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Translation Inhibitors Cause Abnormalities in Ribosome Profiling Experiments

ABSTRACT: 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

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Vadim Gladyshev

PROVIDER: E-GEOD-59573 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Translation Inhibitors Cause Abnormalities in Ribosome Profiling Experiments

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

Ribosome Profiling Reveals Pervasive Translation Outside of Annotated Protein-Coding Genes

Project description:Ribosome profiling suggests that ribosomes occupy many regions of the transcriptome thought to be non-coding, including 5' UTRs and lncRNAs. Apparent ribosome footprints outside of protein-coding regions raise the possibility of artifacts unrelated to translation, particularly when they occupy multiple, overlapping open reading frames (ORFs). Here we show hallmarks of translation in these footprints: co-purification with the large ribosomal subunit, response to drugs targeting elongation, trinucleotide periodicity, and initiation at early AUGs. We develop a metric for distinguishing between 80S footprints and nonribosomal sources using footprint size distributions, which validates the vast majority of footprints outside of coding regions. We present evidence for polypeptide production beyond annotated genes, including induction of immune responses following human cytomegalovirus (HCMV) infection. Translation is pervasive on cytosolic transcripts outside of conserved reading frames, and direct detection of this expanded universe of translated products enables efforts to understand how cells manage and exploit its consequences. Ribosome profiling to verify that true ribosome footprints shift in response to different elongation inhibitors (CHX vs Emetine) and co-purify with an affinity-tagged large ribosomal subunit (bound vs input)

2014-08-21 | E-GEOD-60095 | biostudies-arrayexpress

Ribosome Profiling Reveals Pervasive Translation Outside of Annotated Protein-Coding Genes

2014-08-21 | GSE60095 | GEO

Ribosome profiling of Japanese encephalitis virus infected Neuro2a cells

Project description:Ribosome profiling (Ribo-Seq) and RNA-Seq analysis of mouse neuroblastoma (Neuro2a) cells infected with Japanese Encephalitis Virus (JEV) P20778 Vellore strain. At 18 h post infection (p.i.), infected cells were treated with either cycloheximide (translation elongation inhibitor) or in combination with harringtonine (translation initiation inhibitor). Ribo-Seq libraries were prepared using a broad range of fragment lengths (25 to 70 nucleotides) and deep sequenced. This allowed for estimation of ribosomal frameshifting efficiency and discovery of a novel upstream open reading frame (uORF) in JEV along with perturbations in ribosome-associated tRNA levels upon JEV infection.

2021-07-05 | E-MTAB-9352 | biostudies-arrayexpress

Ribosome Profiling of Mouse Embryonic Stem Cells Reveals the Complexity of Mammalian Proteomes

Project description:The ability to sequence genomes has far outstripped approaches for deciphering the information they encode. Here we present a suite of techniques, based on ribosome profiling (the deep-sequencing of ribosome-protected mRNA fragments), to provide genome-wide maps of protein synthesis as well as a pulse-chase strategy for determining rates of translation elongation. We exploit the propensity of harringtonine to cause ribosomes to accumulate at sites of translation initiation together with a machine learning algorithm to define protein products systematically. Analysis of translation in mouse embryonic stem cells reveals thousands of strong pause sites and novel translation products. These include amino-terminal extensions and truncations and upstream open reading frames with regulatory potential, initiated at both AUG and non-AUG codons, whose translation changes after differentiation. We also define a new class of short, polycistronic ribosome-associated coding RNAs (sprcRNAs) that encode small proteins. Our studies reveal an unanticipated complexity to mammalian proteomes. Examination of translation in mouse embryonic stem cells and during differentiation into embryoid bodies

2011-11-03 | E-GEOD-30839 | biostudies-arrayexpress

Dynamic O-GlcNAcylation of eIF3a controls translation reinitiation during integrated stress response

Project description:In eukaryotic cells, many mRNAs possess upstream open reading frames (uORFs) in addition to the main coding region. After uORF translation, the ribosome could either recycle at the stop codon or resume scanning for downstream start codons in a process known as reinitiation. Accumulating evidence suggests that some initiation factors, including eIF3, linger on the early elongating ribosome, forming an eIF3-80S complex. Very little is known how the eIF3 is carried along with the 80S during elongation and its implications in subsequent translation reinitiation. Here we report that eIF3a undergoes dynamic O-GlcNAc modification in response to nutrient starvation. Stress-induced de-O-GlcNAcylation promotes eIF3 retention on the elongating ribosome and facilitates reinitiation at downstream start codons. Eliminating the modification site from eIF3a via genome editing induces ATF4 reinitiation under the nutrient rich condition. Our findings illustrate a mechanism in balancing ribosome recycling and reinitiation, thereby linking integrated stress response and translational reprogramming.

2021-11-01 | GSE181040 | GEO

Time resolved ribosome profiling study of oxygen and glucose deprivation of rat pheochromocytoma cells

Project description:Oxygen and glucose metabolism plays a pivotal role in many (patho)physiological conditions. In particular, oxygen and glucose deprivation (OGD) occurs during ischemia and stroke, resulting in extensive tissue injury and cell death. We applied time-resolved ribosome profiling technique to assess early events at the level of gene expression in rat pheochromocytoma PC12 cells during short-term OGD. Most substantial alterations in transcripts levels and their translation were seen to occur in the first 20 minutes of OGD. The rapid adaptation of translation apparatus to OGD is global and involves altered elongation and initiation rates. We also observed salient and reproducible alterations in ribosome densities of individual mRNAs such as increased translation of particular upstream Open Reading Frames (uORFs); induced site-specific arrests of the ribosomes and synthesis of extended protein isoforms. Ribosome profiling (with mRNA-seq sequencing) was carried out at 0,20,40 and 60 minutes of OGD. Two biological replicates were used.

2015-04-10 | E-GEOD-60752 | biostudies-arrayexpress

Ribosome Profiling of Mouse Embryonic Stem Cells Reveals the Complexity of Mammalian Proteomes

2011-11-03 | GSE30839 | GEO

Start codon-associated ribosomal frameshifting mediates nutrient stress adaptation

Project description:A translating ribosome is typically thought to follow the reading frame defined by the selected start codon. Using super-resolution ribosome profiling, here we report pervasive out-of-frame translation from the start codon. Unlike programmable frameshifting during elongation, start codon-associated ribosome frameshifting (SCARF) stems from the slippage of the initiating ribosome. Using a massively paralleled reporter assay, we uncovered sequence elements acting as SCARF enhancers or repressors, implying that start codon recognition is coupled with reading frame fidelity. This finding explains thousands of mass spectrometry spectra unannotated from human proteome. Mechanistically, we find that the eukaryotic initiation factor 5B (eIF5B) maintains the reading frame fidelity during the transition from initiation to elongation. Intriguingly, amino acid starvation induces SCARF by proteasomal degradation of eIF5B. The stress-induced SCARF products provide a degradative source to mitigate amino acid scarcity during starvation. Our findings illustrate the beneficial effect of divergent translation in nutrient stress adaptation.

2023-03-01 | GSE184825 | GEO

Protein synthesis rates and ribosome occupancies reveal determinants of translation elongation rates

Project description:Although protein synthesis dynamics has been studied both with theoretical models and by profiling ribosome footprints, the determinants of ribosome flux along open reading frames (ORFs) are not fully understood. Combining measurements of protein synthesis rate with ribosome footprinting data, we here inferred translation initiation and elongation rates for over a thousand ORFs in exponentially-growing wildtype yeast cells. We found that the amino acid composition of synthesized proteins is as important a determinant of translation elongation rate as parameters related to codon and tRNA adaptation. We did not find evidence of ribosome collisions curbing the protein output of yeast transcripts, either in high translation conditions associated with exponential growth, or in strains in which deletion of individual ribosomal protein genes leads to globally increased or decreased translation. Slow translation elongation is characteristic of RP-encoding transcripts, which have markedly lower protein output compared to other transcripts with equally high ribosome densities.

2019-09-10 | PXD014357 | Pride

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