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

Abstract

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.

Transcriptome-wide Analysis of Roles for tRNA Modifications in Translational Regulation

“…Wild-type and mutant strains were grown to mid-log phase in rich media and processed for ribosome profiling, with biological duplicates for each strain. As the use of the translation inhibitor cycloheximide can affect the distribution of ribosomes across open reading frames (Gerashchenko and Gladyshev, 2014; Hussmann et al, 2015), we also generated replicate datasets for six mutants collected without the use of cycloheximide. Tables S1–S4 provide complete datasets for mRNA, ribosome occupancy, and translational efficiency, as well as the six no cycloheximide replicates.…”

Ribosome Footprint Profiling of Translation throughout the Genome

“…If the drug does not block initiation, ribosomes will accumulate particularly at start codons (Ingolia et al, 2011). Reversible inhibitors, such as cycloheximide, seem to allow slow, concentration-dependent elongation prior to lysis (Gerashchenko and Gladyshev, 2014; Hussmann et al, 2015). Collectively, these effects can distort codon-level ribosome profiles substantially.…”

“…Fortunately, these drug effects do not impact expression measurements, which rely only on transcript-level ribosome occupancy (Ingolia et al, 2011; Weinberg et al, 2016). Cycloheximide does not create or destroy ribosome footprints in the middle of a reading frame—it merely redistributes them (Hussmann et al, 2015). Many studies employ inhibitor-free lysis to avoid this redistribution (Lareau et al, 2014; Weinberg et al, 2016).…”

“…Such experiments may depend on the capture and profiling of specific ribosome sub-populations, where conclusions may depend on the quality of the purification. More broadly, answering these questions relies on interpreting codon- and nucleotide-level details of ribosome occupancy profiles, which are particularly sensitive to perturbations during harvesting (Gerashchenko and Gladyshev, 2014; Hussmann et al, 2015) and sequence-dependent biases in library generation (Artieri and Fraser, 2014). Analyses can be hampered by the small absolute number of footprints at individual positions and confounded by correlations such as the link between codon usage bias and expression.…”

“…These diverse analytical approaches have reached differing conclusions about the impact of codon and amino acid usage on translation speed. Estimates of per-codon elongation are particularly sensitive to the impact of cycloheximide and presumably to other perturbations (Hussmann et al, 2015). Actual differences in ribosome occupancy profiles driven by such artifacts may underlie much of the disagreement between studies, and resolving these disagreements requires some undisputable signature of slow elongation.…”