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.
Highlights
Ribosome profiling measures genome-wide translation dynamics at sub-codon resolution
We found that CHX-mediated biases related to rare codons are absent from commonly studied model organisms including two yeast species. These findings show that the effects of CHX are species-specific and do not affect the measurements of translation dynamics in many commonly used model organisms except for baker’s yeast
To comprehensively analyze the effect of CHX on human cells, we focused on HEK 293T cells as a well-established model in ribosome profiling
Summary
Ribosome profiling measures genome-wide translation dynamics at sub-codon resolution. Ribosome profiling studies have reached different conclusions on, e.g., the identification of ORFs in zebrafish[9,10,15], the influence of wobble base-pairing on elongation speed[16,17], or the relationship between elongation rates and tRNA abundance[13,18] These discrepancies are in part caused by adaptations to the original protocols to match the needs of individual laboratories[4,10,19,20,21,22]. CHX appears to reversibly interact with ribosomes, allowing them to move away from their initial position on mRNA33 This movement depends on codon identity, altering the outcomes of studies that require codon-level resolution for occupancy and translation-speed measurements[33]. High concentrations of CHX inhibit elongation, but not initiation[29] leading to ribosome accumulation at the start codon following CHX pretreatment[5]
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