The Ccr4a (CNOT6) and Ccr4b (CNOT6L) deadenylase subunits of the human Ccr4-Not complex contribute to the prevention of cell death and senescence

A key step in cytoplasmic mRNA degradation is the shortening of the poly(A) tail, which involves several deadenylase enzymes. Relatively little is known about the importance of these enzymes for the cellular physiology. Here we focused on the role of the highly similar Ccr4a (CNOT6) and Ccr4b (CNOT6L) deadenylase subunits of the Ccr4-Not complex. In addition to a role in cell proliferation, Ccr4a and Ccr4b play a role in cell survival, in contrast to the Caf1a (CNOT7) and Caf1b (CNOT8) deadenylase subunits or the CNOT1 and CNOT3 noncatalytic subunits of the Ccr4-Not complex. Underscoring the differential contributions of the deadenylase subunits, we found that knockdown of Caf1a/Caf1b or Ccr4a/Ccr4b differentially affects the formation of cytoplasmic foci by processing-body components. Furthermore, we demonstrated that the amino-terminal leucine-rich repeat (LRR) domain of Ccr4b influenced its subcellular localization but was not required for the deadenylase activity of Ccr4b. Moreover, overexpression of Ccr4b lacking the LRR domain interfered with cell cycle progression but not with cell viability. Finally, gene expression profiling indicated that distinct gene sets are regulated by Caf1a/Caf1b and Ccr4a/Ccr4b and identified Ccr4a/Ccr4b as a key regulator of insulin-like growth factor-binding protein 5, which mediates cell cycle arrest and senescence via a p53-dependent pathway.     

The Anti-Proliferative Activity of BTG/TOB Proteins Is Mediated via the Caf1a (CNOT7) and Caf1b (CNOT8) Deadenylase Subunits of the Ccr4-Not Complex

The human BTG/TOB protein family comprises six members (BTG1, BTG2/PC3/Tis21, BTG3/Ana, BTG4/PC3B, TOB1/Tob, and TOB2) that are characterised by a conserved BTG domain. This domain mediates interactions with the highly similar Caf1a (CNOT7) and Caf1b (CNOT8) catalytic subunits of the Ccr4-Not deadenylase complex. BTG/TOB proteins have anti-proliferative activity: knockdown of BTG/TOB can result in increased cell proliferation, whereas over-expression of BTG/TOB leads to inhibition of cell cycle progression. It was unclear whether the interaction between BTG/TOB proteins and the Caf1a/Caf1b deadenylases is necessary for the anti-proliferative activity of BTG/TOB. To address this question, we further characterised surface-exposed amino acid residues of BTG2 and TOB1 that mediate the interaction with the Caf1a and Caf1b deadenylase enzymes. We then analysed the role of BTG2 and TOB1 in the regulation of cell proliferation, translation and mRNA abundance using a mutant that is no longer able to interact with the Caf1a/Caf1b deadenylases. We conclude that the anti-proliferative activity of BTG/TOB proteins is mediated through interactions with the Caf1a and Caf1b deadenylase enzymes. Furthermore, we show that the activity of BTG/TOB proteins in the regulation of mRNA abundance and translation is dependent on Caf1a/Caf1b, and does not appear to require other Ccr4-Not components, including the Ccr4a (CNOT6)/Ccr4b (CNOT6L) deadenylases, or the non-catalytic subunits CNOT1 or CNOT3.     

An unconventional human Ccr4-Caf1 deadenylase complex in nuclear cajal bodies

mRNA deadenylation is a key process in the regulation of translation and mRNA turnover. In Saccharomyces cerevisiae, deadenylation is primarily carried out by the Ccr4p and Caf1p/Pop2p subunits of the Ccr4-Not complex, which is conserved in eukaryotes including humans. Here we have identified an unconventional human Ccr4-Caf1 complex containing hCcr4d and hCaf1z, distant human homologs of yeast Ccr4p and Caf1p/Pop2p, respectively. The hCcr4d-hCaf1z complex differs from conventional Ccr4-Not deadenylase complexes, because (i) hCaf1z and hCcr4d concentrate in nuclear Cajal bodies and shuttle between the nucleus and cytoplasm and (ii) the hCaf1z subunit, in addition to rapid deadenylation, subjects substrate RNAs to slow exonucleolytic degradation from the 3' end in vitro. Exogenously expressed hCaf1z shows both of those activities on reporter mRNAs in human HeLa cells and stimulates general mRNA decay when restricted to the cytoplasm by deletion of its nuclear localization signal. These observations suggest that the hCcr4d-hCaf1z complex may function either in the nucleus or in the cytoplasm after its nuclear export, to degrade polyadenylated RNAs, such as mRNAs, pre-mRNAs, or those RNAs that are polyadenylated prior to their degradation in the nucleus.     

The Not4 E3 Ligase and CCR4 Deadenylase Play Distinct Roles in Protein Quality Control

Eukaryotic cells control their proteome by regulating protein production and protein clearance. Protein production is determined to a large extent by mRNA levels, whereas protein degradation depends mostly upon the proteasome. Dysfunction of the proteasome leads to the accumulation of non-functional proteins that can aggregate, be toxic for the cell, and, in extreme cases, lead to cell death. mRNA levels are controlled by their rates of synthesis and degradation. Recent evidence indicates that these rates have oppositely co-evolved to ensure appropriate mRNA levels. This opposite co-evolution has been correlated with the mutations in the Ccr4-Not complex. Consistently, the deadenylation enzymes responsible for the rate-limiting step in eukaryotic mRNA degradation, Caf1 and Ccr4, are subunits of the Ccr4-Not complex. Another subunit of this complex is a RING E3 ligase, Not4. It is essential for cellular protein solubility and has been proposed to be involved in co-translational quality control. An open question has been whether this role of Not4 resides strictly in the regulation of the deadenylation module of the Ccr4-Not complex. However, Not4 is important for proper assembly of the proteasome, and the Ccr4-Not complex may have multiple functional modules that participate in protein quality control in different ways. In this work we studied how the functions of the Caf1/Ccr4 and Not4 modules are connected. We concluded that Not4 plays a role in protein quality control independently of the Ccr4 deadenylase, and that it is involved in clearance of aberrant proteins at least in part via the proteasome.     

Structural Model of the Human BTG2–PABPC1 Complex by Combining Mutagenesis,NMR Chemical Shift Perturbation Data and Molecular Docking

Degradation of cytoplasmic mRNA in eukaryotes involves the shortening and removal of the mRNA poly(A) tail by poly(A)-selective ribonuclease (deadenylase) enzymes. In human cells, BTG2 can stimulate deadenylation of poly(A) bound by cytoplasmic poly(A)-binding protein PABPC1. This involves the concurrent binding by BTG2 of PABPC1 and the Caf1/CNOT7 nuclease subunit of the Ccr4-Not deadenylase complex. To understand in molecular detail how PABPC1 and BTG2 interact, we set out to identify amino acid residues of PABPC1 and BTG2 contributing to the interaction. To this end, we first used algorithms to predict PABPC1 interaction surfaces. Comparison of the predicted interaction surface with known residues involved in the binding to poly(A) resulted in the identification of a putative interaction surface for BTG2. Subsequently, we used pulldown assays to confirm the requirement of PABPC1 residues for the interaction with BTG2. Analysis of RNA-binding by PABPC1 variants indicated that PABPC1 residues required for interaction with BTG2 do not interfere with poly(A) binding. After further defining residues of BTG2 that are required for the interaction with PABPC1, we used information from published NMR chemical shift perturbation experiments to guide docking and generate a structural model of the BTG2-PABPC1 complex. A quaternary poly(A)-PABPC1-BTG2-Caf1/CNOT7 model showed that the 3′ end of poly(A) RNA is directed towards the catalytic centre of Caf1/CNOT7, thereby providing a rationale for enhanced deadenylation by Caf1/CNOT7 in the presence of BTG2 and PABPC1.     

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.     

Specific roles for the Ccr4-Not complex subunits in expression of the genome

In this work we used micro-array experiments to determine the role of each nonessential subunit of the conserved Ccr4-Not complex in the control of gene expression in the yeast Saccharomyces cerevisiae. The study was performed with cells growing exponentially in high glucose and with cells grown to glucose depletion. Specific patterns of gene deregulation were observed upon deletion of any given subunit, revealing the specificity of each subunit''s function. Consistently, the purification of the Ccr4-Not complex through Caf40p by tandem affinity purification from wild-type cells or cells lacking individual subunits of the Ccr4-Not complex revealed that each subunit had a particular impact on complex integrity. Furthermore, the micro-arrays revealed that the role of each subunit was specific to the growth conditions. From the study of only two different growth conditions, revealing an impact of the Ccr4-Not complex on more than 85% of all studied genes, we can infer that the Ccr4-Not complex is important for expression of most of the yeast genome.     

RNA decay machines: Deadenylation by the Ccr4–Not and Pan2–Pan3 complexes

Shortening and removal of the 3′ poly(A) tail of mature mRNA by poly(A)-specific 3′ exonucleases (deadenylases) is the initial and often rate-limiting step in mRNA degradation. The majority of cytoplasmic deadenylase activity is associated with the Ccr4–Not and Pan2–Pan3 complexes. Two distinct catalytic subunits, Caf1/Pop2 and Ccr4, are associated with the Ccr4–Not complex, whereas the Pan2 enzymatic subunit forms a stable complex with Pan3. In this review, we discuss the composition and activity of these two deadenylases. In addition, we comment on generic and specific mechanisms of recruitment of Ccr4–Not and Pan2–Pan3 to mRNAs. Finally, we discuss specialised and redundant functions of the deadenylases and review the importance of Ccr4–Not subunits in the regulation of physiological processes. This article is part of a Special Issue entitled: RNA Decay mechanisms.