Mistranslating the genetic code with leucine in yeast and mammalian cells

ABSTRACT

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 tRNA^Leu variants that mis-incorporate Leu at phenylalanine or tryptophan codons. Expression of each tRNA^Leu 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 tRNA^Leu 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 tRNA^Leu 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 tRNA^Leu mutants with synonymous anticodons and for different tRNA^Leu isoacceptors with the same anticodon. A comparison to tRNA^Ala 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.

KEYWORDS:

• Genetic code
• mistranslation
• neuroblastoma cells
• protein synthesis
• Transfer RNA
• translation fidelity
• yeast

Acknowledgments

We are grateful to Josh Isaacson, Matthew Berg, Justin Ye, and Ilka Heinemann for insightful discussion and suggestions. We also thank Paula Pittock (Biological Mass Spectrometry Laboratory, University of Western Ontario) for assistance with identification of mistranslated peptides. In addition, we thank Kathleen Collins (University of California, Berkeley) for providing OTTR-seq reagents and protocol.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

All data are available in the figures, tables, supplementary information, and Supplementary Data Files 1–4.

All MS/MS data files are available:

https://figshare.com/articles/dataset/Mass_spec_tRNALeu_2023_zip/23284907/1

https://bioinfor.sharefile.com/d-sb232430cb59d423c99b1cb5e3b492e58

OTTR-seq data files are available at NCBI BioProject database

http://www.ncbi.nlm.nih.gov/bioproject/1066329

SubmissionID:SUB14155213

BioProject ID: PRJNA1066329

Supplemental data

Supplemental data for this article can be accessed online at https://doi.org/10.1080/15476286.2024.2340297

Additional information

Funding

The work was supported from the Natural Sciences and Engineering Research Council of Canada [04282 and 580241 to P.O., 07046 to C.J.B]; Canada Research Chairs [232341 to P.O.]; and the Canadian Institutes of Health Research [165985 to P.O.]. Support for HM, PPC, and T.M.L. provided by the National Instituties of Health, National Human Genome Research Institute [R01HG006753 to T.M.L.].