Abstract
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.
Highlights
Ribosome profiling has revolutionized the study of translation by providing a genome-wide, single-nucleotide resolution view of this process[1]
S. cerevisiae cells display an asymmetric ribosome distribution across coding sequences, with a broad peak of higher ribosome occupancy in the initial ~300–400 nucleotides of the coding sequence[1, 9, 14, 20] that is strongly enhanced by different stresses[1, 8, 12]. We investigated this phenomenon in S. pombe in two ways: first, by calculating the ratio between footprints in nucleotides 10 to 400 and 401 to 800 (Fig. 3a–c, the first 9 nucleotides were not considered to avoid biases created by the accumulation of ribosomes at initiating AUG); second, by examining the behaviour of a metagene representing genome-wide ribosome density along coding sequences (Fig. 3d and Supplementary Fig. S5)
We report that in S. pombe CHX has only minor effects in overall ribosome density in coding sequences (>95% of genes are unaffected, Fig. 1), to the situation in mammalian cells
Summary
Ribosome profiling has revolutionized the study of translation by providing a genome-wide, single-nucleotide resolution view of this process[1]. The key assumption of ribosome profiling is that the distribution of ribosomes on mRNAs at the time of RNase digestion faithfully reproduces their location in vivo To ensure that this is the case, numerous studies have used inhibitors of translation elongation, typically cycloheximide (CHX), to ‘freeze’ ribosomes in their in vivo distribution[6, 7]. The increases caused by oxidative stress, heat shock and amino acid starvation were found to be CHX-dependent[8] This suggested a model in which intermediate concentrations of the drug are slow to act (possibly due to limiting diffusion); as translation initiation is not inhibited, newly-initiating ribosomes would continue translating until they encounter the drug, artefactually increasing ribosome density in the first few hundred nucleotides of the coding sequence[8]. A broad peak of ribosome density at the 5′ of coding sequences is clearly present in unstressed cells that have not been treated with CHX1, 14, it is only apparent when cells are flash-frozen[1]
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