N1-Methylpseudouridine directly modulates translation dynamics

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Data availability

All next-generation sequencing data files have been deposited at the Gene Expression Omnibus under accession GSE309271. The cryo-EM density maps of the ribosome complexes have been deposited in the Electron Microscopy Data Bank under accession numbers EMD-55091 and EMD-55083. Atomic coordinates and structure factors have been deposited in the PDB under accession codes 9SPI and 9SPF.

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Acknowledgements

We thank I. Ulitsky, S. Schwartz and the members of the Stern-Ginossar laboratory for reading the manuscript; and Y. Lubelsky for technical assistance. B.R. is supported by the Ariane de Rothschild Women’s Doctoral Program. K.S.R. is supported by the Senior-Postdoctoral Fellowship from the Weizmann Institute of Science. A.Y. holds the Martin S. and Helen Kimmel Professorial Chair at the Weizmann Institute of Science. Work in the Stern-Ginossar group is supported by a European Research Council consolidator grant (CoG-2019-864012). This project was funded by the Abisch-Frenkel RNA Therapeutics Center.

Author information

Author notes

  1. These authors contributed equally: Batsheva Rozman, Karin Broennimann, K. Shanmugha Rajan

Authors and Affiliations

  1. Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel

    Batsheva Rozman, Aharon Nachshon, Tamar Arazi & Noam Stern-Ginossar

  2. Department of Immunology and Regenerative Biology, Weizmann Institute of Science, Rehovot, Israel

    Karin Broennimann

  3. Department of Chemical and Structural Biology, The Weizmann Institute of Science, Rehovot, Israel

    K. Shanmugha Rajan, Chiranjeet Saha, Ada Yonath & Anat Bashan

  4. Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel

    Vishnu Mohan & Tamar Geiger

  5. Department of Microbiology and Immunology, College of Medicine, University of Illinois Chicago, Chicago, IL, USA

    Clayton J. Wollner & Justin M. Richner

  6. Architecture et Réactivité de l’ARN, Université de Strasbourg, Institut de biologie moléculaire et cellulaire du CNRS, Strasbourg, France

    Eric Westhof

  7. Engineering Research Center of Clinical Functional Materials and Diagnosis & Treatment. Devices of Zhejiang Province, Wenzhou Institute, University of Chinese Academy of Sciences, Wenzhou, China

    Eric Westhof

Authors

  1. Batsheva Rozman
  2. Karin Broennimann
  3. K. Shanmugha Rajan
  4. Aharon Nachshon
  5. Chiranjeet Saha
  6. Tamar Arazi
  7. Vishnu Mohan
  8. Tamar Geiger
  9. Clayton J. Wollner
  10. Justin M. Richner
  11. Eric Westhof
  12. Ada Yonath
  13. Anat Bashan
  14. Noam Stern-Ginossar

Contributions

B.R. and N.S.-G. conceived and designed the project. B.R., K.B. and T.A. performed the molecular biology experiments. K.S.R., C.S., E.W., A.Y. and A.B. acquired and interpreted the cryo-EM data. B.R. and A.N. analysed the deep sequencing data. V.M. and T.G. conducted and interpreted the MS measurements. C.J.W. and J.M.R. provided critical reagents and protocols. B.R., K.S.R. and N.S.-G. wrote the manuscript with critical input from all of the authors.

Corresponding authors

Correspondence to Batsheva Rozman or Noam Stern-Ginossar.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature thanks Jeff Coller, Nahum Sonenberg and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Modified mRNA has increased ribosome load.

a, TapeStation analysis of IVT spike mRNAs containing UTP, Ψ, or m1Ψ. b, Dot blot of IVT Spike mRNAs containing UTP, Ψ, or m1Ψ, probed with the J2 antibody to detect dsRNA, shown before and after cellulose cleaning. c, Quantification of Spike (relative to gapdh) and phosphorylated eIF2α (relative to eIF2α levels) measured by western blot (Fig. 1b) error bars show s.d. of two replicates. d, Scatter plots showing transcript expression in biological replicates of RNA-seq (top panels) and ribosome profiling (bottom panels). Spearman correlation is depicted at the top of each graph. e, Density plot showing ribosome footprint read length distributions. f, Meta gene analysis of ribosome profiling libraries around the Start codon and Stop codons. Each frame (−1, 0, + 1) is labelled in red, grey, or black bars correspondingly. g, Cumulative plots depicting read lengths for human or spike footprints. h, Stacked box plots depicting proportion of reads that start (left panels) or end (right panels) with each nucleotide for spike ribosome footprints. i,j, Polysome profiles of 293T cells transfected with UTP or m1Ψ Spike IVT mRNAs (i) or with UTP or m1Ψ luciferase IVT mRNAs (j). Profiles depict averaged absorbance across three biological replicates and normalized to minimum absorbance values. X-axis depicts the 12 fractions that were collected. k,l, The relative distribution of luciferase mRNA (k) and ANXA5 mRNA, endogenous control transcript (l), across polysome fractions. RNA from 12 gradient fractions (presented in j) and quantified by RT-qPCR. Bars represent the mean and s.d. of three biological replicates. m, Volcano plots of differentially expressed genes (mRNA) and differentially translated genes (ribosome footprints) in cells transfected with UTP or modified spike or luciferase mRNAs. eIF2α phosphorylation dependent genes17 (yellow) and ISGs (magenta) are highlighted. Y-axis denotes -log10 p.values and X-axis shows log2 transformed fold change values. n,o, Bar plots showing ribosome footprint reads mapping to the 3′UTR (n) or to Stop codon (o) of spike and luciferase mRNAs, normalized to reads mapping to the respective CDS. Individual data points represent each replicate. P-value calculated using standard two-sided t-test.

Extended Data Fig. 2 Modified nucleotides slow translation elongation in specific sequence context.

a, Ribosome footprint profiles along a segment of the luciferase mRNA from UTP or m1Ψ modified transcripts. Bars depict read coverage at every nucleotide. Grey squares highlight differences in profiles between samples. b,c, PCA analysis of codon level ribosome densities in spike (b) and luciferase (c) IVT mRNA containing either UTP Ψ and m1Ψ (spike) or UTP and m1Ψ (luciferase) at 24 h post transfection into 293T cells. d-j, Boxplot depicting the log2 -transformed fold change of relative ribosome densities of modified mRNA compared to non-modified in codons along luciferase mRNA (d,e,g,i and j) or spike mRNA (f) binned by number of uridines in the A-site (d), by the location of uridine in the A-site (e, f and g), in the endogenous transcript Actin b (h), by the location of uridine in the P-site codon (i) by location of uridine in the A-site codon and P-site codons (j). V represents non-uridine bases. p.values were calculated using two-sided standard t-test. k, Heatmap showing the −log10-transformed P values from nucleotide enrichment tests at each position (E,P,A site) on luciferase mRNA where ribosome densities differ significantly between m1Ψ and UTP. Positive values indicate enrichment whereas negative values indicate depletion compared to UTP. l, Rate of ribosome depletion. The codon position of 50% ribosome depletion is plotted as a function of harringtonine treatment time (seconds) for cellular transcripts in cells transfected with UTP luciferase mRNA (red) or m1Ψ modified luciferase mRNA (blue). m, Metagene analysis of run-off elongation at distinct time points (treatment time is denoted in Fig legend : 0, 60, 90, 120, 150 s) for cellular genes (solid line) and luciferase gene (dashed line) from UTP luciferase (red, right panel) or m1Ψ luciferase (blue, left panel) mRNAs transfected cells. Ribosome read densities are shown as ratios relative to the untreated 0-timepoint and curves were smoothed using a locally weighted regression with a smoothing parameter (span) of 0.5.

Extended Data Fig. 3 Cryo-EM data processing pipeline used to reconstruct EM maps.

Flow chart presenting the outline of the cryo-EM data processing pipeline used to reconstruct the EM map. The number of micrographs, particles, 3D classes obtained, and the resolution of corresponding maps are indicated. “Gold standard” FSC curves for the consensus EM map (black), unmasked map (green), masked map (blue), and phase randomized masked map (red) are presented. Surface rendering and cross-section of the cryo-EM density maps are coloured according to local resolution distribution. a, Uridine-PRE ribosomes. b, m1Ψ-PRE ribosomes.

Extended Data Fig. 4 Cryo-EM analysis of Uridine-PRE and m1Ψ-PRE ribosome structures.

a, Examples of RNA modification visualized in the P-site tRNAArg. The EM map and the corresponding model are superimposed. The identity of the RNA modification and its PDB ligand ID is indicated. b,c, Superimposition of the mRNA nucleotides in Human Uridine-PRE, m1Ψ-PRE, and Yeast PRE complexes. The distances between the phosphate group near the A/P kink are indicated. b, Comparison between m1Ψ-PRE and Uridine-PRE complexes. c,  Comparison between m1Ψ-PRE with hygromycin and yeast-PRE (PDB: 8CDL) without hygromycin complexes. d–f, Comparison of the distances between SSU rRNA nucleotides and the A-site mRNA nucleotides in Human Uridine-PRE, m1Ψ-PRE, and Yeast-PRE (PDB: 8CDL) complexes. The distances between the A-site nucleotide and SSU rRNA nucleotides are indicated. (d) m1Ψ-PRE complex, (e) Uridine-PRE complex, and (f) Yeast-PRE complex. g, Sugar puckers of the two Uridine nucleotides in Uridine-PRE complex. The cryo-EM density of the Uridine in +3 and +5 positions is shown. h, i, Codon-anticodon base-pairing in the Uridine-PRE complex. (h) Watson Crick (WC) base-pair interactions in the P-site and A-site nucleotides of the Uridine-PRE complex. The EM map of mRNA and tRNA nucleotides in the P-site and A-site is shown to indicate the nucleobase anti-conformation of G(+ 2) and anti-conformation of A35, respectively. The distances of the observed hydrogen bonds between the codon-anticodon are indicated. (i) Same as in (h) for m1Ψ -PRE complex. The EM map of mRNA and tRNA nucleotides in the P-site and A-site is shown to indicate the nucleobase syn-conformation of G(+ 2) and syn-conformation of A35, respectively.

Extended Data Fig. 5 Enhanced translation initiation underlies increased expression of m1Ψ-modified mRNAs.

a, Western blot showing PKR expression levels in WT 293T compared to EIF2AK2-KO 293T cells. β-actin is shown for loading control. b, Western blot analysis for eIF2α phosphorylation levels in WT 293T (left panel) or EIF2AK2-KO 293T (right panel) cells transfected with UTP or m1Ψ modified mRNA. dsRNA was used as a positive control for PKR induction. Vinculin is shown. c, Western blot analysis for ATF4 levels in cells transfected with UTP or m1Ψ modified mRNA and treated with ISRIB. Tunicamycin was used as a control for ATF4 induction and ISRIB activity, vinculin loading control is shown. d, Relative spike mRNA levels at various time points following transfection with mRNAs containing UTP, Ψ, or m1Ψ. Levels were measured by RT-qPCR and normalized to housekeeping gene (ANXA5). Data represent mean and s.d. of three biological replicates. e, Bar plots depicting firefly luciferase luminescence normalized to Renilla control of UTP (red) or m1Ψ modified (blue) mRNA prepared with two different capping mechanisms – vaccinia virus capping enzyme (VCE, left) or co-transcriptional cap1 analog (AG, right). Error bars depict s.d. of three biological replicates. f, Bar plot depicting luciferase mRNA levels in cytosol fraction compared to total cell lysate as measured by RT-qPCR and normalized to ANXA5 gene. Data represent average and s.d. of 3 biological replicates. Statistical significance was calculated by two-sided standard t. test. g, Relative U99 RNA expression normalized to ANXA5 gene as measured by RT-qPCR from each fraction (total, cytosol, membrane-associated) in cells transfected with UTP or m1Ψ luciferase IVT mRNAs after sub cellular cell fractionation. Error bars depict s.d. of three biological replicates. High levels of the nuclear U99 in the membrane-associated compartment illustrate successful fractionation. h, Western blot analysis of luciferase expression at indicated time points following cycloheximide (CHX) treatment in cells transfected with either UTP or m1Ψ-modified luciferase mRNA. i, Quantification of luciferase protein levels normalized to vinculin loading control from panel h. Values are plotted as a function of time post CHX treatment (hours) and fitted to an exponential decay model. Protein half-lives derived from the fitted curves are shown below the graph. j, Scatter plots depicting ribosome densities, calculated as the ratio of ribosome footprints to mRNA levels, for human genes (grey) and no uridine in both A and P sites along the spike mRNA (red). The left panel compares cells transfected with UTP or Ψ-modified spike mRNA, and the right panel compares cells transfected with UTP and m1Ψ-modified spike mRNA. k, Bar plot showing CCND1 endogenous mRNA levels (control) in input and eIF4E-IP samples from cells transfected with UTP- or m1Ψ-modified luciferase mRNAs, measured by RT-qPCR. Error bars show s.d. of 3 biological replicates. Statistical significance was calculated using linear regression. Fold change between input and IP for each sample is indicated on the graph.

Extended Data Fig. 6 Slowed elongation on m1Ψ-modified mRNA does not substantially alter translation fidelity or frameshifting.

a, Western blot showing immunoprecipitation of Spike protein using anti-Sars-Cov2 spike glycoprotein antibody. Lanes 1,3,5 correspond to input samples and 2,4,6 correspond to immunoprecipitated samples. anti-Gapdh is presented as loading control. b, Bar plot depicting mean and s.d. of four biological replicates of peptide abundance measured by mass spectrometry showing the ratio of the intensities of all miscoded spike peptides. P-values were calculated by two sided standard t-test. c, Proportion of ribosome footprint reads in each of the 3 reading frames on spike and human mRNAs, averaged for each of the two biological replicates. Error bars show s.d. d, The distribution of Z-scores in simulated data enforcing +1 frameshifting, 1% (solid line), 5% (dashed line). The dots depict the computed Z-scores of frame shifting signals from uridine-containing (yellow) or non-uridine-containing (grey) slippery sites. The lines represent the median Z-score of slippery sites in each group. e, Density plots of the calculated distributions of median Z. scores for reads mapped to +1 reading frame of simulated data enforcing either 1% (solid line) or 5% (dashed lines) frameshifting. The red dots depicts the observed Z-score of windows downstream to m1Ψ-slowed elongation sites and the red line shows the median value of these scores.

Extended Data Fig. 7 Synonymous recoding tunes the extent of the m1Ψ-dependent boost in protein expression.

a, Bar plots showing Spike protein levels quantified from western blot analysis and normalized to gapdh, in biological duplicates. Error bars show s.d. of duplicates (western blot shown in Fig. 5a). b, Average spike mRNA levels for spike and spike-minU UTP or m1Ψ modified mRNA transfected to cells and analysed by RT-qPCR. Gapdh mRNA was used for normalization. Eror bars depict s.d. of two biological duplicates. c, Bar plots of average luciferase mRNA levels for UTP or m1Ψ modified luciferase mRNAs; MinU, WT and MaxU analysed by RT-qPCR and normalized to gapdh. Error bars depict s.d. of three biological replicates. d, Gating strategy used to determine the live, single-cell, GFP positive population for all flow cytometry analyses of GFP and recoded-GFP. e, Microscopy images showing GFP fluorescence in cells transfected with IVT GFP or recoded GFP mRNAs containing either UTP or m1Ψ at 24 h post-transfection (n = 3 biologically independent experiments). Representative fields are shown. Scale bar: 100 μm. f, Bar plots of relative GFP mRNA levels measured by RT-qPCR and normalized to gapdh for cells transfected with UTP and m1Ψ modified GFP or recoded-GFP mRNAs. Error bars depict s.d. of three biological replicates. g, Heatmap depicting -log10 p-values of Fisher exact test of ribosome densities measured for m1Ψ compared to UTP luciferase mRNA for each nucleotide at specific position in the E, A, and P-sites. h, Scatter plots depicting ribosome densities, calculated as the ratio of ribosome footprints to mRNA levels in cells transfected with UTP or m1Ψ-modified WT luciferase (left panel) and MinU luciferase (right panel), for human genes (grey) and luciferase mRNA (pink), excluding uridine containing codons.

Extended Data Fig. 8 Quality control of the synonymous luciferase mRNA library.

a, Polysome profiles of 293T PKR KO cells transfected with the UTP or m1Ψ IVT mRNA luciferase mRNA library in biological triplicates normalized to minimum absorbance values. X-axis and grey lines depict the 5 fractions that were collected. b, Scatter plot comparing read counts for mRNA sequences with duplicate barcodes (BC1 vs. BC2). Each point represents a sequence, showing high correspondence between barcode replicates (R² = 0.88, p < 2.2 × 10–16). The red dashed line indicates the linear regression fit. c, Dendrogram of hierarchical clustering based on library read counts, showing strong grouping of biological replicates. UTP samples are shown in red and m1Ψ samples in blue. d, Principal component analysis (PCA) of read counts across sequences, shown per sample (UTP-red and m1Ψ-blue), replicate and polysome fraction. Fraction identity is indicated by point shading, ranging from total lysate to non-ribosomal, ribosomal subunits (40S, 60S, 80S), light polysomes, and heavy polysomes. e, Histogram showing polysome enrichment score with four quartile cut offs depicted as red dashed vertical lines. f, Scatter plots showing the relationship between individual sequence features and the polysome enrichment score. Each point represents a synonymous sequence variant, and the black line indicates the linear regression fit.

Extended Data Table 1 Cryo-EM data collection, model refinement and validation statistics

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Supplementary information

Supplementary Fig. 1

Western blot source images. The boxes indicate regions shown in the main or extended data figures, as indiciated above the gels.

Reporting Summary

Supplementary Table 1

Sequences: table of all of the sequences used in this study, including sequence features such as CDS length and percentage uridine for each sequence. Fluc_library: table of all synonymous sequences used in the luciferase library polysome profiling experiment with their sequence features and polysome enrichment score.

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Rozman, B., Broennimann, K., Rajan, K.S. et al. N1-Methylpseudouridine directly modulates translation dynamics. Nature (2026). https://doi.org/10.1038/s41586-025-09945-5

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