Genetic Evidence Supports a Role for the Yeast CCR4-NOT Complex in Transcriptional Elongation

Corresponding author: Clyde L. Denis, Department of Biochemistry and Molecular Biology, Rudman Hall, University of New Hampshire, Durham, NH 03824., cldenis{at}christa.unh.edu (E-mail)

Communicating editor: M. HAMPSEY

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The CCR4-NOT complex is involved in the regulation of gene expression both positively and negatively. The repressive effects of the complex appear to result in part from restricting TBP access to noncanonical TATAA binding sites presumably through interaction with multiple TAF proteins. We provide here genetic evidence that the CCR4-NOT complex also plays a role in transcriptional elongation. First, defects in CCR4-NOT components as well as overexpression of the NOT4 gene elicited 6-azauracil (6AU) and mycophenolic acid sensitivities, hallmarks of transcriptional elongation defects. A number of other transcription initiation factors known to interact with the CCR4-NOT complex did not elicit these phenotypes nor did defects in factors that reduced mRNA degradation and hence the recycling of NTPs. Second, deletion of ccr4 resulted in severe synthetic effects with mutations or deletions in the known elongation factors RPB2, TFIIS, and SPT16. Third, the ccr4 deletion displayed allele-specific interactions with rpb1 alleles that are thought to be important in the control of elongation. Finally, we found that a ccr4 deletion as well as overexpression of the NOT1 gene specifically suppressed the cold-sensitive phenotype associated with the spt5-242 allele. The only other known suppressors of this spt5-242 allele are factors involved in slowing transcriptional elongation. These genetic results are consistent with the model that the CCR4-NOT complex, in addition to its known effects on initiation, plays a role in aiding the elongation process.

EUKARYOTIC gene expression is characterized by the interaction of a number of factors and large protein complexes, each of which may have multiple roles in the formation of mRNA. For example, TAF_II proteins are components of both TFIID that is involved in transcription initiation and the SAGA complex that is proposed to affect chromatin accessibility (GRANT et al. 1997 ; STERNER et al. 1999 ). TFIID has also been shown to recruit a factor important for polyadenylation of mRNA (DANTONEL et al. 1997 ), suggesting a role for TFIID in 3' end formation of mRNA. Individual subunits of TFIID have multiple functions. For instance, TAF_II250 can acetylate and ubiquitinate histones as well as regulate TBP access to DNA (MIZZEN et al. 1996 ; LIU et al. 1998A ; PHAM and SAUER 2000 ). The TFIIH complex, in addition to its known roles in initiation and in promoter clearance (KIM et al. 2000 ), aids in DNA excision repair (DRAPKIN et al. 1994 ). The carboxy-terminal domain (CTD) of RNA polymerase II enjoys multiple functions both in initiation and in postinitiation processes such as mRNA capping, splicing, and polyadenylation (MCCRACKEN et al. 1997 ; HIROSE and MANLEY 1998 ; HIROSE et al. 1999 ; KOMARNITSKY et al. 2000 ; SCHROEDER et al. 2000 ). In addition, TFIIF is important for both initiation and elongation of mRNA (TAN et al. 1995 ; FRANCOIS et al. 1998 ). It is clear, therefore, that the multiple protein interactions possible for these proteins and protein complexes can engender many and varied roles for these factors. We report genetic evidence that the CCR4-NOT complex in addition to its known roles in transcriptional initiation has a function in transcriptional elongation.

The CCR4-NOT complex affects gene expression both positively and negatively (DENIS 1984 ; DENIS and MALVAR 1990 ; SAKAI et al. 1992 ; COLLART and STRUHL 1993 , COLLART and STRUHL 1994 ; LIU et al. 1998B ). The repressive effects of the complex have been linked to restricting TBP access to noncanonical TATAA sequences (COLLART and STRUHL 1994 ; COLLART 1996 ), apparently through the interaction with multiple TAF proteins (BADARINARAYANA et al. 2000 ; LEMAIRE and COLLART 2000 ), and more recently to effects on the degradation of mRNA (TUCKER et al. 2001 ). However, the partial nonoverlap of phenotypes observed when different components of the CCR4-NOT complex are deleted (BAI et al. 1999 ) and the positive role for the complex in gene expression (DENIS 1984 ; LIU et al. 1998B ) suggest additional functions for the CCR4-NOT complex.

The CCR4-NOT complex is present in at least two forms in yeast, 1.9 x 10⁶ daltons (1.9 MD) and 1.0 MD (LIU et al. 1997 , LIU et al. 1998B ; BAI et al. 1999 ). The smaller, core 1.0-MD complex has been well characterized and consists of CCR4, CAF1, the five NOT proteins (NOT1–5), and two new proteins, CAF40 and CAF130 (DRAPER et al. 1994 , DRAPER et al. 1995 ; LIU et al. 1998B ; BAI et al. 1999 ; J. CHEN, Y.-C. CHIANG, J. RAPPSILBER, P. RUSSELL, M. MANN and C. L. DENIS, unpublished data). Although the 1.9-MD complex remains uncharacterized, several proteins such as DBF2, MOB1, CAF4, and CAF16 have been linked to interactions with CCR4-NOT proteins and are likely components of this larger complex (LIU et al. 1997 ; KOMARNITSKY et al. 1998 ; LIU et al. 2001 ). The arrangement of the proteins in the 1.0-MD CCR4-NOT complex have been analyzed (LIU et al. 1998B ; BAI et al. 1999 ; MAILLET et al. 2000 ). CCR4 and CAF1 bind to a central region of NOT1 whereas NOT2, NOT5, and NOT4 associate through the C terminus of NOT1. CAF40 and CAF130, although their locations are less clear, associate with a different part of the complex than that with which either CCR4 and CAF1 or NOT2, NOT5, and NOT4 are associated (J. CHEN, Y.-C. CHIANG, J. RAPPSILBER, P. RUSSELL, M. MANN and C. L. DENIS, unpublished data). Because NOT2 and NOT5 appear primarily responsible for TFIID interactions (BADARINARAYANA et al. 2000 ; LEMAIRE and COLLART 2000 ), CCR4 may be making contacts and playing roles in the cell other than that evinced by NOT2 and NOT5.

A number of factors have been shown to play possible roles in eukaryotic transcriptional elongation (CHAVEZ and AGUILERA 1997 ; UPTAIN et al. 1997 ; HARTZOG et al. 1998 ; WADA et al. 1998 ; CHAVEZ et al. 2000 ; COSTA and ARNDT 2000 ). In yeast defects in many of these factors affecting elongation such as SPT5, SPT4, SPT6, RTF1, RPB2, RPB1, TFIIS, and ELP1 elicit 6-azauracil (6AU) and mycophenolic sensitive phenotypes (ARCHAMBAULT et al. 1992 ; EXINGER and LACROUTE 1992 ; POWELL and REINES 1996 ; HARTZOG et al. 1998 ; LENNON et al. 1998 ; OTERO et al. 1999 ; COSTA and ARNDT 2000 ). 6AU sensitivities result from lowering of GTP and/or UTP levels in the cell that is presumed to impair elongation (EXINGER and LACROUTE 1992 ). We have observed that a deletion of CCR4 or other components of the CCR4-NOT complex gives rise to a 6AU sensitive phenotype. In confirmation of a role for CCR4 in elongation, the ccr4 deletion causes synthetic and allele-specific phenotypes with several known defects in elongation factors. Moreover, a ccr4 deletion or overexpression of NOT1 suppresses an spt5-242 defect that has been shown previously to be suppressed by the slowing of elongation. These and other results suggest a role for components of the CCR4-NOT complex in controlling transcriptional elongation.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Yeast strains and growth conditions:
Yeast strains are listed in Table 1. Strains were grown on YEP plates (1% yeast extract/2% Bacto-peptone/2% agar) supplemented with either 2% glucose (YD plates) or 3% glycerol (YG plates). Minimal plates were prepared lacking uracil as described (BADARINARAYANA et al. 2000 ) and were supplemented with 100 µg/ml 6AU, 40 µg/ml mycophenolic acid, or 600 µg/ml guanine. IMPDH assays were conducted as previously described for ADH assays (LIU et al. 1998B ), except the reaction conditions contained 20 mM IMP, 50 mM glutathione, and 50 mM Tris, pH 7.5 instead of 0.3 M ethanol and 50 mM pyrophosphate buffer. Standard errors of the mean were <20% in all cases.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Defects in CCR4-NOT complex components elicit 6AU sensitive phenotypes:
It was previously observed that a ccr4 deletion could give rise to 6AU sensitivity (CHANG et al. 1999 ). We examined this further by analyzing a number of different strains and their corresponding ccr4 derivatives for growth on medium containing 6AU. In each case, a ccr4 defect resulted in 6AU sensitivity (data not shown), indicating that 6AU sensitivity was another phenotype, like caffeine sensitivity, cold sensitivity, and glycerol 37° sensitivity, that was associated with a ccr4 disruption (DENIS and MALVAR 1990 ; LIU et al. 1997 ). We also examined whether defects in other components of the CCR4-NOT complex elicited 6AU sensitivity (Fig 1). In addition to ccr4, we found that caf1, not1-2, not2-1, not4, and not5 mutations each gave rise to a 6AU phenotype (Fig 1). While a not3 deletion did not result in a 6AU sensitive phenotype (Fig 1), the not3-2 allele did (data not shown). This is in agreement with previous results showing that the not3-2 allele generally results in more severe phenotypes than the not3 deletion (LIU et al. 1998B ; our unpublished data). As 6AU sensitivity in yeast has been generally correlated with defects in transcriptional elongation, although not exclusively so, these results suggest that the CCR4-NOT complex plays a role in elongation in addition to its known role in controlling initiation (DENIS and MALVAR 1990 ; SAKAI et al. 1992 ; COLLART and STRUHL 1994 ).

View larger version (64K): In this window In a new window Download PPT slide   Figure 1. Defects in CCR4-NOT components result in 6AU sensitivity. All yeast strains were grown on minimal medium lacking uracil (-6AU) that was supplemented with 100 µg/ml 6AU (+6AU). Growth was monitored after 5 days at 34° although similar results were obtained at 30° and 25°. All strains were isogenic to either EGY188 (wt) or KY803 (whose growth was identical to EGY188). It should be noted that all strains grew well on minimal medium containing both 6AU and uracil.

6AU sensitivity appears to arise from 6AU lowering the levels of GTP and UTP in the cell (EXINGER and LACROUTE 1992 ). Provision of excess guanine has been found to overcome 6AU sensitivity (EXINGER and LACROUTE 1992 ). Similarly, we found that excess guanine could rescue the 6AU sensitive phenotype observed by defects in CCR4-NOT genes (data not shown). Mycophenolic acid similarly acts to decrease GTP levels in the cell and ccr4, caf1, and not4 deletions also resulted in sensitivity to mycophenolic acid (data not shown), indicating that it was not some specific interaction between 6AU and defects in the CCR4-NOT complex components that elicited the above described phenotypes. Defects in other factors known to associate with the 1.9-MD CCR4-NOT complex but which are not components of the 1-MD CCR4-NOT complex, such as CAF4, CAF16, and DBF2, did not elicit a 6AU sensitive phenotype (Fig 1; data not shown). These data indicate that it is the functionality of the core CCR4-NOT components that are important to the 6AU sensitivity phenotype.

The 6AU sensitivity observed by defects in CCR4-NOT proteins was not, however, found to occur when other known factors involved in transcriptional initiation were deleted. Deletion of PAF1 or CDC73, whose protein products have been shown to be part of an RNA polymerase II complex containing CCR4, did not elicit a 6AU phenotype (CHANG et al. 1999 ; data not shown). Similarly, deletion of RNA polymerase II holoenzyme components, SRB5, SRB9, SRB10, SRB11, GAL11, or SIN4, or SAGA components ADA2 or GCN5 failed to result in 6AU sensitivity (data not shown).

We further examined whether defects in factors that cause a decreased rate of RNA degradation and hence a reduction in recycling of NTPs could result in a 6AU sensitivity phenotype. Defects in proteins known to be involved in mRNA degradation, such as XRN1 (MUHLRAD et al. 1995 ), or the exosome components SKI8, SKI6, SKI2, or SKI3 (VAN HOOF et al. 2000 ) did not result in 6AU sensitivity phenotypes (data not shown).

Disruption in CCR4-NOT function has been previously observed either by deleting specific components of the complex or by overexpressing an individual component of the complex (BADARINARAYANA et al. 2000 ). This latter phenomenon probably results from disturbing the balance within the complex and thereby interrupting its functional contacts (BADARINARAYANA et al. 2000 ). We consequently tested the effect of overproducing each of the components of the CCR4-NOT complex on sensitivity to 6AU. Only NOT4 overexpression resulted in poor growth on medium containing 6AU, although it had no major effect on growth on medium not containing 6AU (data not shown). These results suggest that it is the functionality and potentially the balance of components within the CCR4-NOT complex that is critical to the 6AU sensitivity phenotype.

The ccr4 mutation displays synthetic interactions with defects in the RPB2, TFIIS, and SPT16 elongation factors:
If CCR4 were to be involved in transcriptional elongation processes, defects in CCR4 might be expected to result in synergistic defects or lethalities when combined with defects in other known elongation factors. Previously we had shown that combining the ccr4 deletion with that of hprl, encoding a factor known to control elongation in yeast (CHAVEZ and AGUILERA 1997 ; CHAVEZ et al. 2000 ; Y. CUI and C. L. DENIS, unpublished data), results in a synthetic lethality (CHANG et al. 1999 ). We extended these results by analyzing the effect of ccr4 in combination with defects in elongation factors RPB2, TFIIS, and SPT16 (a component of the FACT elongation complex; ORPHANIDES et al. 1999 ).

The rpb2-10 and, to a lesser degree, the rpb2-4 allele have been shown to slow RNA polymerase II elongation in vitro (POWELL and REINES 1996 ). They and the rpb2-7 allele (which had no effect on in vitro elongation) all result in 6AU sensitive phenotypes (POWELL and REINES 1996 ). When ccr4 was combined with each of these three alleles, rpb2-10 ccr4- and rpb2-4 ccr4-containing strains displayed reduced growth at 30° and an inability to grow at 39°, normally a permissive temperature for ccr4 or rpb2 mutants (Fig 2). The rpb2-7 allele when combined with ccr4 did not cause reduced growth at 30°, but did exhibit a temperature-sensitive phenotype at 37°, slightly lower than that observed for ccr4 rpb2-4 or ccr4 rpb2-10 combinations (Fig 2). The temperature-sensitivity phenotypes observed when the rpb2 alleles were combined with ccr4 were found, however, to be suppressed by growth on minimal medium, suggesting that slowing growth allowed the cell to overcome the block caused by the combination of ccr4 and rpb2 defects (Fig 2). Moreover, each of the three rpb2 alleles when combined with ccr4 resulted in a failure to grow on nonfermentative carbon sources (Fig 2). This latter phenotype is not a result of permanent damage to the mitochondria since revertants capable of growing on nonfermentable carbon sources were obtained in ccr4 rpb2-10 and ccr4 rpb2-4 backgrounds (data not shown). We also observed that the rpb2-10 ccr4-containing strain was capable of growth on medium containing 6AU after 7 days whereas even after 7 days of growth the strains harboring the rpb2-4 ccr4 or rpb2-7 ccr4 alleles remained 6AU sensitive (data not shown). This latter phenomenon suggests an allele-specific interaction between loss of CCR4 function and the rpb2-10 allele.

View larger version (29K): In this window In a new window Download PPT slide   Figure 2. Synthetic interaction between ccr4 and rpb2, dst1, and spt16 defects. Yeast strains were grown on YD, YG, or ura- plates at the temperatures indicated. wt, strain Z96 (isogenic to DY103 except it lacks the URA3 plasmid); all other strains were isogenic to DY103 except spt16 (H154) and spt16 ccr4 (H154-1a).

We extended these analyses by analyzing the effect of combining ccr4 with a deletion in DST1, the gene encoding TFIIS. As observed for the rpb2 alleles, dstl when combined with ccr4 resulted in 39° temperature sensitivity (Fig 2), a phenotype that was suppressed by growth on minimal medium (data not shown), and a nonfermentative growth defect (Fig 2). Similarly, we observed that a ccr4 deletion when combined with the spt16-197 allele gave rise to a 34° temperature-sensitive phenotype that was not observed with either ccr4 or spt16-197 alone (Fig 2). However, combining ccr4 with defects in elongation factors did not always result in severe phenotypes. No augmentation of phenotype was observed when ccr4 was combined with elp1, spt4, or the spt5 alleles, spt5-8 or spt5-242 (data not shown). These above results indicate two important points. First, combining ccr4 with mutations in RPB2, DST1, or SPT16 that are known or presumed to be defective in elongation results in more severe phenotypes. Combining ccr4 with the rpb2-3 allele that affects transcriptional initiation events (LEE et al. 1998 ) did not result in synergistic effects (data not shown). Second, ccr4 displays an allele-specific effect with the rpb2-10 allele, suggesting a functional link between the corresponding proteins. These results support a role for CCR4 in transcript elongation.

rpb1 alleles involved in elongation display allele-specific interactions with ccr4:
Two rpb1 alleles (rpb1-221 and rpb1-244) that have been previously described cause synthetic defects with a dst1 deletion (HARTZOG et al. 1998 ). These rpb1 alleles also suppress the cold-sensitive defect associated with the spt5-242 allele, confirming a role for these alleles in elongation processes controlled by the SPT4-SPT5 complex (HARTZOG et al. 1998 ). We subsequently examined the effect of combining a ccr4 deletion with the rpb1-221 and rpb1-244 alleles. We found that combining a ccr4 deletion with the rpb1-244 allele resulted in a severity of phenotypes greater than that for ccr4 combined with rpb1-221 (Fig 3). Strains containing ccr4 rpb1-244 displayed weaker growth at 30° and no growth at 37° (Fig 3) and were unable to grow on nonfermentative carbon sources (data not shown). The poor viability of rpb1-244 with ccr4 is similar to that observed between rpb1-244 and a dst1 deletion (HARTZOG et al. 1998 ). We also observed that the ccr4 rpb1-244 synthetic lethalities were completely relieved by reintroduction of a wild-type CCR4 gene into the strain. In addition, when ccr4 was combined with the rpb1-1 allele, known to affect initiation of transcription (HOLSTEGE et al. 1998 ), no synergistic effects were obtained (data not shown).

View larger version (19K): In this window In a new window Download PPT slide   Figure 3. Allele-specific interactions between the ccr4 and rpb1 allele. Yeast strains were grown on YD plates at the temperatures indicated. wt, strain FY1642; rpb1-244, strain GHY-149; rpb1-221, strain FY1638; ccr4 derivatives were isogenic to the above three strains.

The spt5-242 cold-sensitive allele is suppressed by ccr4 or overexpression of NOT1:
The above described rpb1-244 and rpb1-221 alleles were initially identified as suppressors of the cold-sensitive phenotype associated with spt5-242. Similarly, the rpb2-10 allele, which causes an in vitro defect for transcriptional elongation (POWELL and REINES 1996 ), is also capable of suppressing spt5-242 (HARTZOG et al. 1998 ). Slowing of elongation in vivo by the addition of 6AU also suppressed spt5-242. Because of the known role of human homologs of SPT5 in elongation (WADA et al. 1998 ), it was postulated that slowing of elongation allows rescue of the spt5-242 elongation defect (HARTZOG et al. 1998 ). We therefore tested if a ccr4 deletion or overexpression of individual CCR4-NOT complex components could also rescue the spt5-242 cold-sensitive phenotype. As summarized in Table 2, the spt5-242 cold-sensitive phenotype was capable of being suppressed by a ccr4 deletion. Also, overexpression of the NOT1 gene specifically suppressed the spt5-242 phenotype (Table 2). Overexpression of the NOT1 gene resulted in about three- to fourfold more NOT1 protein in the cell than normally was present (data not shown). These results support the model that CCR4 and the NOT proteins affect transcriptional elongation.

One simple model for the effect of ccr4 on elongation would be that components of the CCR4-NOT complex, as transcriptional regulators, affect the expression of the rate-limiting enzyme IMPDH in the synthesis of GTP (GLESNE et al. 1991 ; SHAW and REINES 2000 ). Decreasing GTP levels would be expected to slow elongation. Because of the existence of four separate genes encoding IMPDH (SHAW and REINES 2000 ), we chose to quantitate the effect of CCR4-NOT defects directly on IMPDH enzyme levels in the cell. IMPDH enzyme levels (100 milliunits/mg in wild type) were found, however, to be relatively unchanged with the ccr4 (120 milliunits/mg IMPDH), not1-2 (100 milliunits/mg), not2-1 (80 milliunits/mg), or not4 (120 milliunits/mg) alleles, although a caf1 deletion did result in a twofold drop in IMPDH enzyme levels to 56 milliunits/mg.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Genetic evidence for CCR4 involvement in transcriptional elongation:
We have provided genetic evidence that supports a role for CCR4 and components of the CCR4-NOT complex in regulating transcriptional elongation. This novel role for these proteins is supported by several observations. First, defects in nearly all of the individual components of the 1.0-MD core CCR4-NOT complex as well as overexpression of NOT4 elicited 6AU and mycophenolic sensitive phenotypes. These phenotypes were not generally associated with defects in other factors involved in transcriptional initiation and mRNA degradation, or with CCR4-NOT complex components (CAF4, CAF16, and DBF2) not part of the 1.0-MD CCR4-NOT complex. Second, deletion of ccr4 resulted in severe synthetic effects with defects in several known elongation factors: hprl, rpb2, rpb1, dst1, and spt16. Because the biochemical mechanism of action by several of these elongation factors remains largely unknown, it is difficult to ascertain at what step or pathway CCR4 is involved. The spectrum of synthetic defects observed with ccr4 suggests that the CCR4 protein may play a role in a novel aspect of this regulation.

Third, a ccr4 deletion displayed an allele-specific interaction with the rpb1-244 allele that has been suggested to play a role in elongation (HARTZOG et al. 1998 ). This result suggests a functional interaction between CCR4 and RPB1 in terms of elongation and is supported by the previous observations that CCR4 is a component of the PAF1-containing RNA polymerase II transcription complex (CHANG et al. 1999 ) and that components of the 1.9-MD CCR4-NOT complex display multiple physical interactions with the SRB9-11 proteins of the RNA polymerase II holoenzyme (LIU et al. 2001 ). Finally, we showed that a ccr4 deletion or overexpression of NOT1 suppressed the cold-sensitive phenotype associated with the spt5-242 allele, suggesting that they slow the rate of elongation (HARTZOG et al. 1998 ). The particular effect of NOT1 overexpression may result from its role as the scaffold for the CCR4-NOT complex and thereby affect the integrity of the complex by its overexpression (BAI et al. 1999 ).

While the above evidence supports a direct role for CCR4-NOT proteins in affecting some aspect of elongation, it remains possible that the described interactions result from indirect effects of CCR4-NOT factors on transcription initiation processes. Although it is difficult to formally eliminate this alternative explanation, the above multiple correlations between ccr4 and elongation defects and the observation that the CCR4-NOT proteins do not significantly affect the overall enzyme levels of IMPDH in the cell makes this suggestion seem unlikely. In addition, whereas it has been suggested that the dst1 deletion causes sensitivity to 6AU due to its effect on the transcription of the SSM1 gene (SHIMOARAISO et al. 2000 ), we have found that ccr4 has no effect on SSM1 mRNA synthesis (H. BAKER and C. L. DENIS, unpublished data).

Relationship of CCR4-NOT function in initiation to that of elongation:
The CCR4-NOT proteins have been implicated in the control of transcriptional initiation by a number of studies (DENIS and MALVAR 1990 ; SAKAI et al. 1992 ; COLLART and STRUHL 1994 ). The most critical of this evidence is the enhanced transcription from the TATAA-less promoter at HIS3 (COLLART and STRUHL 1993 , COLLART and STRUHL 1994 ), the suppression by ccr4 and caf1 of spt10-enhanced ADH2 expression but not of ADR1^c-enhanced ADH2 expression (DENIS 1984 ; DRAPER et al. 1995 ), and the effect of CCR4-NOT proteins on promoters placed in front of different reporter genes (LIU et al. 1998B ). Moreover, ccr4 does not affect the degradation rate of ADH2 mRNA or elongation through the ADH2 gene (Y. CUI and C. L. DENIS, unpublished data), indicating that the effects of ccr4 on ADH2 expression must be at the level of initiation of transcription. In addition, the CCR4-NOT proteins exhibit multiple contacts to proteins playing important roles in controlling initiation (TFIID, ADA2, and SRB9-11; BENSON et al. 1998 ; LEE et al. 1998 ; BADARINARAYANA et al. 2000 ; LEMAIRE and COLLART 2000 ; LIU et al. 2001 ).

Yet, the multiple roles played by other initiation factors in such processes as DNA repair, promoter clearance, transcriptional elongation, polyadenylation, and 3' end formation suggests that factors controlling initiation can be utilized in other facets of DNA/RNA metabolism. In addition to the genetic evidence described herein, the CCR4-NOT proteins display several characteristics suggestive of roles in aspects of RNA formation other than that of initiation. First, CCR4 is part of the PAF1-RNA polymerase II complex (CHANG et al. 1999 ), which contains HPR1, a protein involved in elongation rather than in initiation (CHAVEZ and AGUILERA 1997 ; CHAVEZ et al. 2000 ; our unpublished data). Second, CCR4-NOT components interact with the subset of proteins SRB9, -10, and -11 of the RNA polymerase II holoenzyme (LIU et al. 2001 ). While this complex does function in initiation, the importance of SRB10 in phosphorylation of RNA polymerase II (HENGARTNER et al. 1998 ) and the dependency of elongation on this phosphorylation (PAYNE et al. 1989 ), suggests a critical role for SRB10 in creating a competent elongating form of RNA polymerase II. Although the SRB9, -10, and -11 proteins generally act as repressors, which has been linked to preinitiation control of RNA polymerase II, they can also function as activators (HOLSTEGE et al. 1998 ; LIU et al. 2001 ) and may be involved in another aspect of RNA formation. As suggested previously (AKHTAR et al. 1996 ), activators or coactivators like the CCR4-NOT proteins could aid in setting up processive polymerases at the promoter and thereby generate more active or increased numbers of elongating polymerases.

Third, components of the CCR4-NOT complex appear to display functions linked to direct RNA/DNA contacts. CCR4 and CAF1 display sequence homology and enzymatic activities related to exo- and endonucleases (MOSER et al. 1997 ; DLAKIC 2000 ; TUCKER et al. 2001 ; J. CHEN, Y.-C. CHIANG and C. L. DENIS, unpublished data) and NOT4 contains a putative RNA binding domain (ALBERT et al. 2000 ; our unpublished data). It is, therefore, likely that these proteins are involved directly in contributing to some facet of RNA synthesis, degradation, or monitoring. Such interactions are consistent with possible roles of these proteins in several aspects of elongation.

Recently, another presumed initiation factor, RTF1, has been found to display multiple genetic interactions with elongation factors and display 6AU sensitive phenotypes (COSTA and ARNDT 2000 ). The intrinsic overlap between initiation and elongation suggests that identifying factors like RTF1 and CCR4-NOT proteins that could act in both processes will not be unique to these factors. These proteins could act to affect promoter clearance, elongation procession, rescue of stalled complexes, or interaction with chromatin rearrangement factors. Identification of specific target genes controlled at the level of elongation by these factors would be one step toward elucidating their precise mechanisms of action.

	ACKNOWLEDGMENTS

We thank F. Winston, A. Aguilera, and G. Hartzog for their generous provision of yeast strains used in this work. We are also indebted to D. Reines for providing strains and valuable discussions that aided this study, and to D. Reines, G. Hartzog, and K. Arndt for comments in regard to the manuscript. The secretarial skills of B. Lauze are also appreciated. This research was supported by National Institutes of Health grant GM41215. This is publication no. 2077 from the New Hampshire Agricultural Experiment Station.

Manuscript received January 5, 2001; Accepted for publication March 15, 2001.

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