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.

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

10.1101/2022.01.07.475042 ◽

2022 ◽

Author(s):

Marion A. L. Picard ◽

Fiona Leblay ◽

Cecile Cassan ◽

Mathilde Decourcelle ◽

Anouk Willemsen ◽

...

Keyword(s):

Gene Expression ◽

Antibiotic Resistance ◽

Codon Usage ◽

Heterologous Gene Expression ◽

Human Cells ◽

Heterologous Gene ◽

Hek293 Cells ◽

Translation Efficiency ◽

Cellular Phenotype ◽

A Genome

Redundancy in the genetic code allows for differences in transcription and/or translation efficiency between mRNA molecules carrying synonymous polymorphisms, with potential phenotypic impact at the molecular and at the organismal level. A combination of neutral and selective processes determines the global genome codon usage preferences, as well as local differences between genes within a genome and between positions along a single gene. The relative contribution of evolutionary forces at shaping codon usage bias in eukaryotes is a matter of debate, especially in mammals. The main riddle remains understanding the sharp contrast between the strong molecular impact of gene expression differences arising from codon usage preferences and the thin evidence for codon usage selection at the organismal level. Here we report a multiscale analysis of the consequences of alternative codon usage on heterologous gene expression in human cells. We generated synonymous versions of the shble antibiotic resistance gene, fused to a fluorescent reporter, and expressed independently them in human HEK293 cells. We analysed: i) mRNA-to-DNA and protein-to-mRNA ratios for each shble version; ii) cellular fluorescence, using flow cytometry, as a proxy for single cell-level construct expression; and iii) real-time cell proliferation in absence or presence of antibiotic, as a proxy for the cellular fitness. Our results show that differences in codon usage preferences in our focal gene strongly impacted the molecular and the cellular phenotype: i) they elicited large differences in mRNA and in protein levels, as well in mRNA-to-protein ratio; ii) they introduced splicing events not predicted by current algorithms; iii) they lead to reproducible phenotypic heterogeneity as different multimodal distributions of cellular fluorescence EGFP; iv) they resulted in a trade-off between burden of heterologous expression and antibiotic resistance. While certain codon usage-related variables monotonically correlated with protein expression, other variables (e.g. CpG content or mRNA folding energy) displayed a bell-like behaviour. We interpret that codon usage preferences strongly shape the molecular and cellular phenotype in human cells through a direct impact on gene expression.

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Identification of a sequence motif upstream of the Drosophila Dopa decarboxylase gene that enhances heterologous gene expression

Genome ◽

10.1139/g94-075 ◽

1994 ◽

Vol 37 (4) ◽

pp. 526-534 ◽

Cited By ~ 2

Author(s):

Ross B. Hodgetts ◽

Millan S. Patel ◽

Jana Piorecky ◽

Andrew D. Swan ◽

Charlotte A. Spencer

Keyword(s):

Gene Expression ◽

Reporter Gene ◽

Heterologous Gene Expression ◽

Regulatory Element ◽

Regulatory Region ◽

Laboratory Strain ◽

Heterologous Gene ◽

Dopa Decarboxylase ◽

Sequence Motif ◽

Cis Acting

In this paper we have examined the role that element S, a DNA sequence motif found approximately 215 bp upstream of the Dopa decarboxylase (Ddc) gene, might play in regulating Ddc expression. Nearly identical versions of the element are present upstream of four other Drosophila genes. For two of these, the element appears to be an important component of the upstream regulatory region, since mutations in it reduce expression of the downstream gene. Because an element S polymorphism differentiates the Ddc+ allele of an inbred laboratory strain from the Ddc+4 allele present in a strain isolated from the wild, we decided to test the activity of both forms. Oligonucleotides containing Ddc+ or Ddc+4 versions of element S were synthesized and their ability to drive the expression of an heterologous (Adh) reporter gene at the second molt was examined. Transgenic larvae carrying the element S – Adh fusion constructs consistently exhibited Adh levels that were elevated 1.5-fold above those seen in control organisms. We have also determined the effects of element S in white prepupae and once again, ADH expression levels were significantly above controls in both groups of transformants carrying the element S construct. The results point to a functional role for element S. Since reporter gene expression in third instar larvae was restricted to tissues where ADH is normally found, we conclude that element S is not involved in directing the tissue specificity of Ddc expression. However, its ability to enhance heterologous gene expression suggests that it may be the fifth in the set of cis-acting sequences in the complex regulatory domain known to specify epidermal Ddc expression during development.Key words: Drosophila, Dopa decarboxylase, regulatory element, reporter gene, transgenic flies.

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Variations in codon usage are not correlated with heterologous gene expression in Saccharomyces cerevisiae and Escherichia coli

Journal of Biotechnology ◽

10.1016/0168-1656(88)90030-2 ◽

1988 ◽

Vol 7 (1) ◽

pp. 1-9 ◽

Cited By ~ 19

Author(s):

Joachim F. Ernst ◽

Eric Kawashima

Keyword(s):

Gene Expression ◽

Escherichia Coli ◽

Saccharomyces Cerevisiae ◽

Codon Usage ◽

Heterologous Gene Expression ◽

Heterologous Gene

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Optimization of Heterologous Gene Expression for In Vitro Evolution

BioTechniques ◽

10.2144/01303bm01 ◽

2001 ◽

Vol 30 (3) ◽

pp. 474-476 ◽

Cited By ~ 5

Author(s):

Ichiro Matsumura ◽

Mark J. Olsen ◽

Andrew D. Ellington

Keyword(s):

Gene Expression ◽

Heterologous Gene Expression ◽

Heterologous Gene ◽

In Vitro Evolution

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Massively parallel gene expression variation measurement of a synonymous codon library

BMC Genomics ◽

10.1186/s12864-021-07462-z ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Alexander Schmitz ◽

Fuzhong Zhang

Keyword(s):

Gene Expression ◽

Codon Usage ◽

Single Cells ◽

Massively Parallel ◽

Protein Abundance ◽

Translation Efficiency ◽

Gene Expression Variation ◽

Expression Variation ◽

Change In Mean ◽

Adaptation Index

Abstract Background Cell-to-cell variation in gene expression strongly affects population behavior and is key to multiple biological processes. While codon usage is known to affect ensemble gene expression, how codon usage influences variation in gene expression between single cells is not well understood. Results Here, we used a Sort-seq based massively parallel strategy to quantify gene expression variation from a green fluorescent protein (GFP) library containing synonymous codons in Escherichia coli. We found that sequences containing codons with higher tRNA Adaptation Index (TAI) scores, and higher codon adaptation index (CAI) scores, have higher GFP variance. This trend is not observed for codons with high Normalized Translation Efficiency Index (nTE) scores nor from the free energy of folding of the mRNA secondary structure. GFP noise, or squared coefficient of variance (CV2), scales with mean protein abundance for low-abundant proteins but does not change at high mean protein abundance. Conclusions Our results suggest that the main source of noise for high-abundance proteins is likely not originating at translation elongation. Additionally, the drastic change in mean protein abundance with small changes in protein noise seen from our library implies that codon optimization can be performed without concerning gene expression noise for biotechnology applications.

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CdSe/ZnS Core-Shell-Type Quantum Dot Nanoparticles Disrupt the Cellular Homeostasis in Cellular Blood–Brain Barrier Models

International Journal of Molecular Sciences ◽

10.3390/ijms22031068 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1068

Author(s):

Katarzyna Dominika Kania ◽

Waldemar Wagner ◽

Łukasz Pułaski

Keyword(s):

Gene Expression ◽

Quantum Dot ◽

Blood Brain Barrier ◽

Protein Gene ◽

Brain Barrier ◽

Core Shell ◽

Cellular Homeostasis ◽

Blood Brain ◽

Shell Type ◽

The Impact

Two immortalized brain microvascular endothelial cell lines (hCMEC/D3 and RBE4, of human and rat origin, respectively) were applied as an in vitro model of cellular elements of the blood–brain barrier in a nanotoxicological study. We evaluated the impact of CdSe/ZnS core-shell-type quantum dot nanoparticles on cellular homeostasis, using gold nanoparticles as a largely bioorthogonal control. While the investigated nanoparticles had surprisingly negligible acute cytotoxicity in the evaluated models, a multi-faceted study of barrier-related phenotypes and cell condition revealed a complex pattern of homeostasis disruption. Interestingly, some features of the paracellular barrier phenotype (transendothelial electrical resistance, tight junction protein gene expression) were improved by exposure to nanoparticles in a potential hormetic mechanism. However, mitochondrial potential and antioxidant defences largely collapsed under these conditions, paralleled by a strong pro-apoptotic shift in a significant proportion of cells (evidenced by apoptotic protein gene expression, chromosomal DNA fragmentation, and membrane phosphatidylserine exposure). Taken together, our results suggest a reactive oxygen species-mediated cellular mechanism of blood–brain barrier damage by quantum dots, which may be toxicologically significant in the face of increasing human exposure to this type of nanoparticles, both intended (in medical applications) and more often unintended (from consumer goods-derived environmental pollution).

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Production of foreign proteins in the yeast Pichia pastoris

Canadian Journal of Botany ◽

10.1139/b95-336 ◽

1995 ◽

Vol 73 (S1) ◽

pp. 891-897 ◽

Cited By ~ 46

Author(s):

James M. Cregg ◽

David R. Higgins

Keyword(s):

Gene Expression ◽

Pichia Pastoris ◽

Heterologous Gene Expression ◽

Expression System ◽

Methylotrophic Yeast ◽

Culture Broth ◽

Heterologous Gene ◽

Foreign Gene ◽

Heterologous Proteins ◽

Yeast Pichia Pastoris

The methanol-utilizing yeast Pichia pastoris has been developed as a host system for the production of heterologous proteins of commercial interest. An industrial yeast selected for efficient growth on methanol for biomass generation, P. pastoris is readily grown on defined medium in continuous culture at high volume and density. A unique feature of the expression system is the promoter employed to drive heterologous gene expression, which is derived from the methanol-regulated alcohol oxidase I gene (AOX1) of P. pastoris, one of the most efficient and tightly regulated promoters known. The strength of the AOX1 promoter results in high expression levels in strains harboring only a single integrated copy of a foreign-gene expression cassette. Levels may often be further enhanced through the integration of multiple cassette copies into the P. pastoris genome and strategies to construct and select multicopy cassette strains have been devised. The system is particularly attractive for the secretion of foreign-gene products. Because P. pastoris endogenous protein secretion levels are low, foreign secreted proteins often appear to be virtually the only proteins in the culture broth, a major advantage in processing and purification. Key words: heterologous gene expression, methylotrophic yeast, Pichia pastoris, secretion, glycosylation.

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