Ccr4p is the catalytic subunit of a Ccr4p/Pop2p/Notp mRNA deadenylase complex in Saccharomyces cerevisiae

The major pathways of mRNA turnover in eukaryotic cells are initiated by shortening of the poly(A) tail. Recent work has identified Ccr4p and Pop2p as components of the major cytoplasmic deadenylase in yeast. We now demonstrate that CCR4 encodes the catalytic subunit of the deadenylase and that Pop2p is dispensable for catalysis. In addition, we demonstrate that at least some of the Ccr4p/Pop2p-associated Not proteins are cytoplasmic, and lesions in some of the NOT genes can lead to defects in mRNA deadenylation rates. The Ccr4p deadenylase is inhibited in vitro by addition of the poly(A) binding protein (Pab1p), suggesting that dissociation of Pab1p from the poly(A) tail may be rate limiting for deadenylation in vivo. In addition, the rapid deadenylation of the COX17 mRNA, which is controlled by a member of the Pumilio family of deadenylation activators Puf3p, requires an active Ccr4p/Pop2p/Not deadenylase. These results define the Ccr4p/Pop2p/Not complex as the cytoplasmic deadenylase in yeast and identify positive and negative regulators of this enzyme complex.     

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 Ccr4--not complex

The Ccr4-Not complex is a unique, essential and conserved multi-subunit complex that acts at the level of many different cellular functions to regulate gene expression. Two enzymatic activities, namely ubiquitination and deadenylation, are provided by different subunits of the complex. However, studies over the last decade have demonstrated a tantalizing multi-functionality of this complex that extends well beyond its identified enzymatic activities. Most of our initial knowledge about the Ccr4-Not complex stemmed from studies in yeast, but an increasing number of reports on this complex in other species are emerging.In this review we will discuss the structure and composition of the complex, and describe the different cellular functions with which the Ccr4-Not complex has been connected in different organisms. Finally, based upon our current state of knowledge, we will propose a model to explain how one complex can provide such multi-functionality. This model suggests that the Ccr4-Not complex might function as a “chaperone platform”.     

RNA-binding protein Khd1 and Ccr4 deadenylase play overlapping roles in the cell wall integrity pathway in Saccharomyces cerevisiae

The Saccharomyces cerevisiae RNA-binding protein Khd1/Hek2 associates with hundreds of potential mRNA targets preferentially, including the mRNAs encoding proteins localized to the cell wall and plasma membrane. We have previously revealed that Khd1 positively regulates expression of MTL1 mRNA encoding a membrane sensor in the cell wall integrity (CWI) pathway. However, a khd1Δ mutation has no detectable phenotype on cell wall synthesis. Here we show that the khd1Δ mutation causes a severe cell lysis when combined with the deletion of the CCR4 gene encoding a cytoplasmic deadenylase. We identified the ROM2 mRNA, encoding a guanine nucleotide exchange factor (GEF) for Rho1, as a target for Khd1 and Ccr4. The ROM2 mRNA level was decreased in the khd1Δ ccr4Δ mutant, and ROM2 overexpression suppressed the cell lysis of the khd1Δ ccr4Δ mutant. We also found that Ccr4 negatively regulates expression of the LRG1 mRNA encoding a GTPase-activating protein (GAP) for Rho1. The LRG1 mRNA level was increased in the ccr4Δ and khd1Δ ccr4Δ mutants, and deletion of LRG1 suppressed the cell lysis of the khd1Δ ccr4Δ mutant. Our results presented here suggest that Khd1 and Ccr4 modulate a signal from Rho1 in the CWI pathway by regulating the expression of RhoGEF and RhoGAP.     

Deadenylation of cytoplasmic mRNA by the mammalian Ccr4-Not complex

The Ccr4-Not complex is one of the major deadenylase factors present in eukaryotic cells. This multi-subunit protein complex is composed of at least seven stably associated subunits in mammalian cells including two enzymatic deadenylase subunits: one DEDD (Asp-Glu-Asp-Asp)-type deadenylase (either CNOT7/human Caf1/Caf1a or CNOT8/human Pop2/Caf1b/Calif) and one EEP (endonuclease-exonuclease-phosphatase)-type enzyme (either CNOT6/human Ccr4/Ccr4a or CNOT6L/human Ccr4-like/Ccr4b). Here, the role of the human Ccr4-Not complex in cytoplasmic deadenylation of mRNA is discussed, including the mechanism of its recruitment to mRNA and the role of the BTG/Tob proteins.     

The yeast Ccr4-Not complex controls ubiquitination of the nascent-associated polypeptide (NAC-EGD) complex

In this work, we determine that the Saccharomyces cerevisiae Ccr4-Not complex controls ubiquitination of the conserved ribosome-associated heterodimeric EGD (enhancer of Gal4p DNA binding) complex, which consists of the Egd1p and Egd2p subunits in yeast and is named NAC (nascent polypeptide-associated complex) in mammals. We show that the EGD complex subunits are ubiquitinated proteins, whose ubiquitination status is regulated during cell growth. Egd2p has a UBA domain that is not essential for interaction with Egd1p but is required for stability of Egd2p and Egd1p. Ubiquitination of Egd1p requires Not4p. Ubiquitination of Egd2p also requires Not4p, an intact Not4p RING finger domain, and all other subunits of the Ccr4-Not complex tested. In the absence of Not4p, Egd2p mislocalizes to punctuate structures. Finally, the EGD complex can be ubiquitinated in vitro by Not4p and Ubc4p, one of the E2 enzymes with which Not4p can interact. Taken together our results reveal that the EGD ribosome-associated complex is ubiquitinated in a regulated manner, and they show a new role for the Ccr4-Not complex in this ubiquitination.