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

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.Author Summary: Ribosome profiling measures the precise locations of millions of actively translating ribosomes on mRNAs. In theory, the frequency with which ribosomes are observed positioned over each type of codon can be used to quantify the speed with which each codon is translated. In practice, ribosome profiling experiments in yeast that use translation inhibitors to arrest translation before measuring the positions of ribosomes report very different apparent translation speeds for each codon than experiments that do not use inhibitors. To explain this inconsistency, we show that a previously unappreciated mechanism causes experiments using translation inhibitors to not measure ribosomes at each position on mRNAs in proportion to the actual amount of time spent there in vivo. Understanding this mechanism reveals that experiments without inhibitors more accurately measure translation dynamics and provides guidance for the design and interpretation of future ribosome profiling experiments.