N1-Methylpseudouridine Directly Modulates Translation Dynamics

N1-Methylpseudouridine Directly Modulates Translation Dynamics | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Biological Sciences - Article N1-Methylpseudouridine Directly Modulates Translation Dynamics Noam Stern-Ginossar, Batsheva Rozman, Karin Broennimann, K Shanmugha Rajan, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5873147/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 14 Jan, 2026 Read the published version in Nature → Version 1 posted You are reading this latest preprint version Abstract The remarkable effectiveness of mRNA vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted synthetic mRNA as a promising technology. A critical feature of this approach is the incorporation of the modified ribonucleotide N1-methylpseudouridine (m¹Ψ), which enhances antigen expression while reducing immunogenicity. However, a comprehensive understanding of how this modification influences translation remains incomplete. Here, we analyze translation at sub-codon resolution using ribosome profiling and demonstrate that m¹Ψ increases ribosome density on synthetic mRNAs. This elevated ribosome load, together with the correlated increase in protein production, occurs independently of intrinsic cellular immunity or eIF2α phosphorylation. Our data reveal that m¹Ψ directly slows ribosome elongation in specific sequence contexts while concurrently enhancing translation initiation. Cryo-electron microscopy shows that m¹Ψ-modified mRNAs alter interactions within the ribosome decoding center, providing a mechanistic basis for slowed elongation dynamics. Furthermore, by introducing synonymous mutations that disrupt modification-mediated changes in elongation, we show that the m¹Ψ-dependent effect on protein output can be modulated, and that its impact is strongest in mRNAs containing non-optimal codons with uridines at the wobble position. Together, these findings demonstrate that m¹Ψ directly modulates translation elongation and initiation, thereby enhancing translational capacity and increasing protein output from synthetic mRNAs in defined sequence contexts. Biological sciences/Biotechnology/Nucleic-acid therapeutics Biological sciences/Molecular biology/Translation Full Text Additional Declarations There is NO Competing Interest. Table 1 is available in the Supplementary Files section. Supplementary Files TABLE1natureformat.xlsx Table 1 FACSanalysis28925.pdf FACS analysis Cite Share Download PDF Status: Published Journal Publication published 14 Jan, 2026 Read the published version in Nature → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5873147","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Biological Sciences - Article","associatedPublications":[],"authors":[{"id":534016119,"identity":"f6607553-3c6f-4e0a-9d8a-7da1be3dce89","order_by":0,"name":"Noam 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A critical feature of this approach is the incorporation of the modified ribonucleotide N1-methylpseudouridine (m¹Ψ), which enhances antigen expression while reducing immunogenicity. However, a comprehensive understanding of how this modification influences translation remains incomplete. Here, we analyze translation at sub-codon resolution using ribosome profiling and demonstrate that m¹Ψ increases ribosome density on synthetic mRNAs. This elevated ribosome load, together with the correlated increase in protein production, occurs independently of intrinsic cellular immunity or eIF2α phosphorylation. Our data reveal that m¹Ψ directly slows ribosome elongation in specific sequence contexts while concurrently enhancing translation initiation. Cryo-electron microscopy shows that m¹Ψ-modified mRNAs alter interactions within the ribosome decoding center, providing a mechanistic basis for slowed elongation dynamics. Furthermore, by introducing synonymous mutations that disrupt modification-mediated changes in elongation, we show that the m¹Ψ-dependent effect on protein output can be modulated, and that its impact is strongest in mRNAs containing non-optimal codons with uridines at the wobble position. Together, these findings demonstrate that m¹Ψ directly modulates translation elongation and initiation, thereby enhancing translational capacity and increasing protein output from synthetic mRNAs in defined sequence contexts.","manuscriptTitle":"N1-Methylpseudouridine Directly Modulates Translation Dynamics","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-24 03:03:15","doi":"10.21203/rs.3.rs-5873147/v1","editorialEvents":[],"status":"published","journal":{"display":false,"email":"[email protected]","identity":"nature","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"nature","sideBox":"Learn more about [Nature](http://www.nature.com/nature/)","snPcode":"","submissionUrl":"","title":"Nature","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"48b34df4-27a5-4aa0-901f-39fa752cfb02","owner":[],"postedDate":"October 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":56780739,"name":"Biological sciences/Biotechnology/Nucleic-acid therapeutics"},{"id":56780740,"name":"Biological sciences/Molecular biology/Translation"}],"tags":[],"updatedAt":"2026-01-15T08:10:18+00:00","versionOfRecord":{"articleIdentity":"rs-5873147","link":"https://doi.org/10.1038/s41586-025-09945-5","journal":{"identity":"nature","isVorOnly":false,"title":"Nature"},"publishedOn":"2026-01-14 05:00:00","publishedOnDateReadable":"January 14th, 2026"},"versionCreatedAt":"2025-10-24 03:03:15","video":"","vorDoi":"10.1038/s41586-025-09945-5","vorDoiUrl":"https://doi.org/10.1038/s41586-025-09945-5","workflowStages":[]},"version":"v1","identity":"rs-5873147","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5873147","identity":"rs-5873147","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}