CPEB is a specificity factor that mediates cytoplasmic polyadenylation during Xenopus oocyte maturation

Cell, Vol. 79, 617-627,

November

16, 1994, Copyright

0 1994 by Cell Press

CPEB Is a Specificity Factor That Mediates Cytoplasmic Polyadenylation during Xenopirs Oocyte Maturation Laura E. Hake and Joel D. Richter Worcester Foundation for Experimental Shrewsbury, Massachusetts 01545

Biology

Summary The translational activation of several maternal mRNAs during Xenopus oocyte maturation is stimulated bycytoplasmic poly(A) elongation, which requires the uri: dine-rich cytoplasmic polyadenylation element (CPE) and the hexanucleotide AAUAAA. Here, we have enriched a CPE-binding protein (CPEB) by single-step RNA affinity chromatography, have obtained a CPEB cDNA, and have assessed the role of CPEB in cytoplasmic polyadenylation. The 62 kDa CPEB contains two RNArecognition motifs, and within this region, it is 62% identical to orb, an oocyte-specific RNA-binding protein from Drosophila. CPEB mRNA and protein are abundant in oocytes and are not detected in embryos beyond the gastrula stage. During oocyte maturation, CPEB is phosphorylated at a time that corresponds with the induction of polyadenylation. Immunodepletion of CPEB from polyadenylation-proficient egg extracts renders them incapable of adenylating exogenous RNA. Partial restoration of polyadenylation in depleted extracts is achieved by the addition of CPEB, thus demonstrating that this protein is required for cytoplasmic polyadenylation.

Introduction Early animal development is in large part programmed by maternal mRNAs that are synthesized and stored in the cytoplasm of growing oocytes. These mRNAs are not translated en masse at any one time, but instead are expressed in a sequence-specific manner at precise stages and, in some cases, precise places in the maturing oocyte or early embryo. One prevalent form of translational regulation of maternal mRNAs is cytoplasmic poly(A) elongation, which occurs in several marine invertebrates (Rosenthal and Ruderman, 1987; reviewed by Jackson and Standart, 1990; Standart and Dale, 1993), mammals (Vassalli et al., 1989; Huarte et al., 1992; Gebauer et al., 1994) Drosophila melanogaster (Schafer et al., 1990; Wharton and Struhl, 1991) and Xenopus laevis (Dworkin et al., 1985; McGrew et al., 1989; Fox et al., 1989; Paris and Phillippe, 1990; Paris and Richter, 1990; Simon et al., 1992; Bouvet et al., 1994; Stebbins-Boaz and Richter 1994; Sheets et al., 1994). Elucidation of many of the key features of polyadenylation and resulting effects on translation have come from studies of this process during oocyte maturation in Xenopus and mouse (reviewed by Wickens, 1992; Richter, 1993; Wormington, 1993). Oocytes contain several translationally dormant mRNAs that have relatively short poly(A) tails that range in size from fewer than 15 to as many as 90 residues (McGrew et al., 1989;

Parisand Phillippe, 1990; Parisand Richter, 1990). Following the induction of maturation, two sequence elements in the3’untranslated regions(3’UTRs)of responsivemRNAs promote the polymerization of 3’ poly(A) to about 150 residues (McGrew et al., 1989; Fox et al., 1989; Paris et al., 1991; Sheets et al., 1994) or more (Vassalli et al., 1989). Once the process of polyadenylation begins, or following the acquisition of a long poly(A) tail, the mRNA assembles into polysomes (McGrew et al., 1989; Paris and Richter, 1990; Vassalli et al., 1989). The sequences that mediate poly(A) elongation are the near-ubiquitous hexanucleotide AAUAAA, also notable for its role in nuclear pre-mRNA cleavage and polyadenylation, and a U-rich structure termed the cytoplasmic polyadenylation element (CPE), which generally resides within 50 nt 5’ of the hexanucleotide (McGrew et al., 1989; Fox et al., 1989; McGrew and Richter 1990; Paris and Richter, 1990; Paris et al., 1991; Huarte et al., 1992; Gebauer et al., 1994). Although CPEs have the general structure of UUUUUAAU, they can vary considerably from this motif, and this may influence the exact time or extent of polyadenylation (Paris and Richter, 1990; Sheets et al., 1994). Indeed, at least two RNAs contain CPEs consisting of oligo (U), which promote polyadenylation and translation during embryogenesis rather than oocyte maturation (Simon et al., 1992; Simon and Richter, 1994). Although the delineation of the cis elements that mediate cytoplasmic polyadenylation was relatively straight forward, an analysis of the proteins that control this process has proceeded more slowly. Generally, two approaches have been taken. One has been to identify factors involved with nuclear polyadenylation that might also contribute to cytoplasmic polyadenylation. In this regard, Bilger et al. (1994) have mixed asolution of bovine proteins containing cleavage- and polyadenylation-specificity factor (CPSF), a protein complex that mediates nuclear polyadenylation, with bovine poly(A) polymerase and have shown that these stimulate CPE-dependent polyadenylation in vitro. However, the extent to which this reflects cytoplasmic poly(A) elongation during Xenopus oocyte maturation or embryogenesis is unclear and would seem unlikely to fully explain the discrimination among mRNAs that are polyadenylated in a stage-specific manner. An alternative approach has been to identifythefactor(s) that interacts with the specificity element for cytoplasmic polyadenylation, which is the CPE. Gel shift analyses using egg extracts have shown that specific factors do indeed recognize RNAs containing the CPE (Paris and Richter, 1990; Fox et al., 1992), but they could not be identified by this method. Ultraviolet (UV) cross-linking experiments, however, have been more successful and have shown that there are at least two proteins that interact with RNAs containing CPEs (McGrew and Richter, 1990; Paris et al., 1991). One of these, a 58 kDa protein that cross-links specifically to RNA containing the CPE UUUUUAAU, is especially intriguing because it is phosphorylated during oocyte maturation, perhaps by ~34~~@, and this appears to be necessary for poly(A) addition (Paris et al., 1991).

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Using antibodies raised against bacterially expressed CPEB, we show that immunodepletion of egg extracts results in the complete loss of polyadenylation activity. Supplementation of the depleted extract with CPEB synthesized in a reticulocyte lysate partially restores activity, thus demonstrating that this protein is an essential component of the cytoplasmic polyadenylation apparatus. Results

12

Figure 1. Specific 84 RNA

Interaction

3

4

5

678

of a 62 kDa Protein

with the CPE of

(A) The sequences of the RNAs containing part of the 84 3’ untranslated region (3’ UTR) and of an RNA-containing part of a mutated 84 RNA 3’ UTR (B4-M2) are shown. The CPE of 84 RNA and the corresponding mutated sequence in B4-M2 RNA are italicized and in bold type; the polyadenylation hexanucleotides are boxed. (B) Radiolabeled 84 or B4-M2 RNAs were incubated in Xenopus oocyte extract and were then irradiated with UV light. The RNAs were then digested with RNase A, and the proteins that became radioactive by label transfer were visualized after SDS-polyacrylamide gel electrophoresis by autoradiography. In lanes 1 and 2, radiolabeled 84 and B4-M2 RNA, respectively, were cross-linked. For the remaining lanes, labeled 84 RNA and l-, lo-, and IOO-fold molar excess of unlabeled 84 RNA (lanes 3, 4, and 5) or l-, lo-, and lOO-fold molar excess of unlabeled B4-M2 RNA (lanes 6, 7, and 6) were added to oocyte extract prior to UV irradiation. CPEB denotes a 62 kDa protein that specifically interacts with CPE-containing RNA. Molecular size markers (in kilodaltons) are indicated to the left of the figure.

Using a one-step RNA affinity chromatography procedure, we now report the isolation, cloning, and characterization of this CPE-binding protein. The protein, henceforth referred to as CPEB, interacts specifically with the sequence UUUUUAAU, has two RNA recognition motifs (RRMs), and has significant homology to orb, a Drosophila oocyte-specific RNA-binding protein (Lantz et al., 1992). CPEB RNA and protein levels are developmentally regulated in that they are prevalent in oocytes, decline precipitously in eggs, and are not detected after gastrulation.

In a previous report, it was shown that a 58 kDa protein could be photocross-linked to a CPE-containing RNA in Xenopus oocyte and egg extracts (Paris et al., 1991). Moreover, point mutations in the CPE reduced not only polyadenylation activity in egg extracts, but also commensurately reduced cross-linking of the 58 kDa protein (Paris et al., 1991). To confirm and extend these observations, we performed an additional series of UV cross-linking experiments with the RNAs shown in Figure 1A. B4 RNA, which is efficiently polyadenylated in egg extracts, contains a CPE (UUUUUAAU) whereas B4-M2, which is not polyadenylated in egg extracts, contains a substitution for this sequence (Paris and Richter, 1990). When synthesized in vitro in the presence of [32PjUTP and photocrosslinked in Xenopus oocyte extracts, B4 RNA was crosslinked by three prevalent proteins of approximately 54,56, and 62 kDa (Figure 1 B, lane 1). When the slight differences in the mobilities of molecular mass marker proteins are taken into account, the 62 kDa protein migrated to the same relative position as the 58 kDa protein identified by Paris et al. (1991). Also as shown by Paris et al. (1991), this same protein was not detected when B4-M2 RNA was used asacross-linking substrate (Figure 1 B, lane 2). When labeled B4 RNA was mixed with a lo- or a lOO-fold molar excess of unlabeled 84 RNA, the detection of the 62 kDa protein by UV cross-linking was drastically reduced (Figure lB, lanes 3-5). However, there was no specific decrease in the cross-linking of the 62 kDa protein when unlabeled B4-M2 RNAwas used in the competition (Figure 1 B, lanes 6-8). Direct bindingofthe62 kDaprotein totheCPEsequence was assessed by UV cross-linking with 3zP-labeled RNase Tl fragments of 84 RNA and RNA oligonucleotides (data not shown). Both an RNase Tl fragment containing the sequence UUUUUAAUG and an RNA oligonucleotide of the sequence GGUUUUUAAUAG efficiently cross-linked the 62 kDa protein (data not shown). Conversely, a 17 nt B4 RNA RNase Tl fragment, which does not contain a CPE sequence, did not bind this protein. Thus, the 62 kDa protein binds directly to the CPE sequence and is henceforth referred to as CPEB (CPE-binding protein). Isolation of the CPEB by Affinity Chromatography To isolate CPEB, we have used single-step RNA affinity chromatography. In vitro transcribed B4 and B4-M2 RNAs containing poly(A) tails (B4A and B4-M2A) were biotinylated posttranscriptionally and incubated with Xenopus oocyte protein extract under conditions that promote binding of CPEB to its substrate RNA. The RNA-protein complexes were captured with avidin bound to Sephacryl

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‘gEEij gz .o 3

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Figure 2. Analysisofthe Proteins Isolated by RNAAffinityChromatogwhy The proteins retained on two RNA affinity matrices are presented. Lane 4 shows the protein retained on a B4A RNA (CPE+) matrix, and lane 5 shows the proteins retained on a B4-M2A RNA (CPE-) matrix. A protein of 62 kDa denoted as CPEB is bound specifically to the CPE+ RNA. The unfractionated oocyte extract that was applied to the RNA affinity matrices is shown in lane 3. For comparison, the UV cross-linked pattern of proteins interacting with CPE+ (B4A) RNA and CPE- (B4-M2A) RNA are shown in lanes 1 and 2, respectively. The asterisk indicates the position of poly(A)-binding protein.

beads, and specifically bound proteins were eluted and analyzed asshown in Figure 2. Although the starting material (total nonyolk oocyte protein) was very heterogenous (Figure 2, lane 3) the material retained by the RNA affinity columns was quite restricted. Fewer than eight proteins bound to either B4A (Figure 2, lane 4) or B4-M2A (lane 5) RNAs as detected by silver staining. One protein of 62 kDa bound to B4A RNA but not B4-M2A RNA. The migration of this protein in SDS-polyacrylamide gels matched exactly the migration of UV cross-linked CPEB (Figure 2, compare lanes 1,2, and 4). Thus, we considered the 62 kDa protein to be a good candidate for CPEB. We also note that a protein (asterisk, Figure 2) that was bound to both the B4A and the B4-M2A columns had a molecular size of -72 kDa, which is similar to that of Xenopus poly(A)-binding protein (Zelus et al., 1989). Subsequent microsequencing of several tryptic peptides showed that this was the case (data not shown). The proteins retained by the B4A column (Figure 2, lane 4) were electrophoresed in an SDS-polyacrylamide (10%) gel and transferred to a polyvinylidene difluoride (PVDF) membrane, and the protein that migrated with asize corresponding to 62 kDa was excised and digested in situ with

trypsin. Of the several tryptic peptide fragments that were subsequently sequenced, three provided unambiguous sequences of 12, 5, and 10 residues. Based on this information, three pairs of oligonucleotides wereprepared and used in the polymerase chain reaction (PCR) with DNA isolated from a Xenopus stage VI oocyte cDNA library. One combination of primers consistently yielded a 900 bp fragment that, when cloned and sequenced, contained a sequence identical to the third tryptic peptide fragment. This, therefore, confirmed that a cDNA corresponding to the 62 kDa protein isolated by affinity chromatography had been obtained. The PCR amplification product was subsequently used in library screening to obtain a cDNA containing an open reading frame (ORF) that encoded a protein with a molecular weight close to that empirically determined for CPEB. The amino acid sequence derived from this ORF is presented in Figure 3A. The positions of the three original tryptic peptides are underlined. In addition, there is a single putative ~34~~~~ kinase phosphorylation site (box in Figure 3A; Moreno and Nurse, 1990). Further analysis of this sequence reveals the presence of two RRMs (brackets over lightly shaded rectangles in Figure 3B and delineated in greater detail in Figure 3C using the nomenclature of Kenan et al., 1991). The carboxy-terminal portion of CPEB that contains the two RRMs is 62% identical to the Drosophila orb protein (Figure 3C), an RNA-binding protein thought to be involved in RNA localization (Lantz et al., 1994; Christersen and McKearin, 1994). The same region of the Xenopus protein is 33% identical to a Caenorhabditis elegans 40.2 kDa protein (Figure 3C) of unknown function (C40Hl .l; GenBank accession number Z19154; Wilson et al., 1994). Another interesting feature of the sequence comparison is that all three proteins have conserved spacings of several cysteine and histidine residues that reside outside of the RRMs (black vertical lines, Figure 38; closed circles, Figure 3C). Cloned CPEB Binds the UUUUUAAU CPE Sequence To confirm that the cDNA does indeed encode a protein that binds to RNA containing a CPE (Paris et al., 1991), it was ligated to DNA for a Myc epitope tag and cloned behind a T7 promoter. Oocytes were then injected with in vitro synthesized Myc-CPEB RNA, incubated overnight to allow for sufficient translation, and used to prepare a protein extract. 3zP-labeled B4A (containing a CPE) and B4-M2A (lacking a CPE) RNAs were then added, and the mixture was irradiated with UV light. Figure 4 shows that although these two RNAs were cross-linked by several similar proteins, a 62 kDa species cross-linked only to B4A RNA (compare lanes 1 and 2). Moreover, labeled MycCPEB was immunoselected with anti-Myc antibody from extracts of oocytes injected with B4A (Figure 4, lane 3) but not B4-M2A (lane 4) RNA. For comparison, lanes 5 and 6 of Figure 4 show that the cross-linking pattern of proteins from noninjected oocytes is identical to that from RNA-injected oocytes (lanes 1 and 2). In addition, antibodies raised against a bacterially expressed glutathione S-transferase (GST)-CPEB fusion protein immunoprecipitated the endogenous CPEB protein that has been cross-

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A

MAFPLKDDLGRAKDCWGCPSDTP~STCSN~IFRRINAMLDNSLDFTGVCTTPNTKGKCEHLQDYQDTE

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link labeled to B4A RNA in oocyte extracts (Figure 4, lane 7) and the phosphorylated form of the protein in egg extracts (Paris et al., 1991; Figure 4, lane 9). No labeled proteins were immunoprecipitated by anti-CPEB when B4M2 RNA was used as the labeling substrate (Figure 4, lanes 8 and 10). These data demonstrate that the cDNA we have isolated does indeed encode CPEB.

Figure3. CPEB Amino Acid Sequence and Comparison with Drosophila Orb and C. elegans C40Hl .l (A) Predicted amino acid sequence of CPEB. The largest ORF of the isolated CPEB cDNA would encode a protein of 566 amino acids with a predicted size of 62 kDa. The positions of the three tryptic peptide sequences used to generate oligonucleotides for PCR are underlined and marked as peptides 1,2, and 3. A putative ~34~~ kinase phosphorylation site is boxed. The asterisk is the stop codon. (B) Schematic diagram of CPEB protein structure compared with that of orb and C40Hl .I. Two RRM domains (residues 314-396 and 431-511 for CPEB) are present in the carboxyl termini of all three proteins. The conserved spacing of cysteine and histidine residues are indicated by the C/H label and the vertical black lines within the schematic. The rectangles with hatched lines, diagonal lines, or very dark stippling represent the remaining coding regions of each protein that share no similarity. (C) Amino acid comparison of the CPEB carboxy1 terminus (amino acids 277-566) with Drosophila orb (amino acids 540-624) and C. elegans C40Hl .I (amino acids 42-326). The region of CPEB that matches most closely with these proteins includes the RRMs, but also extends beyond them. The boxes indicate identical amino acids shared among the proteins. The solid circles denote conserved cysteine and histidine residues. RNP, beta, Ip, and alpha, followed by numbers, refer to the different ribonucleoprotein motifs, p-pleated sheets, loop domains, and a helices that comprise the RRMs (see Kenan et al., 1991). The plus signs indicate conserved hydrophobic residues, and the asterisks denote conserved solventexposed positions.

Developmental Expression of CPEB To examine the levels of CPEB mRNA during development, total RNA was isolated from stage VI oocytes, ovulated eggs, and blastula, gastrula, neurula, and tail bud stage embryos and probed with labeled CPEB cDNA (Figure 5A). A 5.1 kb CPEB mRNA was detected at high levels in oocytes, which declined by about 75% in eggs. This

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Figure 4 The Recombinant CPEB Protein Recognizes Containing RNA, and Antibodies against It lmmunoprecipitate enous CPEB

CPEEndog-

A Myc epitope-tagged CPEB RNA was injected into stage VI Xenopus oocytes that were incubated overnight to allow translation of the injected RNA. Protein extracts from these injected oocytes, as well as other noninjected oocytes, were used in UV cross-linking assays with radiolabeled B4A (CPE+; lanes 1,3,5,7, and 9) and B4-M2A (CPE-; lanes 2,4,6,8, and 10) RNAs. After cross-linking, some of the extract derived from injected oocytes was used to immunoselect Myc epitopetagged CPEB (lanes 3 and 4). All cross-linked proteins were resolved by SDS-polyacrylamide gel electrophoresis and visualized on a phosphorimager. Lanes 1 and 2 show total cross-linked proteins to B4A and B4-M2A RNAs from an extract derived from injected oocytes, and lanes 3 and 4 are proteins selected by an anti-c-Myc monoclonal antibody. Lanes 5 and 6 show total protein cross-linked to B4A and B4-M2A RNA?., respectively, from a noninjected control. Antisera against recombinant CPEB was used to immunoprecipate endogenous CPEB that was cross-link-labeled to either B4A (lanes 7 and 9) or B4-M2A (lanes 8 and IO) RNA in oocyte (lanes 7 and 8) or egg extract (lane 9 and 10). The location of CPEB is noted. The asterisk indicates the position of poly(A)-binding protein.

level was maintained to the gastrula stage, after which it was no longer detected. The relative level of CPEB protein during development was examined by Western blot analysis using rabbit antisera generated against a GST-CPEB fusion protein produced in bacteria (Figure 58). Like CPEB mRNA, CPEB protein was present at high levels in stage VI oocytes and declined by nearly 75% in eggs. By comparison with known amounts of the GST-CPEB fusion protein, there is approximately 3-4 ng of endogenous CPEB per oocyte and 1 ng per egg (data not shown). The protein was detected in embryos up to the gastrulastage and was absent thereafter (Figure 58). In addition, CPEB from eggs had a slightly slower mobility relative to that from oocytes. This is most likely due to phosphorylation of the protein in eggs as shown by Paris et al. (1991; see below). An unexpected

12 Figure 5. Developmental mRNA

Distribution

56

7

8

of the CPEB Protein

and CPEB

(A) RNA was isolated from stage VI oocytes (0), eggs(E), and blastula (B), gastrula (G), neurula (N), and tail bud (T) stage embryos, electrophoresed in a 1% agarose gel and blotted to nylon membrane, and hybridized with 3*P-labeled CPEB cDNA. The positions of the 18s and 28s ribosomal RNAs are indicated as markers to the right of the figure. (B) Protein extract (30 wg) from stage VI oocytes (0), eggs (E), 4-cell (4 cell), early blastula (EB), late blastula (LB), gastrula (G), neurula (N), and tail bud (T) stage embryos was resolved by electrophoresis, blotted to nitrocellulose, and probed with rabbit anti-CPEB antisera and then HRP-labeled goat anti-rabbit IgG. The HRP was detected by enhanced chemiluminescence. (C) Cytoplasmic (CM) and germinal vesicle (GV) fractions from five stage VI oocytes were manually separated under a dissecting microscope and analyzed for CPEB protein by a Western blot as described in (B). The location of CPEB is noted.

finding is that CPEB in four-cell and later embryos migrated faster than it did in eggs, which suggests that it was dephosphorylated at this time. The significance of this observation remains to be determined. Finally, we have fractionated oocytes into nuclear and cytoplasmic compartments and have determined the relative distribution of CPEB. The Western blot in Figure 5C shows that greater than 95% of this protein was cytoplasmic. CPEB Is Phosphorylated during Oocyte Maturation Paris et al. (1991) showed that CPEB from egg extracts had a slower electrophoretic mobility than that from oocyte extracts. Moreover, oocyte CPEB could be induced to have a slower mobility when the extracts were supplemented with baculovirus-expressed cyclin or ~34~~~~ kinase. Because the slow mobility could be reversed by treatment with phosphatase, they inferred that phosphory-

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observation , it is interesting to note that CPEB contains a consensus p34cdc2 phosphorylation site (see Figure 3).

Hours of incubation Progesterone

IP Of 32P-labeled CPEB

- CPEB

-A 100

Polyadenylation of B4 3’ UTR

-A0

1234567 Figure 6. Phosphorylation of CPEB during lates with the Increase in Polyadenylation

Oocyte

Maturation

Corre-

Stage VI Xenopus oocytes were incubated overnight in medium containing 100 @/ml of [3zP]orthophosphate and were then induced to mature with progesterone. At the indicated times, the oocytes were homogenized and CPEB was immunoselected and resolved by SDS gel electrophoresis and visualized on a phosphorimager (top). In parallel, other oocytes were injected with [“P]B4 mRNA, whose polyadenylation was monitored by urea-polyacrylamide gel electrophoresis and autoradiography (bottom). The approximate number of adenosine residues added to the RNA is indicated to the right of the figure.

lation was responsible for the altered mobility of this protein. It was also clear that polyadenylation took place only when CPEB was phosphorylated. To confirm directly that CPEB is phosphorylated and that this correlates with polyadenylation in vivo, we incubated oocytes with [32P]orthophosphate and then induced them to mature with progesterone. CPEB was then immunoselected and resolved by SDS-polyacrylamide gel electrophoresis and visualized on a phosphorimager. In parallel, other oocytes were injected with [3ZP]B4 RNA, whose polyadenylation was analyzed during maturation. Figure 6 (top) shows that CPEB was indeed phosphorylated during maturation and reached a maximum level 4 hr afterthe addition of progesterone (germinal vesicle breakdown, as assessed by the appearance of a white spot at the animal pole, was first detected 6 hr after progesterone treatment). This phosphorylation also correlated with the maximum polyadenylation of 84 RNA, which occurred 4-6 hr after progesterone treatment (Figure 6, lanes 5 and 6). We also note that a low level of polyadenylation occurred even in the absence of progesterone (Figure 6, lane 7), which has been observed before (Paris et al., 1991). Thus, the phosphorylation of endogenous CPEB during oocyte maturation suggests that it could be important for its function. In light of this

CPEB Is Required for Cytoplasmic Polyadenylation Although the data presented thus far and also in Paris et al. (1991) show clearly that the protein we have isolated and cloned binds to the CPE, its role in cytoplasmic polyadenylation has not been conclusively demonstrated. To investigate this, we have used antisera generated against bacterially expressed CPEB to immunodeplete the protein from egg extracts that are normally competent for polyadenylation. This immunodepleted extract was then assayed for its ability to polyadenylate 84 RNA. As shown in Figure 7A, immunodepletion of CPEB rendered the extract incapable of polyadenylating radiolabeled 84 RNA (compare lane 1 with nondepleted extracts in lanes 4 and 5). Extracts that were immunodepleted with preimmune sera (Figure 7A, lane 2) or nonspecific IgG (lane 3) showed no diminution of polyadenylation activity. The efficiency of the immunodepletion of CPEB from egg extracts is shown in Figure 7B. Following depletion with anti-CPEB antibody, no detectable CPEB, as determined by Western blotting, remained in the supernatant, which was the fraction used for the polyadenylation assay (Figure 7B, lane 1). As expected, this protein was present in the pellet fraction, which contained the CPEB antibody bound to protein A-Sepharose beads (Figure 7B, lane 4). In contrast with these results, CPEB was readily detected in the supernatants following mock depletions with preimmune sera or nonspecific IgG (Figure 78, lanes 2 and 3) and was not present in the protein A-Sepharose bead fraction (lanes 5 and 6). We also note that the heavy bands in lanes 4-6 represent some contaminating heavy chain IgG. Thus, these results strongly indicate the necessity for CPEB in cytoplasmic polyadenylation. In a final experiment, we have attempted to restore polyadenylation activity to a CPEB-depleted extract by supplementing it with exogenous CPEB. CPEB RNA was translated in a reticulocyte lysate and was added to a CPEB-depleted extract. Following a 15 min incubation, radiolabeled 84 RNA wasadded and polyadenylation was assessed. Figure 7C shows that no polyadenylation occurred in the extract depleted with anti-CPEB antibody compared with a mock depletion with preimmune serum (compare lane 1 with lane 5). However, when the CPEBdepleted extract was supplemented with 2 or 4 ~1 of reticulocyte lysate containing newly synthesized CPEB, some polyadenylation activity was restored (Figure 7C, lanes 2 and 3). No polyadenylation was restored when 4 ~1 of reticulocyte lysate not primed with CPEB mRNA was added to the depleted extract (Figure 7C, lane 4). Because the CPEB mRNA-primed lysate did not completely restore polyadenylation activity, we suspected that the reticulocyte lysate might have some inhibitory effect. The addition of 4 ~1 of reticulocyte lysate to a mock depleted extract did indeed lower overall polyadenylation activity (Figure 7C, compare lanes 5 and 6). Thus, this result, together with those presented in Figures 7A and 78, demonstrates the requirement for CPEB in cytoplasmic polyadenylation.

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lmmunodepleted samples supernatant

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7. Polyadenylation

in Egg Extracts

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(A) Egg extra&were immunodepleted at 4°C with the following antisera bound toprotein A-Sepharose: anti-CPEB (lane l), preimmune sera (lane 2) and a nonspecific IgG (lane 3). Two other samples were

Cytoplasmic polyadenylation during Xenopus oocyte maturation requires two cis-acting sequences, the nearubiquitous polyadenylation hexanucleotide and aspecificity sequence termed the CPE. In this study, we have isolated, cloned, and characterized a 62 kDa RNA-binding protein that binds to the CPE sequence. This protein, CPEB, is required for cytoplasmic polyadenylation because immunodepletion of the protein from egg extracts renders them incapable of polyadenylating exogenous B4 RNA, a model substrate for this process. At least partial restoration of polyadenylation activity is achieved by supplementing the depleted extract with in vitro synthesized CPEB. The timing of phosphorylation of CPEB during oocyte maturation correlates with the activation of polyadenylation, although it is unknown whether phosphorylation of CPEB alone is sufficient. Structure of CPEB and Its Comparison with Other Proteins Sequencing of a CPEB cDNA showed the longest ORF toencodeaproteinof 568aminoacidswith asizeof 82,431 daltons. The protein, which contains two FIRMS, shows significant homology to only two other proteins, Drosophila orb (Lantz et al., 1992) and C40Hl .l, a C. elegans ORF that was revealed in the genome sequencing project (Wilson et al., 1994). Considering the evolutionary divergence of these three organisms, this similarity is suggestive of a common function or a common target RNA for these proteins. Orb appears to have a role in the development of the 16-cell cyst, the differentiation of the egg chamber, and the establishment of the dorsoventral and anteroposterior axis during oogenesis (Lantz et al., 1994; Christersen and McKearin 1994) and may affect these processes through involvement in the localization of mRNAs to specific regions in the developing oocyte. For example, mutations in the orb gene prevent the appropriate localization of Bicaodal-D and oskar, gurken and hu-Ii tai shao mRNAs. In addition, orb function may not be limited to localization alone, but could involve other aspects of mRNA utilization such as anchoring the RNA to a specific location or by influencing message abundance or translation (Lantz et al 1994; Christersen and McKearin 1994). Because the similarity between CPEB and orb is highest in the putative

not incubated with any antibody, although one was incubated at 4OC (lane 4) while the other was not (lane 5). 84 RNA was added to each extract after depletion and incubated for 1 hr. The RNA was extracted and analyzed by electrophoresis in urea-polyacrylamide gels. (6) The protein supernatants (lanes 1-3) and the protein A-Sepharose beads (lanes 4-6) from each immunodepleted sample in (A) were analyzed by Western blotting for the presence of CPEB. (C) Egg extracts were immtmodepleted with anti-CPEB antibody or preimmune sera as described in (A).’ These extracts were supplemented with the indicated amount of rabbit reticulocyte lysate that had been primed with CPEB RNA (lanes 2 and 3). Some extracts were also supplemented with reticulocyte lysate that had not been primed with RNA (lanes 4 and 6). Labeled 84 RNA then was added to all extracts and polyadenylation was determined as described previously. The extent of polyadenylation is indicated (A,-A,,,&

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RNA-binding domain, it is tempting to speculate that orb may bind to CPE-like sequences. Interestingly, a CPE-like sequence is present in the 3’UTR of oskar RNA (Ephrussi et al., 1991). CPEB, orb, and C40Hl.l also share highly conserved spacing of cysteine and histidine residues on the carboxyterminal side of the RRMs. The coordination of a metal ion by these residues could lead to the formation of either a catalytic domain or a structure that then interacts with other proteins or with nucleic acids (Berg 1990; reviewed in Coleman 1992). A variety of RNA-binding proteins containing zinc-finger domains have been identified in Xenopus (Theunissen et al., 1992; Andreazzoli et al., 1993 and references therein) as well as other organisms (Wang et al., 1992; Brottier et al., 1992), although no close match was found between the motif described herein and those reported in the literature. Developmental Regulation and Localization of CPEB In Xenopus, cytoplasmic polyadenylation is known to occur during oocyte maturation (McGrew et al., 1989; Fox et al., 1989) and early embryonic development at least up to the midblastula stage (Simon et al., 1992). CPEB is present in embryos up to the gastrula stage, and therefore could be important for polyadenylation during these times. Indeed, B4 RNA, whose CPE is bound by CPEB, is polyadenylated in early embryos as well as in the maturing oocyte (Stebbins-Boaz and Richter, 1994). However, other RNAs that are polyadenylated during embryogenesis but not oocyte maturation, such as Cl1 and C12, contain aCPE different from that for B4 RNA (Simon et al., 1992; Simon and Richter, 1994) and are not bound by CPEB, but rather by other proteins that have been identified (Simon and Richter, 1994) and partially purified (Simon and Richter, unpublished data). Thus, the CPEB described in this study may be one of several functionally related proteins whose activity regulates cytoplasmic polyadenylation at different times of development. CPEB Mediates Cytoplasmic Polyadenylation We have shown that immunodepletion-of CPEB from egg extracts ablates polyadenylation activity. Moreover, polyadenylation activity is partially restored when the depleted extract is supplemented with a reticulocyte lysate primed with CPEB mRNA. Thus, CPEB is a critical component for cytoplasmic polyadenylation during Xenopus oocyte maturation. Why does in vitro synthesized CPEB not restore polyadenylation activityto preimmunodepletion levels? There are several possibilities. It is apparent that unprimed reticulocyte lysate is itself inhibitory to polyadenylation in extracts (Figure 7C, lane 6). This may be due to the activation of the micrococcal nuclease present in the reticulocyte lysate by the calcium in the egg extract. It is also possible that CPEB is not appropriately posttranslationally processed, folded, or both during in vitro translation. Indeed, the peptide sequence analysis suggests that metal coordination could be necessary for formation of an active protein. Attempts have been made to synthesize the functional pro-

tein in bacteria; however, addition of E. coli-expressed CPEB to depleted extract did not restore polyadenylation activity (data not shown). This result is not surprising given the high degree of insolubility of this protein when overexpressed in bacteria. Finally, CPEB could interact with another factor(s) that is essential for polyadenylation and is removed during immunodepletion (see below). Nonetheless, the data in Figure 7 show clearly that CPEB depletion destroys polyadenylation activity and that it can be at least partially restored by the subsequent addition of CPEB. The Function of CPEB in Polyadenylation CPEB is bound to RNA irrespective of whether there is active polyadenylation (Paris et al., 1991). In addition, it does not require the AAUAAA sequence to bind, although this sequence is known to be required for both nuclear and cytoplasmic polyadenylation (reviewed by Wahle and Keller 1992; Wickens 1992). Therefore, CPEB, while necessary for cytoplasmic polyadenylation, must act in concert with other proteins to form active polyadenylation complexes. Recently, Bilger et al. (1994) have shown that partially purified bovine cleavage- and polyadenylationspecificity factor (CPSF), a complex of least three proteins known to recognize AAUAAA and effect nuclear polyadenylation (Bienroth et al., 1991; Wahle and Keller, 1992), in combination with bovine poly(A) polymerase, can stimulate polyadenylation in a CPE-dependent fashion in vitro. Although the extent to which CPSF might be present in the oocyte cytoplasm is not clear, nor is whether oocyte CPSF and poly(A) polymerase would have the same activity as their bovine counterparts if assayed in concentrations similar to those present in vivo, the data of Bilger et al. (1994) conceivably complement those presented in this study. For example, CPEB could beviewed as a specificity factor that acts to recruit or stabilize factors that promote poly(A) addition, such as CPSF. Such a model could help explain why different mRNAs are polyadenylated at different times of development. That is, different CPEBs could recruit CPSF to different transcripts. Indeed, Cl1 and Cl2 RNAs, which are polyadenylated in early Xenopus embryos, contain poly(U) CPEs that are bound by different proteins than the one described here (Simon et al., 1992; Simon and Richter, 1994). In contrast, CPEB does bind to cdk2 RNA, which contains a CPE identical to that in B4 RNA and which is polyadenylated during oocyte maturation (Stebbins-Boaz and Richter, 1994). An alternative possiblity that should be considered is that CPEB interacts directly with a poly(A) polymerase, as has recently been shown to occur with the UlA protein (Gunderson et al., 1994). Although in the case of UlA this interaction prevents polyadenylation, it is possible that with CPEB the interaction is stimulatory. Irrespective of how CPEB promotes cytoplasmic polyadenylation, it is likely to do so by interacting with other proteins, which will be the focus of future investigations. Experimental

Procedures

Plasmid Construction and RNA Synthesis The plasmid used to generate 84 mRNA (~84; beginning at base 15, the sequence shown in Figure 1A contains bases 1128-1158 of the

Polyadenylation 625

during

Xenopus

Oocyte

Maturation

84 3’ UTR sequence presented in Smith et al., 1988) was created by subcloning the Hindlll-Xbal fragment of plasmid pGblB4 (Paris and Richter, 1990) into the Hindlll-Xbal site of the pSP64 poly(A) plasmid (Promega, Madison, WI). The plasmid used to generate the B4-M2 RNA (pB4-M2) was made in a similar manner; however, the HindlllXbal fragment of pGb/B4-M2 (Paris and Richter, 1990) was used. In vitro transcription (Riboprobe Gemini System, Promega) from these two plasmids linearized with Xbal generated RNAs of 44 nt. RNAs containing poly(A) tails (B4A and B4-M2A) were synthesized by linearizing the above plasmids with EcoRI. In addition to the 44 nt present in 84 and B4-M2 RNAs, B4A and B4-M2A contained 28 nt of vector sequence and 30 nt of 3’ terminal poly(A). Radiolabeled transcripts were synthesized by inclusion of [@P]UTP in the transcription reaction. Plasmid pCPEB was made by ligating the 1871 nt EcoRl fragment of the purifed hgtl0 clone (see below) into the EcoRI-digested pBluescript SK(+) vector (Stratagene, La Jolla, CA). The pGST-CPEB fusion was created by ligating the Ncol-Xhol fragment of pCPEB in frame with GST into a similarly digested pGEX-KG vector (Guan and Dixon, 1991). The pMyc-CPEB fusion was created by PCR amplification with a 5’ primer containing the Myc epitope tag (bold type; Evan et al., 1985) and the first 21 bp of the CPEB coding region [5’-GTGCTCTAGATGGAACAGAAGCTGATTAGCGAAGAAGATCTGAATATGGCCTTCCCACTGAAAGAT-31 and a 3’ primer located within the CPEB coding region, 3’ of an Nhel site. The resulting PCR fragment was then digested with Xbal and Nhel and inserted into the pCPEB vector that had been previously digested with those enzymes to remove the original Xbal-Nhel fragment. All cloned DNAs generated by P’CR were sequenced (Sequenase Version 2; United Stated Biochemical, Cleveland, OH). UV Cross-Linking Assays and Competition for Binding UV cross-linking assays were performed essentially as described by Paris et al. (1991) with the inclusion of 5 mM EDTA and 5 mglml heparin todecrease nonspecific background. Sampleswereanalyzed bySDSpolyacrylamide (10%) gel electrophoresis. Competition in the UV cross-linking assays was performed by preincubation of the reactions with a I-, IO-, or 1 OO-fold molar excess of unlabeled RNA corresponding to either 84 or B4-M2 for 15 min prior to the addition of radiolabeled 84 RNA. Preparation of Extracts and Polyadenylation Assays Oocytes from one Xenopus female were defolliculated by collagenase digestion (1.5 mglml in OR2 buffer [Hollinger and Corton, 1980]), homogenized by four strokes in a Dounce homogenizer, and then centrifuged at 10,000 rpm in a Beckman SW41 rotor for 15 min. The clear cytoplasmic supernatant, between the pigment pellet and lipid overlay, was carefully removed and frozen immediately at -8OOC. The protein concentration as determined by Bradford assay (Bradford, 1976) was 25 mglml. Small scale extracts from small numbersof oocytes, eggs, or subcellular fractions were made by adding 1 vol of 1 x XB+I (XB: 100 mM KCI, 0.1 mM CaC12, 1 mM MgCl*, IO mM K-HEPES [pH 7.61, and 50 mM sucrose; +I: 10 pglml each of leupeptin, chymostatin, and pepstatin) buffer, repeated pipeting and centrifugation (5 min, 14 K rpm, 4OC) to pellet the pigment granules. The clear cytoplasmic layer was saved for analysis. Polyadenylation competent egg extracts were made by the method described by McGrew and Richter (1990) following the procedure of Murray and Kirschner (1989). Polyadenylation assays were performed precisely as described by Paris et al. (1991). Affinity Chromatography The protocol developed here for affinity chromatography was based on the parameters detailed by Ruby et al. (1991). Large scale in vitro transcription reactions were performed with the Megascript transcription kit from Ambion (Ambion, Austin, TX) with EcoRI-linearized pB4 and pB4-M2. N6-(6-aminohexyl) ATP (GIBCO BRL, Bethesda, MD) was used in a 1:l ratio with ATP in the transcription reaction. Transcribed RNA was then biotinylated by incubation with 2 mM NHS-SS-biotin (Pierce, Rockford, IL) as described by Ruby et al. (1991). Approximately 600 pg of biotinylated RNA were incubated with 300 mg of oocyte protein extract in XBK+B buffer (XBK: XB as above except

with 50 mM KCI; +B: 5 mM EDTA, 5 mglml heparin) at a final total protein concentration of 12 mglml for 1 hr at 23OC with moderate agitation. Biotin-Sephacryl beads saturated with succinylated avidin (Vector Laboratories, San Mateo, CA) as detailed by Rubyet al. (1991), were added at a concentration of 100 pl of packed beads per 1 ml of extract/RNA solution. The beads with protein-RNA complexes were then washed four times with 10 vol of XBK+B buffer containing 0.5% Nonidet P-40 to remove nonspecifically bound proteins. Proteins that were specifically bound were eluted from the beads by washing two times with one packed bead volume of XBK+B buffer containing 0.1 mglml RNase A. Protein Sequencing, PCR, and cDNA Library Screening Eluates from affinity chromatography were electrophoresed in a 13 x 15 cm SDS-polyacrylamide (8.5%) gel for 5 hr at 35 mA. Proteins in the gel were transferred to a PVDF membrane by electroblotting with 30 V at 4OC for 16 hr. A PVDF strip containing CPEB, visualized by Ponceau S staining, was excised from the blot and processed for microsequencing in the Worcester Foundation Protein Chemistry Facility. In brief, tryptic peptide fragments of the protein (Fernandez et al., 1992) were analyzed by HPLC, and the peaks were sequenced by automated Edman degradation on an Applied Biosystems 477A Sequencer. Three peptide sequences were obtained as delineated in ,#igure 3. Sense (Is, 2s, and 3s) and antisense (la, 2a, and 3a) degenerate oligonucleotides containing inosine in the positions of maximal degeneracy were synthesized based on the peptide sequences (Figure 3, peptides 1, 2, and 3) and used in PCR. A 900 bp PCR product that wasconsistentlyamplified with primers Is and Pawas subcloned using the TA cloning kit (Invitrogen, San Diego, CA) and sequenced. This product was used to screen a hgtl0 stage VI oocyte cDNA library as described by Sambrook et al. (1989). Three of 24 primary positives were plaque purified, and their EcoRl inserts were subcloned into pBluescript SK(+) and sequenced. Protein and nucleic acid sequence management was performed with the Wisconsin Package Version 7.3 maintained by the Genetics Computer Group, Incorporated (Madison, WI). Data base comparisons were performed with the BLAST algorithm developed by the National Center for Biotechnology Information (Altschul et al., 1990). Analysis of Myc-CPEB Injected into Oocytes RNA transcribed in vitro from pMyc-CPEB linearized at the EcoRV site was injected into each of 150 manually defolliculated stage VI oocyies that were then incubated overnight at 18°C to allow translation of the injected RNA. Small scale extracts were then made as described above, and the cytoplasmic layer was used in UV cross-linking assays with 32P-labeled B4A or B4-M2A RNA. One-half of each UV cross-linked sample was then run directly, and the other one-half was used in an immunoprecipation with anti-Myc monoclonal antisera (Oncogene Sciences, Uniondale, NY) as described above. Samples were analyzed by electrophoresis in acrylamide-SDS (8.5%) gels, and the dried gel was exposed to a phosphorimager screen for 2 days. Hybridization Conditions and Northern Blot Analysis Nylon membranes (Northern blots) or nitrocellulose filters (library screening) were hybridized following the procedure of Church and Gilbert (1984). Random prime labeled (Prime-it kit: Stratagene) probe concentrations ranged from l-l 0 x 1 O7 cpmlml with a specific activity of l-10 x 10’ cpmlbg. Washed membranes were exposed to Kodak XAR film for l-7 days. RNA for Northern blot analysis was extracted using the RNASTAT60 reagent and following the instructions of the manufacturer (Tel-Test “B,” Incorporated, Friendswood, TX). Each RNA (10 pg) was electrophoresed in 1% agarosegels, blotted to nylon membrane, and hybridized as described above. Western Blot Analysis, Immunoprecipitation, and lmmunodepletion Western blot analysis of proteins was performed essentially as described by Harlow and Lane (1988) with polyclonal rabbit anti-CPEB. The anti-CPEB antibodies were raised by injection of GST-CPEB fusion protein (GST gene fusion system; Pharmacia, Piscataway, NJ) into rabbits (East-Acres Biologicals, Southbridge, MA). Goat anti-rabbit

Cell 626

HRP was used as the secondary antibody and was visualized by enhanced chemikrminescence (Renaissence; DuPont New England Nuclear, Boston, MA). lmmunoprecipitations were performed with antibodies coupled to protein A-Sephacryl beads with DMP (Harlow and Lane 1988) using the NET-gel buffer procedure as described (Sambrook et al., 1989). For immunodepletions, 1 vol of egg extract was added to one packed bead volume of protein A-Sepharose beads with coupled IgG and incubated with moderate agitation for 30 min at 4OC. The beads were then centrifuged, and the supernatant was transferred to another aliquot of packed beads. This was similarly incubated. The final depleted supernatant was then held on ice until use. IgG coupled to beads included anti-CPEB, preimmune sera, and nonspecific goat antimouse total IgG. Expression of CPEB In Vitro In vitro transcribed CPEB mRNA was translated in nuclease-treated reticulocye lysates (Promega) for 3-4 hr at 30%. The concentration of translated product was determined by Western blot analysis with anti-CPEB as described above and comparison with known concentrations of the bacterially expressed protein. In Vivo Labeling of CPEB with Orthophosphate and Polyadenylation Stage VI oocytes from one mature female Xenopus were incubated overnight in 1 x OR2 buffer containing 100 uCi/ml of [32P]orthophosphate and 50 pglml streptomyocin. The next morning, progesterone was added to induce maturation. Oocytes were removed from incubation at 0, 1, 2, 3, 4, and 6 hr, rinsed two times with Ix OR2 and frozen on dry ice. After all samples had been collected, small scale cytoplasmic extracts were made. Endogenous CPEB was immunoprecipitated from the clear cytoplasmic layer. lmmunoprecipitated complexes on protein A beads were washed four times with 10 vol of 1 x NET buffer, 2 x SDS sample buffer was added, and the samples were analyzed by SDS-PAGE. Oocytes from the same female as above were incubated in parallel, but without 32P in the medium. In vitro transcribed3*P-labeled 84 RNA (-20 nl of a 2 mglml solution) was injected into each of 100 oocytes. Following the addition of progesterone, samples were taken in parallel with the orthophosphate-labeled oocytes and were similarly stored on dry ice. RNA from each set of six oocytes by extraction with p-aminosalicylic acid (PAS), SDS, and phenol/chloroform (Kirby 1965) and analyzed by electrophoresis on 8% acrylamide-urea (6 M) gels. Acknowledgments We thank Dr. John Leszyk for amino acid sequencing and the W. M. Keck Foundation for support of the Worcester Foundation Protein Chemistry Facility. We also thank Dr. Jack Dixon for the pGST-KG vector. We are grateful to Drs. Acacia Alcivar-Warren, Barbara Stebbins-Boaz, and Fatima Gebauer for critical reading of the manuscript. This work was supported by grants from the National Institutes of Health. L. E. H. wassupported by a National Institutesof Health institutional postdoctoral training grant and a National Research Service Award postdoctoral fellowship. Received

July 8, 1994; revised

September

9, 1994.

Bilger, A., Fox, C. A., Wahle, E., and Wickens, M. (1994). Nuclear polyadenylation factors recognize cytoplasmic polyadenylation elements Genes Dev. 8, 1106-l 116. Bouvet, P., Omilli, F.,Arlot-Bonnemains. Y., Legagneux, V., Roghi, C., Bassez, T., and Osborne, H. 8. (1994). The deadenylation conferred by the 3’ untranslated region of a developmentally controlled mRNA in Xenopus embryos is switched to polyadenylation by deletion of a short sequence element. Mol. Cell. Biol. 74, 1893-1900. Bradford, M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254. Brottier, P., Nandi, P., Bremont, M., and Cohen, J. (1992). Bovine rotavirus segment 5 protein expressed in the baculovirus system interacts with zinc and RNA. J. Gen. Virol. 73, 1931-1938. Christersen, L. B., and McKearin, D. M. (1994). orb is required for anteroposterior and dorsoventral patterning during Drosophila oogenesis. Genes Dev. 8, 614-628. Church, G. M., and Gilbert, W. (1984). Natl. Acad. Sci. USA 87, 1991-1995.

Genomic

sequencing.

Proc.

Coleman, J. E. (1992). Zinc proteins: enzymes, storage proteins, transcription factors and replication proteins. Annu. Rev. Biochem. 67, 897-946. Dworkin, M. B., Shrutkowski, A., and Dworkin-Rastl, E. (1985). Mobilization of specific maternal RNA species into polysomes after fertilization in Xenopus laevis. Proc. Natl. Acad. Sci. USA 82, 7636-7640. Ephrussi, A., Dickinson, L. K., and Lehmann, R. (1991). oskar organizes the germ plasm and directs localization of the posterior determinant nanos. Cell 66, 37-50. Evan, G. I., Lewis, G. K., Ramsay, G., and Bishop, J. M. (1985). Isolation of monoclonal antibodies specific for human c-myc protooncogene product. Mol. Cell. Biol. 5, 3610-3616. Fernandez J., DeMott, M., Atherton, D., Mische, S. M. (1992). Internal protein sequence analysis: enzymatic digestion for less than 1Oug of protein bound to polyvinylidene diflouride or nitrocellulose membranes Anal. Biochem. 207, 255-284. Fox, C. A., Sheets, M. D., and Wickens, M. (1989). Poly(A) addition during maturation of frog oocytes: distinct nuclear and cytoplasmic activities and regulation by the sequence UUUUUAU. Genes Dev. 3, 2151-2162. Fox, C. A., Sheets, M. D., Wahle, E., and Wickens, M. (1992). Polyadenylation of maternal mRNA during oocyte maturation: poly(A) addition in vitro requires a regulated RNA binding activity and a poly(A) polymerase. EMBO J. 77, 5021-5032. Gebauer, F., Xu, W., Cooper, G. M., and Richter, J. D. (1994). Translational control by cytoplasmic polyadenylation of c-mos mRNA is necessary for oocyte maturation in the mouse. EMBO J., in press. Guan, K., and Dixon, J. E. (1991). Eukaryotic proteins expressed in Eschericbia co/i: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. Anal. Biothem. 792,262-267. Gunderson, S. I., Beyer, K., Martin, G., Keller, W., Boelens, W. C., and Mattaj, I. W. (1994). The human UlA snRNP protein regulates polyadenylation via a direct interaction with poly(A) polymerase. Cell 76,531-541.

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Accession

The GenBank accession this paper is U14169.

Number number

for the CPEB sequence

reported

in