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Codon usage influences fitness through RNA toxicity

Pragya Mittal, James Brindle, Julie Stephen, Joshua B. Plotkin, Grzegorz Kudla
doi: https://doi.org/10.1101/344002
Pragya Mittal
1MRC Human Genetics Unit, IGMM, University of Edinburgh, Edinburgh, Scotland, UK
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James Brindle
1MRC Human Genetics Unit, IGMM, University of Edinburgh, Edinburgh, Scotland, UK
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Julie Stephen
1MRC Human Genetics Unit, IGMM, University of Edinburgh, Edinburgh, Scotland, UK
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Joshua B. Plotkin
2University of Pennsylvania, Philadelphia, USA
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Grzegorz Kudla
1MRC Human Genetics Unit, IGMM, University of Edinburgh, Edinburgh, Scotland, UK
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Abstract

Many organisms are subject to selective pressure that gives rise to unequal usage of synonymous codons, known as codon bias. To experimentally dissect the mechanisms of selection on synonymous sites, we expressed several hundred synonymous variants of the GFP gene in Escherichia coli, and used quantitative growth and viability assays to estimate bacterial fitness. Unexpectedly, we found many synonymous variants whose expression was toxic to E. coli. Unlike previously studied effects of synonymous mutations, the effect that we discovered is independent of translation, but it depends on the production of toxic mRNA molecules. We identified RNA sequence determinants of toxicity, and evolved suppressor strains that can tolerate the expression of toxic GFP variants. Genome sequencing of these suppressor strains revealed a cluster of promoter mutations that prevented toxicity by reducing mRNA levels. We conclude that translation-independent RNA toxicity is a previously unrecognized obstacle in bacterial gene expression.

Significance statement Synonymous mutations in genes do not change protein sequence, but they may affect gene expression and cellular function. Here we describe an unexpected toxic effect of synonymous mutations in Escherichia coli, with potentially large implications for bacterial physiology and evolution. Unlike previously studied effects of synonymous mutations, the effect that we discovered is independent of translation, but it depends on the production of toxic mRNA molecules. We hypothesize that the mechanism we identified influences the evolution of endogenous genes in bacteria, by imposing selective constraints on synonymous mutations that arise in the genome. Of interest for biotechnology and synthetic biology, we identify bacterial strains and growth conditions that alleviate RNA toxicity, thus allowing efficient overexpression of heterologous proteins.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted June 11, 2018.
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Codon usage influences fitness through RNA toxicity
Pragya Mittal, James Brindle, Julie Stephen, Joshua B. Plotkin, Grzegorz Kudla
bioRxiv 344002; doi: https://doi.org/10.1101/344002
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Codon usage influences fitness through RNA toxicity
Pragya Mittal, James Brindle, Julie Stephen, Joshua B. Plotkin, Grzegorz Kudla
bioRxiv 344002; doi: https://doi.org/10.1101/344002

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