The non-prion SUP35 preexists in large chaperone-containing molecular complexes.

Prions, misfolded proteins that aggregate, cause an array of progressively deteriorating conditions to which, currently, there are no effective treatments. The presently accepted model indicates that the soluble non-prion forms of prion-forming proteins, such as the well-studied SUP35, do not exist in large aggregated molecular complexes. Here, we show using analytical ultracentrifugation with fluorescent detection that the non-prion form of SUP35 exists in a range of discretely sized soluble complexes (19S, 28S, 39S, 57S, and 70S-200S). Similar to the [PSI+] aggregated complexes, each of these [psi-] complexes associates at stoichiometric levels with a large variety of molecular chaperones: HSP70 proteins comprise the major component. Another yeast prion-forming protein, RNQ1 (known to promote the production of the prion SUP35 state), is also present in SUP35 complexes. These results establish that the non-prion SUP35, like its prion form, is predisposed to form large molecular complexes containing chaperones and other prion-forming proteins. These results agree with our previous studies on the huntingtin protein. That the normal forms for aggregation-prone proteins may preexist in large molecular complexes has important ramifications for the progression of diseases involving protein aggregation.

Defining the protein complexome of translation termination factor eRF1: Identification of four novel eRF1-containing complexes that range from 20S to 57S in size.

The eukaryotic eRF1 translation termination factor plays an important role in recognizing stop codons and initiating the end to translation. However, which exact complexes contain eRF1 and at what abundance is not clear. We have used analytical ultracentrifugation with fluorescent detection system to identify the protein complexome of eRF1 in the yeast Saccharomyces cerevisiae. In addition to eRF1 presence in translating polysomes, we found that eRF1 associated with five other macromolecular complexes: 77S, 57S, 39S, 28S, and 20S in size. Generally equal abundances of each of these complexes were found. The 77S complex primarily contained the free 80S ribosome consistent with in vitro studies and did not appear to contain significant levels of the monosomal translating complex that co-migrates with the free 80S ribosome. The 57S and 39S complexes represented, respectively, free 60S and 40S ribosomal subunits bound to eRF1, associations not previously reported. The novel 28S and 20S complexes (containing minimal masses of 830 KDa and 500 KDa, respectively) lacked significant RNA components and appeared to be oligomeric, as eRF1 has a mass of 49 KDa. The majority of polysomal complexes containing eRF1 were both substantially deadenylated and lacking in closed-loop factors eIF4E and eIF4G. The thirteen percent of such translating polysomes that contained poly(A) tails had equivalent levels of eIF4E and eIF4G, suggesting these complexes were in a closed-loop structure. The identification of eRF1 in these unique and previously unrecognized complexes suggests a variety of new roles for eRF1 in the regulation of cellular processes.

The RRM1 domain of the poly(A)-binding protein from Saccharomyces cerevisiae is critical to control of mRNA deadenylation.

The poly(A)-binding protein PAB1 from the yeast Saccharomyces cerevisiae plays an important role in controlling mRNA deadenylation rates. Deletion of either its RRM1 or proline-rich domain (P domain) severely restricts deadenylation and slows mRNA degradation. Because these large deletions could be having unknown effects on the structure of PAB1, different strategies were used to determine the importance of the RRM1 and P domains to deadenylation. Since the P domain is quite variable in size and sequence among eukaryotes, P domains from two human PABPCs and from Xenopus were substituted for that of PAB1. The resultant PAB1 hybrid proteins, however, displayed limited or no difference in mRNA deadenylation as compared with PAB1. In contrast to the P domain, the RRM1 domain is highly conserved across species, and a systematic mutagenesis of the RRM1 domain was undertaken to identify its functional regions. Several mutations along the RNA-binding surface of RRM1 inhibited deadenylation, whereas one set of mutations on its exterior non-RNA binding surface shifted deadenylation from a slow distributive process to a rapid processive deadenylation. These results suggest that the RRM1 domain is the more critical region of PAB1 for controlling deadenylation and consists of at least two distinguishable functional regions.

Mass spectrometric identification of proteins that interact through specific domains of the poly(A) binding protein.

Poly(A) binding protein (PAB1) is involved in a number of RNA metabolic functions in eukaryotic cells and correspondingly is suggested to associate with a number of proteins. We have used mass spectrometric analysis to identify 55 non-ribosomal proteins that specifically interact with PAB1 from Saccharomyces cerevisiae. Because many of these factors may associate only indirectly with PAB1 by being components of the PAB1-mRNP structure, we additionally conducted mass spectrometric analyses on seven metabolically defined PAB1 deletion derivatives to delimit the interactions between these proteins and PAB1. These latter analyses identified 13 proteins whose associations with PAB1 were reduced by deleting one or another of PAB1's defined domains. Included in this list of 13 proteins were the translation initiation factors eIF4G1 and eIF4G2, translation termination factor eRF3, and PBP2, all of whose previously known direct interactions with specific PAB1 domains were either confirmed, delimited, or extended. The remaining nine proteins that interacted through a specific PAB1 domain were CBF5, SLF1, UPF1, CBC1, SSD1, NOP77, yGR250c, NAB6, and GBP2. In further study, UPF1, involved in nonsense-mediated decay, was confirmed to interact with PAB1 through the RRM1 domain. We additionally established that while the RRM1 domain of PAB1 was required for UPF1-induced acceleration of deadenylation during nonsense-mediated decay, it was not required for the more critical step of acceleration of mRNA decapping. These results begin to identify the proteins most likely to interact with PAB1 and the domains of PAB1 through which these contacts are made.

Identification of CCR4 and other essential thyroid hormone receptor co-activators by modified yeast synthetic genetic array analysis.

Identification of thyroid hormone receptor (TR) co-regulators has enhanced our understanding of thyroid hormone (TH) action. However, it is likely that many other co-regulators remained unidentified, and unbiased methods are required to discover these proteins. We have previously demonstrated that the yeast Saccharomyces cerevisiae is an excellent system in which to study TR action, and that defined TR signaling complexes in a eukaryotic background devoid of complicating influences of mammalian cell co-regulators can be constructed and analyzed for endogenous yeast genes, many of which are conserved in mammals. Here, a modified synthetic genetic array analysis was performed by crossing a yeast strain that expressed TRbeta1 and the co-activator GRIP1/SRC2 with 384 yeast strains bearing deletions of known genes. Eight genes essential for TH action were isolated, of which 4 are conserved in mammals. Examination of one, the yeast CCR4 and its human homolog CCR4/NOT6 (hCCR4), confirmed that (i) transfected CCR4 potentiates a TH response in cultured cells more efficiently than established TR co-activators and (ii) knockdown of CCR4 expression strongly inhibited a TH response (>80%). TH treatment promoted rapid and sustained hCCR4 recruitment to the TH-responsive deiodinase 1 promoter and TR co-localizes with hCCR4 in the nucleus and interacts with hCCR4 in 2-hybrid and pull-down assays. These findings indicate that a modified yeast synthetic genetic array strategy is a feasible method for unbiased identification of conserved genes essential for TR and other nuclear receptor hormone functions in mammals.

PAB1 self-association precludes its binding to poly(A), thereby accelerating CCR4 deadenylation in vivo.

The mRNA deadenylation process, catalyzed by the CCR4 deadenylase, is known to be the major factor controlling mRNA decay rates in Saccharomyces cerevisiae. We have identified the proline-rich region and RRM1 domains of poly(A) binding protein (PAB1) as necessary for CCR4 deadenylation. Deletion of either of these regions but not other regions of PAB1 significantly reduced PAB1-PAB1 protein interactions, suggesting that PAB1 oligomerization is a required step for deadenylation. Moreover, defects in these two regions inhibited the formation of a novel, circular monomeric PAB1 species that forms in the absence of poly(A). Removal of the PAB1 RRM3 domain, which promoted PAB1 oligomerization and circularization, correspondingly accelerated CCR4 deadenylation. Circular PAB1 was unable to bind poly(A), and PAB1 multimers were severely deficient or unable to bind poly(A), implicating the PAB1 RNA binding surface as critical in making contacts that allow PAB1 self-association. These results support the model that the control of CCR4 deadenylation in vivo occurs in part through the removal of PAB1 from the poly(A) tail following its self-association into multimers and/or a circular species. Known alterations in the P domains of different PAB proteins and factors and conditions that affect PAB1 self-association would, therefore, be expected to be critical to controlling mRNA turnover in the cell.

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 caf1Delta, but not that of ccr4Delta, 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 ccr4Delta, severely blocked deadenylation when coupled with a caf1 deletion. These results indicate that both CAF1 and factors involved in translation are required for deadenylation.

Identification of a 57S translation complex containing closed-loop factors and the 60S ribosome subunit.

In eukaryotic translation the 60S ribosome subunit has not been proposed to interact with mRNA or closed-loop factors eIF4E, eIF4G, and PAB1. Using analytical ultracentrifugation with fluorescent detection system, we have identified a 57S translation complex that contains the 60S ribosome, mRNA, and the closed-loop factors. Previously published data by others also indicate the presence of a 50S-60S translation complex containing these same components. We have found that the abundance of this complex increased upon translational cessation, implying formation after ribosomal dissociation. Stoichiometric analyses of the abundances of the closed-loop components in the 57S complex indicate this complex is most similar to polysomal and monosomal translation complexes at the end of translation rather than at the beginning or middle of translation. In contrast, a 39S complex containing the 40S ribosome bound to mRNA and closed-loop factors was also identified with stoichiometries most similar to polysomal complexes engaged in translation, suggesting that the 39S complex is the previously studied 48S translation initiation complex. These results indicate that the 60S ribosome can associate with the closed-loop mRNA structure and plays a previously undetected role in the translation process.

In vivo evidence that defects in the transcriptional elongation factors RPB2, TFIIS, and SPT5 enhance upstream poly(A) site utilization.

While a number of proteins are involved in elongation processes, the mechanism for action of most of these factors remains unclear primarily because of the lack of suitable in vivo model systems. We identified in yeast several genes that contain internal poly(A) sites whose full-length mRNA formation is reduced by mutations in RNA polymerase II subunit RPB2, elongation factor SPT5, or TFIIS. RPB2 and SPT5 defects also promoted the utilization of upstream poly(A) sites for genes that contain multiple 3' poly(A) signaling sequences, supporting a role for elongation in differential poly(A) site choice. Our data suggest that elongation defects cause increased transcriptional pausing or arrest that results in increased utilization of internal or upstream poly(A) sites. Transcriptional pausing or arrest can therefore be visualized in vivo if a gene contains internal poly(A) sites, allowing biochemical and genetic study of the elongation process.

Ccr4-not complex mRNA deadenylase activity contributes to DNA damage responses in Saccharomyces cerevisiae.

DNA damage checkpoints regulate gene expression at the transcriptional and post-transcriptional level. Some components of the yeast Ccr4-Not complex, which regulates transcription as well as transcript turnover, have previously been linked to DNA damage responses, but it is unclear if this involves transcriptional or post-transcriptional functions. Here we show that CCR4 and CAF1, which together encode the major cytoplasmic mRNA deadenylase complex, have complex genetic interactions with the checkpoint genes DUN1, MRC1, RAD9, and RAD17 in response to DNA-damaging agents hydroxyurea (HU) and methylmethane sulfonate (MMS). The exonuclease-inactivating ccr4-1 point mutation mimics ccr4Delta phenotypes, including synthetic HU hypersensitivity with dun1Delta, demonstrating that Ccr4-Not mRNA deadenylase activity is required for DNA damage responses. However, ccr4Delta and caf1Delta DNA damage phenotypes and genetic interactions with checkpoint genes are not identical, and deletions of some Not components that are believed to predominantly function at the transcriptional level rather than mRNA turnover, e.g., not5Delta, also lead to increased DNA damage sensitivity and synthetic HU hypersensitivity with dun1Delta. Taken together, our data thus suggest that both transcriptional and post-transcriptional functions of the Ccr4-Not complex contribute to the DNA damage response affecting gene expression in a complex manner.

The CCR4-NOT complex plays diverse roles in mRNA metabolism.

It is increasingly clear that the synthesis of eukaryotic mRNA involves an integrated series of events involving large multisubunit protein complexes. The evolutionarily conserved CCR4-NOT complex of proteins has been found to be involved in several aspects of mRNA formation, including repression and activation of mRNA initiation, control of mRNA elongation, and the deadenylation and subsequent degradation of mRNA. Its roles in such diverse processes make the CCR4-NOT complex central to the regulation of mRNA metabolism. In this review we describe the CCR4-NOT complex, its constituents, and its organization, discussing both the well characterized yeast proteins and their higher eukaryotic orthologs. The known biochemical roles of the individual components and of the complex are described with particular emphasis on the two known functions of the complex, repression of TFIID action and deadenylation of mRNA. Finally, the functional diversity of the CCR4-NOT complex is related to its mediating responses from a number of cellular signaling pathways.

Multiple discrete soluble aggregates influence polyglutamine toxicity in a Huntington's disease model system.

Huntington's disease (HD) results from expansions of polyglutamine stretches (polyQ) in the huntingtin protein (Htt) that promote protein aggregation, neurodegeneration, and death. Since the diversity and sizes of the soluble Htt-polyQ aggregates that have been linked to cytotoxicity are unknown, we investigated soluble Htt-polyQ aggregates using analytical ultracentrifugation. Soon after induction in a yeast HD model system, non-toxic Htt-25Q and cytotoxic Htt-103Q both formed soluble aggregates 29S to 200S in size. Because current models indicate that Htt-25Q does not form soluble aggregates, reevaluation of previous studies may be necessary. Only Htt-103Q aggregation behavior changed, however, with time. At 6 hr mid-sized aggregates (33S to 84S) and large aggregates (greater than 100S) became present while at 24 hr primarily only mid-sized aggregates (20S to 80S) existed. Multiple factors that decreased cytotoxicity of Htt-103Q (changing the length of or sequences adjacent to the polyQ, altering ploidy or chaperone dosage, or deleting anti-aging factors) altered the Htt-103Q aggregation pattern in which the suite of mid-sized aggregates at 6 hr were most correlative with cytotoxicity. Hence, the amelioration of HD and other neurodegenerative diseases may require increased attention to and discrimination of the dynamic alterations in soluble aggregation processes.

Stoichiometry and Change of the mRNA Closed-Loop Factors as Translating Ribosomes Transit from Initiation to Elongation.

Protein synthesis is a highly efficient process and is under exacting control. Yet, the actual abundance of translation factors present in translating complexes and how these abundances change during the transit of a ribosome across an mRNA remains unknown. Using analytical ultracentrifugation with fluorescent detection we have determined the stoichiometry of the closed-loop translation factors for translating ribosomes. A variety of pools of translating polysomes and monosomes were identified, each containing different abundances of the closed-loop factors eIF4E, eIF4G, and PAB1 and that of the translational repressor, SBP1. We establish that closed-loop factors eIF4E/eIF4G dissociated both as ribosomes transited polyadenylated mRNA from initiation to elongation and as translation changed from the polysomal to monosomal state prior to cessation of translation. eIF4G was found to particularly dissociate from polyadenylated mRNA as polysomes moved to the monosomal state, suggesting an active role for translational repressors in this process. Consistent with this suggestion, translating complexes generally did not simultaneously contain eIF4E/eIF4G and SBP1, implying mutual exclusivity in such complexes. For substantially deadenylated mRNA, however, a second type of closed-loop structure was identified that contained just eIF4E and eIF4G. More than one eIF4G molecule per polysome appeared to be present in these complexes, supporting the importance of eIF4G interactions with the mRNA independent of PAB1. These latter closed-loop structures, which were particularly stable in polysomes, may be playing specific roles in both normal and disease states for specific mRNA that are deadenylated and/or lacking PAB1. These analyses establish a dynamic snapshot of molecular abundance changes during ribosomal transit across an mRNA in what are likely to be critical targets of regulation.

Characterization of mutations in NOT2 indicates that it plays an important role in maintaining the integrity of the CCR4-NOT complex.

The NOT2 protein is a component of the CCR4-NOT complex that plays multiple roles in the regulation of mRNA production in the yeast Saccharomyces cerevisiae. We have identified four novel not2 mutations and have characterized these and two previously described alleles as to the means by which they affect CCR4-NOT function. While two of the not2 alleles, not2-4 (carrying a G31R alteration) and not2::L9P, resulted in severe growth defects and caused a not phenotype at the HIS3 locus, these phenotypes appear to arise from partially different effects. The not2::L9P mutation resulted in complete loss of the 1.9x10(6)Da (1.9MDa) CCR4-NOT complex, and the not2::L9P protein displayed increased ability to associate with the NOT5 protein. In contrast, the not2-4 allele destabilized the CCR4-NOT complex to a lesser extent and had no effect on NOT5 association with NOT2. Instead, as previously reported, it displayed defective interactions with ADA2, a component of the SAGA complex. The not2::R165G also abrogated NOT2 ability to interact with ADA2 but had little effect on the integrity of the CCR4-NOT complex. Alterations in NOT2 contacts to ADA2, therefore, do not necessarily result in effects on the CCR4-NOT complex nor result in severe growth defects. We also observed that the four NOT2 N-terminal mutations affected NOT5 association with the CCR4-NOT complexes, suggesting that it is the N terminus of NOT2 that contacts and stabilizes NOT5 interactions. These results indicate that it is the loss of the integrity of the CCR4-NOT complex which leads to severe not2 phenotypes and that the NOT2 contacts to ADA2 play a lesser role in NOT2 function.

Only a subset of the PAB1-mRNP proteome is present in mRNA translation complexes.

We have previously identified 55 nonribosomal proteins in PAB1-mRNP complexes in Saccharomyces cerevisiae using mass spectrometric analysis. Because one of the inherent limitations of mass spectrometry is that it does not inform as to the size or type of complexes in which the proteins are present, we consequently used analytical ultracentrifugation with fluorescent detection system (AU-FDS) to determine which proteins are present in the 77S monosomal translation complex that contains minimally the closed-loop structure components (eIF4E, eIF4G, and PAB1), mRNA, and the 40S and 60S ribosomes. We assayed by AU-FDS analysis 33 additional PAB1-mRNP factors but found that only five of these proteins were present in the 77S translation complex: eRF1, SLF1, SSD1, PUB1, and SBP1. eRF1 is involved in translation termination, SBP1 is a translational repressor, and SLF1, SSD1, and PUB1 are known mRNA binding proteins. Many of the known P body/stress granule proteins that associate with the PAB1-mRNP were not present in the 77S translation complex, implying that P body/stress granules result from significant protein additions after translational cessation. These data inform that AU-FDS can clarify protein complex identification that remains undetermined after typical immunoprecipitation and mass spectrometric analyses.

SPT5 affects the rate of mRNA degradation and physically interacts with CCR4 but does not control mRNA deadenylation.

The CCR4-NOT complex has been shown to have multiple roles in mRNA metabolism, including that of transcriptional elongation, mRNA transport, and nuclear exosome function, but the primary function of CCR4 and CAF1 is in the deadenylation and degradation of cytoplasmic mRNA. As previous genetic analysis supported an interaction between SPT5, known to be involved in transcriptional elongation, and that of CCR4, the physical association of SPT5 with CCR4 was examined. A two-hybrid screen utilizing the deadenylase domain of CCR4 as a bait identified SPT5 as a potential interacting protein. SPT5 at its physiological concentration was shown to immunoprecipitate CCR4 and CAF1, and in vitro purified SPT5 specifically could bind to CAF1 and the deadenylase domain of CCR4. We additionally demonstrated that mutations in SPT5 or an spt4 deletion slowed the rate of mRNA degradation, a phenotype associated with defects in the CCR4 mRNA deadenylase complex. Yet, unlike ccr4 and caf1 deletions, spt5 and spt4 defects displayed little effect on the rate of deadenylation. They also did not affect decapping or 5' - 3' degradation of mRNA. These results suggest that the interactions between SPT5/SPT4 and the CCR4-NOT complex are probably the consequences of effects involving nuclear events and do not involve the primary role of CCR4 in mRNA deadenylation and turnover.

Use of the novel technique of analytical ultracentrifugation with fluorescence detection system identifies a 77S monosomal translation complex.

A fundamental problem in proteomics is the identification of protein complexes and their components. We have used analytical ultracentrifugation with a fluorescence detection system (AU-FDS) to precisely and rapidly identify translation complexes in the yeast Saccharomyces cerevisiae. Following a one-step affinity purification of either poly(A)-binding protein (PAB1) or the large ribosomal subunit protein RPL25A in conjunction with GFP-tagged yeast proteins/RNAs, we have detected a 77S translation complex that contains the 80S ribosome, mRNA, and components of the closed-loop structure, eIF4E, eIF4G, and PAB1. This 77S structure, not readily observed previously, is consistent with the monosomal translation complex. The 77S complex abundance decreased with translational defects and following the stress of glucose deprivation that causes translational stoppage. By quantitating the abundance of the 77S complex in response to different stress conditions that block translation initiation, we observed that the stress of glucose deprivation affected translation initiation primarily by operating through a pathway involving the mRNA cap binding protein eIF4E whereas amino acid deprivation, as previously known, acted through the 43S complex. High salt conditions (1M KCl) and robust heat shock acted at other steps. The presumed sites of translational blockage caused by these stresses coincided with the types of stress granules, if any, which are subsequently formed.

PUF3 acceleration of deadenylation in vivo can operate independently of CCR4 activity, possibly involving effects on the PAB1-mRNP structure.

The evolutionarily conserved PUF proteins stimulate CCR4 mRNA deadenylation through binding to 3' untranslated region sequences of specific mRNA. We have investigated the mechanisms by which PUF3 in Saccharomyces cerevisiae accelerates deadenylation of the COX17 mRNA. PUF3 was shown to affect PAN2 deadenylation of the COX17 mRNA independent of the presence of CCR4, suggesting that PUF3 acts through a general mechanism to affect deadenylation. Similarly, eIF4E, the cap-binding translation initiation factor known to control CCR4 deadenylation, was shown to affect PAN2 activity in vivo. PUF3 was found to be required for eIF4E effects on COX17 deadenylation. Both eIF4E and PUF3 effects on deadenylation were shown, in turn, to necessitate a functional poly(A)-binding protein (PAB1) in which removal of the RRM1 (RNA recognition motif 1) domain of PAB1 blocked both their effects on deadenylation. While removal of the proline-rich region (P domain) of PAB1 substantially reduces CCR4 deadenylation at non-PUF3-controlled mRNA and correspondingly blocked eIF4E effects on deadenylation, PUF3 essentially bypassed this P domain requirement. These results indicate that the PAB1-mRNP structure is critical for PUF3 action. We also found that multiple components of the CCR4-NOT deadenylase complex, but not PAN2, interacted with PUF3. PUF3 appears, therefore, both to act independently of CCR4 activity, possibly through effects on PAB1-mRNP structure, and to be capable of retaining the CCR4-NOT complex.

Genome wide expression analysis of the CCR4-NOT complex indicates that it consists of three modules with the NOT module controlling SAGA-responsive genes.

Of the nine known members of the CCR4-NOT complex, CCR4/CAF1 are most important in mRNA deadenylation whereas the NOT1-5 proteins are most critical for transcriptional repression. Whole genome microarray analysis using deletions in seven of the CCR4-NOT genes was used to determine the overall mRNA expression patterns that are affected by members of the yeast CCR4-NOT complex. Under glucose conditions, ccr4 and caf1 displayed a high degree of similarity in the manner that they affected gene expression. In contrast, the not deletions were similar in the way they affected genes, but showed no correlation with that of ccr4/caf1. A number of groups of functionally related proteins were specifically controlled by the CCR4/CAF1 or NOT modules. Importantly, the NOT proteins preferentially affected SAGA-controlled gene expression. Also, both the CCR4/CAF1 and NOT group of proteins shared much greater similarities in their effects on gene expression during the stress of glucose deprivation. BTT1, a member of the nascent polypeptide association complex that binds the ribosome, was shown to be a tenth member of the CCR4-NOT complex, interacting through CAF130. Microarray analysis indicated that BTT1 and CAF130 correlate very highly in their control of gene expression and preferentially repress genes involved in ribosome biogenesis. These results indicate that distinct portions of the CCR4-NOT complex control a number of different cellular processes.

CCR4, a 3'-5' poly(A) RNA and ssDNA exonuclease, is the catalytic component of the cytoplasmic deadenylase.

The CCR4-NOT complex from Saccharomyces cerevisiae is a general transcriptional regulatory complex. The proteins of this complex are involved in several aspects of mRNA metabolism, including transcription initiation and elongation and mRNA degradation. The evolutionarily conserved CCR4 protein, which is part of the cytoplasmic deadenylase, contains a C-terminal domain that displays homology to an Mg2+-dependent DNase/phosphatase family of proteins. We have analyzed the putative enzymatic properties of CCR4 and have found that it contains both RNA and single-stranded DNA 3'-5' exonuclease activities. CCR4 displays a preference for RNA and for 3' poly(A) substrates, implicating it as the catalytic component of the cytoplasmic deadenylase. Mutations in the key, conserved catalytic residues in the CCR4 exonuclease domain abolished both its in vitro activities and its in vivo functions. Importantly, CCR4 was active as a monomer and remained active in the absence of CAF1, which links CCR4 to the remainder of the CCR4-NOT complex components. These results establish that CCR4 and most probably other members of a widely distributed CCR4-like family of proteins constitute a novel class of RNA-DNA exonucleases. The various regulatory effects of the CCR4-NOT complex on gene expression may be executed in part through these CCR4 exonuclease activities.