The Not3/5 subunit of the Ccr4-Not complex: A central regulator of gene expression that integrates signals between the cytoplasm and the nucleus in eukaryotic cells

The Ccr4-Not complex is a conserved multi-subunit complex in eukaryotes that carries 2 enzymatic activities: ubiquitination mediated by the Not4 RING E3 ligase and deadenylation mediated by the Ccr4 and Caf1 orthologs. This complex has been implicated in all aspects of the mRNA life cycle, from synthesis of mRNAs in the nucleus to their degradation in the cytoplasm. More recently the complex has also been implicated in many aspects of the life cycle of proteins, from quality control during synthesis of peptides, to assembly of protein complexes and protein degradation. Consistently, the Ccr4-Not complex is found both in the nucleus, where it is connected to transcribing ORFs, and in the cytoplasm, where it was revealed to be both associated with translating ribosomes and in RNA processing bodies. This functional and physical presence of the Ccr4-Not complex at all stages of gene expression raises the question of its fundamental role. This review will summarize recent evidence designing the Not3/5 module of the Ccr4-Not complex as a functional module involved in coordination of the regulation of gene expression between the nucleus and the cytoplasm.     

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”.     

Carbon source dependent dynamics of the Ccr4-Not complex in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

We have investigated the composition of the conserved Ccr4-Not complex during different physiological states of Saccharomyces cerevisiae. Major changes were found, most notably in the expression of the central scaffold protein Not1p, which was strongly reduced in the absence of glucose. The low expression of Not1p was also evident from the inability of Pop2p to co-purify Not1p in cells from cultures lacking glucose. However, Not1p was still essential under conditions of low expression. The downregulation of Not1p indicates that many of the Ccr4-Not complex components are likely to have roles outside of the complex. We suggest that the use of different carbon sources will be a good starting point to unravel these functions.     

The FgNot3 Subunit of the Ccr4-Not Complex Regulates Vegetative Growth,Sporulation, and Virulence in Fusarium graminearum

The Ccr4-Not complex is evolutionarily conserved and important for multiple cellular functions in eukaryotic cells. In this study, the biological roles of the FgNot3 subunit of this complex were investigated in the plant pathogenic fungus Fusarium graminearum. Deletion of FgNOT3 resulted in retarded vegetative growth, retarded spore germination, swollen hyphae, and hyper-branching. The ΔFgnot3 mutants also showed impaired sexual and asexual sporulation, decreased virulence, and reduced expression of genes related to conidiogenesis. Fgnot3 deletion mutants were sensitive to thermal stress, whereas NOT3 orthologs in other model eukaryotes are known to be required for cell wall integrity. We found that FgNot3 functions as a negative regulator of the production of secondary metabolites, including trichothecenes and zearalenone. Further functional characterization of other components of the Not module of the Ccr4-Not complex demonstrated that the module is conserved. Each subunit primarily functions within the context of a complex and might have distinct roles outside of the complex in F. graminearum. This is the first study to functionally characterize the Not module in filamentous fungi and provides novel insights into signal transduction pathways in fungal development.     

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.