Essential functions of the CNOT7/8 catalytic subunits of the CCR4-NOT complex in mRNA regulation and cell viability
Essential functions of the CNOT7/8 catalytic subunits of the CCR4-NOT complex in mRNA regulation and cell viability
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DOI：
10.1080/15476286.2019.1709747
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发表时间：
2020-01-13
期刊：
影响因子：
4.1
通讯作者：
Suzuki, Toru
中科院分区：
文献类型：
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作者：
Mostafa, Dina;Takahashi, Akinori;Suzuki, Toru
缩短mRNA poly（A）尾（去腺苷酸化）以触发其衰变主要由CCR 4-NOT去腺苷酶复合物介导。虽然已经在哺乳动物CCR 4-NOT复合物中鉴定了四个催化亚基（hocT 6、6L 7和8），但它们各自的生物学作用尚未完全了解。在这项研究中，我们解决了hocT 7/8的原代小鼠胚胎成纤维细胞（MEFs）的活力的贡献。我们发现缺乏hocT 7/8表达的MEF [Cnot 7/8-双敲除（dKO）MEF]经历细胞死亡，而缺乏hocT 6/6L表达的MEF（Cnot 6/6l-dKO MEF）保持存活。免疫共沉淀分析显示，在Cnot 7/8-dKO MEFs中，hocT 6/6L也不存在于CCR 4-NOT复合物中。相比之下，hocT 7或hocT 8仍然与Cnot 6/61-dKO MEF中的CCR 4-NOT复合物中的其他亚基相互作用。在Cnot 7/8-dKO MEFs中缺乏催化活性的hocT 7突变体的外源表达不能恢复细胞活力，即使hocT 6/6L在一定程度上存在于CCR 4-NOT复合物中，证实hocT 7/8对于活力是必需的。大量聚腺苷酸尾分析显示，具有较长聚腺苷酸尾的mRNA在Cnot 7/8-dKO MEF中比在Cnot 6/61-dKO MEF中更多。与延长的poly（A）尾一致，在Cnot 7/8-dKO MEF中比在Cnot 6/61-dKO MEF中更多的mRNA被上调和稳定。重要的是，Cnot 6/6l-dKO小鼠是可行的，并正常生长至成年。总的来说，hocT 7/8催化亚基对于去腺苷化是必需的，这是维持细胞活力所必需的，而hocT 6/6L不是。
Shortening of mRNA poly(A) tails (deadenylation) to trigger their decay is mediated mainly by the CCR4-NOT deadenylase complex. While four catalytic subunits (CNOT6, 6L 7, and 8) have been identified in the mammalian CCR4-NOT complex, their individual biological roles are not fully understood. In this study, we addressed the contribution of CNOT7/8 to viability of primary mouse embryonic fibroblasts (MEFs). We found that MEFs lacking CNOT7/8 expression [Cnot7/8-double knockout (dKO) MEFs] undergo cell death, whereas MEFs lacking CNOT6/6L expression (Cnot6/6l-dKO MEFs) remain viable. Co-immunoprecipitation analyses showed that CNOT6/6L are also absent from the CCR4-NOT complex in Cnot7/8-dKO MEFs. In contrast, either CNOT7 or CNOT8 still interacts with other subunits in the CCR4-NOT complex in Cnot6/6l-dKO MEFs. Exogenous expression of a CNOT7 mutant lacking catalytic activity in Cnot7/8-dKO MEFs cannot recover cell viability, even though CNOT6/6L exists to some extent in the CCR4-NOT complex, confirming that CNOT7/8 is essential for viability. Bulk poly(A) tail analysis revealed that mRNAs with longer poly(A) tails are more numerous in Cnot7/8-dKO MEFs than in Cnot6/6l-dKO MEFs. Consistent with elongated poly(A) tails, more mRNAs are upregulated and stabilized in Cnot7/8-dKO MEFs than in Cnot6/6l-dKO MEFs. Importantly, Cnot6/6l-dKO mice are viable and grow normally to adulthood. Taken together, the CNOT7/8 catalytic subunits are essential for deadenylation, which is necessary to maintain cell viability, whereas CNOT6/6L are not.