TY - JOUR
T1 - Essential functions of the CNOT7/8 catalytic subunits of the CCR4-NOT complex in mRNA regulation and cell viability
AU - Mostafa, Dina
AU - Takahashi, Akinori
AU - Yanagiya, Akiko
AU - Yamaguchi, Tomokazu
AU - Abe, Takaya
AU - Kureha, Taku
AU - Kuba, Keiji
AU - Kanegae, Yumi
AU - Furuta, Yasuhide
AU - Yamamoto, Tadashi
AU - Suzuki, Toru
N1 - Funding Information:
This work was supported by the Japan Society for the Promotion of Science [21229006]; Japan Society for the Promotion of Science [18K06975]; Japan Society for the Promotion of Science [17K07292]; Japan Society for the Promotion of Science [18K07079]. We thank J Miyazaki for the CAG-Cre mice and T Fujiwara for providing the CNOT7 mutant cDNAs. We also thank the DNA Sequencing Section in Okinawa Institute of Science and Technology Graduate University (OIST) for preparing sequence libraries and for performing Hiseq sequencing.
Publisher Copyright:
© 2020, © 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
PY - 2020/3/3
Y1 - 2020/3/3
N2 - 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.
AB - 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.
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U2 - 10.1080/15476286.2019.1709747
DO - 10.1080/15476286.2019.1709747
M3 - Article
C2 - 31924127
AN - SCOPUS:85078625776
SN - 1547-6286
VL - 17
SP - 403
EP - 416
JO - RNA Biology
JF - RNA Biology
IS - 3
ER -