Restrictions in amino acid availability revealed by differential ribosome codon reading
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE59821 | GEO | 2016/02/14
SECONDARY ACCESSION(S): PRJNA256314
REPOSITORIES: GEO
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Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series
2016-02-14 | E-GEOD-59821 | biostudies-arrayexpress
Project description:Cancer cells modulate their metabolic networks to support cell proliferation and a higher demand of building blocks. These changes may restrict the availability of certain amino acids for protein synthesis, which can be utilized for cancer therapy. However, little is known about the amino acid demand changes occurring during aggressive and invasive stages of cancer. Recently, we developed diricore, an approach based on ribosome profiling that can uncover amino acid limitations. Here, we applied diricore to a cellular model in which epithelial breast cells respond rapidly to TGFβ1, a cytokine essential for cancer progression and metastasis, and uncovered shortage of leucine. Further analyses indicated that TGFβ1 treatment of human breast epithelial cells reduces the expression of SLC3A2, a subunit of the leucine transporter, which diminishes leucine uptake and inhibits cell proliferation. Thus, we identified a specific amino acid limitation associated with the TGFβ1 response, a vulnerability that might be associated with aggressiveness in cancer.
| S-EPMC5376977 | biostudies-literature
Project description:It is common wisdom that codon usage bias has evolved in the selection for efficient translation, in which highly expressed genes are encoded predominantly by optimal codons. However, a growing body of evidence suggests regulatory roles for non-optimal codons in translation dynamics. Here we report that in mammalian cells, non-optimal codons play a critical role in promoting selective mRNA translation during amino acid starvation. During starvation, in contrast to genes encoding ribosomal proteins whose translation is highly sensitive to amino acid deprivation, translation of genes involved in the cellular protein degradation pathways remains unaffected. We found that these two gene groups bear different codon composition, with non-optimal codons being highly enriched in genes encoding the ubiquitin-proteasome system. Supporting the selective tRNA charging model originally proposed in Escherichia coli, we demonstrated that tRNA isoacceptors decoding rare codons are maintained in translating ribosomes under amino acid starvation. Finally, using luciferase reporters fused with endogenous gene-derived sequences, we show that codon optimality contributes to differential mRNA translation in response to amino acid starvation. These results highlight the physiological significance of codon usage bias in cellular adaptation to stress.
| S-EPMC5066624 | biostudies-literature
Project description:This SuperSeries is composed of the SubSeries listed below.
2023-09-13 | GSE234803 | GEO
Project description:Limitation for amino acids is thought to regulate translation in mammalian cells primarily by signaling through the kinases mTORC1 and GCN2. We find that a selective loss of arginine tRNA charging during limitation for arginine regulates translation through ribosome pausing at two of six arginine codons. Surprisingly, limitation for leucine, an essential and abundant amino acid in protein, results in little or no ribosome pausing. Chemical and genetic perturbation of mTORC1 and GCN2 signaling revealed that their robust response to leucine limitation prevents ribosome pausing, while an insufficient response to arginine limitation leads to loss of tRNA charging and ribosome pausing. Ribosome pausing decreases protein production and triggers premature ribosome termination without reducing mRNA levels. Together, our results suggest that amino acids that are not optimally sensed by the mTORC1 and GCN2 pathways still regulate translation through an evolutionarily conserved mechanism based on codon-specific ribosome pausing.
| S-EPMC6516488 | biostudies-literature
Project description:Twenty Fmoc-protected trinucleotide phosphoramidites representing a complete set of codons for the natural amino acids were chemically synthesized for the first time. A pool of these reagents was incorporated into oligonucleotides at substoichiometric levels to generate two libraries of variants that randomly carry either few or many codon replacements on a region encoding nine amino acids of the bacterial enzyme TEM-1 beta-lactamase. Assembly of the libraries was performed in a completely automated mode through a simple modification of ordinary protocols. This technology eliminates codon redundancy, stop codons and enables complete exploration of sequence space for single, double and triple mutations throughout a protein region spanning several residues. Sequence analysis of many non-selected clones revealed a good incorporation of the trinucleotides, producing combinations of mutations quite different from those obtained using conventional degenerate oligonucleotides. Ceftazidime-selection experiments yielded several never before reported variants containing novel amino acid combinations in the beta-lactamase omega loop region.
| S-EPMC534637 | biostudies-literature
Project description:Limitation for amino acids is thought to regulate translation in mammalian cells primarily by signaling through the kinases mTORC1 and GCN2. We find that limitation for the amino acid arginine causes a selective loss of tRNA charging, which regulates translation through ribosome pausing at two of six arginine codons. Interestingly, limitation for leucine, an essential and abundant amino acid in protein, results in little or no ribosome pausing. Chemical and genetic perturbation of mTORC1 and GCN2 signaling revealed that their robust response to leucine limitation prevents ribosome pausing, while an insufficient response to arginine limitation led to loss of arginine tRNA charging and ribosome pausing. Codon-specific ribosome pausing decreased protein production and triggered premature ribosome termination without significantly reducing mRNA levels. Together, our results suggest that amino acids which are not optimally sensed by the mTORC1 and GCN2 pathways still regulate translation through an evolutionarily conserved mechanism based on synonymous codon usage.
2018-07-19 | GSE113751 | GEO
Project description:Continuing our investigation of the tRNA genes and gene products in Mycoplasma mycoides, we report the sequence of the gene for tRNALeu (CAA) as well as partial primary structures of the following tRNAs: Leu (CAA), Leu (UAG), Arg (UCU), Thr (AGU) and Ile (CAU). It is suggested that in M. mycoides, at least some of the family codon boxes are read by only one tRNA each, using an unconventional method which does not discriminate between the nucleotides in the third codon position. M. mycoides is the first free-living organism known to use an unconventional method of this kind.
| S-EPMC1138444 | biostudies-other
Project description:BackgroundGlobal residue-specific amino acid mutagenesis can provide important biological insight and generate proteins with altered properties, but at the risk of protein misfolding. Further, targeted libraries are usually restricted to a handful of amino acids because there is an exponential correlation between the number of residues randomized and the size of the resulting ensemble. Using GFP as the model protein, we present a strategy, termed protein evolution via amino acid and codon elimination, through which simplified, native-like polypeptides encoded by a reduced genetic code were obtained via screening of reduced-size ensembles.Methodology/principal findingsThe strategy involves combining a sequential mutagenesis scheme to reduce library size with structurally stabilizing mutations, chaperone complementation, and reduced temperature of gene expression. In six steps, we eliminated a common buried residue, Phe, from the green fluorescent protein (GFP), while retaining activity. A GFP variant containing 11 Phe residues was used as starting scaffold to generate 10 separate variants in which each Phe was replaced individually (in one construct two adjacent Phe residues were changed simultaneously), while retaining varying levels of activity. Combination of these substitutions to generate a Phe-free variant of GFP abolished fluorescence. Combinatorial re-introduction of five Phe residues, based on the activities of the respective single amino acid replacements, was sufficient to restore GFP activity. Successive rounds of mutagenesis generated active GFP variants containing, three, two, and zero Phe residues. These GFPs all displayed progenitor-like fluorescence spectra, temperature-sensitive folding, a reduced structural stability and, for the least stable variants, a reduced steady state abundance.Conclusions/significanceThe results provide strategies for the design of novel GFP reporters. The described approach offers a means to enable engineering of active proteins that lack certain amino acids, a key step towards expanding the functional repertoire of uniquely labeled proteins in synthetic biology.
| S-EPMC2859931 | biostudies-literature
Project description:The vacuolar H(+)-ATPase (V-ATPase) is an ATP-dependent proton pump composed of a peripheral ATPase domain (V1) and a membrane-integral proton-translocating domain (V0) and is involved in many normal and disease processes. An important mechanism of regulating V-ATPase activity is reversible assembly of the V1 and V0 domains. Increased assembly in mammalian cells occurs under various conditions and has been shown to involve PI3K. The V-ATPase is necessary for amino acid-induced activation of mechanistic target of rapamycin complex 1 (mTORC1), which is important in controlling cell growth in response to nutrient availability and growth signals. The V-ATPase undergoes amino acid-dependent interactions with the Ragulator complex, which is involved in recruitment of mTORC1 to the lysosomal membrane during amino acid sensing. We hypothesized that changes in the V-ATPase/Ragulator interaction might involve amino acid-dependent changes in V-ATPase assembly. To test this, we measured V-ATPase assembly by cell fractionation in HEK293T cells treated with and without amino acids. V-ATPase assembly increases upon amino acid starvation, and this effect is reversed upon readdition of amino acids. Lysosomes from amino acid-starved cells possess greater V-ATPase-dependent proton transport, indicating that assembled pumps are catalytically active. Amino acid-dependent changes in both V-ATPase assembly and activity are independent of PI3K and mTORC1 activity, indicating the involvement of signaling pathways distinct from those implicated previously in controlling assembly. By contrast, lysosomal neutralization blocks the amino acid-dependent change in assembly and reactivation of mTORC1 after amino acid starvation. These results identify an important new stimulus for controlling V-ATPase assembly.
| S-EPMC4646367 | biostudies-literature