Project description:Usage of synonymous codons represents a characteristic pattern of preference in each organism. It has been inferred that such bias of codon usage has evolved as a result of adaptation for efficient synthesis of proteins. Here we examined synonymous codon usage in genes of the fission yeast Schizosaccharomyces pombe, and compared codon usage bias with expression levels of the gene. In this organism, synonymous codon usage bias was correlated with expression levels of the gene; the bias was most obvious in two-codon amino acids. A similar pattern of the codon usage bias was also observed in Saccharomyces cerevisiae, Arabidopsis thaliana, and Caenorhabditis elegans, but was not obvious in Oryza sativa, Drosophila melanogaster, Takifugu rubripes and Homo sapiens. As codons of the highly expressed genes have greater influence on translational efficiency than codons of genes expressed at lower levels, it is likely that codon usage in the S. pombe genome has been optimized by translational selection through evolution. Relative amounts of mRNA for each ORF were measured by DNA microarray using genomic DNA as a reference, and the copy number of mRNA was calculated using an estimate of the total mRNA number in the cell as 100,000 copies.
Project description:We have previously shown that the yeast homolog of the RNA-binding vigilin proteins – Scp160p – is involved in enhancing translation efficiency in the context of codon usage. In the current study, we investigated the influence of Scp160p on the biology of polyQ reporters which differ in the codon usage of their polyQ regions. We observe that Scp160p facilitates aggregation of the polyQ reporters independent of codon usage. To explore if Scp160p might also facilitate the aggregation of endogenous polyQ-containing proteins in the yeast proteome, we combined filter trap binding and dimethyl labeling mass spectrometry to assess the aggregation state of the proteome in scp160Δ cells. Filter trap binding allows the isolation of protein aggregates which are SDS-resistant (a feature of polyQ aggregates) from wild-type and scp160Δ cells. Dimethyl labeling with nanoLC-MS/MS provided a quantitative comparison of the amount of aggregated proteins isolated by filter trap binding.
Project description:Usage of synonymous codons represents a characteristic pattern of preference in each organism. It has been inferred that such bias of codon usage has evolved as a result of adaptation for efficient synthesis of proteins. Here we examined synonymous codon usage in genes of the fission yeast Schizosaccharomyces pombe, and compared codon usage bias with expression levels of the gene. In this organism, synonymous codon usage bias was correlated with expression levels of the gene; the bias was most obvious in two-codon amino acids. A similar pattern of the codon usage bias was also observed in Saccharomyces cerevisiae, Arabidopsis thaliana, and Caenorhabditis elegans, but was not obvious in Oryza sativa, Drosophila melanogaster, Takifugu rubripes and Homo sapiens. As codons of the highly expressed genes have greater influence on translational efficiency than codons of genes expressed at lower levels, it is likely that codon usage in the S. pombe genome has been optimized by translational selection through evolution.
Project description:Differences in codon frequency between genomes, genes, or positions along a gene, modulate transcription and translation efficiency, leading to phenotypic and functional differences. Integrative studies quantifying the phenotypic consequences of codon usage bias at different molecular and cellular levels in human cells are lacking. Here, we present a multiscale analysis of the effects of synonymous codon recoding during heterologous gene expression in human cells. 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 and in protein levels, as well in mRNA-to-protein ratio; (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.
Project description:In this study, we combined quantitative proteomics and systematic codon usage analysis of translated mRNA, polysome and ribosome sequencing to uncover VARS translational targets.
Project description:Saccahromycopsis schoenii belongs to a genus of yeasts that have the ability to attack and kill other yeast and fungi. De novo genomic sequencing and genome assembly suggests that S. schoenii might belong to the CTG clade. To examine whether it translated CTG codons to leucine (standard codon usage) or serine (alternative codon usage), we analysed its proteome during growth on full media. To see if translation is changed during nutritional stress or during predation on a prey cell (Saccharomyces cerevisiae), we analysed and quantified its proteome during these conditions compared to its proteomic expression in full media.