Biology subjects

Demange, A.

Publications and source records attributed to Demange, A..

2 recordsLinked to original sources

bioRxiv2022

The non-neutral, multi-level, phenotypic impact of synonymous mutations revealed through heterologous gene expression in human cells.

Differences in codon frequency between genomes, genes, or positions along a gene, modulate transcription and translation efficiency, leading to phenotypic and functional differences. Here, we present a multiscale analysis of the effects of synonymous codon recoding during heterologous gene expression in human cells, quantifying the phenotypic consequences of codon usage bias at different molecular and cellular levels, with an emphasis on translation elongation. Six synonymous versions of an antibiotic resistance gene were generated, fused to a fluorescent reporter, and independently expressed in HEK293 cells. Multiscale phenotype was analysed by means of: quantitative transcriptome and proteome assessment, as proxies for gene expression; cellular fluorescence, as a proxy for single-cell level expression; and real-time cell proliferation in absence or presence of antibiotic, as a proxy for the cell fitness. We show that differences in codon usage bias strongly impact the molecular and cellular phenotype: (i) they result in large differences in mRNA levels and in protein levels, leading to differences of over fifteen times in translation efficiency; (ii) they introduce unpredicted splicing events; (iii) they lead to reproducible phenotypic heterogeneity; and (iv) they lead to a trade-off between the benefit of antibiotic resistance and the burden of heterologous expression. In human cells in culture, codon usage bias modulates gene expression by modifying mRNA availability and suitability for translation, leading to differences in protein levels and eventually eliciting functional phenotypic changes. IMPORTANCESynonymous codons encode for the same amino acid, but they are not neutral regarding gene expression or protein synthesis. Bias between synonymous codons have evolved naturally and are also applied in biotechnology protein production. We have studied the multilevel impact of codon usage on a human cell system. We show that differences in codon usage lead to transcriptomic, proteomic and functional changes, modulating gene expression and cellular phenotype.

systems biology↗

bioRxiv2021

Human cellular homeostasis buffers trans-acting translational effects of heterologous gene expression with very different codon usage bias

The frequency of synonymous codons in protein coding genes is non-random and varies both between species and between genes within species. Whether this codon usage bias (CUBias) reflects underlying neutral mutational processes or is instead shaped by selection remains an open debate, especially regarding the role of selection for enhanced protein production. Variation in CUBias of a gene (be it natural synonymous mutations or biotechnological synonymous recoding) can have an enormous impact on its expression by diverse cis-acting mechanisms. But expression of genes with extreme CUBias can also lead to strong phenotypic effects by altering the overall intracellular translation homeostasis via competition for ribosomal machinery or tRNA depletion. In this study, we expressed at high levels in human cells six different synonymous versions of a gene and used matched transcriptomic and proteomic data to evaluate the impact of CUBias of the heterologous gene on the translation of cellular transcripts. Our experimental design focused specifically on differences during translation elongation. Response to expression of the different synonymous sequences was assessed by various approaches, ranging from analyses performed on a per-gene basis to more integrated approaches of the cell as a whole. We observe that the transcriptome displayed substantial changes as a result of heterologous gene expression by triggering an intense antiviral and inflammatory response, but that changes in the proteomes were very modest. Most importantly we notice that changes in translation efficiency of cellular transcripts were not associated with the direction of the CUBias of the heterologous sequences, thereby providing only limited support for trans-acting effects of synonymous changes. We interpret that, in human cells in culture, changes in CUBias can lead to important cis-acting effects in gene expression, but that cellular homeostasis can buffer the phenotypic impact of overexpression of heterologous genes with extreme CUBias.

cell biology↗