Biochemical Identification of Xenopus Pumilio as a Sequence-specific Cyclin B1 mRNA-binding Protein That Physically Interacts with a Nanos Homolog, Xcat-2, and a Cytoplasmic Polyadenylation Element-binding Protein*

Shingo NakahataDagger , Yoshinao Katsu§, Koichi MitaDagger , Kunio Inoue||, Yoshitaka Nagahama§, and Masakane YamashitaDagger **

From the Dagger  Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, the || Graduate School of BioSciences, Nara Institute of Science and Technology, Ikoma 630-0101, and the § Laboratory of Reproductive Biology and the  Center for Integrative Bioscience, National Institute for Basic Biology, Okazaki 444-8585, Japan

Received for publication, November 21, 2000, and in revised form, March 5, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Translational activation of dormant cyclin B1 mRNA stored in oocytes is a prerequisite for the initiation or promotion of oocyte maturation in many vertebrates. Using a monoclonal antibody against the domain highly homologous to that of Drosophila Pumilio, we have shown for the first time in any vertebrate that a homolog of Pumilio is expressed in Xenopus oocytes. This 137-kDa protein binds to the region including the sequence UGUA at nucleotides 1335-1338 in the 3'-untranslated region of cyclin B1 mRNA, which is close to but does not overlap the cytoplasmic polyadenylation elements (CPEs). Physical in vitro association of Xenopus Pumilio with a Xenopus homolog of Nanos (Xcat-2) was demonstrated by a protein pull-down assay. The results of immunoprecipitation experiments showed in vivo interaction between Xenopus Pumilio and CPE-binding protein (CPEB), a key regulator of translational repression and activation of mRNAs stored in oocytes. This evidence provides a new insight into the mechanism of translational regulation through the 3'-end of mRNA during oocyte maturation. These results also suggest the generality of the function of Pumilio as a translational regulator of dormant mRNAs in both invertebrates and vertebrates.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The final inducer of oocyte maturation is the maturation-promoting factor (MPF),1 which consists of Cdc2 and cyclin B. MPF is stored in immature oocytes as an inactive form (called pre-MPF), although its amount differs from species to species (1, 2). In Xenopus (as well as fish and mammals except for mice), the initiation of oocyte maturation requires proteins (called "initiators") newly synthesized by translational activation of dormant mRNAs stored in oocytes (masked mRNAs). Mos functions as an initiator with the aid of mitogen-activated protein kinase (MAPK) (3-9). The Cdc2 molecule of pre-MPF is phosphorylated on threonine 14/tyrosine 15 by Myt1 and threonine 161 by cyclin-dependent kinase-activating kinase. The Mos/MAPK pathway probably leads to the activation of Cdc25, which dephosphorylates threonine 14/tyrosine 15 via the polo-like kinase (10-12), and to the inhibition of Myt1 through the activation of p90rsk (13, 14).

Enhanced cyclin B synthesis is a ubiquitous event that occurs during oocyte maturation in all species examined so far, and it is indispensable for initiating oocyte maturation in fish and amphibians except Xenopus, in which pre-MPF is absent in immature oocytes (15). The neosynthesized cyclin B is also required for leading oocyte maturation beyond germinal vesicle breakdown (GVBD) in mammals, in which pre-MPF in immature oocytes is only sufficient for inducing GVBD (16, 17). In contrast, it has been shown that newly synthesized cyclins upon progesterone stimulation are not required for initiating oocyte maturation in Xenopus (18, 19). Like c-mos mRNA, however, translation of cyclin B1 mRNA is activated during Xenopus oocyte maturation (20, 21). Artificial translational activation of endogenous cyclin B1 mRNA brings about GVBD without progesterone and Mos (22, 23). Moreover, the finding that progesterone induces cyclin B1 synthesis without any activities of Mos, MAPK, and MPF strongly suggests that progesterone-induced cyclin B1 synthesis is a physiological trigger of pre-MPF activation (24). Taken together, the results of past studies strongly suggest that the translational activation of cyclin B1 mRNA universally plays a key role in initiation of oocyte maturation in lower vertebrates, including Xenopus (25), and an understanding of its control mechanisms during oocyte maturation is therefore of particular importance.

Over the past decade, the mystery of how translationally repressed mRNAs in immature oocytes are released from masking at the appropriate time during oocyte maturation has been partially solved. Translational control of masked mRNAs stored in oocytes is mediated by the U-rich motif U4-6A1-2U, named cytoplasmic polyadenylation element (CPE), present in their 3'-untranslated region (3'-UTR) (for review see Ref. 26). The CPE sequence is the target site for CPE-binding protein (CPEB), which facilitates polyadenylation of the target mRNA during oocyte maturation (27). In addition to the involvement in translational activation, CPEB has recently been shown to mediate the masking of cyclin B1 mRNA (22, 23, 28). Thus, CPEB seems to have a dual function: to maintain maternal mRNAs, including those of cyclin B1, in a dormant state in immature oocytes and to activate their translation via polyadenylation during oocyte maturation. Nevertheless, many issues still remain to be elucidated. For example, although both c-mos and cyclin B1 mRNAs hold the CPEs in their 3'-UTR, the following findings indicate that their translational activation is controlled by different mechanisms: 1) translational activation of c-mos mRNA precedes that of cyclin B1 mRNA (29); 2) in contrast to c-mos mRNA, the protein level of cyclin B1 can increase in the absence of polyadenylation or cap-ribose methylation of mRNA and is independent of MAPK activity (24, 30, 31); 3) a reporter mRNA carrying the cyclin B1 3'-UTR is translationally repressed when injected into oocytes, whereas that carrying the c-mos 3'-UTR is not (23); and 4) the injection of high concentrations of the CPE induces unmasking of cyclin B1 mRNA but not of c-mos mRNA (22). Therefore, regulatory elements other than CPEs and CPEB must operate for the coordinated activation of masked mRNAs to promote oocyte maturation.

In early Drosophila embryos, Pumilio represses translation of maternal hunchback (hb) mRNA, in conjunction with Nanos (32-34). The translational repression is due to the direct binding of Pumilio to two copies of a bipartite sequence, the nanos response elements (NREs), located in the 3'-UTR of hb mRNA (35-38). Loss-of-function mutations of pumilio and nanos induce the premature expression of cyclin B protein in pole cells (39). Deletion from cyclin B mRNA of cis-elements (named TCEs) resembling the NREs in hb mRNA results in a phenotype similar to that caused by pumilio and nanos mutations (40). These results strongly suggest that Pumilio, in cooperation with Nanos, is involved in translational repression of cyclin B mRNA through the NRE-like sequence TCE, although it remains to be elucidated biochemically whether or not the direct binding of Pumilio to cyclin B mRNA brings about its translational repression.

Our goal is to elucidate the molecular mechanism of translational activation of dormant cyclin B1 mRNA upon hormonal stimulation, which is prerequisite for initiating oocyte maturation in fish and amphibians, probably inclusive of Xenopus (25). NRE-like motifs are found in the 3'-UTR of cyclin B1 mRNA in fish and amphibians, including goldfish (41), medaka (42), zebrafish,2 Xenopus (43), and Rana (44). It is therefore expected that a homolog of Pumilio or its related protein(s) binds directly to cyclin B1 mRNA to control its translation. To date, however, there has been no report on protein expression of Pumilio homologs in vertebrates, although cDNAs probably encoding human homologs have been isolated in a cDNA project (45, 46). In the present study, we biochemically characterized a Xenopus Pumilio protein, and we investigated its involvement in the translational control of cyclin B1 mRNA. We isolated a cDNA clone encoding a protein homologous (78% identity) to Drosophila Pumilio in the domain that defines the Pumilio family. A monoclonal antibody raised against this domain has revealed for the first time in any vertebrate that Xenopus oocytes have a protein homologous to Pumilio (named XPum for Xenopus Pumilio), which has an apparent molecular mass of 137 kDa and binds to the NRE of Drosophila hb mRNA, as in the case of the Drosophila counterpart. Using UV cross-linking assays with various RNA probes, we have also provided the first biochemical evidence for the sequence-specific direct binding of Pumilio to the 3'-UTR of cyclin B1 mRNA. Moreover, the results of protein pull-down and co-immunoprecipitation assays indicated that XPum associates with a Xenopus homolog of Nanos, Xcat-2 (47), and CPEB.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Animals and Oocytes-- Xenopus laevis was purchased from a dealer (Hamamatsu Seibutsu Kyozai, Shizuoka, Japan) and maintained in laboratory aquaria at 20 °C. Full-grown immature oocytes at stage VI (48) were isolated with forceps or 0.2% collagenase (Wako, Osaka, Japan) in modified Barth's saline buffered with Hepes (MBS-H) (49) and induced to mature in vitro by cultivating in MBS-H supplemented with 10 µg/ml progesterone.

cDNA Cloning and Recombinant Proteins-- A cDNA clone encoding the conserved region of Pumilio was isolated from ovary RNA by reverse transcription-polymerase chain reaction (RT-PCR) using two primers, Pum1 (GAYCARCAYGGITCICGITTYATICA) and Pum4R (TARTTIGCRTAYTGRTCYTTCATCAT). Pum1 and Pum4R correspond to the highly conserved amino acid sequences in Pumilio, DQHGSRFIQ and MMKDQYANY, respectively. The sequence isolated by RT-PCR (named Pum1-4R) was extended in both 5' and 3' directions by 5' and 3' RACE systems (Life Technologies, Inc., Rockville, MD) according to the manufacturer's instructions using the following primers: XlPumGSP1 (GCTGCCAAACTCAAA), XlPumGSP2 (CATAAGCTGGTAAGCTGCTTG), and XlPumGSP3 (GCTGCTTGAGGATCTCGTTGAACACCA) for 5' RACE, and XlPum3'F1 (CACATGCATCTCGTACGGAAAGGGCCATGC) and XlPum3'F2 (GGGCCATGCTGATAGACGAGGTGTGCACAA) for 3' RACE.

To construct a glutathione S-transferase (GST) fusion protein, the cDNA Pum1-4R was amplified by PCR with a 5'-primer (TGGAATTCTAGACCARCAYGGGTCGCGG) and a 3'-primer (TCGAATTCGCRTAYTGGTCTTTCATCAT). The PCR product was digested with EcoRI and ligated into EcoRI-cut pGEX-KG (50). GST fusion protein (GST-XPum1-4R) was expressed in Escherichia coli TB1 and purified by SDS-polyacrylamide gel electrophoresis followed by electroelution in Tris-glycine buffer without SDS, as described previously (41).

A cDNA clone encoding Xcat-2 (47) was isolated from a lambda  ZAP II library constructed from Xenopus eggs. The open reading frame (ORF) was amplified by a XNos2-F primer (CGGAATTCTAATGGATGGCGGTCTCTGC) and a T7 primer (TAATACGACTCACTATAGGG). The PCR product was digested with EcoRI and XhoI, and ligated into EcoRI/XhoI-cut pGEX-KG to produce a GST fusion protein (GST-Xcat-2). E. coli-produced GST-Xcat-2 or GST was gel-purified as described above.

To construct a histidine-tagged CPEB, the ORF of Xenopus CPEB (27) was amplified by PCR using a 5'-primer (GGAATTCCCATGGCCTTCCCACTGAAAGAT) and a 3'-primer (ATGCTCGAGGCTGGAGTCACGACTTTTCTG). The PCR product was digested with EcoRI and XhoI and ligated into the same cloning site of pET21c (Novagen, Madison, WI). Histidine-tagged Xenopus CPEB (T7-XlCPEB-His) was expressed in E. coli BL21(DE3)pLysS and purified by SDS-polyacrylamide gel electrophoresis. A histidine-tagged version (T7-Xdazl-His) of Xenopus Daz-like protein (51) was similarly produced by inserting the ORF into the EcoRI-site of pET21c (52).

Production of Antibodies-- The recombinant protein GST-XPum1-4R was injected into mice to produce monoclonal antibodies according to the procedures described previously (53). To obtain antibodies specific to XPum1-4R, hybridoma clones were screened with XPum1-4R cleaved from GST with thrombin (50). One clone (Pum2A5, IgG1 with a kappa  light chain) was selected to detect XPum by immunoprecipitation and immunoblotting. Monoclonal antibodies against Xenopus CPEB were raised against T7-XlCPEB-His (54). One clone (XlCPEB12, IgG1 with a kappa  light chain) was used in this study.

Immunoprecipitation and Immunoblotting-- Oocytes were homogenized with a pestle (Pellet Pestle, Kontes, Vineland, NJ) in 1 µl/oocyte of ice-cold extraction buffer (EB; 20 mM Hepes, pH 7.5, 100 mM beta -glycerophosphate, 15 mM MgCl2, 5 mM EGTA, 1 mM dithiothreitol, 100 µM (p-amidinophenyl)methanesulfonyl fluoride, 3 µg/ml leupeptin) and centrifuged at 15,000 × g for 10 min at 4 °C. The extracts were subjected to immunoprecipitation followed by immunoblotting as described previously (53).

Affinity Chromatography and Northwestern Blotting-- The ovary (100 mg) was homogenized in 250 µl of buffer A (10 mM Tris-HCl, pH 7.5, 83 mM KCl, 17 mM NaCl). Extracts were loaded onto a poly(U)-Sepharose column (Amersham Pharmacia Biotech, Tokyo, Japan). After washing with buffer A, bound proteins were step-eluted with buffer A containing 100-400 mM KCl, precipitated with 15% trichloroacetic acid, washed with acetone, and analyzed by immunoblotting with anti-XPum antibody. Oligo(dT)-cellulose chromatography and Northwestern blotting were performed as described previously (55).

For RNA-affinity chromatography, RNA (500 µg) was coupled with 1 ml of cyanogen bromide-activated Sepharose (Amersham Pharmacia Biotech). The beads suspended in binding buffer (10 mM Hepes, pH 7.2, 100 mM KCl, 3 mM MgCl2, 5% glycerol, 1 mM dithiothreitol) were incubated with 500 µl of oocyte extracts for 1 h at 4 °C. After washing the beads with binding buffer, bound proteins were step-eluted with 200-1000 mM KCl and analyzed by anti-XPum immunoblotting.

Protein Pull-down Assay-- For GST pull-down, ~5 µg of gel-purified GST-Xcat-2 or GST as a control was incubated with 20 µl of glutathione-Sepharose (Amersham Pharmacia Biotech) for 30 min on ice. After removal of unbound proteins, 20 µl of oocyte extracts was mixed with the beads in a binding buffer (50 mM Tris-HCl, pH 7.5, 100 mM KCl, 1 mM MgCl2, 80 mM beta -glycerophosphate, 0.2% sodium azide, 0.1% Nonidet P-40) and incubated for 12 h at 4 °C. Following washing six times with binding buffer, the beads were boiled in SDS sample buffer, and solubilized proteins were analyzed by anti-XPum immunoblotting. For histidine pull-down, ~1 µg of Xenopus CPEB (T7-XlCPEB-His) or Daz-like protein (T7-Xdazl-His) as a control was bound to 10 µl of nickel-nitrilotriacetic acid agarose (Qiagen, Tokyo, Japan). After washing with EB, the beads were incubated with 100 µl of oocyte extracts in EB for 12 h at 4 °C and analyzed with anti-XPum immunoblotting, as in the case of GST pull-down assay.

Plasmid Construction and RNA Synthesis-- A cDNA encoding the full-length Xenopus cyclin B1 (43) in pBluescript II SK(+) was utilized as the original template for producing various probes used in this study (this cDNA being named BDelta 0). For a Northwestern assay and RNA-affinity chromatography, a 3' part of cyclin B1 (nucleotides 1137-1395, named 3'-cyclin B) was constructed as follows: BDelta 0 was digested with EcoRI and NdeI, and the larger fragment was isolated, filled in with a Klenow fragment of DNA polymerase I, and self-ligated. An RNA probe for a 5' part of Xenopus cyclin B1 (nucleotides 1-206, named 5'-cyclin B) was produced by linearizing BDelta 0 in pGEM1 (a gift from Dr. Hideki Kobayashi, Kyushu University) with StyI and transcribing with T7 RNA polymerase as described below.

For a UV cross-linking assay and an injection experiment, a cDNA fragment including the entire 3'-UTR of cyclin B1 (nucleotides 1245-1395, named B3') was created by PCR using BDelta 0 as a template with primer 1 (CGGAATTCGAGGTCAGATGTTGTTGTGG) and primer 2 (CGGAATTCCATGTTAAAATGAGCTTTAT). The PCR product was digested with EcoRI and ligated into EcoRI-cut pBluescript II SK(+). A plasmid with the T7-sense oriented insert was further used as a PCR template to produce various deletion and substitution mutants.

For deletion mutants of B3' (see Figs. 4B and 5A for structural details and nomenclature of probes), PCR products obtained with the following primer sets were ligated into pGEM-T Easy Vector (Promega, Madison, WI): probe 1-118, primer 1 and primer 3 (CCGGATCCACATTAAAAACACAATACACTAT); 19-118, primer 4 (GGAAATGGCCCGCCCACTC) and primer 3; 53-118, primer 5 (TGGCATTCCAATTGTGTA) and primer 3; 76-118, primer 6 (GGCACCATGTGCTTCTGT) and primer 3; 95-151, primer 7 (AATAGTGTATTGTGTTTT) and T3 primer (ATTAACCCTCACTAAAG); 105-151, primer 8 (TGTGTTTTTAATGTTTTACTGG) and T3 primer; 119-151, primer 9 (TTTACTGGTTTTAATAAAGCTC) and T3 primer.

Substitution mutants were produced by PCR with the following primers (mutation sites are underlined; see Figs. 4B and 5A for structural details): probe sub13-18 (CAGATGTGCACCAGGAAA), sub63-68 (TGGCATTCCATCATCTTATTGTTGGC), sub70-74 (CCAATTGTGTTCATCTGGCACCATG), sub82-86 (GGCACCCCCCCCTTCTGTAAATAGTGT), sub91-94 (GGCACCATGTGCTTCGTGCAATAGTGT), sub103-108 (ATAGTGTTCATCTTTTTTAATGTGGATCCGG), mut105-108 (ACGCTTTTTAATGTTTTACTGG), mut109-113 (TGTGGGGGGAATGTTTTACTGGTT), and mut114-119 (TGTGTTTTTGAACGCTTACTGGTTT). Using a cDNA of the NRE sequence in Drosophila hb mRNA (ATTATTTTGTTGTCGAAAATTGTACATAAGCC), we also produced a deletion mutant (named Mut1) bearing the sequence ATTATTTTGTTGTCGA and a substitution mutant (named Mut2) bearing the sequence ATTATTTTGTTGTCGAAAATacgcCATctGCC (The NRE consensus bases are underlined, and the mutated bases are written in lowercase letters; cf., Fig. 3A).

For in vitro transcription, the DNA templates were linearized with BamHI. Radiolabeled RNA probes were prepared using an RNA transcription kit (Stratagene, La Jolla, CA), with 25 µCi of [32P]UTP (400 Ci/mmol; Amersham Pharmacia Biotech). Unlabeled transcripts were synthesized in the presence of 400 µM UTP. Large-scale transcription reactions were performed using a MEGAscript T7 kit (Ambion, Austin, TX). All in vitro synthesized RNAs were phenol/chloroform-extracted, ethanol-precipitated, and dissolved in distilled water.

UV Cross-linking-- UV cross-linking assays were carried out essentially as described by Walker et al. (56). Briefly, reactions were performed in 96-well microtiter plates (Falcon 3911, Becton Dickinson, Franklin Lakes, NJ). In each 10-µl reaction, probe RNA (60-100,000 cpm) was incubated with 2 µl of extract for 15 min at room temperature and irradiated in a UV cross-linker (XL-1000, Funakoshi, Tokyo, Japan) at an energy setting of 860 mJ/cm2. A competition assay was performed by preincubation of the reactions with a 25-, 50-, or 100-fold molar excess of unlabeled RNA for 5 min prior to the addition of radiolabeled RNA.

In Vitro Selection-- RNA sequences required for interaction with Xenopus Pumilio were determined by an in vitro selection assay using RNAs having a random sequence of 25 nucleotides and a baculovirus-expressed Xenopus Pumilio protein fused with GST (GST-XPum-Bac). GST-XPum-Bac was produced as follows. A DNA fragment including the ORF of GST was created by PCR using pGEX-KG (50) as a template with a 5'-primer (GAAGATCTGTCATGTCCCCTATACTAGGTTA) and a 3'-primer (TCCGGGAGCTGCATGTGTCAG). The PCR product was digested with BglII and HindIII and ligated into BamHI/HindIII-cut pFastBac1 (Life Technologies, Inc.) to generate pFastBac-GST. A 2.0-kb EcoRI fragment of the XPum cDNA (see "Results") was inserted into the same cloning site of pFastBac-GST. The plasmid DNA was transformed into DH10Bac cells (Life Technologies, Inc.). After transposition, high-molecular-weight DNA was isolated and transfected into Spodoptera frugiperda (Sf21) cells using Cellfectin (Life Technologies, Inc.) according to the manufacturer's instructions. The cells were harvested 4 days after transfection, and GST fusion protein was purified on glutathione-Sepharose (Amersham Pharmacia Biotech) as described above.

In vitro selection assays were carried out according to Sakashita et al. (57). Briefly, the starting RNA source was synthesized from a mixture of DNA templates containing a T7 promoter and a random sequence of 25 nucleotides, using a MEGAscript T7 kit (Ambion). A total of five rounds of selection and amplification were performed. The final cDNA products were subcloned into pBluescript II SK(+) and sequenced.

Oocyte Injection-- Manually isolated stage VI oocytes were injected with 70 ng of competitor RNAs (in 40 nl of distilled water). After incubation of the injected oocytes in MBS-H for 24 h at 18 °C, 10 oocytes were homogenized, subjected to Suc1-precipitation to partially purify the Cdc2-cyclin B complex (58), and analyzed by immunoblotting with a monoclonal antibody that recognizes amphibian cyclin B1 (Bufo 2F5) (59). The remaining oocytes were boiled, cut with a razor, and scored for the percentage of GVBD.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Presence of a Pumilio Homolog in Xenopus Oocytes-- Drosophila Pumilio and a Caenorhabditis elegans Pumilio homolog, FBF, are members of the Pumilio-homology domain (Pum-HD) family, also known as Puf (for Pumilio and FBF) family (60, 61). The sequence in the C-terminal region of the Pum-HD family is highly conserved in many species, including human homologs (DDBJ/EMBL/GenBankTM accession numbers KIAA0099 and KIAA0235) deduced from their cDNAs (45, 46). We therefore designed oligonucleotide primers corresponding to this region to isolate a cDNA clone encoding Xenopus Pumilio by RT-PCR. We initially obtained an 800-bp cDNA fragment (Pum1-4R) that encodes a nucleotide sequence highly homologous to Drosophila Pumilio. We then extended this fragment by 5' and 3' RACE and finally obtained a 2.0-kb sequence (DDBJ/EMBL/GenBankTM accession number AB045628) containing a domain equivalent to those of Drosophila Pumilio (78% identity) and the human homolog KIAA0099 (95% identity) (Fig. 1). This domain is known as the diagnostic hallmark of the Pum-HD family and is defined by the presence of eight copies of an imperfect repeat sequence, comprising a specific RNA-binding domain (60-62) (Fig. 1). Based on the existence of this feature in the obtained clone (Fig. 1), we identify it as a Xenopus homolog of Pumilio. Although the known Pum-HD proteins share few sequences outside the Pum-HD (60), considerable sequence conservation is seen between the Xenopus and the human homologs (69% and 81% identity in the N-terminal and the C-terminal regions, respectively). This finding and biochemical characterization of the protein encoded by this cDNA (see later sections) also confirmed that the obtained cDNA encodes a Xenopus Pumilio homolog. From the lengths of those found in other species, the obtained cDNA apparently lacked the 5' sequence. However, we did not persist with isolation of the full-length cDNA, because the obtained clone was long enough to judge it as a Xenopus Pumilio homolog and to produce recombinant proteins necessary for raising specific antibodies and characterizing the encoded protein.


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Fig. 1.   Sequence alignment of Pumilio homologs. Eight repetitive motifs (1-8) with flanking residues at both the N- and C-terminal ends of Pumilio homologs from Drosophila (GenBankTM accession X62589) (79), Xenopus (accession AB045628) (this study), and human (accession D43951) (46) are shown. Identical residues are marked (#).

Using a monoclonal antibody raised against the recombinant protein encoded by the cDNA Pum1-4R, we investigated the existence of Pumilio homolog proteins in Xenopus oocytes. A single 137-kDa protein, the size of which is comparable to those of Drosophila Pumilio (157 kDa) and the human homolog KIAA0099 (>128 kDa), was recognized specifically by the antibody in both immature and mature oocyte extracts (Fig. 2A). The 137-kDa protein was also detected by immunoprecipitation (Fig. 2B). Because this protein exhibits biochemical characteristics similar to those of Drosophila Pumilio, as described below, we refer to this as Xenopus Pumilio (XPum). No apparent differences in protein content or electrophoretic mobility of XPum were detected between immature and mature oocytes (Fig. 2B). XPum was present throughout oogenesis, although its apparent concentration decreased during the accumulation of yolk (Fig. 2C).


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Fig. 2.   Biochemical characterization of XPum. A and B, presence of XPum in immature and mature oocytes. Extracts from ~1/8 immature stage VI oocytes (lanes 1 and 3) and progesterone-matured oocytes (lanes 2 and 4) were immunoblotted with (lanes 1 and 2) or without (lanes 3 and 4) anti-XPum antibody (A). Bands found in lanes 3 and 4 are nonspecific signals due to the secondary antibody. Extracts from 25 immature stage VI oocytes (lanes 1 and 3) and progesterone-matured oocytes (lanes 2 and 4) were immunoprecipitated with anti-XPum antibody and then immunoblotted with (lanes 1 and 2) or without (lanes 3 and 4) the same antibody (B). Anti-XPum immunoprecipitate without oocyte extract (lane 5) shows the positions of immunoglobulins (Ig). C, XPum expression during oogenesis. Extracts from oocytes at stages I-VI and progesterone-matured oocytes (M) were applied to each lane (3.7 µg of protein/lane, equivalent to 1.7 oocytes at stage VI) and immunoblotted with anti-XPum antibody. Nonspecific bands that were also detected in the control blots without the primary antibody are indicated. D and E, association of XPum with mRNAs. Proteins retained on an oligo(dT)-cellulose column (D) or poly(U)-Sepharose column (E) were step-eluted with 10%, 25%, and 60% formamide or 100, 200, and 400 mM KCl, respectively. The eluted proteins were immunoblotted with anti-XPum antibody. F and G, binding of XPum to cyclin B1 mRNA. RNA-affinity chromatography with full-length cyclin B1 mRNA (F). Oocyte extracts were incubated with Sepharose coupled with (+) or without (-) cyclin B1 mRNA. Bound proteins were eluted stepwise with 250, 500, and 1000 mM KCl, and immunoblotted with anti-XPum antibody. Note the reduction in XPum protein content in the supernatant of mRNA-coupled beads (Sup, +). RNA-affinity chromatography with a 5'-terminal fragment (nucleotides 1-206, 5' cyclin B) or a 3'-terminal fragment (nucleotides 1137-1395, 3' cyclin B) of cyclin B1 mRNA (G). Eluates were assayed as in F. H, Northwestern blotting of oocyte extracts fractionated by oligo(dT)-cellulose chromatography (25% formamide-eluate) using a 3'-terminal fragment of cyclin B1 mRNA (lane 1). The same membrane was probed with anti-XPum antibody (lane 2). The cyclin B1 mRNA-bound 137-kDa protein (arrow in lane 1) is identical to the anti-XPum reactive 137-kDa protein (XPum). Abundant 56-kDa Y-box proteins (FRGY2) were also detected on the Northwestern blot.

XPum as a Cyclin B1 mRNA-binding Protein-- To test the capability of endogenous XPum to bind to mRNAs in oocytes, we first employed oligo(dT)-cellulose and poly(U)-Sepharose affinity chromatography. The anti-XPum immunoblots clearly showed that XPum was enriched in 10-25% of formamide eluates from an oligo(dT)-cellulose column (Fig. 2D) and in 400 mM KCl eluate from a poly(U)-Sepharose column (Fig. 2E), suggesting that this protein is physically associated with mRNAs in oocytes. We then analyzed the interaction between XPum and cyclin B1 mRNA by cyclin B1 mRNA-affinity chromatography. XPum was bound to the resin coupled with cyclin B1 mRNA but not to the control resin without mRNA (Fig. 2F). Similar experiments using resins coupled with either a 5'- or a 3'-fragment of cyclin B1 mRNA showed that XPum had an affinity for the 3'-fragment but not for the 5'-fragment (Fig. 2G).

The interaction between XPum and cyclin B1 mRNA was finally confirmed by Northwestern blotting. Oocyte extracts eluted from an oligo(dT)-cellulose column were probed with the 3'-fragment of cyclin B1 mRNA. The blot showed that, in addition to abundant 56-kDa Xenopus Y-box protein (FRGY2) that can bind to mRNAs in a sequence-nonspecific manner (63, 64), a 137-kDa protein recognized cyclin B1 mRNA (Fig. 2H, lane 1). Anti-XPum immunostaining of the identical blot demonstrated that the 137-kDa cyclin B1 mRNA-recognizing protein is XPum itself (Fig. 2H, lane 2).

Identification of the XPum-binding Site in Cyclin B1 mRNA-- To determine the cyclin B1 mRNA sequence required for the binding of XPum, we carried out UV cross-linking assays with various RNA probes. First, we confirmed that XPum can bind to the wild-type NRE, but not to two mutant NREs, of hb mRNA (Fig. 3, A and B), similar to Drosophila Pumilio (38). The results of UV cross-linking assays with the 3' and 5' sequences of cyclin B1 mRNA also confirmed the results of mRNA-affinity chromatography showing that XPum binds to the 3' region of cyclin B1 mRNA but not to the 5' region (Fig. 3B). The binding of XPum to the labeled B3' probe was inhibited by unlabeled B3' or wild-type NRE but not by mutant NRE (Fig. 3C), indicating that the XPum recognition sequence in the cyclin B1 3'-UTR resembles that of NRE.


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Fig. 3.   Binding of XPum to the NRE of Drosophila hb mRNA. A, sequence of the wild-type hb NRE (WT) and two mutant NREs (Mut1 and Mut2). Lowercase letters designate nucleotides that were changed in Mut2. Sequences conserved among the NREs of hb mRNAs are underlined (35). B, UV cross-linking assay with the wild-type hb NRE (WT), two mutant hb NREs (Mut1 and Mut2), cyclin B1 5' sequence (B5'), and cyclin B1 3' sequence (B3'). Arrows indicate XPum. C, RNA competition assay. Oocyte extracts were incubated with radiolabeled B3' RNA in the presence of a 25-, 50-, or 100-fold molar excess of unlabeled competitor RNAs as indicated.

To map the XPum-binding site in cyclin B1 mRNA more precisely, various deletion and substitution mutants of B3' RNA were used for UV cross-linking (Fig. 4). Among five NRE-like elements present in B3' RNA (Fig. 4A), neither deletion nor substitution of the three elements in the 5'-half affected the binding of XPum to the probes (Fig. 4, B and C). These results indicated that the XPum-binding site resides in a 43-nucleotide stretch from positions 76 to 118, harboring two NRE-like elements; the first element consists of the tetranucleotide UGUA located at positions 91-94 and the second one is the sequence AUUGU at positions 103-108 (Fig. 4A). Mutation of the second element, AUUGU, to UCAUC (sub103-108) had no effect on the binding, as in the case of mutation in an NRE-unrelated sequence in the same area (sub82-86). However, mutation of the first element, UGUA, to GUGC (sub91-94) completely abolished the binding of XPum (Fig. 4C). It is therefore likely that the UGUA sequence at positions 91-94 (nucleotides 1335-1338) is essential for the XPum binding. The finding that deletion or mutation of the UGUA sequence of hb mRNA prevented the binding of XPum (Fig. 3, A and B) also supports this notion.


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Fig. 4.   Identification of the XPum-binding sequence. A, the sequence of B3' RNA. Nucleotides are numbered from the base 1245 of cyclin B1 cDNA sequence to the last nucleotide before the poly(A) tract. The termination codon UGA is marked by double underlining and the NRE-like elements (cf., Fig. 3A) are underlined. B, schematic diagrams of RNA probes and summary of the ability of RNA probes to bind XPum. Potential XPum-binding sites (NRE-like sequences) in B3' RNA are shown by arrowheads. RNA probes are indicated by the nucleotide numbers (The first nucleotide of B3' is designated as number 1 and thus 1-151 RNA is identical to B3'), and their relative positions are shown as open bars. Substituted mutant regions are indicated by solid bars. The XPum-binding ability is shown as similar (+) or significantly reduced (-) relative to B3'. C, UV cross-linking assays (summarized in B) with the RNA probes indicated. The arrow shows XPum.

As a further test of the sequence requirement for XPum-RNA interaction, we carried out in vitro selection experiments using an RNA population with 25 nucleotides of a randomized sequence and GST-fused XPum produced in baculovirus-infected insect cells. After a total of five cycles of selection, the sequences recognized by XPum were determined (Table I). The highly conserved sequence is represented by the tetranucleotide UGUA. Moreover, in 11 of 15 UGUA sites, the consensus sequence is followed by an A residue at the 3'-end, which closely matches the sequence at positions 1335-1339 of cyclin B1 mRNA (Fig. 4A). Thus, it is most likely that the UGUA(A) sequence at positions 1335-1338 (1339) is a component important for the XPum binding.

                              
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Table I
In vitro selected RNA sequences by XPum
The first and last four nucleotides in the sequences are derived from the primers used in the PCR amplification step. The UGUA sequence is in boldface and the A residue following the UGUA sequence is underlined.

Sequence Elements Responsible for Masking Cyclin B1 mRNA-- Endogenous cyclin B1 mRNA can be translationally activated by injection into oocytes of a high concentration of the cyclin B1 3'-UTR that includes the CPEs, probably due to displacement of putative masking factors bound to the CPEs (22, 23). Because the cyclin B1 3'-UTR comprises the XPum binding site, as well as the CPEs, we investigated whether injection of excess amounts of the XPum-binding sequence can activate endogenous cyclin B1 mRNA (Fig. 5). Consistent with the previous finding (22), when B3' RNA was injected into Xenopus oocytes, cyclin B1 protein accumulated in the absence of progesterone and more than 75% of the oocytes underwent GVBD (Fig. 5B). In contrast, 76-118 RNA, which can bind to XPum (Fig. 5C), stimulated neither cyclin B1 synthesis nor GVBD (Fig. 5, B and D), indicating that the XPum-binding sequence itself is unable to titrate masking proteins required for translational repression of cyclin B1 mRNA.


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Fig. 5.   Identification of the masking element in the 3'-UTR of cyclin B1 mRNA. Oocytes were injected with various RNAs containing the XPum-binding site and/or the CPEs to determine the ability of each RNA to induce translational activation of cyclin B1 mRNA and GVBD. A, partial sequence of B3', showing the XPum-binding site in a black box, two CPEs (CPE1 and CPE2) in white boxes, and polyadenylation signal (AAUAAA) in boldface. Schematic diagrams of RNA probes and summary of the ability of RNA probes to bind to XPum and to induce GVBD are also shown. The XPum-binding site (XPB, shaded box), CPE1 and CPE2 (open boxes), and substituted mutant regions (solid bars) in the probes are indicated. The XPum-binding ability is shown as similar (+) or significantly reduced (-) relative to B3'. Percentage of GVBD induced by the injection of each RNA is shown as - (less than 10%), -* (10-20%), and + (more than 70%). ND, not determined. B, protein expression of cyclin B1 in oocytes injected with either the RNAs indicated or distilled water (Water), uninjected immature oocytes (Ni), and progesterone-induced mature oocytes (Nm). Suc1 precipitates were analyzed by anti-cyclin B1 immunoblotting. C, UV cross-linking with the RNA probes indicated. D, GVBD in oocytes injected with the RNAs indicated. Data are shown as the average of three independent experiments using 35-45 oocytes in each experiment.

To identify the cis-element responsible for masking cyclin B1 mRNA, we continued similar injection experiments using several deletion and substitution mutants (Fig. 5A). The shortest RNA that retained the GVBD-inducing ability equivalent to B3' comprised the 3'-terminal one-third of B3' (105-151 RNA, Fig. 5D). This RNA harbors two CPEs, named CPE1 at nucleotides 1353-1360 and CPE2 at nucleotides 1371-1377 (Fig. 5A) (21). Injection of 119-151 RNA, which lacks CPE1 but retains CPE2, did not induce GVBD (Fig. 5, A and D), suggesting that CPE1, but not CPE2, is involved in masking. Experiments with substituted mutant RNAs (mut105-108 carrying a mutation in four nucleotides upstream of CPE1, mut109-113 with a mutation in the 5'-half of CPE1, and mut114-119 with a mutation in the 3'-half of CPE1) showed that any mutations near or in CPE1 hampered the GVBD-inducing activity of 105-151 RNA (Fig. 5, A and D), indicating the necessity of CPE1 and its neighboring sequences for masking cyclin B1 mRNA. The necessity of the sequences near CPE1 was also indicated by the finding that 76-118 RNA, which contains complete CPE1 but only an additional two nucleotides on its 3'-side, had little activity for unmasking cyclin B1 mRNA and inducing GVBD (Fig. 5, A, B, and D).

XPum-interacting Proteins-- Given that Pumilio regulates translation of mRNAs with the aid of Nanos in Drosophila (37, 39), we analyzed the interaction between XPum and Xcat-2, a Xenopus homolog of Nanos (47), by protein pull-down assays using recombinant Xcat-2 and endogenous XPum. XPum bound to GST-Xcat-2 but not to GST as a control (Fig. 6A), indicating that XPum interacts with Xcat-2 at least in vitro.


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Fig. 6.   Association of XPum with Xcat-2 (A) and CPEB (B-D). A, GST-Xcat-2 pull-down assay. Beads prebound to E. coli-expressed GST (lane 1), GST-Xcat-2 (lane 2), or no protein (lane 3) were incubated with immature oocyte extracts, and bound proteins were analyzed by anti-XPum immunoblotting. B, T7-XlCPEB pull-down assay. Immature oocyte extracts were mixed with beads prebound to T7-Xdazl-His (lane 1) or T7-XlCPEB-His (lane 2), and examined by anti-XPum immunoblotting. C, co-immunoprecipitation assay. Untreated (lanes 2 and 4) or SDS-treated (lane 6) immature oocyte extracts, mature oocyte extracts (lane 5), and extraction buffer alone (lanes 1 and 3) were subjected to immunoprecipitation with control antibody (lanes 1 and 2) or anti-XlCPEB antibody (lanes 3-6). The precipitates were analyzed by immunoblotting with anti-XPum and anti-CPEB antibodies. D, immature oocyte extracts treated without (lane 1) or with (lane 2) RNase and extraction buffer alone (lane 3) were immunoprecipitated with anti-CPEB antibody, then immunoblotted with anti-XPum and anti-CPEB antibodies.

The injection experiments reported above (Fig. 5) strongly suggested that CPEB plays an essential role in masking and unmasking cyclin B1 mRNA. We therefore investigated the interaction between XPum and CPEB by protein pull-down assays. It was found that XPum bound to the beads prebound to T7-XlCPEB-His but not to those prebound to T7-Xdazl-His as a control (Fig. 6B), indicating the ability of the recombinant CPEB to associate with XPum. To know their interaction in vivo, we performed co-immunoprecipitation assays with a monoclonal antibody raised against Xenopus CPEB (XlCPEB12), which recognizes a 62-kDa CPEB (and an additional 64-kDa phosphorylated CPEB in mature oocytes) with high specificity both by immunoprecipitation and immunoblotting (Fig. 6C). XPum, as well as CPEB, was detected in anti-CPEB immunoprecipitates from immature and mature oocyte extracts but not in anti-goldfish cyclin B1 (B63) (41) immunoprecipitates as a control (Fig. 6C). Treatment of oocyte extracts with SDS (0.25% at final concentration) prior to immunoprecipitation caused the loss of XPum, but not CPEB, in anti-CPEB immunoprecipitates (Fig. 6C), confirming that XPum was not precipitated directly with the anti-CPEB antibody but was precipitated through an interaction with CPEB. Treatment of extracts with RNase A (0.2 mg/ml, 5 min, at room temperature) did not prevent the precipitation of XPum by the anti-CPEB antibody (Fig. 6D), ruling out the possibility that co-precipitation of XPum and CPEB was mediated by cyclin B1 mRNA to which they simultaneously bind. Anti-CPEB immunoblotting of anti-XPum immunoprecipitates gave essentially the same results (data not shown). These results provide evidence of physical interaction between XPum and CPEB in vivo for the first time in any species.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have focused on Pumilio as one of the possible translational regulators of cyclin B1, because the 3'-UTR of cyclin B1 mRNA has sequences resembling those required for translational repression by Pumilio in Drosophila. We report here the following main findings: 1) A 137-kDa Xenopus homolog of Pumilio (XPum) exists in oocytes and eggs, the first demonstration of Pumilio protein expression in vertebrates; 2) XPum binds to the cyclin B1 3'-UTR containing the UGUA at nucleotides 1335-1338; 3) The sequence responsible for masking cyclin B1 mRNA, as determined by translational activation of endogenous cyclin B1 mRNA by injection of various cis-elements, is located around CPE1 (nucleotides 1353-1360), close to but different from the XPum-binding site; 4) XPum physically associates with a Xenopus Nanos homolog, Xcat-2, at least in vitro and with CPEB in vivo.

The amino acid sequence alignment of Pumilio homologs exhibits a high degree of conservation in the Pum-HD (Fig. 1), which is known to have a dual function, not only recognizing the NREs but also interacting with some component of the translational machinery (36, 37, 65). The sequence similarity is suggestive of a common function of Pumilio. Indeed, our results showed that XPum can bind to the NRE of hb mRNA, the sequence recognized by the Drosophila counterpart. Moreover, the present UV cross-linking assays using endogenous Pumilio protein and various mutant probes indicated that the Pumilio-binding site contains the sequence UGUA, which is identical to the sequence determined by gel-shift experiments with recombinant Pumilio proteins from human and Drosophila (60). Recent studies have also demonstrated that yeast Pumilio-related proteins play a role in negative control of translation by recognizing a cis-element similar to that for Drosophila, Xenopus, and human Pumilio homologs (66, 67). Taken together, the results strongly suggest that the function of Pumilio as a translational regulator and its cis-element of the target RNAs are widely conserved in evolution.

Drosophila Pumilio represses translation of maternal hb mRNA by direct binding to the NRE in its 3'-UTR (36, 38). The results of genetic analyses also suggest that Pumilio, in cooperation with Nanos, controls translation of cyclin B mRNA in the migrating pole cells (39), although there has been no direct biochemical evidence for the binding of Pumilio to cyclin B mRNA in any species to date. The results of the present study have provided the first biochemical evidence of a Pum-binding site in cyclin B1 mRNA as well as evidence of the binding of endogenous Pumilio to cyclin B1 mRNA in a sequence-specific manner. These findings led us to verify the possibility that, as in Drosophila, XPum plays an essential role in translational control of cyclin B1 mRNA during oocyte maturation. To this end, we injected various cis-elements into oocytes in the expectation that, if the injected sequence is responsible for masking cyclin B1 mRNA, it will titrate the masking proteins and thereby induce translational activation of endogenous cyclin B1 as already demonstrated (22, 23). We confirmed that the injection of large amounts of cyclin B1 3'-UTR into Xenopus oocytes induced cyclin B1 synthesis and GVBD. We also demonstrated that the cis-element necessary for translational repression of cyclin B1 mRNA exists in the neighborhood of CPE1 (nucleotides 1353-1360) but not in CPE2 (nucleotides 1371-1377). Injection of the XPum-binding sequence, however, induced neither translational activation of endogenous cyclin B1 mRNA nor GVBD (Fig. 5). These results seem to imply that the binding of Pumilio to cyclin B1 mRNA is not responsible for masking cyclin B1 mRNA. Nevertheless, this suggestion needs to be proved by experimental approaches other than the injection of cis-elements, because it has been reported that injection of large amounts of CPE-containing RNA into mouse oocytes does not induce translational activation of cyclin B1 mRNA despite the involvement of CPEB in both the repression and the stimulation of cyclin B1 mRNA in this species, as in the case of Xenopus (28). In cooperation with CPEB as a major control element, XPum might contribute as a fine tuner of cyclin B1 mRNA translation. This notion is also supported by the present finding that XPum physically interacts with CPEB both in vivo and in vitro (Fig. 6, B-D).

The function of Drosophila Pumilio in control of the translation of mRNAs is dependent on Nanos (68, 69). FBF, a C. elegans Pumilio-related protein, also controls the sperm/oocyte switch in a hermaphrodite germ line by regulating the translation of fem-3 mRNA with interaction with one of the C. elegans Nanos homologs (61, 65). XPum may likewise collaborate with one or more partners to regulate the translation of specific maternal mRNAs (for review see Ref. 70). The most likely candidate for the XPum partner is a Xenopus homolog of Nanos, Xcat-2 protein (71, 72). In fact, we have provided evidence of association between XPum and Xcat-2, at least in vitro (Fig. 6A). However, Xcat-2 protein is reported to be absent in oocytes and eggs despite the presence of its mRNA. The protein appears in accordance with the movement of germ plasm during early embryogenesis (73). Consistent with this, Xcat-2 is undetectable in anti-XPum immunoprecipitates from oocyte extracts.3 Although we cannot exclude the possibility that one or more Nanos homologs other than Xcat-2 collaborate with XPum in oocytes, it remains a mystery whether XPum acts together with a Nanos homolog to govern the translation of mRNAs in oocytes.

The actual biological roles of XPum are completely unknown at present, but we speculate that XPum plays an important role in translational control of cyclin B1 mRNA like in Drosophila (39, 40). CPEB directly binds to maskin, a protein that can also bind directly to the cap-binding translation initiation factor elF-4E, which leads to translational repression. The dissociation of maskin from elF-4E allows elF-4G to bind to elF-4E, which brings elF-3 and the 40 S ribosomal subunit to the mRNA to initiate translation via cap-ribose methylation (30, 74, 75). Recent studies have also shown that a progesterone-induced early phosphorylation of CPEB at serine 174 is catalyzed by Eg2 (76) and that this phosphorylation recruits cleavage and polyadenylation specificity factor into an active cytoplasmic polyadenylation complex (77). Thus, CPEB plays a key role in both translational repression and activation of mRNAs stored in oocytes (for review see Ref. 78). We demonstrated in this study that XPum is physically associated with CPEB in oocytes. In cooperation with CPEB, XPum may control the CPEB/maskin-mediated translational masking and unmasking to assure the highly coordinated successive translational activation of masked mRNAs during oocyte maturation. Further studies are required to understand the biological significance of the interactions among XPum, CPEB, and cyclin B1 mRNA, as well as to elucidate the functions of XPum in oocytes.

    ACKNOWLEDGEMENTS

We thank Dr. Hideki Kobayashi (Kyushu University) for providing the cDNA for Xenopus cyclin B1 in pGEM1 and Dr. Satoru Kobayashi (Tsukuba University) for his helpful discussions. Thanks are also due to Drs. Nancy Standart and Ann Kaminski (University of Cambridge, UK) for technical advice on UV-crosslink and RNA affinity chromatography, respectively.

    FOOTNOTES

* This work was supported by grants-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture of Japan (10160201 to M. Y.) and JSPS-RFTF 96L00401 and BioDesign Program from the Ministry of Agriculture, Forestry and Fisheries, Japan (to Y. N.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB045628.

** To whom correspondence should be addressed: Div. of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan. Tel.: 81-11-706-4454; Fax: 81-11-706-4456; E-mail: myama@sci.hokudai.ac.jp.

Published, JBC Papers in Press, March 29, 2001, DOI 10.1074/jbc.M010528200

2 M. Yamashita (2000) GenBankTM accession number AB040435.

3 S. Nakahata and M. Yamashita, unpublished data.

    ABBREVIATIONS

The abbreviations used are: MPF, maturation-promoting factor; MAPK, mitogen-activated protein kinase; GVBD, germinal vesicle breakdown; CPE, cytoplasmic polyadenylation element; CPEB, CPE-binding protein; UTR, untranslated region; hb, hunchback; NRE, nanos response element; MBS-H, modified Barth's saline buffered with Hepes; RT, reverse transcription; PCR, polymerase chain reaction; GST, glutathione S-transferase; ORF, open reading frame; bp, base pair(s); kb, kilobase(s); RACE, rapid amplification of cDNA ends; XPum, Xenopus Pumilio.

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ABSTRACT
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RESULTS
DISCUSSION
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