Understanding Biases in Ribosome Profiling Experiments Reveals Signatures of Translation Dynamics in Yeast
Understanding Biases in Ribosome Profiling Experiments Reveals Signatures of Translation Dynamics in Yeast
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DOI：
10.1371/journal.pgen.1005732
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发表时间：
2015-12-01
期刊：
影响因子：
4.5
通讯作者：
Press, William H.
中科院分区：
文献类型：
--
作者：
Hussmann, Jeffrey A.;Patchett, Stephanie;Press, William H.
核糖体分析可生成信使 RNA 上活跃翻译核糖体位置的快照。这些快照可用于对翻译动态进行推断。然而，最近的酵母核糖体分析研究对于每个密码子的平均翻译率得出了相互矛盾的结论。一些实验在测量核糖体位置之前使用放线菌酮 (CHX) 来稳定核糖体，这些研究都违反直觉地报告了密码子翻译率与其同源 tRNA 丰度之间存在弱负相关性。相反，一些没有 CHX 进行的实验报告了很强的正相关性。为了解释这一矛盾，我们在使用 CHX 的实验中发现了每种密码子下游核糖体密度的意外模式。这些模式证明，在 CHX 存在的情况下，延伸继续发生，但密码子特异性延伸率显着改变。因此，这些实验中测量的核糖体位置并不反映核糖体在体内每个位置花费的时间量。这些结果表明，使用 CHX 进行酵母实验得出的结论可能需要重新检验。特别是，我们表明，在所有此类实验中，在添加 CHX 破坏这些动态之前，由含量较低的 tRNA 解码的密码子实际上翻译得更慢。
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.