Translational Repression by Deadenylases*

The CCR4-CAF1-NOT complex is a major cytoplasmic deadenylation complex in yeast and mammals. This complex associates with RNA-binding proteins and microRNAs to repress translation of target mRNAs. We sought to determine how CCR4 and CAF1 participate in repression and control of maternal mRNAs using Xenopus laevis oocytes. We show that Xenopus CCR4 and CAF1 enzymes are active deadenylases and repress translation of an adenylated mRNA. CAF1 also represses translation independent of deadenylation. The deadenylation-independent repression requires a 5′ cap structure on the mRNA; however, deadenylation does not. We suggest that mere recruitment of CAF1 is sufficient for repression, independent of deadenylation.

The CCR4-CAF1-NOT complex is a major cytoplasmic deadenylation complex in yeast and mammals. This complex associates with RNA-binding proteins and microRNAs to repress translation of target mRNAs. We sought to determine how CCR4 and CAF1 participate in repression and control of maternal mRNAs using Xenopus laevis oocytes. We show that Xenopus CCR4 and CAF1 enzymes are active deadenylases and repress translation of an adenylated mRNA. CAF1 also represses translation independent of deadenylation. The deadenylation-independent repression requires a 5 cap structure on the mRNA; however, deadenylation does not. We suggest that mere recruitment of CAF1 is sufficient for repression, independent of deadenylation.
The role of CAF1 and CCR4 enzymes in regulation appears to be complex (5). In some systems, deadenylation is required for repression, yet in others it is dispensable (5,6,37). For example, in budding yeast, CAF1 is required for repression of mRNAs targeted by the regulatory protein, PUF5, even though deadenylation of the target is not (24). Similarly, miRNA complexes elicit deadenylation via the CCR4-CAF1 complex, yet deadenylation is not essential for repression (13,22,(37)(38)(39). These findings suggest that the deadenylase enzymes CCR4 and CAF1 may possess a second, deadenylation-independent role in translational repression.
Control of mRNAs is pervasive during early development and often is mediated by changes in poly(A) length (1,3). Maternal mRNAs direct oogenesis and embryogenesis until zygotic transcription begins (1). The temporal control of deadenylation in Xenopus oocytes and embryos is highly regulated; specific mRNAs are deadenylated at precise times throughout the early period of development and lose poly(A) to characteristic extents (9, 40 -42). In Xenopus, the PARN deadenylase, recruited by the 3Ј-UTR binding protein, CPEB, removes poly(A) from target mRNAs (e.g. cyclin B1) prior to maturation (9). It dissociates once oocyte maturation begins, allowing polyadenylation and translation (9). Other deadenylases, including CCR4 and CAF1, likely facilitate the intricacies of deadenylation. The CCR4-NOT complex catalyzes deadenylation of multiple mRNAs in Drosophila embryos and again is recruited by specific regulatory proteins, such as Smaug (7,11).
We sought to determine how CAF1 and CCR4 contribute to repression and participate in the control of maternal mRNAs, focusing on the Xenopus oocyte. Our data reveal that the CAF1 enzyme possesses an intrinsic repression activity, independent of its ability to deadenylate the mRNA. This activity requires the mRNA 5Ј cap structure. We show that mere recruitment of CAF1, even without deadenylation, is sufficient to repress a target mRNA.

EXPERIMENTAL PROCEDURES
DNA Constructs-With pCS2ϩ3HA:MS2, three HA tags were inserted in the BamHI/EcoRI restriction sites of pCS2ϩ vector (addGene). An NcoI restriction site was introduced before the EcoRI site that is in-frame with the HA tags (pCS2ϩ3HA). The MS2 fragment from pET-MS2 was ligated into the NcoI/StuI restriction sites of pCS2ϩ3HA, creating pCS2ϩ3HA:MS2 (47). This vector contains the Sp6 promoter followed by three HA tags, the MS2 coat protein, and the MCS. pCS2ϩ3HA:MS2ϩPL. Additional restriction sites were added to the MCS of pCS2ϩ3HA:MS2. The vector was cleaved with StuI/XhoI. AP005 (sense, cctggacccatcgatgaaggaagatcttcctagactagtctagaac) and AP006 (antisense, tcgagttctagactagtctaggaagatcttccttcatcgatgggtccagg) were annealed, kinased, and inserted into the StuI/XhoI restriction sites creating pCS2ϩ3HA:MS2ϩPL vector. This vector contains the same features as pCS2ϩ3HA:MS2 with ClaI, BglII, and SpeI added in the MCS between StuI/XhoI.
Each plasmid carrying an MS2 fusion contains the SP6 promoter, followed by three HA tags, one copy of the MS2 coat protein, and the protein to be tested. All of the plasmids were linearized with NotI before in vitro transcription.
pLG-MS2 and pLGMS2-LucHS were linearized with BglII and transcribed with T7 RNA polymerase. pLG-MS2ϩA39 and pLGMS2ϩA39-LucHS were linearized with BamHI and transcribed with T7 RNA polymerase. pJSP65 was linearized with SalI and transcribed with Sp6 RNA polymerase. pCSFV-Luc-MS2 was linearized with BglII and transcribed with T7 RNA polymerase.
In Vitro Transcription-Plasmids were linearized with the restriction enzymes indicated above. All of the in vitro transcriptions were described by Kwak et al. (49).
Western Blotting-All Western blotting was done as described by Kwak et al. (49) with the following modifications. The oocytes were lysed in 10 l of PBS plus protease inhibitors (Roche Applied Science) centrifuged at 3000 rpm for 10 min at 4°C, and the supernatants were collected. The lysates from two oocytes were loaded on to 8 -16% SDS/PAGE gels (Lonza). The proteins were analyzed by Western blotting using mouse monoclonal anti-HA tag antibody (HA11 1:1000 dilution, from Covance, Princeton) and anti-actin antibody (actin 1:40,000 dilution, from MP Biomedicals).
Tethered Function Assays-Oocyte injections were performed as described (47,48). The oocytes were collected at the indicated time points.
RNA Extraction and Gel Electrophoresis-Oocyte RNA was prepared by using TRI reagent, following the manufacturer's instructions (Sigma). RNAs were separated on a 6% polyacrylamide gel (short RNAs) and analyzed by autoradiography.
Northern Blots-10 g of total mRNA was separated on a 1% agarose, formaldehyde, 1ϫ MOPS gel. The RNA was transferred by capillary action to activated nylon membrane (Millipore) using 10ϫ SSC. The blots were hybridized in ULTRAhyb TM (Ambion) and washed according to the manufacturer's specifications. The firefly luciferase mRNA was detected with a probe antisense to nucleotides 1131-1640. The probe was transcribed using the T7 megascript kit from Ambion and was internally labeled with [␣-32 P]UTP. Northern blots were exposed to a phosphorimaging screen for 1 day. The screens were scanned on a Strom phosphorimaging (Molecular Dynamics).

Domain Structure of Xenopus CCR4 and CAF1 Homologs-
The Xenopus laevis genome is predicted to encode four CCR4 and two CAF1 homologs. Expressed sequence tag data indicate that each mRNA is expressed in stage VI oocytes. CCR4a and CCR4b contain leucine-rich repeats and a CCR4-deadenylase domain. The Xenopus CCR4 proteins are in the exonucleaseendonuclease phosphatase family and include four amino acids (DEDD) necessary for catalytic activity (Fig. 1A) (5,16). Based on conservation (Fig. 1A), CCR4a and CCR4b appear to be functionally distinct (supplemental Fig. S1 and Fig. 1A). The two CAF1 homologs, CAF1a and CAF1b, belong to the RNase D/DEDD family of endonucleases and possess the characteristic four amino acids necessary for catalytic activity (Fig. 1B) (5,17).
Tethered CCR4 and CAF1 Repress-To test the activities of Xenopus CCR4 and CAF1, CCR4 (CCR4a and CCR4b) or CAF1 (CAF1a and CAF1b) was tethered to firefly luciferase reporter RNAs using the MS2 coat protein ( Fig. 2A). Xenopus oocytes first were injected with mRNAs encoding HA-MS2-CCR4 or HA-MS2-CAF1. These proteins possessed three HA tags followed by the MS2 coat protein and the protein of interest. The oocytes were left for 6 h to allow accumulation of the fusion proteins and then were co-injected with two reporter RNAs: a firefly luciferase mRNA with three MS2 coat protein-binding sites in its 3Ј-UTR and, as a control, a Renilla luciferase mRNA without MS2-binding sites. The firefly luciferase mRNA possessed a 39-nucleotide poly(A) tail. After 16 h, the firefly and Renilla luciferase levels were determined.
To determine whether CCR4 or CAF1 could catalyze deadenylation in oocytes, we injected radiolabeled (130 nucleotides) RNAs with MS2-binding sites and an A 39 tail (Fig. 3A). These RNAs were deadenylated by MS2-CCR4 and MS2-CAF1 but not by MS2-GLD2 D242A or MS2-Xp54 (Fig. 3B). For example, 89% of the RNA tethered to XlCAF1b was fully or partially deadenylated (supplemental Fig. S2). The deadenylases also excise several nucleotides into the RNA "body." 3 We conclude 3 Tethered CAF1 generated RNAs that are 9 -12 nucleotides shorter and 10 -15 nucleotides longer than the A 0 marker. Previous work shows that CAF1 can degrade non-adenosine nucleotides (51,52) and that deadenylation in oocytes results in removal of a few non-adenosine nucleotides from the end of the RNA (40). Partially deadenylated RNAs with oligo(A) tracts of 10 -15 nucleotides have been reported in previous studies (8, 24, 25, 53).  that CCR4 and CAF1 can repress translation of an adenylated mRNA and are active deadenylases. Tethered CAF1 Proteins Repress mRNAs without Poly(A)-To test whether CCR4 and CAF1 repress translation independent of deadenylation, we next assayed their activities on a reporter RNA without a poly(A) tail. Repression activity was expressed as a ratio of firefly to Renilla luciferase activities. CCR4a did not repress firefly expression, whereas CCR4b moderately repressed expression of a nonadenylated firefly mRNA (Fig. 4A). The moderate repression by CCR4b was statistically significant (p value Ͻ 0.002, eight experiments). Surprisingly, both CAF1 homologs repressed the same reporter mRNA by 2-3-fold (Fig. 4A). The reduction was specific; GLD2 D242A had no significant effect (Fig. 4A), and the levels of Renilla luciferase, produced from the mRNA without MS2-binding sites, were unaffected (data not shown). The MS2:CAF1 proteins were similar in activity to MS2:Xp54 (Fig. 4A) (43), and were expressed at comparable levels. The reporter mRNAs were not destabilized by the tethered CAF1 proteins or the controls (Fig. 4B). We conclude that the XlCAF1 can repress translation of a nonadenylated RNA. Repression by XlCAF1 proteins was more robust then by XlCCR4b; therefore, hereafter, we focus on the XlCAF1 proteins.
CAF1 Repression Is Deadenylation-independent-If Xenopus CAF1 proteins repress translation independent of deadenylation, then mutations in the catalytic domain of CAF1 should not disrupt repression. To test this prediction, we generated  missense mutations in the first two amino acids required for catalysis in other CAF1 proteins (Fig. 1B) (31).
Both CAF1 mutants CAF1a and CAF1b repressed expression of a nonadenylated mRNA (Fig. 5, A and B, top panels) and an adenylated mRNA (Fig. 5, A and B, bottom panels). Surprisingly, the CAF1a mutant repressed a mRNA with a poly(A) tail to the same extent as the wild type protein (Fig. 5A, bottom  panel). As expected, the missense mutations abolish catalytic activity of the proteins (Fig. 5, C and D, compare lanes 5 and 6), and the two proteins were expressed at similar levels (Fig. 5, A  and B).
The results of XlCAF1b were similar to those of XlCAF1a but differ in one important respect. Like the XlCAF1a mutant, the XlCAF1b mutant repressed translation with or without a poly(A) tail, was catalytically inactive, and was expressed well (Fig. 5, B and D). However, the XlCAF1b mutant relieved repression by 2-fold on an adenylated mRNA (Fig. 5B, bottom panel; p value Ͻ 0.005, seven experiments). We conclude that both proteins repress translation through a poly(A)-and deadenylation-independent mechanism. XlCAF1b also represses through deadenylation, whereas XlCAF1a does not.
Human CAF1 Proteins Repress Nonadenylated mRNAs-Next, we sought to determine whether the poly(A)-and deadenylation-independent mechanism of repression was specific to Xenopus. To test this, we tethered human CAF1 (hCAF1 and hPOP2). hCAF1 and hPOP2 repressed translation independent of a poly(A) tail (Fig. 6). Similarly, the hCAF1 catalytic mutant repressed an adenylated mRNA to the same extent as the wild type hCAF1 protein (Fig. 6A).
We conclude that the poly(A)-and deadenylation-independent mechanism of repression is not specific to XlCAF1 proteins. Strikingly, hCAF1 does not appear to utilize deadenyla-tion akin to XlCAF1a; however, similar to XlCAF1b, hPOP2 can repress through deadenylation.
Deadenylation Rates and Repression-To examine the kinetics of deadenylation, we analyzed labeled RNAs at various times after they had been injected into oocytes expressing XlCAF1a or XlCAF1b proteins. One hour after injection, the majority of RNA remained adenylated (Fig. 7A, lanes 7, 8, 11, and 12). By 2 h, deadenylation had begun (Fig. 7A, lanes 9 and 13). For example, after 1 h, only 73% of the RNA tethered to XlCAF1a was fully or partially deadenylated; whereas after 2 h, 47% of the RNA was deadenylated or partially deadenylated (data not shown). The rates of deadenylation, as tethered proteins, are low (ϳ0.5 nucleotide/min), as is the default deadenylation pathway during oocyte maturation (40).
The kinetic analysis prompted an additional test of the deadenylation independence of repression activity. If XlCAF1b repressed through a deadenylation-independent mechanism, then an mRNA could be repressed before it was deadenylated. To test this, we measured the level of firefly luciferase 1 h after injection of the mRNA in oocytes expressing XlCAF1b. We   Fig. 7A the majority (86%, data not shown) of the RNA was adenylated. XlCAF1b reduced the expression at the 1-h time point after the firefly luciferase mRNA was injected (Fig. 7B). The reduction was specific because GLD2 D242A, Xp54, and XlCAF1a did not reduce the level of firefly luciferase (Fig. 7B). However, as seen before, after 16 h Xp54, XlCAF1a, and XlCAF1b reduced the level of firefly luciferase (data not shown).

chose 1 h after injection because in
The 7m GpppG Cap Is Required for CAF1 Repression-To assess whether the cap structure was necessary for repression, we used a firefly luciferase mRNA bearing a 5Ј cap structure of ApppG. As expected, the level of translation from an ApppG capped mRNA was reduced by 97% (supplemental Fig. S3A). Strikingly, XlCAF1 proteins did not repress translation of an ApppG capped adenylated mRNA (Fig. 8A, top panel; p value Ͼ 0.5; three experiments). However, the XlCAF1 proteins repressed a reporter mRNA with a 7-methyl GpppG cap (Figs. 2B and 4A; p value of less then or equal to 1.8 ϫ 10 Ϫ6 ; five and eight experiments, respectively). As seen before, Xp54 did not repress the mRNA that contained an ApppG (Fig. 8A) (44). The results were the same with an ApppG capped nonadenylated mRNA (supplemental Fig. S3B).
We next tested whether the reduced translation rate of the ApppG mRNA was enough to alleviate repression by XlCAF1. To do so, we introduced a stem-loop before the translational start of a 7-methyl GpppG mRNA (Fig. 8A). As with the ApppG capped mRNA, the stem-loop reduced translation by 98% (supplemental Fig. S3A). However, unlike the ApppG capped mRNA, the translation of the stemloop mRNA with the 7-methyl GpppG cap was repressed by the XlCAF1 proteins (Fig. 8A, middle panel). Moreover, the missense XlCAF1 mutants repressed the stem-loop mRNA as well (Fig. 8A, middle panel). This reduction was specific because GLD2 D242A does not repress translation (Fig. 8A, middle panel). We conclude that XlCAF1 represses translation at or before recognition of the 7-methyl GpppG cap.
To further probe the mechanism of repression, we used an IRES derived from classical swine flu virus (CSFV). Initiation from the CSFV IRES does not require eIF4F or eIF3 (45,46). We prepared a luciferase mRNA with an IRES and an ApppG cap to avoid degradation when injected into oocytes. XlCAF1 did not repress translation of the IRES-containing reporter (Fig. 8A,  bottom panel). As seen previously, Xp54 did not repress the reporter mRNA with the IRES (Fig. 8A, bottom panel) (44). The IRES was functional, because that mRNA was translated more than 75-fold more efficiently than a replica mRNA lacking the CSFV IRES (data not shown). We conclude that translational repression by XlCAF1 proteins requires the 7-methyl GpppG cap and, likely, the translation initiation factors eIF4F and eIF3.

DISCUSSION
CAF1 proteins possess an intrinsic repression activity independent of deadenylation. By "intrinsic," we imply that the mere presence of a CAF1 protein on an mRNA is sufficient to elicit repression, whether or not deadenylation occurs. Several lines of evidence support this central conclusion. CAF1 proteins repress mRNAs without poly(A) tails, catalytically dead enzymes retain repression activity, and wild type CAF1 represses prior to deadenylation.
Deadenylation-independent repression by CAF1 proteins requires the cap, because it is blocked by a nonfunctional cap analog, ApppG. Repression also appears to require eIF4F and/or eIF3, because it is alleviated by use of the CSFV IRES. CAF1 may interfere with translation by contacting the cap and initiation factors directly or by acting through protein intermediaries. For simplicity, we will refer to CAF1 as a translational repressor, without implying its mechanism of action.
CAF1b possesses a low level of deadenylation-dependent repression activity, whereas CAF1a does not. Human POP2 also shows some deadenylation-dependent repression activity, whereas human CAF1 does not, demonstrating that the difference between CAF1a and CAF1b is conserved. Thus regulatory proteins and miRNAs might differ in activity depending on which isoform they recruit.
In vivo, regulatory factors, including both proteins and miRNAs, recruit CAF1 to mRNAs (7,8,(11)(12)(13)(14)22). We suggest that repression induced by RNA-binding proteins may be due in part to recruitment of CAF1 per se, independent of deadenylation. 3Ј-UTR-bound regulatory proteins, such as the PUF proteins, recruit CAF1 and other components of the NOT-CCR4-CAF1 complex (8). Similarly, Ago proteins, which associate with mRNAs via miRNAs, recruit CAF1 and several other components of the complex (12). miRNAs often (though not always) elicit deadenylation of their targets (37), as do many regulatory proteins (5). Although deadenylation often is required for full repression, that is not universally the case (37). The repression activity of CAF1 may be responsible.
Mere recruitment of CAF1 causes translational repression. In that sense, CAF1 can be viewed as a repressor, independent of its enzymatic activity. From that perspective, our findings reveal an unanticipated mode of mRNA control.