The role of the CNOT1 subunit of the CCR4-NOT complex in mRNA deadenylation and cell viability

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 G₁ fraction was increased in CNOT1-depleted cells. Virtually, the same level of the sub G₁ 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.     

The CCR4-NOT complex is implicated in the viability of aneuploid yeasts

To identify the genes required to sustain aneuploid viability, we screened a deletion library of non-essential genes in the fission yeast Schizosaccharomyces pombe, in which most types of aneuploidy are eventually lethal to the cell. Aneuploids remain viable for a period of time and can form colonies by reducing the extent of the aneuploidy. We hypothesized that a reduction in colony formation efficiency could be used to screen for gene deletions that compromise aneuploid viability. Deletion mutants were used to measure the effects on the viability of spores derived from triploid meiosis and from a chromosome instability mutant. We found that the CCR4-NOT complex, an evolutionarily conserved general regulator of mRNA turnover, and other related factors, including poly(A)-specific nuclease for mRNA decay, are involved in aneuploid viability. Defective mutations in CCR4-NOT complex components in the distantly related yeast Saccharomyces cerevisiae also affected the viability of spores produced from triploid cells, suggesting that this complex has a conserved role in aneuploids. In addition, our findings suggest that the genes required for homologous recombination repair are important for aneuploid viability.     

Insights into the structure of the CCR4-NOT complex by electron microscopy

The CCR4-NOT complex is a deadenylation complex, which plays a major role for mRNA stability. The complex is conserved from yeast to human and consists of nine proteins NOT1-NOT5, CCR4, CAF1, CAF40 and CAF130. We have successfully isolated the complex using a Protein A tag on NOT1, followed by cross-linking on a glycerol gradient. All components of the complex were identified by mass spectrometry. Electron microscopy of negatively stained particles followed by image reconstruction revealed an L-shaped complex with two arms of similar length. The arms form an accessible cavity, which we think could provide an extensive interface for RNA-deadenylation.     

Distinct expression patterns of the subunits of the CCR4-NOT deadenylase complex during neural development

The stability of mRNA influences the dynamics of gene expression. The mammalian CCR4–NOT complex is associated with deadenylase activity, which shortens the mRNA poly(A) tail and thereby contributes to destabilization of mRNAs. The complex consists of at least nine subunits and predominantly forms a 2.0 MDa protein complex in HeLa cells. Accumulating evidence suggests that the CCR4–NOT complex is involved in cell growth and survival; however, the regulatory mechanisms of its biological activity remain obscure. Here, we analyzed the expression levels of the subunits of the CCR4–NOT complex in various mouse tissues and found that they showed distinct expression patterns. CNOT6, 6L, 7, and 10 were expressed nearly ubiquitously, whereas others were expressed in tissue-specific manners, such as those displaying especially high expression in the brain. Furthermore, CNOT2, 3, 6, and 8 were rapidly downregulated during differentiation of neural stem cells. These findings suggest that subunit composition of the CCR4–NOT complex differs among tissues and is altered during neural development, thereby imparting an additional layer of specificity in the control of gene expression.     

Localization of the Rsp5p ubiquitin-protein ligase at multiple sites within the endocytic pathway

The Saccharomyces cerevisiae RSP5 gene encodes an essential HECT E3 ubiquitin-protein ligase. Rsp5p contains an N-terminal C2 domain, three WW domains in the central portion of the molecule, and a C-terminal catalytic HECT domain. A diverse group of substrates of Rsp5p and vertebrate C2 WW-domain-containing HECT E3s have been identified, including both nuclear and membrane-associated proteins. We determined the intracellular localization of Rsp5p and the determinants necessary for localization, in order to better understand how Rsp5p activities are coordinated. Using both green fluorescent protein fusions to Rsp5p and immunogold electron microscopy, we found that Rsp5p was distributed in a punctate pattern at the plasma membrane, corresponding to membrane invaginations that are likely sites of endosome formation, as well as at perivacuolar sites. The latter appeared to correspond to endocytic intermediates, as these structures were not seen in a sla2/end4-1 mutant, and double-immunogold labeling demonstrated colocalization of Rsp5p with the endosomal markers Pep12p and Vps32p. The C2 domain was an important determinant of localization; however, mutations that disrupted HECT domain function also caused mislocalization of Rsp5p, indicating that enzymatic activity is linked to localization. Deletion of the C2 domain partially stabilized Fur4p, a protein previously shown to undergo Rsp5p- and ubiquitin-mediated endocytosis; however, Fur4p was still ubiquitinated at the plasma membrane when the C2 domain was deleted from the protein. Together, these results indicate that Rsp5p is located at multiple sites within the endocytic pathway and suggest that Rsp5p may function at multiple steps in the ubiquitin-mediated endocytosis pathway.     

Interaction between NANOS2 and the CCR4-NOT deadenylation complex is essential for male germ cell development in mouse

Nanos is one of the evolutionarily conserved proteins implicated in germ cell development and we have previously shown that it interacts with the CCR4-NOT deadenylation complex leading to the suppression of specific RNAs. However, the molecular mechanism and physiological significance of this interaction have remained elusive. In our present study, we identify CNOT1, a component of the CCR4-NOT deadenylation complex, as a direct factor mediating the interaction with NANOS2. We find that the first 10 amino acids (AAs) of NANOS2 are required for this binding. We further observe that a NANOS2 mutant lacking these first 10 AAs (NANOS2-ΔN10) fails to rescue defects in the Nanos2-null mouse. Our current data thus indicate that the interaction with the CCR4-NOT deadenylation complex is essential for NANOS2 function. In addition, we further demonstrate that NANOS2-ΔN10 can associate with specific mRNAs as well as wild-type NANOS2, suggesting the existence of other NANOS2-associated factor(s) that determine the specificity of RNA-binding independently of the CCR4-NOT deadenylation complex.