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

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.     

Xenopus CAF1 requires NOT1-mediated interaction with 4E-T to repress translation in vivo

RNA-regulatory factors bound to 3′ UTRs control translation and stability. Repression often is associated with poly(A) removal. The deadenylase CAF1 is a core component of the CCR4–NOT complex. Our prior studies established that CAF1 represses translation independent of deadenylation. We sought the mechanism of its deadenylation-independent repression in Xenopus oocytes. Our data reveal a chain of interacting proteins that links CAF1 to CCR4–NOT and to Xp54 and 4E-T. Association of CAF1 with NOT1, the major subunit of CCR4–NOT, is required for repression by CAF1 tethered to a reporter mRNA. Affinity purification-mass spectrometry and coimmunoprecipitation revealed that at least five members of the CCR4–NOT complex were recruited by CAF1. The recruitment of these proteins required NOT1, as did the ability of tethered CAF1 to repress translation. In turn, NOT1 was needed to recruit Xp54 and 4E-T. We examined the role of 4E-T in repression using mutations that disrupted either eIF4E-dependent or -independent mechanisms. Expression of a 4E-T truncation that still bound eIF4E alleviated repression by tethered CAF1, NOT1, and Xp54. In contrast, a mutant 4E-T that failed to bind eIF4E did not. Repression of global translation was affected only by the eIF4E-dependent mechanism. Reporters bearing IRES elements revealed that repression via tethered CAF1 and Xp54 is cap- and eIF4E-independent, but requires one or more of eIF4A, eIF4B, and eIF4G. We propose that RNA-binding proteins, and perhaps miRNAs, repress translation through an analogous chain of interactions that begin with the 3′ UTR-bound repressor and end with the noncanonical activity of 4E-T.     

CAF1 plays an important role in mRNA deadenylation separate from its contact to CCR4

The CAF1 protein is a component of the CCR4–NOT deadenylase complex. While yeast CAF1 displays deadenylase activity, this activity is not required for its deadenylation function in vivo, and CCR4 is the primary deadenylase in the complex. In order to identify CAF1-specific functional regions required for deadenylation in vivo, we targeted for mutagenesis six regions of CAF1 that are specifically conserved among CAF1 orthologs. Defects in residues 213–215, found to be a site required for binding CCR4, reduced the rate of deadenylation to a lesser extent and resulted in in vivo phenotypes that were less severe than did defects in other regions of CAF1 that displayed greater contact to CCR4. These results imply that CAF1, while affecting deadenylation through its contact to CCR4, has functions in deadenylation separate from its contact to CCR4. Synthetic lethalities of caf1Δ, but not that of ccr4Δ, with defects in DHH1 or PAB1, both of which are involved in translation, further supports a role of CAF1 separate from that of CCR4. Importantly, other mutations in PAB1 that reduced translation, while not affecting deadenylation by themselves or when combined with ccr4Δ, severely blocked deadenylation when coupled with a caf1 deletion. These results indicate that both CAF1 and factors involved in translation are required for deadenylation.     

CCR4-associated factor 1 coordinates the expression of Plasmodium falciparum egress and invasion proteins

Coordinated regulation of gene expression is a hallmark of the Plasmodium falciparum asexual blood-stage development cycle. We report that carbon catabolite repressor protein 4 (CCR4)-associated factor 1 (CAF1) is critical in regulating more than 1,000 genes during malaria parasites' intraerythrocytic stages, especially egress and invasion proteins. CAF1 knockout results in mistimed expression, aberrant accumulation and localization of proteins involved in parasite egress, and invasion of new host cells, leading to premature release of predominantly half-finished merozoites, drastically reducing the intraerythrocytic growth rate of the parasite. This study demonstrates that CAF1 of the CCR4-Not complex is a significant gene regulatory mechanism needed for Plasmodium development within the human host.     

A complex containing the CCR4 and CAF1 proteins is involved in mRNA deadenylation in Drosophila

The CCR4-NOT complex is the major enzyme catalyzing mRNA deadenylation in Saccharomyces cerevisiae. We have identified homologs for almost all subunits of this complex in the Drosophila genome. Biochemical fractionation showed that the two likely catalytic subunits, CCR4 and CAF1, were associated with each other and with a poly(A)-specific 3' exonuclease activity. In Drosophila, the CCR4 and CAF1 proteins were ubiquitously expressed and present in cytoplasmic foci. Individual knock-down of several potential subunits of the Drosophila CCR4-NOT complex by RNAi in tissue culture cells led to a lengthening of bulk mRNA poly(A) tails. Knock-down of two individual subunits also interfered with the rapid deadenylation of Hsp70 mRNA during recovery from heat shock. Similarly, ccr4 mutant flies had elongated bulk poly(A) and a defect in Hsp70 mRNA deadenylation. A minor increase in bulk poly(A) tail length was also observed in Rga mutant flies, which are affected in the NOT2 subunit. The data show that the CCR4-NOT complex is conserved in Drosophila melanogaster and plays a role in general and regulated mRNA deadenylation.