Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, MA 01605, USA
*Author for correspondence (e-mail: joel.richter{at}umassmed.edu)
Accepted April 30, 2001
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SUMMARY |
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Key words: Polyadenylation, Oocyte maturation, CPEB, Mouse, IAK1
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INTRODUCTION |
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While the core features of cytoplasmic polyadenylation have been elucidated, they do not indicate a priori how polyadenylation stimulates translation. The mechanism of translational activation was suggested by initial studies demonstrating that the CPE, which is necessary for polyadenylation during maturation, also mediates translational repression (masking) prior to maturation (de Moor and Richter, 1999). The studies implied that CPEB, the only factor identified that could bind the CPE, activated and repressed translation. How CPEB could act both positively and negatively was indicated when maskin, a CPEB-interacting factor, was identified (Stebbins-Boaz et al., 1999). In oocytes, maskin not only binds CPEB, but also the cap-binding protein eIF4E. This configuration of factors excludes the interaction between eIF4E and eIF4G, which is essential for cap-dependent translation (for a discussion of initiation factors, see) (Gingras et al., 1999). During oocyte maturation at a time commensurate with polyadenylation, the interaction between maskin and eIF4E is disrupted, which presumably then allows eIF4G to bind eIF4E and promote initiation (Stebbins-Boaz et al., 1999). It seems plausible that polyadenylation could be involved in the dissociation of maskin from eIF4E. It should also be borne in mind that polyadenylation might also stimulate translation in a maskin-independent manner, for example, by inducing cap-specific 2'-o-methylation (Kuge and Richter, 1995).
Cytoplasmic polyadenylation also takes place in maturing mouse oocytes, where at least some of the key features are similar to those described in Xenopus. For example, the CPE and AAUAAA drive polyadenylation-induced translation (Vassalli et al., 1989; Huarte et al., 1992, Gebauer et al., 1994; Tay et al., 2000; Oh et al., 2000). In addition, the CPE (also referred to as the ACE, or adenylation control element) also represses translation before maturation (Stutz et al., 1997; Stutz et al., 1998; Tay et al., 2000), as is the case in Xenopus. However, aside from the fact that mouse oocytes contain CPEB (Gebauer and Richter, 1996; Tay et al., 2000), and that polyadenylation is important for meiotic progression (Gebauer et al., 1994; Tay et al., 2000), little is known about the biochemical features of the process in these species.
In this study, we have analyzed the molecules that mediate cytoplasmic polyadenylation in maturing mouse oocytes. All the factors that are involved in this process in Xenopus (CPEB, CPSF, PAP, maskin, IAK1/Eg2) are also present in the cytoplasm of mouse oocytes. After the induction of oocyte maturation, a kinase becomes active that phosphorylates CPEB Ser174, an event that is crucial for cytoplasmic polyadenylation. The injection of a peptide that is known to block the activity of IAK1/Eg2 impedes meiotic progression. Furthermore, the injection of an mRNA encoding a truncated form of CPEB, which acts as a dominant negative mutation because it cannot be phosphorylated by IAK1/Eg2, inhibits cytoplasmic polyadenylation. These data indicate that IAK1/Eg2 and CPEB are essential components for cytoplasmic polyadenylation in mouse oocytes.
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MATERIALS AND METHODS |
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Analysis of polyadenylation
[32P]UTP-labeled murine cyclin 3' UTR (plus or minus the CPE), radioinert Xenopus full-length wild-type CPEB and N CPEB, which lacks residues 1-146 (Mendez et al., 2000a), were synthesized in vitro by T3 or T7 RNA polymerase. Depending on the experiment, the labeled RNA (
6x106 cpm/µl) alone was injected into GV stage oocytes, or was mixed with the unlabeled RNAs (0.7 mg/ml, equal volumes) before injection (
10 pl per injection). The oocytes were cultured in the presence of isobutylmethylxanthine (IBMX) for 6 hours, after which time they were washed in IBMX-free medium and cultured for an additional 16-17 hours. RNA was then extracted from the oocytes according to Tay et al. (Tay et al., 2000), and the RNA was resolved on a 6% polyacrylamide gel containing 8 M urea and phosphorimaged.
Analysis of polyadenylation factors
Whole-mount immunohistochemistry was performed as described by Messinger and Albertini (Messinger and Albertini, 1991). Briefly, oocytes were fixed in 100 mM Pipes, 5 mM MgCl, 2.5 mM EGTA, 0.1% aprotinin, 1 mM DTT, 50% D2O, 1 µM taxol, 0.1% Triton X-100 and 2% formaldehyde for 20 minutes at 37°C, and then blocked in phosphate-buffered saline containing 2% BSA, 2% powdered milk, 2% goat serum and 0.1% Triton X-100 for 30 minutes at room temperature. The antibodies (1:100 dilution) directed against murine CPEB (Tay et al., 2000), Xenopus maskin (Stebbins-Boaz et al., 1999), CPSF100 (Mendez et al., 2000b), IAK1 (Transduction Laboratories) and Xenopus PAP (Gebauer and Richter, 1995) were added to the blocking buffer and the oocytes were incubated at 37°C for 1 hour with gentle rotation. After washing with the blocking solution, the secondary fluorophore-labeled antibody (1:1000), which was either Alexa 488 goat anti-mouse IgG or Alexa 594 goat anti-rabbit IgG (Molecular probes) was added, again in blocking buffer. The oocytes were also simultaneously stained with DAPI. Western blots were probed with the antibodies noted above; for CPEB, 110 GV oocytes were analyzed, 250 oocytes for maskin, 535 oocytes for CPSF100, 180 oocytes for IAK1 and 200 oocytes for poly(A) polymerase.
Analysis of CPEB phosphorylation
Peptides spanning a region from murine CPEB that contains the LDS/TR motif that is recognized by IAK1/Eg2 (RGSRLDTRPILDSRSSC), or a mutant sequence with alanine for serine substitutions (RGSRLDARPILDARSSC), which cannot be phosphorylated by this kinase (Mendez et al., 2000a) were coupled to maleimide-activated ovalbumin (Pierce). The conjugates were added to extracts prepared as follows. 50 GV, GVBD (MI) or MII stage oocytes were frozen and thawed three times in 4.25 µl of buffer (Hampl and Eppig, 1995a) that contained 125 mM Mops, pH 7.2, 300 mM ß-glycerophosphate, 2 mg p-nitrophenylphosphate, 7.5 mM EGTA, 0.5 mM Na3VO4, 75 mM MgCl, 5 mM dithiothreitol, 10 µg/ml each of aprotinin, soybean trypsin inhibitor, pepstatin A and leupeptin, and 25 µg BSA). After brief centrifugation, 10 µl of wild-type or mutant peptide conjugated to ovalbumin was to the supernatant, together with 1 µCi [-32P]ATP 1 mM phenylmethylsulfonyl fluoride, and 0.025 µg protein kinase A inhibitor (final reaction volume of 30 µl). The solution was incubated for 1 hr at 30°C and then analyzed by 12% PAGE.
For the phosphopeptide mapping, purified baculovirus-expressed Xenopus CPEB was added to extracts made as described above, but in this case from 600 MI stage oocytes. The entire extract was then resolved by 8% SDS PAGE and CPEB was electroblotted onto nylon membrane and digested with trypsin. The 2D phosphopeptide mapping procedure has been described (Boyle et al., 1991; Mendez et al., 2000a). In addition, CPEB was phosphorylated in vitro with purified baculovirus expressed Xenopus Eg2, and the phosphopeptide map was similarly derived.
Some oocytes were injected with ovalbumin-coupled wild-type and mutant peptides (as noted above). In this case, the peptides were brought up to a concentration of 5 mg/ml, and
10 pl was injected per oocyte. The oocytes were incubated for up to 10 hours, and were scored for the presence or absence of the first polar body.
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RESULTS |
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In order to define more precisely the target sites on CPEB for the MI kinase activity noted above, 2D phosphopeptide mapping was performed. In this case, the substrate was purified recombinant Xenopus CPEB, because mouse CPEB is insoluble after bacterial expression. At the GV stage, there were relatively few sites of CPEB phosphorylation, and even these were close to background levels (Fig. 3D). At MI, however, several sites were phosphorylated, including one that corresponded to the main LDS/TR phosphorylation site, which is clearly shown by the position of this spot compared with the position of the main site of CPEB phosphorylation when purified baculovirus expressed Eg2 was used as the kinase source (Fig. 3, arrows). To confirm that this phosphopeptide is indeed the same one as that detected when recombinant Eg2 was the kinase source, aliquots of the two reactions were mixed before the 2D mapping (Fig. 3D). Co-migration of the two spots demonstrates that CPEB is in fact phosphorylated on Ser174 by a mouse oocyte kinase. Therefore, mouse oocytes contain a developmentally regulated kinase activity that closely matches that predicted for IAK1/Eg2 in which CPEB is phosphorylated on the critical residue that is necessary for cytoplasmic polyadenylation. In this regard, it is worth noting that in Xenopus oocytes, Eg2 is the only kinase that phosphorylates CPEB Ser 174.
An IAK1/Eg2 blocking peptide inhibits polar body formation
We next examined whether the IAK1/Eg2 kinase activity is involved in oocyte maturation in the mouse. GV stage oocytes were injected with a CPEB-derived peptide (Fig. 3B) that specifically blocks Eg2 kinase activity and thereby inhibits Xenopus oocyte maturation (Mendez et al., 2000a). Fig. 4 shows that this peptide only minimally slowed the rate of first polar body formation compared with uninjected controls. In Xenopus, this wild-type peptide also only slowed, but did not block, meiotic progression (Mendez et al., 2000a). However, in Xenopus, the mutant alanine-containing peptide (Fig. 3B) dramatically inhibited maturation in injected oocytes. The interpretation of these results is that while Eg2 binds both the wild-type and mutant peptides, once the wild-type peptide is phosphorylated, these molecules then dissociate and the kinase is free to phosphorylate another substrate peptide (i.e. the wild-type peptide is a competitive inhibitor). However, because Eg2 cannot phosphorylate the mutant peptide, the molecules remain attached, thereby irreversibly lowering the effective concentration of the kinase (i.e. the mutant peptide is a dominant inhibitor). Therefore, the mutant peptide would be expected to be the most efficacious in abrogating Eg2-dependent meiotic progression, which is the case observed in injected mouse oocytes (Fig. 4).
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DISCUSSION |
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CPEB functions during two distinct phases of meiosis
Although mouse oocytes do not require protein synthesis to enter or complete maturation, polyadenylation-induced translation potentiates the rate of this process. In maturing mouse oocytes, there are two phases of M-phase promoting factor (MPF, a heterodimer of cdc2 and cyclin B) activity that drive the MI and MII transitions. The increase in this activity is regulated at the translational level (Hampl and Eppig, 1995b), which is, at least in part, due to the cytoplasmic polyadenylation of cyclin B1 mRNA (Tay et al., 2000). The findings presented in this study demonstrate that CPEB is responsible for the translation of this mRNA. Another mRNA whose polyadenylation-induced translation is essential for proper meiotic progress encodes Mos, a serine/threonine kinase. In mouse oocytes, Mos is a component of cytostatic factor (CSF), a protein complex that arrests meiosis at MII. The destruction of CSF activity by disruption of the Mos gene results in a high incidence of parthenogenetic activation. In these cases, the now mitotically dividing oocytes occasionally fail to be ovulated and give rise to ovarian cysts and tumors (Colledge et al., 1994; Hashimoto et al., 1994; Choi et al., 1996). While mouse oocytes contain some Mos protein, they also contain dormant CPE-containing Mos mRNA that, upon the induction of maturation, undergoes polyadenylation-induced translation. The destruction of Mos mRNA (OKeefe et al., 1989) or the abrogation of Mos mRNA polyadenylation (Gebauer et al., 1994) prevents Mos synthesis, inhibits proper MII arrest and can occasionally lead to parthenogenesis. Therefore, the translational activation of Mos mRNA, by CPEB-mediated polyadenylation, is necessary for the late phase of oocyte meiosis.
CPEB function is also crucial at the pachytene stage of prophase I. CPEB-deficient female mice are sterile because they lack ovaries altogether, or contain only malformed ovaries. Late stage embryos (18.5 dpc) contain ovaries, but they are devoid of oocytes. Earlier embryonic ovaries (16.5 dpc) do contain oocytes, but their chromatin is fragmented and/or dispersed, suggesting a defect in synapsis or chromosome pairing. While such oocytes contain wild-type levels of mRNAs that encode two key components of the synaptonemal complex (synaptonemal complex proteins 1 and 3), they are not translated. These observations, plus the fact that these mRNAs contain CPEs in their 3' UTRs that are bound by CPEB both in vitro and in vivo, demonstrate that the regulated translation of synaptonemal complex protein mRNAs by CPEB is essential for progression through first meiotic prophase (J. T. and J. D. R., unpublished).
Oh et al. have shown that cytoplasmic polyadenylation of CPE-containing mRNAs occurs after fertilization in the mouse, which suggests that maternally inherited sequences may play an important, but heretofore unknown role in the embryo of this species (Oh et al., 2000; see also Tong et al., 2000). However, whether embryonic polyadenylation is CPEB dependent is unclear because Tay et al. have been unable to detect this protein in the fertilized egg (Tay et al., 2000). Furthermore, if only a small amount of CPEB remains stable after fertilization (e.g. 10%), as is the case in Xenopus (Groisman et al., 2000), then it would not necessarily have been detected, and thus could promote cytoplasmic polyadenylation in the embryo (see below). Clearly, additional experiments are required to determine whether CPEB is involved embryonic polyadenylation.
A conserved signaling pathway promotes oocyte maturation
In Xenopus oocytes exposed to progesterone, the natural inducer of maturation, a transient but essential decrease in cAMP is followed by the activation of Eg2 (Andresson and Ruderman, 1998). Decreased cAMP is also necessary for mouse oocyte maturation, which in this case is followed by the activation of IAK1. It seems likely that IAK1/Eg2 is itself activated by phosphorylation, and the kinase responsible is almost certainly the same in mouse and Xenopus. IAK1/Eg2 then becomes localized at spindles and centrosomes, where it may be involved in the assembly of these structures (Nigg, 2001). However, as noted above, no CPEB or maskin can be detected after fertilization. Therefore, while the signaling events leading to translational activation during oocyte maturation are conserved among vertebrates, the events that control translation after fertilization may be quite different, and could reflect the relative importance of maternally inherited mRNA.
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ACKNOWLEDGMENTS |
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