Project description:Ribosome profiling data reports on the distribution of translating ribosomes, at steady-state, with codon-level resolution. We present a robust method to extract codon translation rates and protein synthesis rates from these data, and identify causal features associated with elongation and translation efficiency in physiological conditions in yeast. We show that neither elongation rate nor translational efficiency is improved by experimental manipulation of the abundance or body sequence of the rare AGG tRNA. Deletion of three of the four copies of the heavily used ACA tRNA shows a modest efficiency decrease that could be explained by other rate-reducing signals at gene start. This suggests that correlation between codon bias and efficiency arises as selection for codons to utilize translation machinery efficiently in highly translated genes. We also show a correlation between efficiency and RNA structure calculated both computationally and from recent structure probing data, as well as the Kozak initiation motif, which may comprise a mechanism to regulate initiation. We test whether tRNA abundance affects elongation or translation efficiency by changing the tRNA levels through deletion or over expression and measuring the ribosomal dwell time at each codon using a robust statistical method that accounts for flow conservation.
Project description:Codon usage bias is a universal feature of eukaryotic and prokaryotic genomes and has been proposed to regulate translation efficiency, accuracy and protein folding based on the assumption that codon usage affects translation dynamics. The role of codon usage in regulating translation, however, is not clear and has been challenged by recent ribosome profiling studies. Here we used a Neurospora cell-free translation system to directly monitor the velocity of mRNA translation. We demonstrated that the use of preferred codons enhances the rate of translation elongation, whereas non-optimal codons slow translation. In addition, codon usage regulates ribosome traffic on the mRNA. These conclusions were supported by ribosome profiling results in vitro and in vivo with substrate mRNAs manipulated to increase signal over background noise. We further show that codon usage plays an important role in regulating protein function by affecting co-translational protein folding. Together, these results resolve a long-standing fundamental question and demonstrate the importance of codon usage on protein folding.
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:The codon usage of mRNAs controls the speed of translation elongation, which is primarily determined by the abundance of cognate tRNAs. By profiling mRNA expression around the cell cycle we found that mRNAs that are relatively upregulate in the G2/M phase are enriched in rare codons. To understand the impact of this codon bias on translation, we have cultured NIH 3T3 cells with different concentrations of fetal calf serum (FCS), 1, 2, 5, and 10%, respectively, to induce distinct proliferation rates and thus distinct proportions of cells in the culture in the G2/M phase. We then estimated the levels of all proteins and mRNAs, and the change in translation efficiency (proteins per mRNA) in highly (10% FCS) relatively to less highly proliferating cells (lower FCS concentrations).
Project description:Familial dysautonomia (FD) results from mutation in IKBKAP/ELP1, a gene encoding the scaffolding protein for the Elongator complex. This highly conserved complex is required for the translation of codon-biased genes in lower organisms. Here we investigate whether Elongator serves a similar function in mammalian peripheral neurons, the population devastated in FD. Using codon-biased eGFP sensors, and multiplexing of codon usage with transcriptome and proteome analyses of over 6,000 genes, we identify two categories of genes, as well as specific gene identities that depend on Elongator for normal expression. Moreover, we show that multiple genes in the DNA damage repair pathway are codon-biased, and that with Elongator loss, their misregulation is correlated with elevated levels of DNA damage. These findings link Elongator's function in the translation of codon-biased genes with both the developmental and neurodegenerative phenotypes of FD, and also clarify the increased risk of cancer associated with the disease.
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:Ribosome profiling data reports on the distribution of translating ribosomes, at steady-state, with codon-level resolution. We present a robust method to extract codon translation rates and protein synthesis rates from these data, and identify causal features associated with elongation and translation efficiency in physiological conditions in yeast. We show that neither elongation rate nor translational efficiency is improved by experimental manipulation of the abundance or body sequence of the rare AGG tRNA. Deletion of three of the four copies of the heavily used ACA tRNA shows a modest efficiency decrease that could be explained by other rate-reducing signals at gene start. This suggests that correlation between codon bias and efficiency arises as selection for codons to utilize translation machinery efficiently in highly translated genes. We also show a correlation between efficiency and RNA structure calculated both computationally and from recent structure probing data, as well as the Kozak initiation motif, which may comprise a mechanism to regulate initiation.
Project description:ϕXacN1 is a novel jumbo myovirus infecting the causative agent of Asian citrus canker, Xanthomonas citri. Its linear 384,670 bp double-stranded DNA genome encodes 592 predicted protein coding genes and shows 65,875 bp direct terminal repeats (DTRs), so far the longest DTRs among sequence phage genomes. The DTRs harbor 56 tRNA genes, corresponding to all 20 amino acids. This is the highest number of tRNA genes reported in a phage genome. Codon usage analyses revealed a propensity that the phage encoded tRNAs target codons that are highly used by the phage but less frequently by its host. The existence of these tRNA genes, additional seven translation-related genes as well as a chaperonin gene found in the ϕXacN1 genome suggests an increased level of independence of phage replication on host molecular machinery and a wide host range. Consistently, ϕXacN1 showed a wider host range than other X. citri phages in an infection test against a panel of X. citri strains. Phylogenetic analyses revealed a clade of phages composed of ϕXacN1 and ten other jumbo phages showing an evolutionary stability in their large genome sizes.