CNOT1 regulates circadian behaviour through <i>Per2</i> mRNA decay in a deadenylation-dependent manner

Circadian clocks are an endogenous internal timekeeping mechanism that drives the rhythmic expression of genes, controlling the 24 h oscillatory pattern in behaviour and physiology. It has been recently shown that post-transcriptional mechanisms are essential for controlling rhythmic gene expression. Controlling the stability of mRNA through poly(A) tail length modulation is one such mechanism. In this study, we show that <i>Cnot1</i>, encoding the scaffold protein of the CCR4-NOT deadenylase complex, is highly expressed in the suprachiasmatic nucleus, the master timekeeper. CNOT1 deficiency in mice results in circadian period lengthening and alterations in the mRNA and protein expression patterns of various clock genes, mainly <i>Per2. Per2</i> mRNA exhibited a longer poly(A) tail and increased mRNA stability in <i>Cnot1^+/−</i> mice. CNOT1 is recruited to <i>Per2</i> mRNA through BRF1 (ZFP36L1), which itself oscillates in antiphase with <i>Per2</i> mRNA. Upon <i>Brf1</i> knockdown, <i>Per2</i> mRNA is stabilized leading to increased PER2 expression levels. This suggests that CNOT1 plays a role in tuning and regulating the mammalian circadian clock.昼夜节律钟（circadian clock）是一类内源性内在计时机制，可驱动基因的节律性表达，调控行为与生理过程中的24小时振荡模式。近期研究证实，转录后调控机制对于控制基因的节律性表达至关重要，其中通过调节多聚腺苷酸尾（poly(A) tail）长度以调控mRNA稳定性，便是此类机制之一。本研究表明，编码CCR4-NOT脱腺苷酶复合物支架蛋白的Cnot1基因，在作为主计时中枢的视交叉上核（suprachiasmatic nucleus）中呈高表达状态。小鼠体内CNOT1缺失会导致昼夜节律周期延长，并改变多种时钟基因的mRNA与蛋白质表达模式，其中以Per2基因的变化最为显著。在Cnot1杂合敲除（Cnot1^+/-）小鼠中，Per2 mRNA的多聚腺苷酸尾更长，且mRNA稳定性更高。CNOT1蛋白可通过BRF1（ZFP36L1）被招募至Per2 mRNA，而BRF1自身的表达振荡与Per2 mRNA呈反相模式。当敲低Brf1基因后，Per2 mRNA的稳定性得以提升，进而导致PER2蛋白的表达水平升高。上述结果表明，CNOT1在哺乳动物昼夜节律钟的校准与调控过程中发挥着关键作用。