The role of the CNOT1 subunit of the CCR4-NOT complex in mRNA deadenylation and cell viability
The role of the CNOT1 subunit of the CCR4-NOT complex in mRNA deadenylation and cell viability
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DOI：
10.1007/s13238-011-1092-4
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发表时间：
2011-09-01
期刊：
影响因子：
21.1
通讯作者：
Yamamoto, Tadashi
中科院分区：
文献类型：
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作者：
Ito, Kentaro;Takahashi, Akinori;Yamamoto, Tadashi
人类 CCR4-NOT 脱腺苷酶复合物由至少九个酶亚基和非酶亚基组成。越来越多的证据表明，非酶亚基参与 mRNA 脱腺苷化的调节，尽管它们的确切作用仍有待确定。在这项研究中，我们通过消除 HeLa 细胞中 CNOT1 亚基的表达来研究其功能。流式细胞术分析显示 CNOT1 耗尽的细胞中亚 G(1) 分数增加。实际上，当用针对所有酶亚基的 siRNA 混合物处理细胞时，可以看到相同水平的 sub G(1) 部分，这表明 CNOT1 耗尽通过破坏 CCR4-NOT 相关去腺苷酶活性来诱导细胞凋亡。进一步分析表明 CNOT1 耗尽会导致其他 CCR4-NOT 亚基数量减少。重要的是，来自 CNOT1 耗尽细胞的 CNOT6L 免疫沉淀相关去腺苷酸酶的比活性低于对照细胞的比活性。据报道，P 体的形成（据报道发生 mRNA 衰减）在 CNOT1 耗尽的细胞中很大程度上受到抑制。因此，CNOT1 在展示 CCR4-NOT 复合物的酶活性方面具有重要作用，因此对于控制 mRNA 脱腺苷化和 mRNA 衰减至关重要。我们进一步表明 CNOT1 耗竭增强了 CHOP mRNA 水平并激活了 caspase-4，这与内质网 ER 应激诱导的细胞凋亡有关。总而言之，CNOT1 的缺失在结构和功能上使 CCR4-NOT 复合物恶化并诱导 mRNA 稳定，从而导致翻译增加，从而导致 ER 应激介导的细胞凋亡。我们得出结论，CNOT1 通过确保 CCR4-NOT 脱腺苷酶的活性来促进细胞活力。
The human CCR4-NOT deadenylase complex consists of at least nine enzymatic and non-enzymatic subunits. Accumulating evidence suggests that the non-enzymatic subunits are involved in the regulation of mRNA deadenylation, although their precise roles remain to be established. In this study, we addressed the function of the CNOT1 subunit by depleting its expression in HeLa cells. Flow cytometric analysis revealed that the sub G(1) fraction was increased in CNOT1-depleted cells. Virtually, the same level of the sub G(1) fraction was seen when cells were treated with a mixture of siRNAs targeted against all enzymatic subunits, suggesting that CNOT1 depletion induces apoptosis by destroying the CCR4-NOT-associated deadenylase activity. Further analysis revealed that CNOT1 depletion leads to a reduction in the amount of other CCR4-NOT subunits. Importantly, the specific activity of the CNOT6L immunoprecipitates-associated deadenylase from CNOT1-depleted cells was less than that from control cells. The formation of P-bodies, where mRNA decay is reported to take place, was largely suppressed in CNOT1-depleted cells. Therefore, CNOT1 has an important role in exhibiting enzymatic activity of the CCR4-NOT complex, and thus is critical in control of mRNA deadenylation and mRNA decay. We further showed that CNOT1 depletion enhanced CHOP mRNA levels and activated caspase-4, which is associated with endoplasmic reticulum ER stress-induced apoptosis. Taken together, CNOT1 depletion structurally and functionally deteriorates the CCR4-NOT complex and induces stabilization of mRNAs, which results in the increment of translation causing ER stress-mediated apoptosis. We conclude that CNOT1 contributes to cell viability by securing the activity of the CCR4-NOT deadenylase.