Ribosome profiling reveals the rhythmic liver translatome and circadian clock regulation by upstream open reading frames

Mammalian gene expression displays widespread circadian oscillations. Rhythmic transcription underlies the core clock mechanism, but it cannot explain numerous observations made at the level of protein rhythmicity. We have used ribosome profiling in mouse liver to measure the translation of mRNAs into protein around-the-clock and at high temporal and nucleotide resolution. Transcriptome-wide, we discovered extensive rhythms in ribosome occupancy, and identified a core set of ~150 mRNAs subject to particularly robust daily changes in translation efficiency. Cycling proteins produced from non-oscillating transcripts revealed thus far unknown rhythmic regulation associated with specific pathways (notably in iron metabolism, through the rhythmic translation of transcripts containing iron responsive elements), and indicated feedback to the rhythmic transcriptome through novel rhythmic transcription factors. Moreover, estimates of relative levels of core clock protein biosynthesis that we deduced from the data explained known features of the circadian clock better than did mRNA expression alone. Finally, we identified uORF translation as a novel regulatory mechanism within the clock circuitry. Consistent with the occurrence of translated uORFs in several core clock transcripts, loss-of-function of Denr, a known regulator of re-initiation after uORF usage and of ribosome recycling, led to circadian period shortening in cells. In summary, our data offer a framework for understanding the dynamics of translational regulation, circadian gene expression, and metabolic control in a solid mammalian organ. A total of 48 mice were entrained under 12hours light:dark conditions for 2 weeks and also collected under 12hours light:dark. Mice were sacrificed every two hours during the 24 hours daily cycle. Two replicates per time point, each replicate is a pool of 2 livers.哺乳动物基因表达存在广泛的节律性振荡。节律性转录是核心生物钟机制的基础，但无法解释蛋白质节律层面的诸多观测结果。我们利用小鼠肝脏中的核糖体谱（ribosome profiling）技术，以高时间分辨率和核苷酸分辨率，全天候监测mRNA向蛋白质的翻译过程。在转录组范围内，我们发现核糖体占位存在广泛的节律性变化，并鉴定出约150个mRNA组成的核心集合，其翻译效率呈现尤为显著的每日波动。从非振荡转录本翻译产生的节律性蛋白，揭示了此前未知的、与特定通路相关的节律性调控机制（尤其在铁代谢通路中：通过含铁应答元件（iron responsive element, IRE）的转录本的节律性翻译实现），并表明可通过新型节律性转录因子对节律性转录组产生反馈调控。此外，我们从数据中推导得到的核心生物钟蛋白生物合成相对水平，相比单纯的mRNA表达水平，更能解释生物钟的已知特征。最后，我们鉴定出上游开放阅读框（uORF, upstream open reading frame）翻译是生物钟环路中的新型调控机制。鉴于多个核心生物钟转录本中存在翻译的uORF，对Denr（已知的uORF翻译后再起始及核糖体回收调控因子）进行功能缺失实验，可导致细胞的生物钟周期缩短。综上，本研究数据为理解实体哺乳动物器官中的翻译调控、节律性基因表达及代谢控制的动态过程提供了研究框架。共计48只小鼠在12小时光照:12小时黑暗的光周期条件下同步驯化2周，并于该光周期下进行采样。在每日24小时周期内，每2小时处死小鼠并采集样本；每个时间点设置2个生物学重复，每个重复由2只小鼠的肝脏混合制成。