Mistranslating the genetic code with leucine in yeast and mammalian cells
Translation fidelity relies on accurate aminoacylation of transfer RNAs (tRNAs) by aminoacyl-tRNA synthetases (AARSs). AARSs specific for alanine (Ala), leucine (Leu), serine, and pyrrolysine do not recognize the anticodon bases. Single nucleotide anticodon variants in their cognate tRNAs can lead t...
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Taylor & Francis Group
2024-12-01
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| Series: | RNA Biology |
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| Online Access: | https://www.tandfonline.com/doi/10.1080/15476286.2024.2340297 |
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| author | Josephine Davey-Young Farah Hasan Rasangi Tennakoon Peter Rozik Henry Moore Peter Hall Ecaterina Cozma Julie Genereaux Kyle S. Hoffman Patricia P. Chan Todd M. Lowe Christopher J. Brandl Patrick O’Donoghue |
| author_facet | Josephine Davey-Young Farah Hasan Rasangi Tennakoon Peter Rozik Henry Moore Peter Hall Ecaterina Cozma Julie Genereaux Kyle S. Hoffman Patricia P. Chan Todd M. Lowe Christopher J. Brandl Patrick O’Donoghue |
| author_sort | Josephine Davey-Young |
| collection | DOAJ |
| description | Translation fidelity relies on accurate aminoacylation of transfer RNAs (tRNAs) by aminoacyl-tRNA synthetases (AARSs). AARSs specific for alanine (Ala), leucine (Leu), serine, and pyrrolysine do not recognize the anticodon bases. Single nucleotide anticodon variants in their cognate tRNAs can lead to mistranslation. Human genomes include both rare and more common mistranslating tRNA variants. We investigated three rare human tRNALeu variants that mis-incorporate Leu at phenylalanine or tryptophan codons. Expression of each tRNALeu anticodon variant in neuroblastoma cells caused defects in fluorescent protein production without significantly increased cytotoxicity under normal conditions or in the context of proteasome inhibition. Using tRNA sequencing and mass spectrometry we confirmed that each tRNALeu variant was expressed and generated mistranslation with Leu. To probe the flexibility of the entire genetic code towards Leu mis-incorporation, we created 64 yeast strains to express all possible tRNALeu anticodon variants in a doxycycline-inducible system. While some variants showed mild or no growth defects, many anticodon variants, enriched with G/C at positions 35 and 36, including those replacing Leu for proline, arginine, alanine, or glycine, caused dramatic reductions in growth. Differential phenotypic defects were observed for tRNALeu mutants with synonymous anticodons and for different tRNALeu isoacceptors with the same anticodon. A comparison to tRNAAla anticodon variants demonstrates that Ala mis-incorporation is more tolerable than Leu at nearly every codon. The data show that the nature of the amino acid substitution, the tRNA gene, and the anticodon are each important factors that influence the ability of cells to tolerate mistranslating tRNAs. |
| format | Article |
| id | doaj-art-b5dfad6187bd4976a2eaefb2e55ed373 |
| institution | DOAJ |
| issn | 1547-6286 1555-8584 |
| language | English |
| publishDate | 2024-12-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | RNA Biology |
| spelling | doaj-art-b5dfad6187bd4976a2eaefb2e55ed3732025-08-20T02:50:08ZengTaylor & Francis GroupRNA Biology1547-62861555-85842024-12-0121150752910.1080/15476286.2024.2340297Mistranslating the genetic code with leucine in yeast and mammalian cellsJosephine Davey-Young0Farah Hasan1Rasangi Tennakoon2Peter Rozik3Henry Moore4Peter Hall5Ecaterina Cozma6Julie Genereaux7Kyle S. Hoffman8Patricia P. Chan9Todd M. Lowe10Christopher J. Brandl11Patrick O’Donoghue12Department of Biochemistry, The University of Western Ontario, London, Ontario, CanadaDepartment of Biochemistry, The University of Western Ontario, London, Ontario, CanadaDepartment of Biochemistry, The University of Western Ontario, London, Ontario, CanadaDepartment of Biochemistry, The University of Western Ontario, London, Ontario, CanadaDepartment of Biomolecular Engineering, Baskin School of Engineering & UCSC Genomics Institute, University of California Santa Cruz, Santa Cruz, CA, USADepartment of Biochemistry, The University of Western Ontario, London, Ontario, CanadaDepartment of Biochemistry, The University of Western Ontario, London, Ontario, CanadaDepartment of Biochemistry, The University of Western Ontario, London, Ontario, CanadaBioinformatic Solutions, Inc, Waterloo, Ontario, CanadaDepartment of Biomolecular Engineering, Baskin School of Engineering & UCSC Genomics Institute, University of California Santa Cruz, Santa Cruz, CA, USADepartment of Biomolecular Engineering, Baskin School of Engineering & UCSC Genomics Institute, University of California Santa Cruz, Santa Cruz, CA, USADepartment of Biochemistry, The University of Western Ontario, London, Ontario, CanadaDepartment of Biochemistry, The University of Western Ontario, London, Ontario, CanadaTranslation fidelity relies on accurate aminoacylation of transfer RNAs (tRNAs) by aminoacyl-tRNA synthetases (AARSs). AARSs specific for alanine (Ala), leucine (Leu), serine, and pyrrolysine do not recognize the anticodon bases. Single nucleotide anticodon variants in their cognate tRNAs can lead to mistranslation. Human genomes include both rare and more common mistranslating tRNA variants. We investigated three rare human tRNALeu variants that mis-incorporate Leu at phenylalanine or tryptophan codons. Expression of each tRNALeu anticodon variant in neuroblastoma cells caused defects in fluorescent protein production without significantly increased cytotoxicity under normal conditions or in the context of proteasome inhibition. Using tRNA sequencing and mass spectrometry we confirmed that each tRNALeu variant was expressed and generated mistranslation with Leu. To probe the flexibility of the entire genetic code towards Leu mis-incorporation, we created 64 yeast strains to express all possible tRNALeu anticodon variants in a doxycycline-inducible system. While some variants showed mild or no growth defects, many anticodon variants, enriched with G/C at positions 35 and 36, including those replacing Leu for proline, arginine, alanine, or glycine, caused dramatic reductions in growth. Differential phenotypic defects were observed for tRNALeu mutants with synonymous anticodons and for different tRNALeu isoacceptors with the same anticodon. A comparison to tRNAAla anticodon variants demonstrates that Ala mis-incorporation is more tolerable than Leu at nearly every codon. The data show that the nature of the amino acid substitution, the tRNA gene, and the anticodon are each important factors that influence the ability of cells to tolerate mistranslating tRNAs.https://www.tandfonline.com/doi/10.1080/15476286.2024.2340297Genetic codemistranslationneuroblastoma cellsprotein synthesisTransfer RNAtranslation fidelity |
| spellingShingle | Josephine Davey-Young Farah Hasan Rasangi Tennakoon Peter Rozik Henry Moore Peter Hall Ecaterina Cozma Julie Genereaux Kyle S. Hoffman Patricia P. Chan Todd M. Lowe Christopher J. Brandl Patrick O’Donoghue Mistranslating the genetic code with leucine in yeast and mammalian cells RNA Biology Genetic code mistranslation neuroblastoma cells protein synthesis Transfer RNA translation fidelity |
| title | Mistranslating the genetic code with leucine in yeast and mammalian cells |
| title_full | Mistranslating the genetic code with leucine in yeast and mammalian cells |
| title_fullStr | Mistranslating the genetic code with leucine in yeast and mammalian cells |
| title_full_unstemmed | Mistranslating the genetic code with leucine in yeast and mammalian cells |
| title_short | Mistranslating the genetic code with leucine in yeast and mammalian cells |
| title_sort | mistranslating the genetic code with leucine in yeast and mammalian cells |
| topic | Genetic code mistranslation neuroblastoma cells protein synthesis Transfer RNA translation fidelity |
| url | https://www.tandfonline.com/doi/10.1080/15476286.2024.2340297 |
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