Ribosome queuing enables non-AUG translation to be resistant to multiple protein synthesis inhibitors
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ABSTRACT:
PROVIDER: PRJNA515197 | ENA |
REPOSITORIES: ENA
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Project description:Aberrant translation initiation at non-AUG start codons is associated with multiple cancers and neurodegenerative diseases. Nevertheless, how non-AUG translation is regulated differently from canonical translation is poorly understood. We thus used start codon-selective reporters and ribosome profiling to characterize how translation from non-AUG start codons responds to protein synthesis inhibitors in human cells. These analyses surprisingly revealed that translation of non-AUG reporters and the endogenous GUG-encoded DAP5 (eIF4G2/p97) mRNA are resistant to cycloheximide (CHX), a translation inhibitor which slows but does not completely abrogate elongation. Our data suggest that slowly elongating ribosomes cause queuing of scanning pre-initiation complexes (PIC), preferentially enhancing otherwise poor recognition of non-AUG start codons. Consistent with this model, limiting PIC formation or scanning sensitizes non-AUG translation to CHX. Moreover, PIC queuing can cause translation from an AUG codon in a poor context to become less sensitive to CHX. We further find that non-AUG translation is resistant to other inhibitors that target ribosomes within the coding sequence, but not those targeting newly initiated ribosomes. In total, these data indicate that ribosome queuing enables mRNAs with poor initiation context, namely those from non-AUG start codons, to be resistant to pharmacological inhibitors.
2019-06-05 | GSE125086 | GEO
Project description: Not available
| S-EPMC6601509 | biostudies-literature
Project description:Techniques for systematically monitoring protein translation have lagged far behind methods for measuring mRNA levels. Here we present a ribosome profiling strategy, based on deep sequencing of ribosome protected mRNA fragments, that enables genome-wide investigation of translation with sub-codon resolution. We used this technique to monitor translation in budding yeast under both rich and starvation conditions. These studies defined the protein sequences being translated and found extensive translational control both for determining absolute protein abundance and for responding to environmental stress. We also observed distinct phases during translation involving a large decrease in ribosome density going from early to late peptide elongation as well as wide-spread, regulated initiation at non-AUG codons. Ribosome profiling is readily adaptable to other organisms, making high-precision investigation of protein translation experimentally accessible. Examine replicates of ribosome footprints and mRNA abundance in biological replicates of log-phase growth and acute amino acid starvation
2009-02-12 | E-GEOD-13750 | biostudies-arrayexpress
Project description:Upon the environmental changes, cells flexibly and rapidly alter the gene expression by the translation control. In plants, the translation of NIP5;1, a boric acid diffusion facilitator, is upregulated through the upstream open reading frame (uORF), which comprises only AUG and stop codons, in response to the shortage of boric acid. However, the molecular details of how the minimum uORF controls the translation of downstream main ORF depending on the boric acid concentration remained unclear. Here we showed that the 80S ribosome (80S) assembled at AUG-stop migrates into the subsequent RNA segment for downstream translation initiation and that the presence of boric acid impedes the process by stable confinement of eukaryotic release factor 1 (eRF1) on 80S on AUG-stop. Ribosome profiling along the in vitro-translated reporter mRNA revealed that the number of the ribosomes on AUG-stop and that along the downstream are anti-correlated in response to boric acid, reflecting the 80S scanning leaving AUG-stop for translation initiation from downstream ORF. Moreover, cryo-electron microscopy (cryo-EM) analysis revealed that, in the presence of boric acid, the initiator methionyl-tRNA (Met-tRNAi) in the P site stably contacts eRF1 in the A site on AUG-stop. In contrast, the absence of boric acid led to weaker eRF1 density, suggesting that eRF1 cannot be stably positioned without boric acid. Consistent with the structural features, the hydrolysis of Met-tRNAi on AUG-stop was accelerated by boric acid, which is likely to be the checkpoint for the subsequent 80S scanning of the downstream. Our results provide a molecular insight into the translation regulation by a minimum and environment-responsive uORF.
2023-09-20 | GSE189222 | GEO
Project description:Ribosome profiling and high-throughput sequencing provide unprecedented opportunities for the analysis of mRNA translation. Using this novel method, several studies have demonstrated the widespread role of short upstream reading frames in translational control as well as slower elongation at the beginning of open reading frames in response to stress. Based on the initial studies, the importance of adding or omitting translation inhibitors, such as cycloheximide, was noted as it markedly affected ribosome coverage profiles. For that reason, many recent studies omitted translation inhibitors in the culture medium. Here, we investigate the influence of ranging cycloheximide concentrations on ribosome profiles in Saccharomyces cerevisiae and demonstrate that increasing the drug concentration can overcome some of the artifacts. We subjected cells to various manipulations and show that neither oxidative stress nor heat shock nor amino acid starvation affect translation elongation. Instead, the observations in the initial studies are the result of cycloheximide-inflicted artifacts. Likewise, we find little support for short upstream reading frames to be involved in wide-spread protein synthesis regulation under stress conditions. Our study highlights the need for better standardization of ribosome profiling methods. Ranging concentrations of cycloheximide and various stress contitions were tested with Ribo-seq
2014-08-01 | E-GEOD-59573 | biostudies-arrayexpress
Project description:In response to environmental changes, cells flexibly and rapidly alter gene expression, through translational controls. In plants, the translation of NIP5;1, a boric acid diffusion facilitator, is upregulated through upstream open reading frames (uORFs) that comprise only AUG and stop codons, in response to a shortage of boric acid in the environment. However, the molecular details of how the minimum uORF controls the translation of the downstream main ORF, in a boric acid concentration-dependent manner, have remained unclear. Here, combining ribosome profiling, TCP-Seq, structural analysis with cryo-electron microscopy, and biochemical assays, we showed that the 80S ribosome (80S) assembled at AUG-stop migrates into the subsequent RNA segment, followed by downstream translation initiation, and that boric acid impedes this process by the stable confinement of eukaryotic release factor 1 (eRF1) on the 80S on AUG-stop. Our results provide molecular insight into translation regulation by a minimum and environment-responsive uORF.
2023-09-19 | GSE223755 | GEO
Project description:Genomic analyses in budding yeast have helped to define the foundational principles of eukaryotic gene expression, but have systematically excluded specific classes of potential coding regions, including those with non-AUG start codons. Without methods to define coding regions empirically, the prevalence of these non-canonical coding regions has been impossible to assess. Here, we applied an experimental approach to globally annotate translation initiation sites in yeast and identified a class of 149 genes that encode N-terminally extended alternate protein isoforms that result from translation initiation at non-AUG codons upstream of the annotated AUG start codon. These alternate isoforms are produced in concert with canonical isoforms and are translated with a high degree of specificity, resulting from initiation at only a small subset of possible start codons in 5’ leader regions. Their translation is enriched during meiosis, and is induced by low eIF5A levels, which are observed in this context. These findings reveal widespread production of non-canonical protein isoforms and, more generally, show unexpected complexity to the rules by which the budding yeast genome is decoded.
2020-07-24 | GSE150375 | GEO
Project description:Techniques for systematically monitoring protein translation have lagged far behind methods for measuring mRNA levels. Here we present a ribosome profiling strategy, based on deep sequencing of ribosome protected mRNA fragments, that enables genome-wide investigation of translation with sub-codon resolution. We used this technique to monitor translation in budding yeast under both rich and starvation conditions. These studies defined the protein sequences being translated and found extensive translational control both for determining absolute protein abundance and for responding to environmental stress. We also observed distinct phases during translation involving a large decrease in ribosome density going from early to late peptide elongation as well as wide-spread, regulated initiation at non-AUG codons. Ribosome profiling is readily adaptable to other organisms, making high-precision investigation of protein translation experimentally accessible.
2009-02-12 | GSE13750 | GEO
Project description:Ribosome profiling and high-throughput sequencing provide unprecedented opportunities for the analysis of mRNA translation. Using this novel method, several studies have demonstrated the widespread role of short upstream reading frames in translational control as well as slower elongation at the beginning of open reading frames in response to stress. Based on the initial studies, the importance of adding or omitting translation inhibitors, such as cycloheximide, was noted as it markedly affected ribosome coverage profiles. For that reason, many recent studies omitted translation inhibitors in the culture medium. Here, we investigate the influence of ranging cycloheximide concentrations on ribosome profiles in Saccharomyces cerevisiae and demonstrate that increasing the drug concentration can overcome some of the artifacts. We subjected cells to various manipulations and show that neither oxidative stress nor heat shock nor amino acid starvation affect translation elongation. Instead, the observations in the initial studies are the result of cycloheximide-inflicted artifacts. Likewise, we find little support for short upstream reading frames to be involved in wide-spread protein synthesis regulation under stress conditions. Our study highlights the need for better standardization of ribosome profiling methods.
2014-08-01 | GSE59573 | GEO
Project description:Ribosome profiling with translation inhibitors reveals pervasive translation in murine ES cells. Ribosome profiling or stranded RNAseq (ribominus) with murine embryonic stem cells treated with either DMD-pateamineA, Puromycin, Harringtonine or vehicle (no drug control). Two replicates per condition.
2016-01-12 | E-GEOD-67741 | biostudies-arrayexpress