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Humans and other commonly used model organisms are resistant to cycloheximide-mediated biases in ribosome profiling experiments.

ABSTRACT: Ribosome profiling measures genome-wide translation dynamics at sub-codon resolution. Cycloheximide (CHX), a widely used translation inhibitor to arrest ribosomes in these experiments, has been shown to induce biases in yeast, questioning its use. However, whether such biases are present in datasets of other organisms including humans is unknown. Here we compare different CHX-treatment conditions in human cells and yeast in parallel experiments using an optimized protocol. We find that human ribosomes are not susceptible to conformational restrictions by CHX, nor does it distort gene-level measurements of ribosome occupancy, measured decoding speed or the translational ramp. Furthermore, CHX-induced codon-specific biases on ribosome occupancy are not detectable in human cells or other model organisms. This shows that reported biases of CHX are species-specific and that CHX does not affect the outcome of ribosome profiling experiments in most settings. Our findings provide a solid framework to conduct and analyze ribosome profiling experiments.

SUBMITTER: Sharma P

PROVIDER: S-EPMC8384890 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Humans and other commonly used model organisms are resistant to cycloheximide-mediated biases in ribosome profiling experiments

Project description:Ribosome profiling measures genome-wide translation dynamics at sub-codon resolution. Cycloheximide (CHX), a widely used translation inhibitor to arrest ribosomes in these experiments, has been shown to induce biases in yeast, questioning its use. However, whether such biases are present in datasets of other organisms including humans is unknown. Here we compare different CHX-treatment conditions in human cells and yeast in parallel experiments using an optimized protocol. We find that human ribosomes are not susceptible to conformational restrictions by CHX, nor does it distort gene-level measurements of ribosome occupancy, measured decoding speed or the translational ramp. Furthermore, CHX-induced codon-specific biases on ribosome occupancy are not detectable in human cells or other model organisms. This shows that reported biases of CHX are species-specific and that CHX does not affect the outcome of ribosome profiling experiments in most settings. Our findings provide a solid framework to conduct and analyze ribosome profiling experiments.

2021-07-28 | GSE136940 | GEO

Humans and other commonly used model organisms are resistant to cycloheximide-mediated biases in ribosome profiling experiments

Project description:Humans and other commonly used model organisms are resistant to cycloheximide-mediated biases in ribosome profiling experiments

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Effects of cycloheximide on the interpretation of ribosome profiling experiments in Schizosaccharomyces pombe.

Project description:Stress conditions lead to global and gene-specific changes in RNA translation. Ribosome profiling experiments have identified genome-wide alterations in the distribution of ribosomes along mRNAs. However, it is contentious whether these changes reflect real responses, or whether they are artefacts caused by the use of inhibitors of translation (notably cycloheximide). To address this issue we performed ribosome profiling with the fission yeast Schizosaccharomyces pombe under conditions of exponential growth (unstressed) and nitrogen starvation (nutritional stress), and both in the presence and absence of cycloheximide. We examined several aspects of the translational response, including density of ribosomal footprints on coding sequences, 5' leader ribosomal densities, distribution of ribosomes along coding sequences, and ribosome codon occupancies. Cycloheximide had minor effects on overall ribosome density, which affected mostly mRNAs encoding ribosomal proteins. Nitrogen starvation caused an accumulation of ribosomes on 5' leaders in both cycloheximide-treated and untreated cells. By contrast, stress-induced ribosome accumulation on the 5' side of coding sequences was cycloheximide-dependent. Finally, codon occupancy showed strong positive correlations in cycloheximide-treated and untreated cells. Our results demonstrate that cycloheximide does influence some of the results of ribosome profiling experiments, although it is not clear if this effect is always artefactual.

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Understanding Biases in Ribosome Profiling Experiments Reveals Signatures of Translation Dynamics in Yeast.

Project description:Ribosome profiling produces snapshots of the locations of actively translating ribosomes on messenger RNAs. These snapshots can be used to make inferences about translation dynamics. Recent ribosome profiling studies in yeast, however, have reached contradictory conclusions regarding the average translation rate of each codon. Some experiments have used cycloheximide (CHX) to stabilize ribosomes before measuring their positions, and these studies all counterintuitively report a weak negative correlation between the translation rate of a codon and the abundance of its cognate tRNA. In contrast, some experiments performed without CHX report strong positive correlations. To explain this contradiction, we identify unexpected patterns in ribosome density downstream of each type of codon in experiments that use CHX. These patterns are evidence that elongation continues to occur in the presence of CHX but with dramatically altered codon-specific elongation rates. The measured positions of ribosomes in these experiments therefore do not reflect the amounts of time ribosomes spend at each position in vivo. These results suggest that conclusions from experiments in yeast using CHX may need reexamination. In particular, we show that in all such experiments, codons decoded by less abundant tRNAs were in fact being translated more slowly before the addition of CHX disrupted these dynamics.

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Nuclease-mediated depletion biases in ribosome footprint profiling libraries.

Project description:Ribosome footprint profiling is a high-throughput sequencing-based technique that provides detailed and global views of translation in living cells. An essential part of this technology is removal of unwanted, normally very abundant, ribosomal RNA sequences that dominate libraries and increase sequencing costs. The most effective commercial solution (Ribo-Zero) has been discontinued as a standalone product and a number of new, experimentally distinct commercial applications have emerged on the market. Here we evaluated several commercially available alternatives designed for RNA-seq of human samples and find them generally unsuitable for ribosome footprint profiling. We instead recommend the use of custom-designed biotinylated oligos, which were widely used in early ribosome profiling studies. Importantly, we warn that depletion solutions based on targeted nuclease cleavage significantly perturb the high-resolution information that can be derived from the data, and thus do not recommend their use for any applications that require precise determination of the ends of RNA fragments.

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Effects of cycloheximide (CHX) on ribosome profiling experiments in Schizosaccharomyces pombe

Project description:CHX is an inhibitor of translation elongation often used in ribosome profiling experiments. There is evidence that CHX treatment of cells may cause artefacts in the distribution of ribosomes on mRNAs. We investigate this possibility in S. pombe by performing ribosome profiling in the presence and absence of this drug.

2017-09-05 | E-MTAB-5794 | biostudies-arrayexpress

Transcriptomic profiling of 39 commonly-used neuroblastoma cell lines.

Project description:Neuroblastoma cell lines are an important and cost-effective model used to study oncogenic drivers of the disease. While many of these cell lines have been previously characterized with SNP, methylation, and/or mRNA expression microarrays, there has not been an effort to comprehensively sequence these cell lines. Here, we present raw whole transcriptome data generated by RNA sequencing of 39 commonly-used neuroblastoma cell lines. These data can be used to perform differential expression analysis based on a genetic aberration or phenotype in neuroblastoma (e.g., MYCN amplification status, ALK mutation status, chromosome arm 1p, 11q and/or 17q status, sensitivity to pharmacologic perturbation). Additionally, we designed this experiment to enable structural variant and/or long-noncoding RNA analysis across these cell lines. Finally, as more DNase/ATAC and histone/transcription factor ChIP sequencing is performed in these cell lines, our RNA-Seq data will be an important complement to inform transcriptional targets as well as regulatory (enhancer or repressor) elements in neuroblastoma.

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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 widespread protein synthesis regulation under stress conditions. Our study highlights the need for better standardization of ribosome profiling methods.

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Corrigendum: Transcriptomic profiling of 39 commonly-used neuroblastoma cell lines.

Project description:This corrects the article DOI: 10.1038/sdata.2017.33.

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Effects of cycloheximide (CHX) on ribosome profiling experiments in Schizosaccharomyces pombe

Project description:Effects of cycloheximide (CHX) on ribosome profiling experiments in Schizosaccharomyces pombe

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