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The tRNASerAGA-2\u20133 variant (G35A) occurs in 2% of the human population and causes mis-incorporation of serine at phenylalanine codons. Here, we developed a dual fluorescent reporter to quantify mis-incorporation levels in live human and murine cells and validated mistranslation by mass spectrometry. In \u03b2-lymphocytes from individuals in the 1000 genomes project, we confirmed the anticipated genotype of cells with A35 minor alleles, and tRNA sequencing demonstrated expression, C32 hypo-modification, and partial 5\u2032-fragmentation of the endogenous mutant tRNASerAAA. Nanoparticle delivery of the fluorescent reporter confirmed serine mis-incorporation in the pan-genome cell lines. The data demonstrate that a natural genome-encoded human tRNA mutant causes mistranslation in cells derived from healthy individuals. Our findings have important implications for translation fidelity in humans and the application of missense suppressor tRNAs to medicine.<\/jats:p>","DOI":"10.1093\/nar\/gkag224","type":"journal-article","created":{"date-parts":[[2026,3,4]],"date-time":"2026-03-04T12:52:39Z","timestamp":1772628759000},"source":"Crossref","is-referenced-by-count":1,"title":["Mistranslation from an endogenous tRNA variant in human pan-genome cell lines"],"prefix":"10.1093","volume":"54","author":[{"given":"Peter","family":"Rozik","sequence":"first","affiliation":[{"name":"Department of Biochemistry, The University of Western Ontario , London, Ontario, N6A 5C1 ,","place":["Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Henry","family":"Moore","sequence":"additional","affiliation":[{"name":"Department of Biomolecular Engineering, Baskin School of Engineering & UCSC Genomics Institute, University of California Santa Cruz , Santa Cruz, CA 95064 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jeremy T","family":"Lant","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, The University of Western Ontario , London, Ontario, N6A 5C1 ,","place":["Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kyle S","family":"Hoffman","sequence":"additional","affiliation":[{"name":"Bioinformatics Solutions Inc. , Waterloo, Ontario, N2L 3K8 ,","place":["Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sarah K","family":"Schultz","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, The University of Western Ontario , London, Ontario, N6A 5C1 ,","place":["Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Baasil","family":"Afzal","sequence":"additional","affiliation":[{"name":"Department of Anatomy and Cell Biology, The University of Western Ontario , London, Ontario, N6A 5C1 ,","place":["Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Patricia P","family":"Chan","sequence":"additional","affiliation":[{"name":"Department of Biomolecular Engineering, Baskin School of Engineering & UCSC Genomics Institute, University of California Santa Cruz , Santa Cruz, CA 95064 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lauren E","family":"Flynn","sequence":"additional","affiliation":[{"name":"Department of Anatomy and Cell Biology, The University of Western Ontario , London, Ontario, N6A 5C1 ,","place":["Canada"]},{"name":"Department of Chemical and Biochemical Engineering, The University of Western Ontario , London, Ontario, N6A 5C1 ,","place":["Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1721-1667","authenticated-orcid":false,"given":"Ilka U","family":"Heinemann","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, The University of Western Ontario , London, Ontario, N6A 5C1 ,","place":["Canada"]},{"name":"Children\u2019s Health Research Institute, London Health Sciences Centre , London, Ontario, N6A 5W9 ,","place":["Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3253-6021","authenticated-orcid":false,"given":"Todd M","family":"Lowe","sequence":"additional","affiliation":[{"name":"Department of Biomolecular Engineering, Baskin School of Engineering & UCSC Genomics Institute, University of California Santa Cruz , Santa Cruz, CA 95064 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9500-8640","authenticated-orcid":false,"given":"Patrick","family":"O\u2019Donoghue","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, The University of Western Ontario , London, Ontario, N6A 5C1 ,","place":["Canada"]},{"name":"Department of Chemistry, The 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