Essential functions of the CNOT7/8 catalytic subunits of the CCR4-NOT complex in mRNA regulation and cell viability

Abstract

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.

The CCR4–NOT complex maintains liver homeostasis through mRNA deadenylation

“…Liver-specific disruption of Cnot1 causes lethal hepatitis associated with elongated mRNA poly(A) tails Suppression of CNOT1 largely abrogated deadenylase activity (Temme et al, 2010;Ito et al, 2011;Nousch et al, 2013;Mostafa et al, 2020), suggesting that CNOT1 is an essential scaffold subunit in the CCR4-NOT complex in vivo. We generated conditional KO mice for Cnot1 (Cnot1 fl/fl mice) by inserting loxP sequences into the Cnot1 gene locus so that exons 20 and 21 were deleted (Fig S1A and B).…”

“…In this study, we provide evidence that the CCR4-NOT deadenylase complex plays critical roles in liver homeostasis. Liver-specific disruption of Cnot1, which encodes a scaffold subunit of the complex, resulted in substantial elongation of bulk RNA poly(A) tails, as in the case of Drosophila S2 cells, Caenorhabditis elegans, and MEFs (Temme et al, 2010;Nousch et al, 2013;Mostafa et al, 2020), further indicating an essential role of CNOT1 in CCR4-NOT complex-mediated mRNA deadenylation in vivo. Consistent with previous reports that poly(A) tails stabilize mRNAs in eukaryotes (Dreyfus & Regnier, 2002;Weill et al, 2012), more than 80% of the mRNAs that we analyzed showed elongated half-lives in livers from Cnot1-LKO mice (Fig 7A).…”

Importance of CNOT8 Deadenylase Subunit in DNA Damage Responses Following Ionizing Radiation (IR)

“…Similarly, microarray analysis showed that CNOT7 and CNOT8, despite high homology, have specific substrates for their enzymatic activity (10,17). Some other studies have suggested, combined depletion of CNOT7 and CNOT8 subunits cause mRNA decay in human HTGM5 cells, reduce cell proliferation in MCF7 breast cancer cells and result in cell viability reduction and cell death in mouse embryonic fibroblasts (MEFs) (11,22,23). Accordingly, it has been suggested that alterations of CNOT7 and CNOT8 expression are relevant to the development and progression of cancer (17,23,24).…”

The Regulatory Properties of the Ccr4–Not Complex

“…For instance, CNOT7 null mice show defects in spermatogenesis, even in the presence of CNOT8, suggesting that CNOT7 and CNOT8 may not be entirely redundant in function [60,61]. In mammalian cells, the complex contains either CNOT7 or CNOT8, suggesting they compete for binding to CNOT1 [18,19].…”

“…These appear to form various heterodimers, CNOT7/CNOT6, CNOT7/CNOT6L, CNOT8/CNOT6, and CNOT8/CNOT6L. Thus, the complex contains either CNOT7 or CNOT8, suggesting they compete for binding to CNOT1 [18,19]. While the E3 ubiquitin ligase Not4 is consistently present in the yeast complex, CNOT4 is not as stably associated as the other subunits in mammalian cells [18].…”