Meiotic maturation of mouse oocytes triggers the translation and polyadenylation of dormant tissue-type plasminogen activator mRNA

The serine protease tissue-type plasminogen activator (t-PA) is synthesized by murine oocytes undergoing meiotic maturation, but not by arrested primary oocytes. Dormant, stable t-PA mRNA accumulates during oocyte growth, so that fully grown, arrested primary oocytes contain in their cytoplasm approximately 10,000 copies of this molecule. Translation of t-PA mRNA is triggered upon resumption of meiosis and is accompanied by a progressive and concerted increase in its size. This structural change can be accounted for by increased polyadenylation at the 3' end of the molecule. Following its translation, t-PA mRNA is degraded; it is no longer detectable in fertilized eggs. The identification of a dormant mRNA in murine oocytes and the demonstration that its translational activation is accompanied by elongation of its poly(A) tail may provide insights into the control of gene expression during meiotic maturation and early mammalian development.     

Regulated Pumilio-2 binding controls RINGO/Spy mRNA translation and CPEB activation

CPEB is a sequence-specific RNA-binding protein that controls the polyadenylation-induced translation of mos and cyclin B1 mRNAs in maturing Xenopus oocytes. CPEB activity requires not only the phosphorylation of S174, but also the synthesis of a heretofore-unknown upstream effector molecule. We show that the synthesis of RINGO/Spy, an atypical activator of cyclin-dependent kinases (cdks), is necessary for CPEB-directed polyadenylation. Deletion analysis and mRNA reporter assays show that a cis element in the RINGO/Spy 3'UTR is necessary for translational repression in immature (G2-arrested) oocytes. The repression is mediated by 3'UTR Pumilio-Binding Elements (PBEs), and by its binding protein Pumilio 2 (Pum2). Pum2 also interacts with the Xenopus homolog of human Deleted for Azoospermia-like (DAZL) and the embryonic poly(A)-binding protein (ePAB). Following the induction of maturation, Pum2 dissociates not only from RINGO/Spy mRNA, but from XDAZL and ePAB as well; as a consequence, RINGO/Spy mRNA is translated. These results demonstrate that a reversible Pum2 interaction controls RINGO/Spy mRNA translation and, as a result, CPEB-mediated cytoplasmic polyadenylation.     

Isolation of novel murine maternal mRNAs regulated by cytoplasmic polyadenylation.

The cytoplasmic polyadenylation element (CPE) is an AU-rich sequence in the 3'-untranslated region of many stored maternal mRNAs. The CPE directs the meiotic maturation-specific cytoplasmic polyadenylation and translational activation of these dormant mRNAs in Xenopus. The work presented here demonstrates that the CPE controls a similar regulation in mouse oocytes and utilizes the information to isolate novel maternal mRNAs by polymerase chain reaction (PCR). A degenerate CPE primer was used in an anchored PCR reaction with cDNAs from primary mouse oocytes. Clones were identified that contained the canonical polyadenylation signal AATAAA. A novel PCR test was then used to determine the polyadenylation state of the respective mRNAs before and after meiotic maturation. Two mRNAs, OM-1 and OM-2, are cytoplasmically polyadenylated upon maturation. Another mRNA is not polyadenylated during maturation, although it contains multiple CPE-like elements, indicating that this sequence element is not sufficient for adenylation during this time. Microinjection into primary oocytes of antisense oligodeoxynucleotides directed against OM-1 destroys the mRNA but does not appear to interfere with maturation in vitro. These experiments identify two novel maternal mRNAs and establish a simple strategy for isolating other maternal messages that control meiotic maturation, fertilization, and early mouse development.     

RINGO/cdk1 and CPEB mediate poly(A) tail stabilization and translational regulation by ePAB

One activity that controls mRNA translation in vertebrate oocytes, embryos, and neurons is cytoplasmic polyadenylation. In Xenopus oocytes, where much of the biochemistry of this process has been elucidated, nuclear pre-mRNAs containing a cytoplasmic polyadenylation element (CPE) in their 3' untranslated regions (UTRs) have long poly(A) tails; once the RNAs are spliced and transported to the cytoplasm, the tails are shortened. Following the resumption of meiosis, the poly(A) tails are lengthened and translation ensues. CPEB is a sequence-specific RNA-binding protein that coordinates these events and does so by binding to the CPE as well as several factors including Gld2, a poly(A) polymerase, and PARN [poly(A)-specific ribonuclease], a deadenylase. Here, we show that ePAB, embryonic poly(A)-binding protein, transiently associates with the polyadenylation complex; it initially interacts with CPEB, but after polyadenylation, it binds the poly(A) tail. ePAB dissociation from CPEB is regulated by RINGO (Rapid Inducer of G(2)/M progression in Oocytes), a cyclin B1-like cofactor that activates cdk1, a protein kinase that phosphorylates CPEB. Subsequent ePAB binding to the poly(A) tail is necessary to protect the homopolymer from degradation by deadenylating enzymes. Poly(A)-bound ePAB also interacts with eIF4G, which instigates translation initiation of CPEB-bound mRNAs.     

Progesterone and insulin stimulation of CPEB-dependent polyadenylation is regulated by Aurora A and glycogen synthase kinase-3

Progesterone stimulation of Xenopus oocyte maturation requires the cytoplasmic polyadenylation-induced translation of mos and cyclin B mRNAs. One cis element that drives polyadenylation is the CPE, which is bound by the protein CPEB. Polyadenylation is stimulated by Aurora A (Eg2)-catalyzed CPEB serine 174 phosphorylation, which occurs soon after oocytes are exposed to progesterone. Here, we show that insulin also stimulates Aurora A-catalyzed CPEB S174 phosphorylation, cytoplasmic polyadenylation, translation, and oocyte maturation. However, these insulin-induced events are uniquely controlled by PI3 kinase and PKC-zeta, which act upstream of Aurora A. The intersection of the progesterone and insulin signaling pathways occurs at glycogen synthase kinase 3 (GSK-3), which regulates the activity of Aurora A. GSK-3 and Aurora A interact in vivo, and overexpressed GSK-3 inhibits Aurora A-catalyzed CPEB phosphorylation. In vitro, GSK-3 phosphorylates Aurora A on S290/291, the result of which is an autophosphorylation of serine 349. GSK-3 phosphorylated Aurora A, or Aurora A proteins with S290/291D or S349D mutations, have reduced or no capacity to phosphorylate CPEB. Conversely, Aurora A proteins with S290/291A or S349A mutations are constitutively active. These results suggest that the progesterone and insulin stimulate maturation by inhibiting GSK-3, which allows Aurora A activation and CPEB-mediated translation.     

Symplekin and multiple other polyadenylation factors participate in 3'-end maturation of histone mRNAs

Most metazoan messenger RNAs encoding histones are cleaved, but not polyadenylated at their 3' ends. Processing in mammalian cell extracts requires the U7 small nuclear ribonucleoprotein (U7 snRNP) and an unidentified heat-labile factor (HLF). We describe the identification of a heat-sensitive protein complex whose integrity is required for histone pre-mRNA cleavage. It includes all five subunits of the cleavage and polyadenylation specificity factor (CPSF), two subunits of the cleavage stimulation factor (CstF), and symplekin. Reconstitution experiments reveal that symplekin, previously shown to be necessary for cytoplasmic poly(A) tail elongation and translational activation of mRNAs during Xenopus oocyte maturation, is the essential heat-labile component. Thus, a common molecular machinery contributes to the nuclear maturation of mRNAs both lacking and possessing poly(A), as well as to cytoplasmic poly(A) tail elongation.     

Expression in in-vivo and in-vitro growing and maturing oocytes: focus on regulation of expression at the translational level

Studies of expression in in-vivo and in-vitro maturing oocytes have the potential to elucidate signalling pathways involved in the intricate crosstalk between the oocyte and its somatic compartment during differentiation and morphogenetic processes, and the origin of disturbances in oocyte maturation possibly involved in reduced fertility. This review summarizes data on expression studies with focus on regulation of expression at the translational level in the maturing oocyte. The regulation of gene expression at the translational level as analysed in in-vitro maturing oocytes is complex and highly conserved between different species. It is characterized by differential degradation, and by storage and recruitment of distinct maternal mRNAs involving conserved consensus sequences in the 5' or 3' untranslated regions (UTRs) of mRNAs. Proteins interacting with such sequences affect the temporal 3' polyadenylation, and bring the 5' and 3' UTRs of mRNAs into close proximity for efficient initiation of translation. Post-translational modifications of mRNA-associated proteins contribute to maturation- and developmentally controlled and to cell cycle-dependent expression. New methodologies for analysis of ovary-specific gene expression and function of genes in oogenesis are also reviewed, e.g. RT-PCR, SAGE-PCR, real-time rapid cycle fluorescence monitored RT-PCR, differential display techniques, and microinjection of anti-sense RNA, double-stranded RNAs or mRNAs expressing green fluorescent protein-tagged proteins into maturing oocytes.     

Maturation-specific polyadenylation: in vitro activation by p34cdc2 and phosphorylation of a 58-kD CPE-binding protein.

During Xenopus oocyte maturation, poly(A) elongation controls the translational recruitment of specific mRNAs that possess a CPE (cytoplasmic polyadenylation element). To investigate the activation of polyadenylation, we have employed oocyte extracts that are not normally competent for polyadenylation. Addition of cell lysates containing baculovirus-expressed cyclin to these extracts induces the polyadenylation of exogenous B4 RNA. The involvement of p34cdc2 kinase in cyclin-mediated polyadenylation was demonstrated by p13-Sepharose depletion; removal of the kinase from oocyte extracts with this affinity matrix abolishes polyadenylation activation. Reintroduction of cell lysates containing baculovirus-expressed p34cdc2, however, completely restores this activity. To identify factors of the polyadenylation apparatus that might be responsible for the activation, we employed UV cross-linking and identified a 58-kD protein that binds the B4 CPE in oocyte extracts. In polyadenylation-proficient egg extracts, this protein has a slower electrophoretic mobility, which suggests a post-translational modification. A similar size shift of the protein is evident in oocyte extracts supplemented with lysates containing baculovirus-expressed cyclin and p34cdc2. This size shift, which is reversed by treatment with acid phosphatase, coincides temporally with cyclin-induced polyadenylation activation. We propose that p34cdc2 kinase activity leads to the phosphorylation of the 58-kD CPE-binding protein and that this event is crucial for the cytoplasmic polyadenylation that occurs during oocyte maturation.     

A novel p34(cdc2)-binding and activating protein that is necessary and sufficient to trigger G(2)/M progression in Xenopus oocytes.

The activation of maturation-promoting factor (MPF) is required for G(2)/M progression in eukaryotic cells. Xenopus oocytes are arrested in G(2) and are induced to enter M phase of meiosis by progesterone stimulation. This process is known as meiotic maturation and requires the translation of specific maternal mRNAs stored in the oocytes. We have used an expression cloning strategy to functionally identify proteins involved in G(2)/M progression in Xenopus oocytes. Here we report the cloning of two novel cDNAs that when expressed in oocytes induce meiotic maturation efficiently. The two cDNAs encode proteins of 33 kD that are 88% identical and have no significant homologies to other sequences in databases. These proteins, which we refer to as p33(ringo) (rapid inducer of G(2)/M progression in oocytes), induce very rapid MPF activation in cycloheximide-treated oocytes. Conversely, ablation of endogenous p33(ringo) mRNAs using antisense oligonucleotides inhibits progesterone-induced maturation, suggesting that synthesis of p33(ringo) is required for this process. We also show that p33(ringo) binds to and activates the kinase activity of p34(cdc2) but does not associate with p34(cdc2)/cyclin B complexes. Our results identify a novel p34(cdc2) binding and activating protein that regulates the G(2)/M transition during oocyte maturation.     

Mammalian poly(A)-binding protein is a eukaryotic translation initiation factor, which acts via multiple mechanisms

Translation initiation is a multistep process involving several canonical translation factors, which assemble at the 5'-end of the mRNA to promote the recruitment of the ribosome. Although the 3' poly(A) tail of eukaryotic mRNAs and its major bound protein, the poly(A)-binding protein (PABP), have been studied extensively, their mechanism of action in translation is not well understood and is confounded by differences between in vivo and in vitro systems. Here, we provide direct evidence for the involvement of PABP in key steps of the translation initiation pathway. Using a new technique to deplete PABP from mammalian cell extracts, we show that extracts lacking PABP exhibit dramatically reduced rates of translation, reduced efficiency of 48S and 80S ribosome initiation complex formation, and impaired interaction of eIF4E with the mRNA cap structure. Supplementing PABP-depleted extracts with wild-type PABP completely rectified these deficiencies, whereas a mutant of PABP, M161A, which is incapable of interacting with eIF4G, failed to restore translation. In addition, a stronger inhibition (approximately twofold) of 80S as compared to 48S ribosome complex formation (approximately 65% vs. approximately 35%, respectively) by PABP depletion suggests that PABP plays a direct role in 60S subunit joining. PABP can thus be considered a canonical translation initiation factor, integral to initiation complex formation at the 5'-end of mRNA.