Translation efficiency
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Gene expression is a fundamental process that enables cells to produce specific proteins in a timely and spatially dependent manner. In eukaryotic cells, the complex organization of the cell body requires precise control of protein synthesis and localization. Certain mRNAs encode proteins with an N-terminal signal sequences that direct the translation apparatus toward a specific organelle. Here, we focus on the mechanisms governing the translation of mRNAs, which encode proteins with an endoplasmic reticulum (ER) signal in human cells. The binding of a signal-recognition particle (SRP) to the translation machinery halts protein synthesis until the mRNA-ribosome complex reaches the ER membrane. The commonly accepted model suggests that mRNA that encodes a protein that contains an ER signal peptide continuously repeats the cycle of SRP binding followed by association and dissociation with the ER. In contrast to the current view, we show that the long mRNAs remain on the ER while being translated. On the other hand, due to low ribosome occupancy, the short mRNAs continue the cycle, always facing a translation pause. Ultimately, this leads to a significant drop in the translation efficiency of small, ER-targeted proteins. The proposed mechanism advances our understanding of selective protein synthesis in eukaryotic cells and provides new avenues to enhance protein production in biotechnological settings.
- Publikační typ
- časopisecké články MeSH
Programmed stop codon readthrough is a post-transcription regulatory mechanism specifically increasing proteome diversity by creating a pool of C-terminally extended proteins. During this process, the stop codon is decoded as a sense codon by a near-cognate tRNA, which programs the ribosome to continue elongation. The efficiency of competition for the stop codon between release factors (eRFs) and near-cognate tRNAs is largely dependent on its nucleotide context; however, the molecular mechanism underlying this process is unknown. Here, we show that it is the translation initiation (not termination) factor, namely eIF3, which critically promotes programmed readthrough on all three stop codons. In order to do so, eIF3 must associate with pre-termination complexes where it interferes with the eRF1 decoding of the third/wobble position of the stop codon set in the unfavorable termination context, thus allowing incorporation of near-cognate tRNAs with a mismatch at the same position. We clearly demonstrate that efficient readthrough is enabled by near-cognate tRNAs with a mismatch only at the third/wobble position. Importantly, the eIF3 role in programmed readthrough is conserved between yeast and humans.
- MeSH
- aminoacyl-tRNA metabolismus MeSH
- elongace translace peptidového řetězce * MeSH
- eukaryotický iniciační faktor 3 metabolismus MeSH
- HeLa buňky MeSH
- iniciace translace peptidového řetězce MeSH
- kvasinky genetika MeSH
- lidé MeSH
- paromomycin farmakologie MeSH
- regulace genové exprese MeSH
- ribozomy účinky léků metabolismus MeSH
- terminační kodon * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Genes encoding the KDM5 family of transcriptional regulators are disrupted in individuals with intellectual disability (ID). To understand the link between KDM5 and ID, we characterized five Drosophila strains harboring missense alleles analogous to those observed in patients. These alleles disrupted neuroanatomical development, cognition and other behaviors, and displayed a transcriptional signature characterized by the downregulation of many ribosomal protein genes. A similar transcriptional profile was observed in KDM5C knockout iPSC-induced human glutamatergic neurons, suggesting an evolutionarily conserved role for KDM5 proteins in regulating this class of gene. In Drosophila, reducing KDM5 changed neuronal ribosome composition, lowered the translation efficiency of mRNAs required for mitochondrial function, and altered mitochondrial metabolism. These data highlight the cellular consequences of altered KDM5-regulated transcriptional programs that could contribute to cognitive and behavioral phenotypes. Moreover, they suggest that KDM5 may be part of a broader network of proteins that influence cognition by regulating protein synthesis.
- MeSH
- aktivace transkripce MeSH
- Drosophila melanogaster genetika metabolismus MeSH
- Drosophila genetika metabolismus MeSH
- histondemethylasy metabolismus genetika MeSH
- lidé MeSH
- mentální retardace genetika metabolismus MeSH
- mitochondrie metabolismus genetika MeSH
- neurony * metabolismus MeSH
- proteiny Drosophily * genetika metabolismus MeSH
- proteosyntéza MeSH
- ribozomální proteiny * genetika metabolismus MeSH
- ribozomy metabolismus genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Reinitiation after translation of short upstream ORFs (uORFs) represents one of the means of regulation of gene expression on the mRNA-specific level in response to changing environmental conditions. Over the years it has been shown-mainly in budding yeast-that its efficiency depends on cis-acting features occurring in sequences flanking reinitiation-permissive uORFs, the nature of their coding sequences, as well as protein factors acting in trans. We earlier demonstrated that the first two uORFs from the reinitiation-regulated yeast GCN4 mRNA leader carry specific structural elements in their 5' sequences that interact with the translation initiation factor eIF3 to prevent full ribosomal recycling post their translation. Actually, this interaction turned out to be instrumental in stabilizing the mRNA·40S post-termination complex, which is thus capable to eventually resume scanning and reinitiate on the next AUG start site downstream. Recently, we also provided important in vivo evidence strongly supporting the long-standing idea that to stimulate reinitiation, eIF3 has to remain bound to ribosomes elongating these uORFs until their stop codon has been reached. Here we examined the importance of eIF3 and sequences flanking uORF1 of the human functional homolog of yeast GCN4, ATF4, in stimulation of efficient reinitiation. We revealed that the molecular basis of the reinitiation mechanism is conserved between yeasts and humans.
- MeSH
- eukaryotický iniciační faktor 3 chemie metabolismus MeSH
- iniciace translace peptidového řetězce * MeSH
- lidé MeSH
- messenger RNA genetika metabolismus MeSH
- otevřené čtecí rámce * MeSH
- proteosyntéza MeSH
- ribozomy metabolismus MeSH
- savci MeSH
- transkripční faktor ATF4 chemie metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Regeneration capacity is reduced as CNS axons mature. Using laser-mediated axotomy, proteomics and puromycin-based tagging of newly-synthesized proteins in a human embryonic stem cell-derived neuron culture system that allows isolation of axons from cell bodies, we show here that efficient regeneration in younger axons (d45 in culture) is associated with local axonal protein synthesis (local translation). Enhanced regeneration, promoted by co-culture with human glial precursor cells, is associated with increased axonal synthesis of proteins, including those constituting the translation machinery itself. Reduced regeneration, as occurs with the maturation of these axons by d65 in culture, correlates with reduced levels of axonal proteins involved in translation and an inability to respond by increased translation of regeneration promoting axonal mRNAs released from stress granules. Together, our results provide evidence that, as in development and in the PNS, local translation contributes to CNS axon regeneration.
- MeSH
- axony fyziologie MeSH
- embryonální kmenové buňky fyziologie MeSH
- kokultivační techniky MeSH
- lidé MeSH
- proteosyntéza fyziologie MeSH
- regenerace nervu fyziologie MeSH
- stárnutí buněk fyziologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Ribosome was long considered as a critical yet passive player in protein synthesis. Only recently the role of its basic components, ribosomal RNAs and proteins, in translational control has begun to emerge. Here we examined function of the small ribosomal protein uS3/Rps3, earlier shown to interact with eukaryotic translation initiation factor eIF3, in termination. We identified two residues in consecutive helices occurring in the mRNA entry pore, whose mutations to the opposite charge either reduced (K108E) or increased (R116D) stop codon readthrough. Whereas the latter increased overall levels of eIF3-containing terminating ribosomes in heavy polysomes in vivo indicating slower termination rates, the former specifically reduced eIF3 amounts in termination complexes. Combining these two mutations with the readthrough-reducing mutations at the extreme C-terminus of the a/Tif32 subunit of eIF3 either suppressed (R116D) or exacerbated (K108E) the readthrough phenotypes, and partially corrected or exacerbated the defects in the composition of termination complexes. In addition, we found that K108 affects efficiency of termination in the termination context-specific manner by promoting incorporation of readthrough-inducing tRNAs. Together with the multiple binding sites that we identified between these two proteins, we suggest that Rps3 and eIF3 closely co-operate to control translation termination and stop codon readthrough.
- MeSH
- eukaryotický iniciační faktor 3 genetika metabolismus MeSH
- geneticky modifikované organismy MeSH
- proteosyntéza genetika MeSH
- ribozomální proteiny genetika fyziologie MeSH
- ribozomy metabolismus MeSH
- RNA transferová metabolismus MeSH
- Saccharomyces cerevisiae - proteiny genetika fyziologie MeSH
- Saccharomyces cerevisiae genetika metabolismus MeSH
- terminace translace peptidového řetězce * genetika MeSH
- terminační kodon metabolismus MeSH
- vazba proteinů MeSH
- vazebná místa genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The growth rate hypothesis (GRH) predicts a positive correlation between growth rate and RNA content because growth depends upon the protein synthesis machinery. The application of this hypothesis to photoautotrophic organisms has been questioned. We tested the GRH on one prasinophycean, Tetraselmis suecica, and one chlorophycean, Dunaliella salina, grown at three sulphate concentrations. Sulphate was chosen because its concentration in the oceans increased through geological time and apparently had a role in the evolutionary trajectories of phytoplankton. Cell protein content and P quota were positively related to the RNA content (r = 0.62 and r = 0.74, respectively). The correlation of the RNA content with growth rates (r = 0.95) indicates that the GRH was valid for these species when growth rates were below 0.82 d(-1) .
- MeSH
- Chlorophyta růst a vývoj metabolismus MeSH
- dusík metabolismus MeSH
- mořská voda chemie MeSH
- oceány a moře MeSH
- proteosyntéza * MeSH
- ribozomy metabolismus MeSH
- RNA metabolismus MeSH
- sírany metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- oceány a moře MeSH
