Transcriptomic, proteomic, and functional consequences of codon usage bias in human cells during heterologous gene expression

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 analyzed 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 protein levels, leading to differences of over 15 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.

Automated summary

Differences in codon frequency have significant effects on transcription and translation efficiency, leading to phenotypic and functional differences. This study presents a multiscale analysis of synonymous codon recoding effects during heterologous gene expression and quantifies the impact of codon usage bias at different molecular and cellular levels. The results demonstrate that codon usage bias strongly influences molecular and cellular phenotype, affecting mRNA and protein levels, leading to reproducible phenotypic heterogeneity and trade-off between antibiotic resistance and heterologous expression burden. In human cells, codon usage bias modulates gene expression by altering mRNA availability and suitability for translation, resulting in protein level differences and functional phenotypic changes.