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Cytoplasmic CstF-77 protein belongs to a maskingcomplex with cytoplasmic polyadenylation

element-binding protein in Xenopus oocytes.Christel Rouget, Catherine Papin, Elisabeth Mandart

To cite this version:Christel Rouget, Catherine Papin, Elisabeth Mandart. Cytoplasmic CstF-77 protein belongs to amasking complex with cytoplasmic polyadenylation element-binding protein in Xenopus oocytes..Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2006,281 (39), pp.28687-98. �10.1074/jbc.M601116200�. �hal-00260282�

https://hal.archives-ouvertes.fr/hal-00260282https://hal.archives-ouvertes.fr

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Cytoplasmic CstF-77 protein belongs to a Masking Complex withCPEB in Xenopus Oocytes

Christel Rouget, Catherine Papin and Elisabeth MandartFrom the Centre de Recherches de Biochimie Macromoléculaire, CNRS, 1919 route de Mende, 34293

Montpellier cedex 05, FranceRunning title: Cytoplasmic CstF-77 in masking mRNA

Address correspondence to Elisabeth Mandart: CRBM, CNRS, 1919 route de Mende, 34293 Montpelliercedex 05, France, Tel (33) 4 67 61 33 39; FAX (33) 4 67 52 15 59; E-mail:elisabeth.mandart@crbm.cnrs.fr

Regulated mRNA translation is ahallmark of oocytes and early embryos, ofwhich cytoplasmic polyadenylation is a majormechanism. This process involves multipleprotein components, including the CPSF(Cleavage and Polyadenylation SpecificityFactor), which is also required for nuclearpolyadenylation. The CstF (Cleavagestimulatory Factor), with CPSF, is required forthe pre-mRNA cleavage before nuclearpolyadenylation. However, some evidencesuggests that the CstF-77 subunit might have afunct ion independent o f nuc learpolyadenylation, which could be related to thecell cycle. As such, we addressed the questionwhether CstF-77 might have a role incytoplasmic polyadenylation. We investigatedthe function of the CstF-77 protein in Xenopusoocytes, and show that CstF-77 has indeed arole in the cytoplasm. The Xenopus CstF-77protein (X77K) localizes mainly to the nucleus,but also in punctuate cytoplasmic foci. We showthat X77K resides in a cytoplasmic complexwi th e IF4E, CPEB (Cytop lasmicPolyadenylation Element Binding protein),CPSF-100 and XGLD2, but is not required forcytoplasmic polyadenylation per se.Impairment of X77K function in ovo leads to anacceleration of the G2/M transition, with apremature synthesis of Mos and AuroraAproteins. However, the kinetic of Mos mRNApolyadenylation is not modified. Furthermore,X77K represses mRNA translation in vitro.These results suggest that X77K could beinvolved in masking of mRNA prior topolyadenylation.

Regulation of gene expression plays acentral role in many cellular functions. In

eukaryotes, formation of the mature 3’ end of amessenger RNA is a two-step reaction thatinvolves cleavage of the nascent transcript andsubsequent polyadenylation in the nucleus(reviewed in 1-3). In mammals, the cleavagereaction requires two sequences, the highlyconserved hexanucleotide AAUAAA located 30-20 nucleotides upstream of the cleavage site, andthe G/U rich sequence that lies downstream of the3’ cleavage site (DSE for Downstream Element).These sequences are bound by differentmultisubunit factors. CPSF binds to theAAUAAA sequence via the CPSF-160 subunit,and CstF recognizes the DSE element via theCstF-64 protein (4,5). CPSF and CstF are knownto interact cooperatively at 3' end signals (6), andallow pre-mRNA cleavage with the cleavagefactors I and II (CFI and CFII), the carboxy-terminal domain of RNA polymerase II and thepoly(A) polymerase (PAP). CPSF consists of the160K, 100K, 73K, 30K and hFip proteins (7-9),and CstF is an heterotrimeric structure composedof the 77K, 64K and 50K proteins (10,11). CstF-77 is the subunit required for integrity of the CstFcomplex. CstF-77 binds to CstF-64, CstF-50 (12)and CPSF-160 (5) and therefore helps to holdtogether the much larger complete polyadenylationcomplex.

CPSF is also involved in cytoplasmicpolyadenylation (13,14), a mechanism ofcontrolling translation which is often used whentranscription is quiescent, such as in oocytes orearly embryos. In Xenopus oocytes, severaldormant mRNAs are stored with short poly(A)tails, which are elongated when oocytes arestimulated by progesterone, provoking theirtranslation.

Cytoplasmic polyadenylation in frogoocytes also requires two sequences on the 3’UTR

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of target mRNAs, recognized by multiple proteincomponents (reviewed in 15). CPEB directly bindsto the CPE (Cytoplasmic PolyadenylationElement) located upstream of the AAUAAAsequence, which is recognized by CPSF, as innuclear polyadenylation (14). CPEB interacts withCPSF, which may help to stabilize its associationwith the AAUAAA element and a cytoplasmicpoly(A) polymerase. GLD2, a poly(A) polymerasewhich is structurally distinguishable from nuclearPAP, was first identified in Caenorhabdit iselegans (16). The Xenopus related protein(XGLD2) was shown to bind to CPEB and CPSFand to participate in cytoplasmic polyadenylation(17,18). Sequences other than the CPE, the PREsequences (Polyadenylation Response Element),regulate early cytoplasmic polyadenylation ofspecific mRNAs including Mos (19,20). TheCPEB protein has been shown to repress mRNAtranslation in immature oocytes and to directcytoplasmic polyadenylation and translationalactivation in maturing oocytes. Indeed, mRNAtranslation repression is controlled by a complexcomposed of CPEB, Maskin and eIF4E. Maskininteracts with CPEB and eIF4E and preventseIF4G binding to eIF4E. After stimulation, CPEBis phosphory la ted and cy top lasmicpolyadenylation ensues, allowing disruption ofMaskin-eIF4E binding and recruitment of eIF4G(21,22).

The yeast Saccharomyces cerevisiae andDrosophila melanogaster homologues of CstF-77are, respectively, the Rna14 and Su(f) proteins.Several genetic and biochemical studies haveshown a role for Rna14 in 3’end processing (23-25), although other studies for Rna14 havesuggested a function independent of nuclearpolyadenylation. Indeed, rna14 mutants can beseparated into two classes: the poly(A)-negativeclass, which contains mutants deficient in mRNA3’ processing, and the poly(A)-positive class,which includes mutants that are not impaired innuclear polyadenylation (26,27). Rna14 and Su(f)localize both to the nucleus and the cytoplasm. Inyeast, this cytoplasmic localization is mainly inmitochondria (26,28). However, no mitochondrialfunction for Rna14 has been described so far. InD.melanogaster , the Su(f) protein has beenimplicated in cell cycle progression (29). InXenopus and human cells, studies have shown thatCstF-77, CstF-64 and CPSF-100 are concentrated

in nuclear domains in Cajal or coiled Bodies (30-33) as well as various components required fortranscription and processing of the three classes ofnuclear transcript (reviewed in 34). However,nothing was reported concerning a possiblecytoplasmic localization of the CstF subunits.

The aim of our work was to study thecytoplasmic function of CstF-77, bearing in mindpossible roles independent of nuclearpolyadenylation that could be related to the cellcycle. We therefore addressed the question ofwhether CstF-77 could have a role in cytoplasmicpolyadenylation. The Xenopus oocyte is anappropriate model to study other functions ofCstF-77, as there is neither transcription nornuclear polyadenylation during oocyte maturation(15). In this report, we identify the Xenopus CstF-77 protein (X77K) and show that it is partiallylocalized in the cytoplasm. We demonstrate thatX77K belongs to a cytoplasmic complex witheIF4E, CPEB, CPSF-100 and XGLD2. However,the protein is not required for cytoplasmicpolyadenylation per se. Inhibition of X77Kfunction in oocytes accelerates meiotic maturationand precociously induces Mos and AuroraAprotein synthesis, without modifying the kinetic ofMos mRNA polyadenylation. Moreover, X77Kinhibits in vitro mRNA translation in a dosedependent manner. These results suggest thatX77K could have a function in mRNA maskingprior to cytoplasmic polyadenylation.

EXPERIMENTAL PROCEDURES

X77K cloning and protein alignment - AXenopus leavis oocyte cDNA library in λgt11from Clontech (ZL5000b) was screened with a1.030 base pair (bp) probe obtained by RT-PCRfrom X e n o p u s RNA with the followingdegenerated oligonucleotides (Eurogentec): 5'-ATGGCYMARGCWTAYGAYTTYGCACT-3'and 5'-ACKCKSGTRTTRTTRTCYTCRTTBAG-3' where Y=C+T, M=A+C, R=A+G, W=A+T,K=T+G, S=C+G and B=T+C+G. Several clonescontaining the cDNA of the Xenopus CstF-77homologue (X77K) were isolated and sequenced.The largest cDNA (about 2.300 bp) was subclonedat the EcoR1 site of the pBluescript II KS. Itcontains an open reading frame encoding a proteinof 719 amino acids. This X77K amino acidsequence was aligned with those of Homo sapiens

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CstF-77 (NP001317), Mus musculus CstF-77(AAH03241), Drosophila melanogaster Su(f)(P25991), Caenorhabditis elegans CstF-77(AAA62311) and Saccharomyces cerevisiaeRNA14 (NP013777) with the ClustalW program.The X77K sequence was submitted to Genbankwith the following accession number: AM071387.

Xenopus oocytes and embryos - Stage VIoocytes were selected after surgical removal ofovaries from mature female Xenopus laevis andtreatment with collagenase (Sigma) at 1mg/ml inOR-2 (5mM Hepes pH 7.2, 82.5mM NaCl, 2mMKCl, 1mM MgCl2). All further manipulations ofoocytes were performed in modified Ringer’sSolution (MMR: 5mM Hepes pH 7.8, 100mMNaCl, 2mM KCl, 1mM MgSO4, 0.1mM EDTA,2mM CaCl2). Progesterone was used at a finalconcentration of 1mM. For protein oocyte extracts,oocytes were homogenized at 5 µl per oocyte inlysis buffer (20mM Tris pH7.5, 50mM NaCl,50mM NaF, 10mM β-glycerophosphate, 5mMNa4P2O7, 1mM Na3VO4, 1mM EDTA, 1mMEGTA, 0.1mM PMSF, plus 10 µg/ml each ofleupeptin, chymostatin, pepstatin and aprotinin) at4°C, centrifuged at 13000 rpm for 3 minutes at4°C and the supernatant was used for furtheranalysis. For manually enucleated oocytes,nucleus were collected and boiled in 5X Laëmmlibuffer and cytoplasms were homogenized at 5 µlper cytoplasm in lysis buffer. In vitro fertilizationand embryo cultivation was carried out asdescribed (35). The quantification of proteins inoocyte and embryo extracts was done by Bioradprotein assay, with quantified BSA as standard.For microinjections, the usual injected volume forantibodies, purified protein or RNA was 20 to 40nl per oocyte and the number of injected oocytes is35 for each condition.

Antibodies and Western blots- The anti-pTpY-MAPK was obtained from New EnglandBiolabs (9106S). The anti-eIF4E was provided byS. Morley or was obtained from mouse antiserausing the same peptide antigen. The anti-CPSF-100 and the anti-Pak5 were provided by E. Wahleand N. Morin’s laboratories respectively. The anti-β-tubulin and anti-HA antibodies were obtainedfrom E7 (Iowa hybridoma bank) and from the12CA5 hybridomas respectively. The anti-RPAand anti-AuroraA were provided by J.M.Lemaître and C. Prigent respectively. The anti-

Mos, anti-ERK and IgG were obtained from SantaCruz (sc-086, sc-94 and sc-2027, respectively).Other antibodies used against GST and Xenopus77K, CPEB and GLD2 are rabbit polyclonalantisera and were affinity purified. The 77K Cterantibody is directed against the 17 amino-acidscarboxyl terminal of the human 77K protein:NH2-VPPVHDIYRARQQKRIR-COOH that waskindly provided by David Bentley, and the 573antibody is directed against the following X77Kpeptide: NH2-LKDDVDRKPEYPKPD-COOH.The anti-CPEB antibody is directed against GST-CPEB fusion protein as described (36). The antiXGLD2 antibody is directed against the GST-XGLD2 2/3 carboxy terminal fusion protein. Formicroinjections, dialyzed antibodies were used at1 µg/µl. Western blots were probed with primaryantibody at 50 ng/ml and the appropriatesecondary antibody horseradish peroxidase (HRP)conjugate diluted according to manufacturerrecommendations (Amersham) and revealed byECL (Amersham).

RNA and recombinant proteins - The Mos3'UTR construct was obtained by inserting theSpe1/Xba1 fragment from the pT7-G UK XMosplasmid kindly provided by N. Sagata (37) intoSpe1/Xba1-digested pCS2 (Dave Tumer,University of Michigan). This 320-nucleotidefragment contains Mos PRE, CPE and AAUAAAsequences. Mos 3'UTR RNA were prepared bylinearizing the pCS2-Mos320 with Xba1 andcarrying out transcription reaction according toPapin and Smith (38). The pCSH vectorcorresponds to the pCS2 vector with two HA tagsinserted in the multiple cloning site. CappedmRNA encoding HA-X77K, HA-XGLD2 andHA-CPEB were prepared by cutting the pCSH-X77K ORF, pCSH-XGLD2 ORF and pCSH-CPEB ORF respectively with Not1 and carryingout transcription reaction.

Immunoprecipitations - Protein oocyteextracts corresponding to 20 oocytes wereperformed in extract buffer (100 mM KCl, 0.1 mMCaCL2, 1mM MgCl2, 10 mM Hepes pH7.6, 50mM sucrose, plus 10 µg/ml each of leupeptin,chymostatin, pepstatin and aprotinin). For someexperiments, the oocyte extracts were treated 10min at room temperature with RNAse A at 0,02mg/ml. Oocyte extracts were incubated with 200ng of antibodies over night at 4°C on wheel,supplemented with 20 µl of Protein A-Sepharose

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beads and mixed for another 45 min at 4°C onwheel. The immunoprecipitates were then washed3 times in XRB A (0.2% Triton X-100, 10 mMTris pH8, 150 mM NaCl and 2 mM EDTA), 3times in XRB B (0.2% Triton X-100, 10 mM TrispH8, 500 mM NaCl and 2 mM EDTA) and 2times in 10 mM Tris pH8, eluted by boiling in 2XLaëmmli buffer, separated by SDS-PAGE andanalyzed by Western blotting.

For immunoprecipitation of radio-labeledX77K, 1 µg of 77K antibody was incubated or notwith 10 µg of its antigen (77K Cter peptidecoupled with thyroglobulin) before being appliedto protein-A Sepharose beads for 30 min at 4°C.The beads were then washed three times withPBS, supplemented with 35S methionine X77Ktranslated in rabbit reticulocyte lysates, and mixed1 h on ice. The immunoprecipitates were thenwashed 3 times in XRB A, 3 times in XRB B, and2 times in 10 mM Tris pH8, eluted by boiling in2X Laëmmli buffer, separated by SDS-PAGE, andanalyzed by autoradiography.

Cap column and GST Pull-Down assay–The cap column assay was performed as described(22). Briefly, 30 oocytes were homogenized inhomogenization buffer (HB), and treated or not 10min at room temperature with RNAse A (0,02mg/ml). The oocyte supernatants weresupplemented with 0.1 mM GTP or 5 mM 7mGTPand applied in batch to a 7mGTP-Sepharose (cap)column (Amersham) that had been equilibratedwith HB plus bovine serum albumin (BSA, 0.1mg/ml). Following mixing for 1h at 4°C ineppendorf, the cap column was extensivelywashed (100 bed volumes) with 0.1 M KCl in 50mM Tris pH 7.5. The bound material was theneluted with 5 mM 7mGTP on column. The eluatesand a fraction of oocyte lysates collected beforeand after mixing with the cap column wereseparated by SDS-PAGE and analyzed by Westernblotting.

The GST pull-down assay was performedas described (39). Briefly, HA-tagged CPEB andHA-tagged XGLD2 were in vitro translated inrabbit reticulocyte lysates (Promega) in thepresence of unlabeled amino acids. GST-X77Kexpressed in Baculovirus and GST expressed inE.coli were purified with glutathione sepharose(Amersham). 5 µg of purified GST-X77K or GSTwere bound to 20 µ l of glutathione beads in

binding buffer. The beads were subsequentlyincubated 2h at room temperature in 400 µl ofbinding buffer plus 5 µg of the HA-CPEB or HA-XGLD2. The beads were then extensively washedwith binding buffer and boiled in 2X Laëmmlibuffer. The eluates and inputs were separated bySDS-PAGE and analyzed by Western blotting.

Polyadenylation assays - Total RNA fromoocytes was extracted using the Mini RNAIsolation IITM kit (Zymo Research) and the PAT(PolyAdenylation Test) assay was carried outaccording to Salles and Strickland (40) with minormodifications. Reverse transcription was done ontotal RNA with oligo(dT)-anchor (dT-PAT) asprimer (5'-GCGAGCTCCGCGGCCGCGT(12)-3').Subsequent PCR was carried out using the dT-PAT primer and a specific upstream primer forM o s 3 ' U T R R N A ( 5 ' -GCACTGAAAATACAAGCAAGGATATG-3').C y c l i n B 1 O R F R N A ( 5 ’ -GTGGAAATGGCCCGCCCACTC-3’), AuroraA3 ’ U T R R N A ( 5 ’ -GGCTGTCACCGTACAACGCTACTTG-3’) andac t in type 5 3 ’UTR RNA (5 ’ -CAAATGTTGCAGGTACACCTG-3’). The PCRproducts were resolved in a 2.5% agarose gel andvisualized with ethidium bromide staining.

For in vitro polyadenylation assays, eggextracts were prepared according to Murray andKirschner (41). Egg extracts were adjusted to 1mM ATP, 1 mM MgCl2, 7.5 mM creatinephosphate and 0.1 mM EGTA pH7.7. 30 ng ofMos 3'UTR RNA was added to the egg extract andthe polyadenylation assay was carried out at 23°Cfor 1h, prior to RNA extraction as described inMcGrew and Richter (42). Mos 3' UTRpolyadenylation analysis was carried out asdescribed above (PAT assay). For depletionexperiments, 2 µg of antibodies were incubated 30min at 4°C with 15 µl of Dynabead protein-Abeads (Dynal). Beads were washed three timeswith PBS plus 100 µg/ml BSA and incubated with15 µ l of egg extract for 30 min at roomtemperature. After precipitation, depleted extractwas collected and used for polyadenylation assayand Western blot analysis. Bead precipitates wereeluted by boiling in 2X Laëmmli buffer andanalyzed by Western blotting.

In vitro translation in Rabbit ReticulocyteLysate (RRL) - The luciferase translation assay

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was performed according to manufacturerrecommendations (Promega). 100 ng of LuciferasemRNA (provided in RRL kit) were added to 10 µlof RRL in presence of [35S]methionine (2µl,1000Ci/mmol, methionine/ cysteine mix,Amersham) with or without the indicatedquantities of GST-77K or GST proteins, for 1.5hat 30°C. The equivalent of 20% of the reactionwas used for autoradiography and western blotanalysis. The amount of proteins added to thereaction was analyzed by Coomassie staining. Theluciferase RNA was analysed by RT-PCR after thereaction. Reverse transcription was done withrandom primers and the amplification with thef o l l o w i n g o l i g o n u c l e a o t i d e s : 5 ’ -AAGCCACCATGGAAGA-3’ and 5 ’ -CTCTAGAATTACACGGCGATC-3’.

Cell culture and immunofluorescence -Mouse NIH 3T3 cells were cultured in DMEMsupplemented with 10% FBS in a 37°C incubatorwith 5% CO2. Xenopus XTC cells were cultured in70% L15 Leibovitch's Media supplemented with10% FBS in hermetic tissue culture flask at 25°C.Cultured cells were fixed using 4%paraformaldehyde (in PBS) for 15 min at roomtemperature. The cells were then permeabilized inPBS containing 1% Triton-X100 for 10 min atroom temperature and blocked in PBS containing0,1% Tween and 3% BSA (blocking reagent).Antibody incubations were done 1h at roomtemperature in blocking reagent followed bysecondary Alexa-546-labeled goat anti-rabbit IgG(Molecular probe) used in identical conditions.DNA was visualized by DAPI staining.

RESULTS

Identification of Xenopus CstF-77homologue - A Xenopus laevis oocyte library wasscreened with a probe made by RT-PCR fromX e n o p u s oocyte RNA and degenerateoligonucleotides. A cDNA encoding the Xenopushomologue of CstF-77 was isolated. This cDNAcontains an open reading frame (ORF) encoding aprotein of 719 amino-acids, with a predictedmolecular weight of 83kDa that we named X77K.We identified a domain organization similar to theknown CstF-77 homologues, with 11 HAT (half aTPR) repeats, a bipartite nuclear localizationsignal (NLS) and a proline-rich domain at the Cterminus (figure 1). Apart from the NLS, these

domains are known to mediate protein-proteininteractions, and the HAT repeats have beendescribed to be specific to a family of proteinsinvolved in RNA processing (11,43). For thehuman CstF-77 protein, the proline-rich domainhas been shown to be necessary both for bindingother CstF subunits and also for self association(12). It is noteworthy that the CstF-77 proteins arehighly conserved, since the amino acid sequenceof X77K shares 94% identity overall with thehuman and mouse CstF-77 proteins, 57% withD.melanogaster protein and 27% with S.cerevisiaeprotein (figure S1).

CstF-77 is localized both in the nucleusand cytoplasm - To study the expression of X77Kprotein, we raised a polyclonal antibody againstthe human C terminal peptide (77K antibody). ByWestern blotting, this antibody recognizes the HA-tagged X77K expressed in oocytes, and thepurified Baculovirus GST-X77K protein (figure2A lanes 2 and 5). It also detects endogenousXenopus CstF-77 protein from oocytes or celllysates (figure 2A lanes 1 and 3 respectively) andendogenous mouse (lane 4) and human protein(not shown). Moreover, the in vitro translatedX77K protein is immunoprecipitated by the 77Kantibody but not by the 77K antibody pre-incubated with the peptide antigen (figure 2B).These results show the specificity of the 77Kantibody for the 77K protein.A Western blot analysis on oocyte and embryolysates shows that X77K is expressed at a constantlevel dur ing X e n o p u s oogenesis andembryogenesis up to stage 40 (figure 3A and 3B).In S. cerevisiae and D. melanogaster, the CstF-77homologues Rna14 and Su(f) respectively localizeboth to the nucleus and cytoplasm (28,44). InXenopus oocytes, the nuclear localization ofX77K has been well determined, where it ispresent in Cajal bodies and lampbrushchromosomes (32). However, nothing has beenreported concerning a possible cytoplasmiclocalization. To investigate whether X77K couldbe cytoplasmic in Xenopus laevis, lysates fromnucleus, cytoplasm or total oocytes were analyzedby Western blotting with 77K antibody. Figure 3Cshows that X77K is mainly nuclear, but is alsopresent in the cytoplasm. We verified thatcytoplasmic extracts were not contaminated withnuclear extracts by probing the membrane for thenuclear protein RPA (Replication Protein A). This

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double localization was confirmed byimmunofluorescence experiments on XenopusXTC and mouse NIH3T3 cell lines (figure 3D):CstF-77 protein is mainly in the nucleus but is alsolocalized in punctuated bodies distributed in thewhole cytoplasm. These results show that theCstF-77 protein is localized both in the nucleusand cytoplasm in vertebrates, as in S. cerevisiaeand Drosophila.As X77K has a functional NLS but partiallylocalizes to the cytoplasm, we investigatedwhether the protein shuttles between the GV(Germinal Vesicle) and the cytoplasm, even if itdoes not present a NES (Nuclear Export Signal).To answer this question, oocytes were treated ornot overnight with leptomycin B, a nuclear exportinhibitor. They were then enucleated, to analyzewhether the protein accumulated in the nucleus inpresence of leptomycin B. In this condition, nochange in the relative levels of X77K between thenucleus and the cytoplasm was observed, showingthat X77K does not shuttle between the twocompartments (figure 3E). Surprisingly, the CPEBprotein was sensitive to leptomycin B andaccumulated in the nucleus (figure 3E). This resultvalidates the leptomycin B treatment and impliesthat CPEB shuttles between the cytoplasm and thenucleus. This observation was not expected sinceCPEB was not detected in the nucleus undernormal conditions (35 and figure 3E) and suggestsnew functions for CPEB in the nucleus, or innucleo-cytoplasmic transport.

X77K is part of a cytoplasmic complexwith CPEB, eIF4E, CPSF-100 and XGLD2 - Aswe asked whether X77K could have a function incytoplasmic polyadenylation, we checked forX77K partners in the cytoplasm. In Xenopusoocytes, CPEB plays a key role in translationalrepression in immature oocytes, and in directingcytoplasmic polyadenynation during oocytematuration. We investigated whether X77K couldbelong to a complex which includes CPEB. Weperformed co-immunoprecipitations from totalstage VI oocyte extracts, treated or not with RNaseA, with antibodies directed against differentproteins known to be in a cytoplasmic complexwith CPEB. In this experiment, X77K, CPEB,eIF4E and CPSF-100 proteins were co-immunoprecipitated with 77K, eIF4E and CPEBantibodies but not with nonspecific IgG (figure4A). As a negative control we used an antibody

directed against the Pak5 (P21 activated kinase)protein. This antibody did not immunoprecipitateany of the proteins tested. Similarly, Pak5 proteinwas not immunoprecipitated by the 77K, eIF4E orCPEB antibodies. These results show that X77Kspecifically interacts with eIF4E, CPEB andCPSF-100 independently of the mRNA.To show that cytoplasmic X77K is involved in thiscomplex, we enucleated oocytes and performedco-immunoprecipitation experiments from oocytecytoplasm extracts treated with RNase A. TheX77K, CPEB and CPSF-100 proteins were co-immunoprecipitated by X77K, eIF4E and CPEBantibodies (figure 4B). These results show thatcytoplasmic X77K belongs to a complex withCPEB, eIF4E and CPSF.It has been reported that XGLD2, a divergentpoly(A) polymerase, directly binds to CPEB, evenin stage VI oocytes (18). We asked whetherXGLD2 was also in the complex with X77K instage VI oocytes. As XGLD2 is expressed at a lowlevel in oocytes (18), immunoprecipitations wereperformed from oocytes overexpressing XGLD2.HA-tagged XGLD2 mRNA was injected intooocytes and, after 14 hours of expression, theoocyte extracts were subjected to differentantibody immunoprecipitations in presence orabsence of RNase A. In these experiments, HA-XGLD2, X77K, eIF4E and CPEB proteins wereco-immunoprecipitated with XGLD2, X77K,eIF4E and CPEB antibodies (figure 4C), showingthat XGLD2 is also part of the complex. It isinteresting to note that two forms of eIF4E wereimmunoprecipitated by the eIF4E antibody butthat only the lower form is mainly co-immunoprecipitated (figure 4A and C).To better characterize the complex and investigatewhether the complex exists in metaphase IIoocytes, we used another approach. Cap columns(7mGTP chromatography), which allow eIF4Erecruitment, were used to identify proteins presentin the complex. Stage VI and metaphase IIextracts, treated (figure 4D lanes 6 and 7) or not(lanes 2 and 3) with RNase, were supplementedwith GTP to reduce non-specific adsorption. As anegative control, some extracts were supplementedwith free cap (7m GTP) (figure 4D lanes 4 and 5).The extracts were then applied on a cap columnand the bound material was eluted with free capand detected by Western blot. A fraction of bothX77K and CPEB from stage VI and metaphase II

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oocytes with or without RNAse was retained onthe column in presence of GTP but not when7mGTP was added in the extracts (figure 4D). Thisexperiment was done using GTP at a lowerconcentration than 7mGTP in the extracts. Toconfirm the specificity of the binding, a controlexperiment was done either, using the sameconcentration of GTP and 7mGTP in the extracts,or, by eluting the bound proteins with GTP or7mGTP (figure S2). We observed that GTP did notcompete for eIF4E, 77K and CPEB binding while7mGTP did (figure S2 compare lane 6 with lane 8)and that GTP only weakly eluted bound proteinscompare to 7mGTP (compare lane 4 with lane 2).Asmall amount of total X77K was retained on theresin (figure 4D and S2), which is coherent withthe fact that only a small fraction of X77K iscytoplasmic and can bind to eIF4E. It isnoteworthy that CPEB was also poorly retained onthe resin (figure 4D and S2), even though it isabundant in the cytoplasm. However, this resultcould be explained by the fact that CPEB, knownto be part of the masking complex, interacts onlytransiently with eIF4E (20). In metaphase IIoocytes, the electrophoretic mobility of CPEB isshifted and the protein is partially degraded (45).However, the same proportion of total CPEB wasretained on the column in stage VI and metaphaseII extracts. As negative controls, we looked forthe binding of ERK and β−tubulin proteins, andobserved, as expected, that they were not retainedon the column (figure 4D). We failed to detectendogenous XGLD2 on the resin, probablybecause of its low expression level.To assess whether the interactions were direct, weperformed GST-pull-down experiments withpurified GST-X77K from Baculovirus and eitherHA-CPEB or HA-XGLD2 proteins translated invitro. Figure 4E shows that GST-X77K bound toHA-CPEB and HA-XGLD2, whereas GST alonedid not. In the same kind of experiment, a directinteraction between GST-X77K and HA-eIF4Ewas not conclusive, due to a high background inthe GST control (data not shown).Altogether, these results show that X77K belongsto a RNA-independent cytoplasmic complex witheIF4E, CPEB, CPSF-100 and XGLD2, and thatX77K is capable of directly interacting with CPEBand XGLD2.

X77K is not required for cytoplasmicpolyadenylation - We showed that X77K binds to

proteins required for cytoplasmic polyadenylationin oocytes. To address whether X77K is requiredfor cytoplasmic polyadenylation, we performed invitro polyadenylation tests in egg extracts thatwere subjected to 77K, or control IgGimmunodepletions. As a control, we also did theexperiment on egg extracts depleted for CPEB, aprotein that is known to be required forcytoplasmic polyadenylation. Figure 5A showsthat immunodepletions of X77K and CPEBcompletely removed the endogenous proteins (leftpanel), which were retained on the antibody beads(right panel). Moreover, even if CPEB wasapparently not depleted by 77K antibody, and viceversa, we observed a co-immunoprecipitationbetween X77K and CPEB in egg extracts (figure5A right panel) that confirmed our results obtainedwith oocyte lysates (figure 4).To do the polyadenylation test, Mos 3'UTR RNA,bearing wild-type CPE and PRE elements, wasincubated for one hour with egg extracts or X77K,CPEB or mock depleted extracts. The total RNAwas isolated at the beginning of the reaction andone hour later. Then, Mos 3’UTR polyadenylationwas checked by PAT (PolyAdenylation Test)assay, to measure the poly(A) tail length. Figure5 B shows that Mos 3'UTR RNA waspolyadenylated in control and X77K depletedextracts, while it was not polyadenylated in CPEBdepleted extracts (compare lanes 2, 4, 6 with lane8). These results show that depletion of X77Kdoes not impair cytoplasmic polyadenylation invitro.To verify that this is also the case in vivo, weperformed a polyadenylation test in oocytes.Oocytes were first injected with Mos 3’UTRRNA. Some of them were subsequently injectedwith 77K, CPEB or nonspecific IgG. One hourlater, the oocytes were induced, or not, to maturewith progesterone, and harvested when 30% of thecontrol oocytes have undergone GVBD (GerminalVesicle Break Down). The total RNA was isolatedand scored for Mos mRNA polyadenylation. Incontrol oocytes, Mos 3’UTR was polyadenylatedonly when the oocytes were submitted toprogesterone (Figure 5C compare lane 2 with lane3) . The change in electrophoretic mobilityobserved was due to the lengthening of thepoly(A) tail, since the addition of oligo (dT) andRNaseH before the PAT assay suppressed themobility shift (figure 5C right panel). As in egg

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extracts, the impairment of X77K function did notmodify the Mos 3’UTR polyadenylation in theoocytes (figure 5C compare lane 6 with lanes 3and 8). In CPEB antibody injected oocytes, Mos3’UTR was polyadenylated to a lesser extent thanin control oocytes (compare lane 10 with lanes 3and 8). This short poly(A) tail of Mos 3’UTRobserved was probably due to a PRE-dependentpolyadenylation. Indeed it has been shown thatMos 3’UTR could be precociously polyadenylatedindependently of CPE/CPEB, but the length of thepoly(A) tail was shorter than in the presence ofCPEB (20). The above experiment shows thatX77K is not required for polyadenylation ofexogenous Mos mRNA.To confirm this result, we performed apolyadenylation test on endogenous mRNAs fromoocytes injected with IgG, 77K or CPEBantibodies, or non-injected (figure 5D). CyclinB1and AuroraA mRNAs were polyadenylated incontrol and 77K antibody injected oocytes, whichis in agreement with the result obtained withexogenous Mos mRNA. In CPEB antibodyinjected oocytes, there is no polyadenylation ofcyclinB1 mRNA that has no PRE sequence, whilewe observed a short poly(A) tail on AuroraAmRNA which bears a PRE element (19). Thepolyadenylation observed with our PAT assay is aspecific reaction, as endogenous actin mRNA,which is not subjected to polyadenylation duringoocyte maturation, is not polyadenylated in ourtest (figure 5D). Furthermore, we observed a weakdeadenylation of actin mRNA from 6 hours ofprogesterone addition, showing that the injectedantibodies did not impair the deadenylationreaction during meiotic maturation. Altogetherthese results obtained in vitro and in ovo show thatX77K is not required for cytoplasmicpolyadenylation.

77K antibody injected into oocytesaccelerates the G2/M transition - During theexperiments of polyadenylation test in oocytes, weobserved that X77K antibody injection acceleratesoocyte maturation. The antibodies were injectedinto and maintained in the cytoplasm (figure 6Ainsert), arguing that 77K antibody affected thefunction of the cytoplasmic X77K protein.Moreover, as maturation is accompanied with acessation of transcription (15), an effect on thenuclear polyadenylation could be ruled out. Werepeated the experiment several times and the 77K

antibody injected oocytes always underwent 50%GVBD one hour or more before the controloocytes (figure 6A), depending on the batch ofoocytes. This was also the case when we injectedanother antibody directed against X77K (the 573antibody) (data not shown). In agreement withthese results, this acceleration was accompaniedby a premature synthesis of Mos protein andactivation of MAP kinase (figure 6B). In 77Kantibody injected oocytes, Mos protein is welldetected 2 hours after progesterone induction,while in control oocytes (uninjected or IgGinjected) it is clearly detected only 5 hours afterprogesterone addition. The MAPK activation,revealed by the bi-phosphorylation of ERK, is alsoearlier in 77K antibody injected oocytes (2 hours)than in control oocytes (4 hours). To investigatewhether downstream events of maturation wereaccelerated, we checked for proteins where thesynthesis is induced at GVBD. In 77K antibodyinjected oocytes, AuroraA synthesis increased 2hours after progesterone induction while, incontrol oocytes (IgG injected and uninjectedoocytes), this increase is well observed at 5 hours(figure 6B). This acceleration of protein synthesisalso occurred for cyclinB1 (data not shown).One supposition for this premature proteinsynthesis was that the impairment of X77Kfunction in oocytes accelerates mRNAcytoplasmic polyadenylation. To test thishypothesis, we performed a time course of Mos3’UTR polyadenylation in 77K antibody injectedoocytes. The oocytes were first injected with Mos3’UTR RNA and then with 77K antibody or IgG.Oocytes were harvested at different times afterprogesterone induction, total RNA was extractedand checked for Mos mRNA polyadenylation. Incontrol oocytes and 77K antibody injectedoocytes, Mos mRNA polyadenylation occurred atthe same time, showing that injection of 77Kantibody does not modify the kinetics ofcytoplasmic polyadenylation (figure 6C). CPEB isknown to belong to a masking complex witheIF4E and Maskin. It has been reported thatmRNA cytoplasmic polyadenylation allows thedisruption of Maskin/eIF4E interaction by eIF4Grecruitment, and translation ensues (22). Thissuggests that the role of cytoplasmicpolyadenylation is to allow the binding of eIF4Gto eIF4E. In 77K antibody injected oocytes, Mos,AuroraA and CyclinB1 proteins were precociously

9

synthesized, while Mos mRNA polyadenylationwas not accelerated. This phenotype, with the factthat X77K belongs to a complex with CPEB andeIF4E, suggests that X77K could have a role inmRNA masking before cytoplasmicpolyadenylation.

X77K represses mRNA translation in vitro- The above results document that X77K functionsin vivo as part of a complex involved in mRNAmasking, thereby preventing a prematuretranslation of protein that must be synthesized at aprecise time. To test a potential function of theX77K protein in mRNA translation inhibition, weperformed an in vitro translation experiment. Inthis assay, we added increasing quantities ofpurified GST-77K or GST proteins in rabbitreticulocyte lysates programmed with a luciferasereporter, and checked for mRNA translation bymeasuring and quantifying 35S methionineincorporation. Figure 7 shows that luciferasemRNA translation was inhibited by addition ofrecombinant X77K in a dose dependent manner,while no translation inhibition was observed withthe GST protein. Importantly, this effect is not dueto destabilization of Luciferase mRNA by theaddition of the recombinant proteins. This result isin agreement with a role for X77K in mRNAmasking.

DISCUSSION

In this study, we identified the XenopusCstF-77 protein (X77K) and showed that the CstF-77 proteins are very well conserved in vertebrates(figure 1 and S1). X77K is expressed in oocytesand embryos at a constant level, and is localizedmainly to the nucleus, but also in discretecytoplasmic foci in vertebrate cells (figure 3).However, X77K does not shuttle between the twocompartments (figure 3E ). The cytoplasmiclocalization, together with other arguments (seeintroduction), suggested a cytoplasmic role forCstF-77 protein. We established that X77K is partof a cytoplasmic complex with eIF4E, CPEB,CPSF and XGLD2 (figure 4) but is not requiredfor cytoplasmic polyadenylation per se (figure 5).Nevertheless, impairment of X77K function inoocytes, accelerates oocyte maturation and proteinsynthesis without modifying the kinetic of MosmRNA polyadenylation (figure 6). Moreover,X77K represses mRNA translation in vitro (figure

7). Altogether, these results support a role forX77K in mRNA masking.

The known function of nuclear CstF-77protein is to enhance cleavage by stabilizing theinteraction between CPSF and AAUAAA in thepre-cleavage complexes (46). We envisage thatcytoplasmic X77K could have the same functionof complex stabilization on cytoplasmic mRNA.This could explain why cytoplasmic X77K is notrequired for cytoplasmic polyadenylation per se,since its nuclear counterpart is required for pre-mRNA cleavage but not for the subsequentpoly(A) addition step of the reaction. In thenucleus, CstF-77 interacts with CPSF via itsCPSF-160 subunit (5). In the cytoplasm, X77Kalso binds to CPSF and CPEB (figure 4B ).Moreover, X77K directly interacts with XGLD2and CPEB in vitro, showing that X77K, CPSF,CPEB and XGLD2 are part of the samecytoplasmic complex. CstF-77 has no RNAbinding domain, thus, one or several bindingpartners must recruit X77K to increase the affinityof the polyadenylation complexes (nuclear orcytoplasmic) on the target RNA. In the nucleus,CstF-64 is the RNA binding protein that targets,with CPSF-160, CstF-77 on the pre-mRNA. In thecytoplasm, CPEB could replace CstF-64 for thisfunction. Moreover, CstF-77 has a functionalNLS, leading to its intranuclear targeting (31 andour unpublished data), and does not shuttlebetween the nucleus and the cytoplasm (figure3E ), which could explain the low level of itscytoplasmic counterpart. This suggests that CstF-77 can stay into the cytoplasm only in interactionwith other proteins, and that a fraction of X77Knewly synthesized can be recruited by CPEB,XGLD2 or CPSF-160 and probably other proteins(see below).

CstF-77 is not the only protein thatbelongs to both nuclear and cytoplasmicpolyadenylation complexes: besides CPSF, shownto be involved in cytoplasmic polyadenylation(14), Symplekin was recently identified as part ofthe cytoplasmic polyadenylation machinery (18).Symplekin is a dual location protein that has beenlocalized to the cytoplasmic plaques of tightjunctions, and in the nucleus where it interactswith CPSF, CstF-77 and CstF-64 (12,47).However, Symplekin does not interact with thefully assembled CstF, suggesting that Symplekinand CstF-77 can compete for the same site in

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CstF-64 (12). CPEB binds both X77K (this study)and Symplekin (18), strengthening the idea thatCPEB could have the same function in thecytoplasm than CstF-64 in the nucleus.

A cytoplasmic localization for the otherCstF subunits has not been described invertebrates, and the S.cerevisiae homologueRNA15 is only nuclear (28). However, we cannotrule out a possible cytoplasmic function for theCstF-64 or CsF-50 subunits, as gametes expressdifferent protein isoforms from those present insomatic cells. Indeed, a second form of CstF-64protein (tau CstF-64), expressed in meiotic andpost meiotic male germ cells, has been described(48). Searches for specific CstF-64 or CstF-50isoforms in oocytes, and their possible functions inthe cytoplasm, have not been tested, due to a lackof purified antibodies recognizing thecorresponding Xenopus proteins.

We observed an acceleration of the G2/Mtransition in 77K antibody injected oocytessubmitted to progesterone. This phenotype isprobably due to premature Mos protein synthesis,that activates the MAPK pathway and MPF(Maturation Promoting Factor). However, the MosmRNA polyadenylation kinetic is not accelerated,suggesting that impairment of X77K functionallows Mos mRNA recruitment on the ribosomeindependently of its polyadenylation state. It hasbeen suggested that the function of cytoplasmicpolyadenylation was to displace eIF4E from amasking complex (22). One possibility is that 77Kantibody, by interacting with the X77K protein,disrupts the masking complex, allowing thebinding of eIF4E to eIF4G, and the recruitment ofthe small ribosomal subunit by the mRNA. Thus,X77K could play a role in mRNA masking prior tocytoplasmic polyadenylation.

However, as X77K represses CPE-independent mRNA translation (figure 7), itprobably has a more general function in mRNAtranslation inhibition. In that case, X77K would betargeted on the mRNA by one or several proteinsother than CPEB. CPSF can recruit X77K on themRNA by itself. Nevertheless, other RNA bindingproteins, in cooperation with CPSF, could alsoaddress X77K on the messenger RNA. Xp54, aubiquitous translational repressor that alsointeracts with CPEB (49), could be one of them.XPumilio (50,51) and XDAZL (52), othertranslational regulators independent of CPE

dependent cytoplasmic polyadenylation, are alsopotential candidates. Thus, CPSF and an X77K-containing complex could interact cooperatively tostabilize a RNA masking complex, until it isdisrupted or modified by a signal allowing mRNAtranslation.

CstF-77 protein is localized in discretecytoplasmic foci in NIH3T3 and XTC cells(Figure 3D). In oocytes, Xp54 and p47, theXenopus and Spisula homologues of the yeastdecapping activator Dhh1, are localized in storedmRNP and bind to CPEB (49,53,54). Moreover, ithas been reported that human CPEB1 expressioninduces the assembly of stress granules, that inturn, recruit the processing bodies (PB) (55), andthat eIF4E is localized in PB (56,57). Thus,proteins initially involved in mRNA translationcan be localized in PB and, inversely, decappingactivator can be stored in mRNP in oocytes.Recently, Brengues et al (58) have shown thatmRNA molecules move from PB to polysomesand vice versa. These data show that stressgranules, PB and stored mRNP are dynamicallylinked sites of mRNP remodeling.

The fact that: 1) X77K belongs to acomplex with eIF4E, CPEB and XGLD2; 2) Theimpairment of 77K function leads to a prematureprotein synthesis in oocytes; 3) RecombinantX77K represses mRNA translation in vitro and 4)we observed CstF-77 in NIH3T3 and XTCcytoplasmic foci, suggest that cytoplasmic CstF-77could have a ubiquitous function in mRNPremodeling events, for the transition of mRNAfrom inactive to active translation.

Acknowledgments- We thank the Thierry Lorcalab for reagents, advice and assistance, Cyril Bernis foregg extracts and Jamal Tazy for luciferaseoligonucleotides. We are grateful to Simon Morley,Jean-Marc Lemaître, Nathalie Morin and ClaudePrigent for the antibodies, Dr N.Sagata for the Mosvector, Dr D. Bentley for the CstF 77 carboxy terminalpeptide and Elmar Wahle and Uwe Kuehn for the giftof unpublished anti CPSF-100 antibody. We also thankA. Bernet, J.M. Donnay, Y. Boublik and G. Herrada fortechnical assistance. We are grateful to D. Fisher forlooking over the English. This work was supported bythe Centre National de la Recherche Scientifique andthe Association pour la Recherche sur le Cancer(contrat numbers 4469 and 3147 to E.M.). CR issupported by the Ministère de l'éducation nationale, de

11

la Recherche et de l'enseignement supérieur and theAssociation pour la Recherche sur le Cancer.

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FIGURE LEGENDS

Fig. 1. A, Amino acid sequence of X77K. The HAT repeats and the Prolin-rich domain are underlinedwith solid and broken lines respectively. The bipartite nuclear localization signal (NLS) is in bold. Aminoacids are numbered on the right. B, Schematic representation of X77K protein.

Fig. 2. 77K antibody characterization. A, Western blot analysis of stage VI oocyte extract (lane 1), HA-tagged X77K expressed in oocytes (lane 2), XTC and NIH (3T3) cell lines (lanes 3 and 4) and purifiedBaculovirus GST-tagged X77K (lane 5) probed with 77K antibody. B, Immunoprecipates from 35Smethionine labeled X77K protein (1ng) with control IgG, or 77K antibody or 77K antibody pre-incubatedwith 77K antigen were analyzed by autoradiography.

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Fig. 3. Endogenous expression of CstF-77 protein. A and B, Immunoblot analysis of Xenopus oocyteextracts (25 µg by lane) A and embryo extracts (12,5 µg by lane) B with 77K and β-tubulin antibodies.The β-tubulin, consistently expressed throughout oocyte maturation and embryogenesis, serves as aloading control. C, Oocytes were manually enucleated and pools of nucleus and cytoplasms werecollected separately. Proteins extracts were analyzed by Western blotting with 77K and RPA antibodies.The equivalent of one oocyte (StVI), one nucleus (N) or one cytoplasm (Cyto) was loaded. TheReplication Protein A (RPA) is exclusively expressed in nucleus and serves as a control of enucleation. D,Immunofluorescence of NIH3T3 and XTC cells. Cells were fixed, stained with 77K antibody and withDAPI and observed by fluorescence microscopy. A nuclear and cytoplasmic expression of endogenous77K is observed in both cell lines. E, X77K localization is not sensitive to leptomycin B. Oocytes weretreated or not over night with leptomycin B (100 nM final) and were manually enucleated. Pools ofnucleus and cytoplasms were collected separately. Proteins extracts were analyzed by Western blottingwith 77K, CPEB, RPA and β-tubulin antibodies. The equivalent of one oocyte (StVI), one cytoplasm(Cyt) or 0.5 nucleus (N) was loaded. The RPA and β-tubulin proteins are respectively expressed innucleus and cytoplasm and serves as enucleation and loading controls.

Fig. 4. X77K is part of a complex with eIF4E, CPEB, CPSF and XGLD2. A, StVI oocyte extracts, treatedor not with RNase A as indicated, were immunoprecipated with 77K, eIF4E, CPEB, Pak5 antibodies orwith control IgG. The immunoprecipitates were analyzed by Western blotting with 77K, CPEB, eIF4E,CPSF-100 and Pak5 antibodies. B, Oocytes were manually enucleated and cytoplasmic extracts weretreated with RNase A and immunoprecipated with 77K, eIF4E, CPEB or control IgG. Theimmunoprecipitates were analyzed by Western blotting with 77K, CPEB and CPSF-100 antibodies. C,Oocytes were injected with mRNA encoding HA-tagged XGLD2 and, after 14 hours of HA-XGLD2expression, protein extracts were treated or not with RNase A and immunoprecipated with differentantibodies as indicated. The immunoprecipitates were analyzed by Western blotting with HA, 77K, eIF4Eand CPEB antibodies. The equivalent of 1 oocyte (A and C) and one cytoplasm (B) was loaded as input.D, Cap column assay. Protein extracts from StVI oocytes or mature oocytes (MII) treated or not withRNase A were supplemented with GTP or 7mGTP and applied to 7mGTP-Sepharose beads (cap column).After beads washes, the bound proteins were eluted with 7mGTP. All of the eluted proteins (B: Bound),the equivalent 1/10 of StVI oocytes or mature oocytes extracts (input StVI or input MII), 1/8 of thesupernatant of protein extracts after cap column binding (U: Unbound) were analyzed by Western blottingwith eIF4E, 77K, CPEB, ERK and β-tubulin antibodies. E, GST pull-down assay. 5 µg of purified GST-X77K or GST were bound to glutathione beads that were subsequently incubated with in vitro translatedHA-tagged CPEB (left panel) or HA-tagged XGLD2 (right panel). The bound proteins were boiled inSDS sample buffer. The eluates and the equivalent of 1/10 of the in vitro translated proteins (input) wereanalyzed by Western blotting with HA and GST antibodies.

Fig. 5. X77K is not required for cytoplasmic polyadenylation. A and B, Polyadenylation assay in eggextracts. A, Egg extracts were immunodepleted with 77K or CPEB antibodies or with control IgG. 1/10 ofthe egg extracts, immunodepleted or not (left panel) and 1/10 of the immunoprecipitates (right panel)were analyzed by Western blotting with 77K or CPEB antibodies. B, Egg extracts, immunodepleted ornot as indicated, were incubated with exogenous Mos 3'UTR RNA. Total RNA extracted at 0 and 1 hourafter Mos 3'UTR RNA addition was submitted to Mos polyadenylation analysis (PAT assay) usingspecific primers. C and D, Polyadenylation assay in oocytes. C, Exogenous Mos mRNA polyadenylation.Left panel, Oocytes were first injected with Mos RNA and 30 min later were injected with 77K, CPEBantibodies or with nonspecific IgG. After 1 hour incubation, maturation was induced or not withprogesterone. Total RNA was extracted from pools of 5 oocytes collected at the time of progesteroneaddition (time point 0h) or when 30% of control oocytes have undergone GVBD (time point 6h). TotalRNA was submitted to Mos polyadenylation analysis. Right panel, total RNA, corresponding to lane 1and 3 of the left panel, were submitted or not to RNaseH treatment before Mos polyadenylation analysis

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(PAT assay). D, Endogenous mRNA polyadenylation. Oocytes were injected with 77K, CPEB antibodiesor with nonspecific IgG. After 1 hour incubation , maturation was induced or not with progesterone. TotalRNA was extracted from pools of 5 oocytes collected at the time of progesterone addition (time point 0h),when 30% of control oocytes have undergone GVBD (time point 6h), and two hours later (time point 8h).Total RNA was submitted to endogenous CyclinB1, AuroraA and actin type5 polyadenylation analysis(PAT assay) using specific primers. C and D, Lane 4 corresponds to RNA from a mix of oocytes injectedwith the different antibodies at time point 0. Fragment size (M) is indicated on the left in base pair (bp).

Fig. 6. Injection of 77K antibody accelerates oocyte maturation. A, Oocytes were injected with 77Kantibody or nonspecific IgG or left uninjected and maturation was induced by progesterone one hour later.The percentage of GVBD was scored at the indicated times and graphed. For each treatment, 35 oocyteswere injected. This graph is representative of six experiments. Insert, 5 hours after 77K antibodyinjection, oocytes were manually enucleated and pools of cytoplasms and nucleus were collectedseparately. Proteins extracts were analyzed by Western blotting with 77K and RPA antibodies. Theequivalent of one uninjected oocyte (StVI), one cytoplasm (cyto) and one nucleus (N) from injectedoocytes was loaded. The Replication Protein A (RPA) serves as a control of enucleation. An exclusivecytoplasmic localization of 77K IgG is observed. B, Western blot analysis of Mos and AuroraA synthesis,activated MAPK (ppERK) and β-tubulin in oocytes collected during an experiment similar to the onedepicted in A. The β-tubulin serves as a loading control. C, Oocytes were first injected with Mos 3’UTRRNA and 30 min later were injected with 77K antibody or nonspecific IgG. 1 hour later, maturation wasinduced with progesterone. Total RNA was extracted from pools of 5 oocytes collected at the indicatedtimes and was submitted to Mos polyadenylation analysis. Fragment size is indicated on the left in basepair (bp).

Fig. 7. X77K protein inhibits mRNA in vitro translation in a dose dependent manner. A, LuciferasemRNA was translated in rabbit reticulocyte lysates (RRL) in presence of 35S methionine alone (Control)or with increasing amount of purified GST-77K or GST as indicated (0.5 µg to 3 µg). 20% of each treatedRRL were analyzed by SDS-PAGE. Translated luciferase was revealed by autoradiography and ß-tubulin,as a loading control, was analyzed by Western blotting. The integrity of luciferase mRNA in presence ofthe recombinant proteins was verified by RT PCR. The increasing amount of GST-77K and GST wereanalyzed by Coomassie staining. Two bands are detected for GST-77K, the lower one corresponding toGST-77K lacking the carboxy terminal end. B, Quantification of luciferase translation by Phosphoimagerwhere control is referred as 100%.

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