Laboratory for Immunogenetics, Center for Integrative Medical Sciences, Riken, Yokohama, Japan.
Cell Signal Unit, Okinawa Institute of Science and Technology Graduate University, Onna, Japan.
RNA Biol. 2022;19(1):234-246. doi: 10.1080/15476286.2021.2021676. Epub 2021 Dec 31.
CCR4-NOT complex-mediated mRNA deadenylation serves critical functions in multiple biological processes, yet how this activity is regulated is not fully understood. Here, we show that osmotic stress induces MAPKAPK-2 (MK2)-mediated phosphorylation of CNOT2. Programmed cell death is greatly enhanced by osmotic stress in CNOT2-depleted cells, indicating that CNOT2 is responsible for stress resistance of cells. Although wild-type (WT) and non-phosphorylatable CNOT2 mutants reverse this sensitivity, a phosphomimetic form of CNOT2, in which serine at the phosphorylation site is replaced with glutamate, does not have this function. We also show that mRNAs have elongated poly(A) tails in CNOT2-depleted cells and that introduction of CNOT2 WT or a non-phosphorylatable mutant, but not phosphomimetic CNOT2, renders their poly(A) tail lengths comparable to those in control HeLa cells. Consistent with this, the CCR4-NOT complex containing phosphomimetic CNOT2 exhibits less deadenylase activity than that containing CNOT2 WT. These data suggest that CCR4-NOT complex deadenylase activity is regulated by post-translational modification, yielding dynamic control of mRNA deadenylation.
CCR4-NOT 复合物介导的 mRNA 去腺苷酸化在多种生物过程中发挥着关键作用，但这种活性是如何被调节的还不完全清楚。在这里，我们显示渗透压应激诱导 MAPKAPK-2 (MK2) 介导的 CNOT2 磷酸化。在 CNOT2 耗尽的细胞中，渗透压应激大大增强了程序性细胞死亡，表明 CNOT2 负责细胞的应激抗性。尽管野生型 (WT) 和非磷酸化的 CNOT2 突变体逆转了这种敏感性，但在磷酸化位点的丝氨酸被谷氨酸取代的磷酸模拟形式的 CNOT2 没有这种功能。我们还表明，在 CNOT2 耗尽的细胞中，mRNA 具有延长的 poly(A) 尾巴，并且引入 CNOT2 WT 或非磷酸化突变体，但不是磷酸模拟 CNOT2，可使它们的 poly(A) 尾巴长度与对照 HeLa 细胞相当。与此一致的是，含有磷酸模拟 CNOT2 的 CCR4-NOT 复合物表现出比含有 CNOT2 WT 的复合物更低的脱腺苷酸酶活性。这些数据表明，CCR4-NOT 复合物脱腺苷酸酶活性受翻译后修饰调节，从而对 mRNA 去腺苷酸化进行动态控制。
