Article

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

European Molecular Biology Laboratory, (EMBL), Heidelberg, Germany.
FEBS letters (Impact Factor: 3.34). 06/2011; 585(14):2182-6. DOI: 10.1016/j.febslet.2011.05.071
Source: PubMed

ABSTRACT 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.

Structured summary of protein interactions
CAF1 physically interacts with CCR4 and NOT1 by tandem affinity purification (View interaction)

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Available from: Bettina Böttcher, Aug 30, 2015
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    • "Architecture of the CCR4–NOT complex. (A,B) Model of the CCR4–NOT complex using X-ray crystal structures docked into a negative-stain EM map of the yeast CCR4–NOT complex (EMDB ID: 1901; Nasertorabi et al., 2011). Crystal structures of CCR4–NOT subunits were docked into the EM map using the UCSF Chimera software based on size and shape considerations and guided by the prediction of Nasertorabi and colleagues. "
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    ABSTRACT: The CCR4-NOT complex is a highly conserved, multifunctional machinery with a general role in controlling mRNA metabolism. It has been implicated in a number of different aspects of mRNA and protein expression, including mRNA degradation, transcription initiation and elongation, ubiquitination, and protein modification. The core CCR4-NOT complex is evolutionarily conserved and consists of at least three NOT proteins and two catalytic subunits. The L-shaped complex is characterized by two functional modules bound to the CNOT1/Not1 scaffold protein: the deadenylase or nuclease module containing two enzymes required for deadenylation, and the NOT module. In this review, we will summarize the currently available information regarding the three-dimensional structure and assembly of the CCR4-NOT complex, in order to provide insight into its roles in mRNA degradation and other biological processes.
    Frontiers in Genetics 05/2014; 5:137. DOI:10.3389/fgene.2014.00137
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    • "In Saccharomyces cerevisiae, two variant complexes with a distinct molecular weight of 1.0 and 1.9 MDa are present (Liu et al., 1997; Liu et al., 1998; Chen et al., 2001). The overall structure of the yeast 1.0 MDa complex is L-shaped with the Caf1 and Ccr4 components located in the hinge connecting the two arms (Nasertorabi et al., 2011). In Homo sapiens, the Ccr4-Not complex has an estimated molecular weight of 1.2 MDa. "
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    ABSTRACT: The shortening of the poly(A) tail of cytoplasmic mRNA (deadenylation) is a pivotal step in the regulation of gene expression in eukaryotic cells. Deadenylation impacts on both regulated mRNA decay as well as the rate of mRNA translation. An important enzyme complex involved in poly(A) shortening is the Ccr4-Not deadenylase. In addition to at least six non-catalytic subunits, it contains two distinct subunits with ribonuclease activity: a Caf1 subunit, characterized by a DEDD (Asp-Glu-Asp-Asp) domain, and a Ccr4 component containing an endonuclease-exonuclease-phosphatase (EEP) domain. In vertebrate cells, the complexity of the complex is further increased by the presence of paralogs of the Caf1 subunit (encoded by either CNOT7 or CNOT8) and the occurrence of two Ccr4 paralogs (encoded by CNOT6 or CNOT6L). In plants, there are also multiple Caf1 and Ccr4 paralogs. Thus, the composition of the Ccr4-Not complex is heterogeneous. The potential differences in the intrinsic enzymatic activities of the paralogs will be discussed. In addition, the potential redundancy, cooperation, and/or the extent of unique roles for the deadenylase subunits of the Ccr4-Not complex will be reviewed. Finally, novel approaches to study the catalytic roles of the Caf1 and Ccr4 subunits will be discussed.
    Frontiers in Genetics 12/2013; 4:296. DOI:10.3389/fgene.2013.00296
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    • "The presence of CNOT10 in organisms other than animals (Figure 3) indicates its loss in fungi and potential functional replacement with Caf130, for which no similar proteins outside the Saccharomycetes could be found. This might go in accordance with the differences observed in Not1; whilst the C-terminus, the binding site of Caf1, Ccr4, and Not2-5 (Basquin et al., 2012), is relatively conserved, the N-terminus - which harbors the Caf40 and CNOT10 /CNOT11, and possibly also the Caf130, binding sites (Nasertorabi et al., 2011; Bawankar et al., 2013) - is reduced in fungi compared to animals (Figure 4A). In the Candida group, the proteins lack approximately 180 amino acids at the N-terminal part compared to S. cerevisiae (Supplemental Figure 2 shows an alignment of Not1 sequences from S. cerevisiae, representative fungal pathogens and humans). "
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    ABSTRACT: The fungal Ccr4-NOT complex has been implicated in orchestrating gene expression networks that impact on pathways key for virulence in pathogenic species. The activity of Ccr4-NOT regulates cell wall integrity, antifungal drug susceptibility, adaptation to host temperature, and the developmental switches that enable the formation of pathogenic structures, such as filamentous hyphae. Moreover, Ccr4-NOT impacts on DNA repair pathways and genome stability, opening the possibility that this gene regulator could control adaptive responses in pathogens that are driven by chromosomal alterations. Here we provide a synthesis of the cellular roles of the fungal Ccr4-NOT, focusing on pathways important for virulence toward animals. Our review is based on studies in models yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, and two species that cause serious human infections, Candida albicans and Cryptococcus neoformans. We hypothesize that the activity of Ccr4-NOT could be targeted for future antifungal drug discovery, a proposition supported by the fact that inactivation of the genes encoding subunits of Ccr4-NOT in C. albicans and C. neoformans reduces virulence in the mouse infection model. We performed bioinformatics analysis to identify similarities and differences between Ccr4-NOT subunits in fungi and animals, and discuss this knowledge in the context of future antifungal strategies.
    Frontiers in Genetics 12/2013; 4:302. DOI:10.3389/fgene.2013.00302
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