e.g., catalase, SOD, etc.) were exceeded. The protective effects of the cellular environment against millimolar H2O2 concentrations is nothing more than the buffering effect of high concentrations of carbohydrates, proteins, lipids, and metabolites, not the result of biologically-relevant specific antioxidant defense mechanisms."}, "author_response": "Review of the Revised Manuscript\nThe authors have addressed some of the concerns raised in the previous round of review. They clarified that xm5h2U is not a novel modification but rather a cellular response to oxidative stress derived from xm5s2U. They also expanded their investigation to include the presence of xm5h2U in the polysomal fraction and attempted to interpret structures using Cryo-EM. Despite these improvements, the manuscript still falls short of the standards required for publication and needs substantial revision in four critical areas.\n\n1. Incomplete acknowledgment of prior work on h2U detection in cells\nIn response to the suggestion that the authors should cite the first report of desulfurated 5-substituted 2-thiouridine forms observed in cellular systems, they did include a reference to the relevant study. However, they failed to emphasize a crucial finding: h2U had already been identified in cells - both under oxidative stress and in conditions without stress, including in cells lacking antioxidant systems.\nSpecifically, the earlier work demonstrated for the first time that mcm5h2U, a desulfurated derivative of mcm5s2U, as well as desulfurated immature forms (e.g., cm5s2U) were present in both yeast and human cells not submitted to oxidative stress. The study included a range of human cell lines, including seven cancer types (HeLa, A375, MCF7, U87, MOLT-4, K562, A431) and the non-cancerous HEK293 line. Notably, the levels of h2U varied between cell types, depending on the efficiency of their antioxidant defense, reaching up to 11%.\nThis essential context (even in short) is missing from the current manuscript.\nFor instance:\nLine 128: “Although desulfurated derivatives such as mcm5h2U have been detected in yeast and human cells under oxidative conditions (Ref 56), their mechanistic roles in translational regulation have not been fully elucidated.”\nIn the Discussion, the authors briefly state that the cited publication used a different detection method but failed to elaborate on what that method entailed. It should be clearly stated that the referenced study estimated modification levels using calibration curves derived from synthetic standards, rather than desulfurated tRNA prepared in-house. This distinction is important for understanding the accuracy and reproducibility of the measurements.\nMoreover, the authors use the phrase ‘clear evidence’ multiple times:\nLine 131: “In this study, we present ‘clear evidence’ of the presence h2U derivatives in human cells and mouse tissues.”\nLine 215: “These results provide ‘clear evidence’ that oxidative desulfuration of 2-thiouridine derivatives naturally occurs in human cultured cells.”\nThis phrasing implicitly undermines the clarity of evidence presented in prior studies (including Ref 56), which had already provided compelling data on the presence of mcm5h2U and related compounds. Such wording could be perceived as dismissive of foundational work in the field. It is essential that the authors discuss their findings in the context of existing literature, presenting facts objectively and respectfully acknowledging prior contributions.\n\n2. Concerns Regarding Oxidative Stress Conditions and Data Consistency\nA significant issue in the current manuscript is the choice of hydrogen peroxide (H₂O₂) concentration used to induce oxidative stress in HeLa cells. The authors applied a concentration of 2 mM H₂O₂, which appears to be inappropriately high given that the reported IC₅₀ for H₂O₂ in HeLa cells is 50 µM (as stated in Sierant et al., Ref. 56). This discrepancy raises concerns about the physiological relevance of the experiment and the integrity of the results.\nThe use of 2 mM H₂O₂ likely exceeds the oxidative stress threshold compatible with cell viability. Such a high concentration may induce oxidation in already dead or dying cells, which then become an in vitro system, compromising the biological interpretation of the results. Importantly, this could explain the inconsistencies observed in the data. For example, the increase in h2U levels was minimal—only a 2% rise—which contradicts the expectation of significant desulfuration under oxidative stress. This low increase raises the possibility of an improperly conducted experiment or issues with the H₂O₂ reagent itself. If the latter is the case, it would cast doubt on the reliability of other experiments using the same oxidative treatment.\nFurthermore, the authors should consider the specific sensitivity of HeLa and other tested cell lines to H₂O₂. It is well-established that the cellular response to oxidative agents varies depending on the antioxidant system of the cell, including glutathione levels and catalase activity. The authors should reference studies such as PMCID: PMC6527626 to support a more nuanced discussion of cellular susceptibility and antioxidant defense variability.\nIn the Discussion section, the authors state: “Approximately 15% of tRNALys in HeLa cells contained mcm5h2U, whereas little was detected in A549 and T24 cells (Figure S3).” However, this observation is not consistent with the findings of Sierant et al. (2024), who demonstrated that A549 cells exhibit a sensitivity to H₂O₂ similar to that of HeLa cells. Based on this, one would expect a comparable degree of mcm5s2U damage and corresponding levels of mcm5h2U in both cell types. The divergence in outcomes suggests either methodological differences that were not disclosed or inconsistencies in experimental execution. This contradiction must be addressed directly by the authors.\nA further point of concern is the apparently redundant experimental design shown in Supplementary Figures S4A and S4B. The data appear nearly identical despite being described as the result of separate oxidations (\"double\" in vitro oxidation of tRNALys). It is unclear why a second oxidation step was necessary if the outcome did not differ from the first one.\n\n3. Structural interpretation of the mcm5s2U-G base pair\nThe structural analysis and discussion related to the mcm⁵s²U-G base pair presented in Figure 7c and its corresponding Supplementary Discussion are both conceptually confusing and technically flawed. Instead of clarifying the structural basis for wobble pairing involving modified uridines, the authors provide a misleading and overly speculative interpretation, lacking consistency with established structural and mechanistic knowledge in the field.\nThe authors attempt to address the reviewer’s earlier comments by providing a detailed discussion of the base pairing behavior of mcm⁵s²U-G within the tRNA–mRNA–ribosome complex.\nUnfortunately, the current explanation reflects a misunderstanding of the role of the wobble position and the behavior of modified uridines during decoding. Hence, the authors' estimation of the “ionized” state of mcm⁵s²U is both unnecessary and misleading. Unlike mnm⁵s²U, which can exist in a zwitterionic form (U), mcm⁵s²U does not ionize under physiological conditions in the same way. Therefore, the concept of an “anionic base pair” is incorrect and should be avoided. As noted in Rozov et al. (Ref. 34), there is no mention of “anionic U-G wobble pairing”; this is a misrepresentation of prior work. If needed, the more accurate terminology is “zwitter ionic U” pairing with G (see Ref. 37), though even this does not apply in the case of mcm⁵s²U.\nIt is actually not clear why the discussion is so extensive, since the structure of this base pairing has already been proposed and a careful comparison shows that this new cryo-TEM structure obtained by the authors is very similar, if not identical, to the structure obtained by X-ray crystallography.\nA reliable mechanistic explanation was first provided by Vendeix et al. (2012), who described a tautomeric enol form (E4) of mcm⁵s²U that forms a C–G-like base pair via three hydrogen bonds, which is very similar to the Watson-Crick geometry. Despite possible discrepancies between their and your experimental conditions, this geometry was consistently observed in both structural studies. Therefore, it is reasonable to consider this structure as the most likely conformation under the current conditions and to recognize it as such in this context.\nA few words of discussion: it must be emphasized that at the ribosome, all codon units are rigidly fixed in the A-site. Two first base pairs, that is N1-N36 and N2-N35 form the codon - anticodon minihelix. For the formation of the third base pair binding, the first anticodon nucleoside (N34) exhibits a degree of spatial flexibility to allow forming the base pair with N3. This flexibility is a defining feature of the nucleosides in wobble position. Modified uridines such as mcm⁵s²U at position 34 are specifically tailored to accommodate this flexibility, enabling them to pair either with A3 (via standard Watson–Crick geometry) or G3 (via alternative, non-canonical arrangements).\nClassical Crick wobble pairs, such as U34-G, in which U binds to G in major groove, are not spatially compatible with the ribosomal decoding center (as demonstrated in the works of Westhof team), prompting the evolution of modified uridines capable of forming base pairs of minor-groove shifted U with G. For G3 decoding, this involves U* adopting an altered structure/tautomer to interact with G - not a shift of the entire base pair, as is mistakenly stated multiple times in the manuscript.\nThus, several misleading or technically incorrect statements should be revised:\nAbstract: “The mcm5h2U • G pair shows a minor-groove shifted U • G wobble geometry”\nIncorrect: it is not the pair that shifts, but rather U that adopts a minor-groove shifted tautomeric structure.\nSuggested revision: “The mcm5h2U • G interaction involves “a minor-groove-shifted U” base pairing with G in a non-standard geometry.” (here it is analogy to a new wobble base pair).\nLine 319: “mcm5s2U34 pairs with G3 of the AAG codon via a minor-groove shifted G-U wobble pairing”\nSuggested revision: “mcm5s2U34 pairs with G3 of the AAG codon via a non-canonical base pairing involving a minor-groove-shifted U.”\nLine 328: “The mcm5h2U-G pair adopts a similar geometry to the minor-groove-shifted U-G wobble geometry.”\nSuggested revision: “The mcm5h2U-G pair adopts a similar geometry to the new wobble base pair mnm5s2U-G, with U⁎ shifted into the minor groove.”\nLine 330: “The 2-thiocarbonyl group of mcm5s2U promotes the acidity of the N3 atom (Ref 37), thereby contributing to the minor-groove-shifted mcm5s2U-G pairing.”\nThis is an oversimplified and partially misleading biochemical interpretation.\nSuggested revision: “The presence of the 2-thiocarbonyl group lowers the pKa of N3 by approximately 1 unit, increasing its proton-dissociation potential; however, minor-groove shifted geometry arises from structural rearrangements primarily due to C5 substitutions, not ionization alone.”\nHydrogen bonding mis-assignments and structural clarifications\nThe hydrogen bond pattern proposed by the authors is not chemically plausible:\n“U4(O)-G1(NH) and U3(NH)-G2(NH )”\nCorrect interpretation based on structural precedents:\no Classical wobble (Crick):\n U2(O) – G1(NH)\n U3(NH) – G6(O)\no New wobble (Rozov & Westhof):\n U4(O) – G1(NH)\n U3(N) – G2(NH₂)\no Alternative non-canonical geometry (suggested to be used here):\n U4(OH) – G6(O)\n U3(N) – G1(NH)\n U2(O) – G2(NH₂)\nThe authors should re-consider the hydrogen bonding network shown in Fig. 7c, ideally providing distance measurements between donors and acceptors to validate the geometry. This would significantly improve the credibility and clarity of the structural data.\n\n4. Implausible Selectivity of Oxidation Products\nA particularly questionable result reported in the manuscript is the observation that under cellular conditions, s²U undergoes only desulfuration to yield h²U, without any formation of the oxidized product U. This finding contradicts all earlier literature concerning the chemical behavior of 2-thiouridine under oxidative conditions (e.g. Rao et al., 1974; Watanabe et al., 1980; Sochacka et al., 2011), which consistently demonstrated that U is a prominent oxidation product of s²U, both in chemical models and in biological systems.\nThe absence of U in the authors' data is not explained or discussed, and given the well-documented oxidative pathways of s²U, it is difficult to accept this result without an in-depth mechanistic rationale or additional experimental validation. As it stands, this contradicts known biochemistry and casts doubt on the specificity or sensitivity of the detection methods used.\n\n5. Minor Corrections\nSeveral editorial issues and ambiguous phrasing remain in the manuscript:\nLine 96:\n“they proposed”\nThis phrase lacks clarity. Please specify who proposed the stated idea — e.g., provide the author(s) or reference directly in the sentence.\nLine 308:\n“resulution”\nThis is a spelling error. Please correct to “resolution.”"}]

Decision letter