The Eukaryotic Ccr4-Not Complex: A Regulatory Platform Integrating mRNA Metabolism with Cellular Signaling Pathways?

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Introduction

Ccr4-Not complexes are multi-subunit protein complexes, which are conserved from yeast to human. These complexes exist in several forms larger than 1 MDa and consist of at least nine core subunits (Ccr4p, Caf1p, Caf40p, Caf130p, Not1-5p in yeast, and CNOT1-10 in humans; see Table I). They play essential roles in the control of gene expression. Genes encoding the core subunits were first revealed by genetic studies of transcription in yeast. In particular, two different approaches, one that led to the identification of Ccr4p and one that led to the identification of the Not proteins, each suggested the existence of a large multisubunit complex. The specificities of the defined complexes were that the former was necessary for nonfermentative gene expression (1, 2, 3), while the latter repressed transcription from promoters that lacked a canonical TATA sequence preferentially (4, 5, 6). Purification of Ccr4p revealed a large number of associated proteins (7), whereas genetic interactions, phenotypic similarities, co-immunoprecipitation, and co-elution from gel filtration columns suggested that the Not proteins were in a large complex (4, 5, 6). It was in 1998 that the group of Clyde Denis finally demonstrated that the Ccr4 and Not complexes were one and the same (8).

Until 2001, namely, three years after the description of the Ccr4-Not complex and many years after the first genetic isolation of its constituents, the Ccr4-Not complex remained a molecular mystery. For a long time, it was considered to be a global transcriptional regulatory complex affecting genes both positively and negatively. Gradually, evidence linking the Not proteins to TFIID and Mediator function and, thus, to transcriptional regulation, accumulated. The link between Ccr4-Not and TFIID complexes was reinforced in 2001 by the finding that Not5p is required for the appropriate recruitment of TAF1 to promoter DNA (9). In the same year, Parker and colleagues discovered that the Ccr4p and Caf1p proteins are the major yeast deadenylase (10), thus attributing an enzymatic activity to the Ccr4-Not complex. However, this activity, surprisingly, contributes to mRNA decay rather than mRNA synthesis and takes place not in the nucleus but in specialized bodies in the cytoplasm (11). While this biochemical activity of Ccr4p and Caf1p has now been defined in quite some detail (10, 12, 13, 14), involvement of the Not proteins in the deadenylation process has not really been demonstrated. Instead, most studies have converged toward models in which Ccr4p and Caf1p, on one hand, and the Not proteins on the other, might be contributing different functions to the cell. In this model, the Ccr4-Not complex might be composed of two functional modules, which are linked to different aspects of mRNA metabolism. In 2002, this picture was modified by the finding that CNOT4, the human ortholog of Not4p, is an ubiquitin E3 ligase (15). This implied involvement in proteasomal protein-degradation pathways. Thus, two distinct enzymatic activities, contributing to very distinct cellular processes, mRNA deadenylation and protein ubiquitylation, are associated within the Ccr4-Not complex. In addition, other subunits, in particular Not2p and Not5p, have very specific interactions with the general transcription factor TFIID 9, 16, 17.

As the story unfolds, the role of the Ccr4-Not complex is extending to other cellular machines. The Dhh1p RNA helicase within the decapping complex is associated with Not1p and is apparently controlled by the Ccr4-Not complex. In addition, the posttranslational modifications of the general stress transcription factor, Msn2p, and its transcriptional activation capacity, is controlled by the Ccr4-Not complex, at least in part through the interaction of the complex with a phosphatase (Collart, unpublished observations). Many other proteins (such as a subunit of the nascent-polypeptide associated complex (18)) involved in other cellular functions have been found to interact with Ccr4-Not complex subunits. However, functional control of these proteins by the Ccr4-Not complex remains to be studied. The emerging picture for the Ccr4-Not complex is that of a regulatory platform, consisting of different functional modules controlling different cellular machines. At present, the exact role of each individual subunit, the dynamics of the interaction between the different subunits, and the reason for the association of all of these subunits in a complex, as well as the control of these dynamics by the status of the cell, remain to be characterized.

In efforts to reconcile genetic identification and interactions with the recent biochemical findings, two reviews on Ccr4-Not proteins appeared last year. These reviews emphasized the original genetic studies (19) or the control of mRNA metabolism by yeast Ccr4-Not subunits (18). In this chapter, we expand this by integrating findings in other eukaryotic systems and by extending the role of Ccr4-Not proteins to other cellular machines and pathways.

Section snippets

The Ccr4-Not Complex: Conserved in Composition and Organization

The Ccr4-Not complex was first identified in yeast cell extracts, in which it forms protein complexes of ∼1.0 MDa and ∼1.9 MDa, as assayed by gel filtration chromatography (8). Protein complexes of similar size have been identified in extracts of human HeLa cells 20, 21; Albert and Timmers, unpublished observations. Involvement of Ccr4-Not core components in the biochemical stability of the yeast complexes has been reviewed in 2003 (18). From these analyses, it can be concluded that the

Interaction of the Core Ccr4-Not Complex with Additional Proteins in Larger Structures

While the core subunits of the 1 MDa Ccr4-Not complex have been defined as proteins that can be co-purified, many other proteins have been identified as interacting with one or the other core subunit. In some studies, it has been proposed that these interacting proteins are components of higher order Ccr4-Not complexes, which have been refractory to purification. The existing data favor a model in which the core Ccr4-Not complex may interact with many different proteins, leading to different,

Role of the Yeast Ccr4-Not Complex in mRNA Metabolism

Strong evidence exists that the Ccr4-Not complex is essential for determining appropriate mRNA levels. Not only were most of the components isolated in yeast using selections for altered mRNA levels, but also mRNA expression profiling has been performed with strains lacking the nonessential subunits of the complex. From this, it has become clear that more than half of the genome displays altered mRNA levels when one or the other component is lacking (Collart, unpublished observations). However,

Not4 Proteins are Protein–Ubiquitin Ligases

Cloning of the NOT4 gene from yeast indicated the presence of a putative Zn-binding module at the highly conserved N-terminus of Not4p (88). Structural analysis of this part of human CNOT4 by NMR methods unambiguously showed the presence of two Zn-ions (33), which are coordinated by eight cysteine-residues organized in a cross-brace manner. This type of Zn-coordination is the hallmark for RING fingers, which most often employ a Cys3His-Cys4 motif for this (89). RING fingers have been implicated

Role of the Ccr4-Not Complex in Protein Modification

The Ccr4-Not complex also seems to be involved in other post-translational modifications. Increased transcriptional activation by Msn2p was demonstrated in all ccr4-not mutants and this was found to correlate with altered post-translational modifications of Msn2p in ccr4-not mutants (95). Interestingly, this is apparently the one phenotype shared by all mutants of the core complex and also by dhh1, and it confers heat shock resisitance to all mutants. The observed changes in Msn2p might reflect

The Ccr4-Not Complex as a Regulatory Platform that Senses Glucose Levels and Stress

As has been described, the Ccr4-Not complex can regulate transcription by interaction with TFIID and possibly SAGA, mRNA decay by interaction with Dhh1p and its mRNA deadenylases, ubiquitylation via its integral E3 ligase, and protein modification by interaction with the Glc7p phosphatase. In addition, there may be other interactions that are functionally relevant as many proteins can interact with subunits of the Ccr4-Not complex. This defines the Ccr4-Not complex as a regulatory platform for

Perspectives

Since 1999, our knowledge about the Ccr4-Not proteins has expanded enormously. The composition of the core Ccr4-Not complex has been elucidated and many interacting proteins have been isolated. Two different enzymatic activities have been attributed to Ccr4-Not subunits. However, these findings do not provide a crystal-clear picture of the molecular and cellular functioning of the Ccr4-Not complex. It has helped, however, to direct experimental efforts. It seems most straightforward to view the

Acknowledgements

We thank Bas Winkler and Cécile Deluen for critical reading of the manuscript. H. Th. M. Timmers was supported by the Netherlands Organisation for Scientific Research (NWO-MW Pionier grant 900–98–142) and the European Commission (RTN2–2001–00026) and M. A. Collart was supported by Swiss National Science Foundation grant 3100A0–100793 and OFES grant 02.0017.

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