Coupling translation termination to RNA surveillance: roles of release factors in quality control and RNA degradation pathways in mammalian mitochondria

Coupling translation termination to RNA surveillance: roles of release factors in quality control and RNA degradation pathways in mammalian mitochondria | 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 Coupling translation termination to RNA surveillance: roles of release factors in quality control and RNA degradation pathways in mammalian mitochondria Joanna Rorbach, Annika Krüger, Daria Kovalchuk, Lukasz Borowski, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7036318/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 Accurate protein synthesis depends on the proper recognition of stop codons by release factors (RFs). The human mitochondrial genome deviates from the standard genetic code by using not only the canonical stop codons UAG and UAA but also two non-canonical stop codons, AGA and AGG. Consequently, human mitochondria employ two release factors—mtRF1a and mtRF1—that recognize canonical and non-canonical stop codons, respectively. Surprisingly, mtRF1 is conserved across vertebrates, despite the absence of non-canonical stop codons in many species. Here, we investigate the function of mtRF1 in Mus musculus, whose mitochondrial open reading frames (mt-ORFs) lack non-canonical stop codons. Using a combination of biochemical approaches and mitoribosome profiling, we demonstrate that mtRF1 retains functional activity as a non-canonical RF, mediating an alternative release mechanism for COX1 via stop codon readthrough. Similarly, in human mitochondria, AGG stop codon readthrough enables ND6 release by mtRF1a in the absence of mtRF1. Our data reveal that mitoribosome stop codon readthrough is a conserved mechanism that compensates for the loss of RFs. While this mechanism facilitates access to alternative stop codons, it is also associated with RNA degradation. A targeted siRNA screen shows that components of the mitochondrial degradosome—including PNPase and SUV3—as well as the mitochondrial poly(A) polymerase MTPAP, participate in this decay pathway. Together, our study provides new insights into mitochondrial translation termination and RNA quality control, revealing stop codon readthrough as a rescue mechanism that enables the completion of mitochondrial protein synthesis when standard termination fails. Biological sciences/Molecular biology/Translation/Ribosome Biological sciences/Molecular biology/RNA metabolism/RNA quality control mitochondrial translation release factors stop codon readthrough mitoribosome quality control RNA decay Full Text Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryData.pdf Supplementary 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-7036318","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":488407149,"identity":"5ad58384-e536-4f2c-a49c-ce0b791dbc80","order_by":0,"name":"Joanna Rorbach","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4klEQVRIiWNgGAWjYBACxgY4kwfEszFgI1VLGmEtSACs5bABQXXM7c3PHhfUMOTzS589+OHnjvPGfAzsDx/gdVjPMXPjGccYLGf25SVL9p65bcbGwGOM1yrGGQlm0jxsDAYGZ3gMpBnbbtsAtbBJ4NeS/k2a5x9Yi/FvxrZzQC3sz3/g15JjJs3bBtZiBrTlANBhDGb4dAD9cqZMmrdPwkCyhy/Nsrct2ZiNmccYr8MM29u3SfN8szHg5+E9fONnm53h/Pb2hx/wamkAU8jGMuN1FgODPAH5UTAKRsEoGAUMDAA30zzn/ZLrfgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-2891-2840","institution":"Karolinska Institutet","correspondingAuthor":true,"prefix":"","firstName":"Joanna","middleName":"","lastName":"Rorbach","suffix":""},{"id":488407150,"identity":"a4491ea4-a242-466f-90ed-68ca7469f4f0","order_by":1,"name":"Annika Krüger","email":"","orcid":"https://orcid.org/0000-0002-2482-0316","institution":"Max Planck Institute Biology of Ageing-Karolinska Institutet Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Annika","middleName":"","lastName":"Krüger","suffix":""},{"id":488407151,"identity":"9baffe10-0815-4d94-82b4-5dd59c6e2136","order_by":2,"name":"Daria Kovalchuk","email":"","orcid":"","institution":"Karolinska Institutet","correspondingAuthor":false,"prefix":"","firstName":"Daria","middleName":"","lastName":"Kovalchuk","suffix":""},{"id":488407152,"identity":"ab9c953c-30f4-4820-ad66-e02b9c7c070b","order_by":3,"name":"Lukasz Borowski","email":"","orcid":"https://orcid.org/0000-0003-3766-0726","institution":"University of Warsaw, Faculty of Biology","correspondingAuthor":false,"prefix":"","firstName":"Lukasz","middleName":"","lastName":"Borowski","suffix":""},{"id":488407153,"identity":"dffeb2b5-c80f-4f29-b0e3-228836aa1b06","order_by":4,"name":"Elisa Angeloni","email":"","orcid":"https://orcid.org/0000-0002-6122-7327","institution":"Institute of Clinical Physiology, National Research Council","correspondingAuthor":false,"prefix":"","firstName":"Elisa","middleName":"","lastName":"Angeloni","suffix":""},{"id":488407154,"identity":"5a1e0135-1e39-4204-8536-4024aaa0cc05","order_by":5,"name":"Erik McShane","email":"","orcid":"https://orcid.org/0000-0002-7806-2063","institution":"Blavatnik Institute, Harvard Medical School","correspondingAuthor":false,"prefix":"","firstName":"Erik","middleName":"","lastName":"McShane","suffix":""},{"id":488407155,"identity":"8d81e4aa-767c-4bf3-b874-3a6ff2566f86","order_by":6,"name":"Giulia Santonoceto","email":"","orcid":"","institution":"Institute of Biochemistry and Biophysics, Polish Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Giulia","middleName":"","lastName":"Santonoceto","suffix":""},{"id":488407156,"identity":"b317d921-359c-4a6b-af4e-29ed7f425dbc","order_by":7,"name":"Fabio Spada","email":"","orcid":"","institution":"Baseclick GmbH","correspondingAuthor":false,"prefix":"","firstName":"Fabio","middleName":"","lastName":"Spada","suffix":""},{"id":488407157,"identity":"6320282f-dce4-491f-956f-8b2ce0290ec6","order_by":8,"name":"Thomas Frischmuth","email":"","orcid":"","institution":"baseclick GmbH,","correspondingAuthor":false,"prefix":"","firstName":"Thomas","middleName":"","lastName":"Frischmuth","suffix":""},{"id":488407158,"identity":"35dbdc01-3ca7-4349-b34c-be3dd8ef9696","order_by":9,"name":"L. 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The human mitochondrial genome deviates from the standard genetic code by using not only the canonical stop codons UAG and UAA but also two non-canonical stop codons, AGA and AGG. Consequently, human mitochondria employ two release factors—mtRF1a and mtRF1—that recognize canonical and non-canonical stop codons, respectively. Surprisingly, mtRF1 is conserved across vertebrates, despite the absence of non-canonical stop codons in many species. Here, we investigate the function of mtRF1 in Mus musculus, whose mitochondrial open reading frames (mt-ORFs) lack non-canonical stop codons. Using a combination of biochemical approaches and mitoribosome profiling, we demonstrate that mtRF1 retains functional activity as a non-canonical RF, mediating an alternative release mechanism for COX1 via stop codon readthrough. Similarly, in human mitochondria, AGG stop codon readthrough enables ND6 release by mtRF1a in the absence of mtRF1. 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