Nucleolar stress generates ribosomal DNA–derived RNA–DNA hybrids that prime innate immunity

Nucleolar stress generates ribosomal DNA–derived RNA–DNA hybrids that prime innate immunity | 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 Article Nucleolar stress generates ribosomal DNA–derived RNA–DNA hybrids that prime innate immunity Ken Takashima, Yohana Mtali, Akter Suraiya, Doreen Emily, Kenichi Miharada, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7795572/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Self- and non-self-nucleic acids are recognized by the innate immune system. Although RNA-DNA hybrids generated through R-loop formation during transcriptional stress have been implicated in innate immune activation, their physiological significance remains incompletely understood. Here, we demonstrate that nucleolar stress caused by dysfunction of nucleolar proteins RRN3 and ribosomal proteins, trigger the cytosolic accumulation of RNA-DNA hybrids originating from ribosomal DNA loci (ribo-RNA-DNA-hybrids). The formation of ribo-RNA-DNA-hybrids depend on XP proteins and partially activates cGAS- and TLR3-pathways, resulting in the activation of NF-κB without triggering IRF3 phosphorylation, leading to hyperinflammatory responses upon secondary stimulation. Importantly, cytosolic ribo-RNA-DNA hybrids increase in cells of Diamond-Blackfan anemia patients, certain cancer cells, myeloid cells stimulated with mRNA-LNP vaccines, correlating with heightened inflammatory responses. These findings reveal a crucial role for nucleolar stress-induced accumulation of cytosolic ribo-RNA-DNA hybrids under pathological conditions, including genetic disorders and tumorigenesis, as well as in the adjuvanticity of mRNA-LNP vaccines. Biological sciences/Immunology/Inflammation Biological sciences/Molecular biology/DNA damage and repair Biological sciences/Immunology/Vaccines/RNA vaccines Biological sciences/Immunology/Innate immunity Biological sciences/Cell biology/Organelles/Nucleus Innate immunity inflammation nucleolus nucleotide excision repair RNA-DNA hybrids cGAS TICAM-1 Diamond Blackfan anemia cancer vaccine Full Text Additional Declarations There is NO Competing Interest. Supplementary Files 251007TakashimaKetal.ExtendedDataforsubmittion.pdf Extended Data Cite Share Download PDF Status: Under Review 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-7795572","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":529623035,"identity":"705d3721-1b7f-4258-8c0c-bf2e77d73dc6","order_by":0,"name":"Ken Takashima","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABFklEQVRIiWNgGAWjYBACCQYGZiB1gAdIH0aXPIBdCxtcC1syRIiNSC1AzGOMrgU7kJzf/Njgx587MrrtZz4b8+bYRfPLNx9g5mGwk2dgPIvVGmk2NuPE3rZnPGZncjcn825Lzp3ZxpYA1JJs2MBwLgGbFjk2BuMDvA2HecwO5G4+zLuNOXfDMR7z3zwMzEDlZwywa2H/fPDPH6CW828eA7XUg7QYAG2px6lFmo3HOJmHDajlRg4z0GGHYVoO49Qi2ZZTbCzbBtLyzNhw7rbjQL+kJTDOMThu2IbDLxKHj2+WfPPnsL3Z+eTHEm+3Vef2Mx8+wPCmolqeXwJ7iOECQCexSZwhRQcY8PeQrGUUjIJRMAqGJQAADcVdWhX+AIoAAAAASUVORK5CYII=","orcid":"https://orcid.org/0009-0006-2194-1426","institution":"Graduate School of Medical Sciences, Faculty of Life Sciences, Kumamoto University","correspondingAuthor":true,"prefix":"","firstName":"Ken","middleName":"","lastName":"Takashima","suffix":""},{"id":529623036,"identity":"968bf31c-22da-4aab-b37d-5153b098b9d5","order_by":1,"name":"Yohana Mtali","email":"","orcid":"","institution":"Graduate School of Medical Sciences, Kumamoto University","correspondingAuthor":false,"prefix":"","firstName":"Yohana","middleName":"","lastName":"Mtali","suffix":""},{"id":529623037,"identity":"9ebc7934-30c4-4934-bc74-c364ab508067","order_by":2,"name":"Akter Suraiya","email":"","orcid":"","institution":"Kumamoto University","correspondingAuthor":false,"prefix":"","firstName":"Akter","middleName":"","lastName":"Suraiya","suffix":""},{"id":529623038,"identity":"aec55401-5b71-4548-8175-1c08bcb52725","order_by":3,"name":"Doreen Emily","email":"","orcid":"","institution":"Kumamoto University","correspondingAuthor":false,"prefix":"","firstName":"Doreen","middleName":"","lastName":"Emily","suffix":""},{"id":529623039,"identity":"de14c6d1-0fa0-4313-894f-ef5ec08f9a2c","order_by":4,"name":"Kenichi Miharada","email":"","orcid":"https://orcid.org/0000-0001-8073-4001","institution":"Kumamoto University","correspondingAuthor":false,"prefix":"","firstName":"Kenichi","middleName":"","lastName":"Miharada","suffix":""},{"id":529623040,"identity":"b958300e-94d2-488b-89b9-b15adce4b4aa","order_by":5,"name":"Hiroyuki Oshiumi","email":"","orcid":"https://orcid.org/0000-0003-1567-8722","institution":"Graduate School of Medical Sciences, Faculty of Life Sciences, Kumamoto University","correspondingAuthor":false,"prefix":"","firstName":"Hiroyuki","middleName":"","lastName":"Oshiumi","suffix":""}],"badges":[],"createdAt":"2025-10-07 04:10:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7795572/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7795572/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":93687792,"identity":"6dc17ef4-5364-41fa-b2c2-63f13fba505a","added_by":"auto","created_at":"2025-10-16 13:34:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":12684606,"visible":true,"origin":"","legend":"","description":"","filename":"251007TakashimaKetal.NucleolusdysfunctionandinnateimmuneprimingVer.R9integratedfinal.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7795572/v1/15abbd2d38ebe52f7c17a38d.pdf"},{"id":93687790,"identity":"d2028cd1-6275-495d-9162-f731bc5c8227","added_by":"auto","created_at":"2025-10-16 13:34:14","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":7639,"visible":true,"origin":"","legend":"","description":"","filename":"NCOMMS2580003.json","url":"https://assets-eu.researchsquare.com/files/rs-7795572/v1/374e112d61a675038e544cee.json"},{"id":93687793,"identity":"9d0c1583-b49f-455b-b053-dc844bfd75ca","added_by":"auto","created_at":"2025-10-16 13:34:15","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2458373,"visible":true,"origin":"","legend":"","description":"","filename":"251007TakashimaKetal.ExtendedDataforsubmittion.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7795572/v1/76e8ebb86b318467ea593a16.pdf"},{"id":93689081,"identity":"af98e579-fb23-468d-839f-7948d78d6760","added_by":"auto","created_at":"2025-10-16 13:50:30","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11299312,"visible":true,"origin":"","legend":"Article File","description":"","filename":"251007TakashimaKetal.NucleolusdysfunctionandinnateimmuneprimingVer.R9integratedfinal.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7795572/v1_covered_f64a97cf-d407-4007-b34d-c8ae0e3f3f50.pdf"},{"id":93687791,"identity":"75e85434-7083-4cb7-b88d-13585e41f612","added_by":"auto","created_at":"2025-10-16 13:34:15","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2458373,"visible":true,"origin":"","legend":"Extended Data","description":"","filename":"251007TakashimaKetal.ExtendedDataforsubmittion.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7795572/v1/692bb105bfc2c6a2fef9dc2c.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Nucleolar stress generates ribosomal DNA–derived RNA–DNA hybrids that prime innate immunity","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Innate immunity, inflammation, nucleolus, nucleotide excision repair, RNA-DNA hybrids, cGAS, TICAM-1, Diamond Blackfan anemia, cancer, vaccine","lastPublishedDoi":"10.21203/rs.3.rs-7795572/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7795572/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Self- and non-self-nucleic acids are recognized by the innate immune system. Although RNA-DNA hybrids generated through R-loop formation during transcriptional stress have been implicated in innate immune activation, their physiological significance remains incompletely understood. Here, we demonstrate that nucleolar stress caused by dysfunction of nucleolar proteins RRN3 and ribosomal proteins, trigger the cytosolic accumulation of RNA-DNA hybrids originating from ribosomal DNA loci (ribo-RNA-DNA-hybrids). The formation of ribo-RNA-DNA-hybrids depend on XP proteins and partially activates cGAS- and TLR3-pathways, resulting in the activation of NF-κB without triggering IRF3 phosphorylation, leading to hyperinflammatory responses upon secondary stimulation. Importantly, cytosolic ribo-RNA-DNA hybrids increase in cells of Diamond-Blackfan anemia patients, certain cancer cells, myeloid cells stimulated with mRNA-LNP vaccines, correlating with heightened inflammatory responses. These findings reveal a crucial role for nucleolar stress-induced accumulation of cytosolic ribo-RNA-DNA hybrids under pathological conditions, including genetic disorders and tumorigenesis, as well as in the adjuvanticity of mRNA-LNP vaccines.","manuscriptTitle":"Nucleolar stress generates ribosomal DNA–derived RNA–DNA hybrids that prime innate immunity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-16 13:34:10","doi":"10.21203/rs.3.rs-7795572/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"29e7ad14-b86a-49a4-bf98-eaebb169085c","owner":[],"postedDate":"October 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":56295728,"name":"Biological sciences/Immunology/Inflammation"},{"id":56295729,"name":"Biological sciences/Molecular biology/DNA damage and repair"},{"id":56295730,"name":"Biological sciences/Immunology/Vaccines/RNA vaccines"},{"id":56295731,"name":"Biological sciences/Immunology/Innate immunity"},{"id":56295732,"name":"Biological sciences/Cell biology/Organelles/Nucleus"}],"tags":[],"updatedAt":"2026-04-15T06:26:40+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-16 13:34:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7795572","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7795572","identity":"rs-7795572","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}