The translation inhibitor cycloheximide affects ribosome profiling data in a species-specific manner

Abstract

Ribosome profiling provides genome-wide snapshots of translation dynamics by determining ribosomal positions at sub-codon resolution. To maintain this positional information, the translation inhibitor cycloheximide (CHX) has been widely used to arrest translating ribosomes prior to library preparation. Several studies have reported CHX-induced biases in yeast data casting uncertainty about its continued use and questioning the accuracy of many ribosome profiling studies. However, the presence of these biases has not been investigated comprehensively in organisms other than Saccharomyces cerevisiae. Here, we use a highly standardized and optimized protocol to compare different CHX-treatment conditions in yeast and human cells. Our data suggest that unlike in S. cerevisiae, translating ribosomes in human cells are not susceptible to conformational restrictions by CHX. Furthermore, CHX-induced codon-specific effects on ribosome occupancy are not detectable in human cells nor in other model organisms including Schizosaccharomyces pombe and Candida albicans. In fact, we find that even in S. cerevisiae most biases can be avoided by omitting CHX pre-treatment, indicating that other parameters of library generation contribute to differences between ribosome profiling experiments. Together our findings provide a framework for researchers who plan their own ribosome profiling experiments or who analyze published datasets to draw judicious conclusions.

Optimization of Ribosome Footprinting Conditions for Ribo-Seq in Human and Drosophila melanogaster Tissue Culture Cells

“…However, it seems likely that cycloheximide treatment has less of an effect in humans and some other organisms, compared to yeast and fly, not impacting transcript-specific ribosome occupancy ( Sharma et al, 2021 ). Both the results presented here in Drosophila and other studies have shown that cycloheximide treatment does not affect either footprint size distribution or framing ( Sharma et al, 2019 ). Snap freezing material is an alternative to ‘trapping’ ribosomes during elongation using cycloheximde, which does not seem to affect framing, read length or distribution.…”

“…Metagene analysis revealed that cycloheximide treatment had a limited effect on footprint length or periodicity with Buffer 1, A-RNaseI (comparing Supplementary Figure S4A and Figure 3A ) and Buffer 2 E-RNAseI (comparing Supplementary Figure S4B and Figure 2B ). The distribution of reads across CDSs is affected by cycloheximide treatment, as previously described in other organisms ( Duncan and Mata, 2017 ; Gerashchenko and Gladyshev, 2014 ; Hussmann et al, 2015 ; Sharma et al, 2019 ). Specifically, cycloheximide treatment results in a build-up of Ribo-Seq reads at the start codon and in the first ∼15 nt of CDSs ( Figure 2C ).…”

Multifaceted deregulation of gene expression and protein synthesis with age

“…Flash-freezing and rapid thawing followed by disruption in the presence of high concentrations of CHX allowed to minimize artifacts associated with the CHX treatment (56). We note that no CHX-induced artifacts were reported for animal tissue samples (57). After homogenization and centrifugation, 250 μL of lysate were supplied with 20 units of SUPERase-In RNase inhibitor and taken for RNA-seq library preparation, and the 500 μL of lysate was brought to 1 mL with lysis buffer and taken for Ribo-seq library preparation.…”

Nuclease-mediated depletion biases in ribosome footprint profiling libraries

“…Other groups have had success using double-strand specific DNase, combined with a high-temperature re-annealing of the final DNA library that only leaves the most abundant sequences (the rRNA fragments) doublestranded (Chung et al 2015), but this method has not to our knowledge been tested on a broad range of fragment lengths, nor with improved library preparation methods such as randomized unique molecular identifiers (McGlincy and Ingolia 2017). Recent reports indicate that optimization of RNase digestion conditions (Sharma et al 2019) as well as judicious choice of RNase enzyme (Gerashchenko and Gladyshev 2017) can also reduce the rRNA content of ribosome profiling libraries, though many procedures using these different nucleases obscure the high resolution information on ribosome position and A site status. Finally, it is important to note that off-target RNaseH activity will not only affect the conclusions of ribosome footprint profiling experiments, but of any protocol that requires precise knowledge of the 5′ and 3′ ends of RNA fragments.…”