Stop-Codon–Mediated Polycistronic Translation Enables Multi-ORF Expression in Mammalian Neurons In Vivo

Stop-Codon–Mediated Polycistronic Translation Enables Multi-ORF Expression in Mammalian Neurons In Vivo | 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 Stop-Codon–Mediated Polycistronic Translation Enables Multi-ORF Expression in Mammalian Neurons In Vivo Shunit Olszakier, Ronit Heinrich, Michael Andreyanov, Hussein Wessal, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8629892/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 10 You are reading this latest preprint version Abstract Multigene expression in mammalian systems remains constrained by the presumed monocistronic nature of translation and by reliance on bulky regulatory elements such as IRESs or self-cleaving peptides. Here we show that canonical stop codons alone are sufficient to drive robust polycistronic protein expression in mammals. We demonstrate efficient bi- and tricistronic expression of full-length, functional open reading frames from a single mRNA, without any auxiliary translational elements, linkers, or sequence modifications. This strategy operates across diverse protein classes and supports downstream translation over intercistronic distances exceeding 2 kb. Crucially, we achieve tricistronic expression in mammalian neurons in vivo following adeno-associated virus delivery to the rat brain. To our knowledge, this is the first demonstration of in vivo expression of three independent protein-coding ORFs from a single mammalian transcript using only canonical stop codons. Leveraging this capability, we introduce Genetically Encoded Functional Rainbow Indicators (GEFRIs), compact tricistronic reporters enabling simultaneous structural labeling and functional calcium imaging from a single viral vector. Together, this work establishes stop-codon–mediated polycistronism as a practical, scarless, and uniquely compact platform for coordinated multi-protein expression in mammalian cells and in vivo . Biological sciences/Biological techniques Biological sciences/Biotechnology Biological sciences/Cell biology Biological sciences/Neuroscience Full Text Additional Declarations No competing interests reported. Supplementary Files Olszakietetal.2026Supplementaryfigures.pdf Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 15 Apr, 2026 Reviews received at journal 23 Mar, 2026 Reviewers agreed at journal 18 Mar, 2026 Reviewers agreed at journal 16 Mar, 2026 Reviewers agreed at journal 16 Mar, 2026 Reviewers invited by journal 06 Mar, 2026 Editor invited by journal 27 Feb, 2026 Editor assigned by journal 24 Feb, 2026 Submission checks completed at journal 21 Jan, 2026 First submitted to journal 21 Jan, 2026 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. 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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-8629892","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":603172346,"identity":"8997f515-4af1-4dd2-a438-0f73e24c7889","order_by":0,"name":"Shunit Olszakier","email":"","orcid":"","institution":"Technion – Israel Institute of Technology","correspondingAuthor":false,"prefix":"","firstName":"Shunit","middleName":"","lastName":"Olszakier","suffix":""},{"id":603172348,"identity":"c165603f-c936-4257-ae33-4cf5205f0b38","order_by":1,"name":"Ronit Heinrich","email":"","orcid":"","institution":"Technion – Israel Institute of 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