Cap ribose methylation of c-mos mRNA stimulates translation and oocyte maturation in Xenopus laevis.

In Xenopus oocytes, progesterone stimulates the cytoplasmic polyadenylation and resulting translational activation of c-mos mRNA, which is necessary for the induction of oocyte maturation. Although details of the biochemistry of polyadenylation are beginning to emerge, the mechanism by which 3' poly(A) addition stimulates translation initiation is enigmatic. A previous report showed that polyadenylation induced cap-specific 2'-O-methylation, and suggested that this 5' end modification was important for translational activation. Here, we demonstrate that injected c-mos RNA undergoes polyadenylation and cap ribose methylation. Inhibition of this methylation by S-isobutylthioadenosine (SIBA), a methyltransferase inhibitor, has little effect on progesterone-induced c-mos mRNA polyadenylation or general protein synthesis, but prevents the synthesis of Mos protein as well as oocyte maturation. Maturation can be rescued, however, by the injection of factors that act downstream of Mos, such as cyclin A and B mRNAs. Most importantly, we show that the translational efficiency of injected mRNAs containing cap-specific 2'-O-methylation (cap I) is significantly enhanced compared to RNAs that do not contain the methylated ribose (cap 0). These results suggest that cap ribose methylation of c-mos mRNA is important for translational recruitment and for the progression of oocytes through meiosis.     

Translational control of cyclin B1 mRNA during meiotic maturation: coordinated repression and cytoplasmic polyadenylation

Translational control is prominent during meiotic maturation and early development. In this report, we investigate a mode of translational repression in Xenopus laevis oocytes, focusing on the mRNA encoding cyclin B1. Translation of cyclin B1 mRNA is relatively inactive in the oocyte and increases dramatically during meiotic maturation. We show, by injection of synthetic mRNAs, that the cis-acting sequences responsible for repression of cyclin B1 mRNA reside within its 3'UTR. Repression can be saturated by increasing the concentration of reporter mRNA injected, suggesting that the cyclin B1 3'UTR sequences provide a binding site for a trans-acting repressor. The sequences that direct repression overlap and include cytoplasmic polyadenylation elements (CPEs), sequences known to promote cytoplasmic polyadenylation. However, the presence of a CPE per se appears insufficient to cause repression, as other mRNAs that contain CPEs are not translationally repressed. We demonstrate that relief of repression and cytoplasmic polyadenylation are intimately linked. Repressing elements do not override the stimulatory effect of a long poly(A) tail, and polyadenylation of cyclin B1 mRNA is required for its translational recruitment. Our results suggest that translational recruitment of endogenous cyclin B1 mRNA is a collaborative effect of derepression and poly(A) addition. We discuss several molecular mechanisms that might underlie this collaboration.     

Translational control by cytoplasmic polyadenylation of c-mos mRNA is necessary for oocyte maturation in the mouse.

The c-mos proto-oncogene product is a key element in the cascade of events leading to meiotic maturation of vertebrate oocytes. We have investigated the role of cytoplasmic polyadenylation in the translational control of mouse c-mos mRNA and its contribution to meiosis. Using an RNase protection assay we show that optimal cytoplasmic polyadenylation of c-mos mRNA requires three cis elements in the 3' UTR: the polyadenylation hexanucleotide AAUAAA and two U-rich cytoplasmic polyadenylation elements (CPEs) located 4 and 51 nucleotides upstream of the hexanucleotide. When fused to CAT coding sequences, the wild-type 3' UTR of c-mos mRNA, but not a 3' UTR containing mutations in both CPEs, confers translational recruitment during maturation. This recruitment coincides with maximum polyadenylation. To assess whether c-mos mRNA polyadenylation is necessary for maturation of mouse oocytes, we have ablated endogenous c-mos mRNA by injecting an antisense oligonucleotide, which results in a failure to progress to meiosis II after emission of the first polar body. Such antisense oligonucleotide-injected oocytes could be efficiently rescued by co-injection of a c-mos mRNA carrying a wild-type 3' UTR. However, co-injection of a c-mos mRNA lacking functional CPEs substantially lowered the rescue activity. These results demonstrate that translational control of c-mos mRNA by cytoplasmic polyadenylation is necessary for normal development.     

c-mos and cdc2 cooperate in the translational activation of fibroblast growth factor receptor-1 during Xenopus oocyte maturation

During oocyte maturation in Xenopus, previously quiescent maternal mRNAs are translationally activated at specific times. We hypothesized that the translational recruitment of individual messages is triggered by particular cellular events and investigated the potential for known effectors of the meiotic cell cycle to activate the translation of the FGF receptor-1 (XFGFR) maternal mRNA. We found that both c-mos and cdc2 activate the translation of XFGFR. However, although oocytes matured by injection of recombinant cdc2/cyclin B translate normal levels of XFGFR protein, c-mos depletion reduces the level of XFGFR protein induced by cdc2/cyclin B injection. In oocytes blocked for cdc2 activity, injection of mos RNA induced low levels of XFGFR protein, independent of MAPK activity. Through the use of injected reporter RNAs, we show that the XFGFR 3' untranslated region inhibitory element is completely derepressed by cdc2 alone. In addition, we identified a new inhibitory element through which both mos and cdc2 activate translation. We found that cdc2 derepresses translation in the absence of polyadenylation, whereas mos requires poly(A) extension to activate XFGFR translation. Our results demonstrate that mos and cdc2, in addition to functioning as key regulators of the meiotic cell cycle, cooperate in the translational activation of a specific maternal mRNA during oocyte maturation.     

The Mos pathway regulates cytoplasmic polyadenylation in Xenopus oocytes.

Cytoplasmic polyadenylation controls the translation of several maternal mRNAs during Xenopus oocyte maturation and requires two sequences in the 3' untranslated region (UTR), the U-rich cytoplasmic polyadenylation element (CPE), and the hexanucleotide AAUAAA. c-mos mRNA is polyadenylated and translated soon after the induction of maturation, and this protein kinase is necessary for a kinase cascade culminating in cdc2 kinase (MPF) activation. Other mRNAs are polyadenylated later, around the time of cdc2 kinase activation. To determine whether there is a hierarchy in the cytoplasmic polyadenylation of maternal mRNAs, we ablated c-mos mRNA with an antisense oligonucleotide. This prevented histone B4 and cyclin A1 and B1 mRNA polyadenylation, indicating that the polyadenylation of these mRNAs is Mos dependent. To investigate a possible role of cdc2 kinase in this process, cyclin B was injected into oocytes lacking c-mos mRNA. cdc2 kinase was activated, but mitogen-activated protein kinase was not. However, polyadenylation of cyclin B1 and histone B4 mRNA was still observed. This demonstrates that cdc2 kinase can induce cytoplasmic polyadenylation in the absence of Mos. Our data further indicate that although phosphorylation of the CPE binding protein may be involved in the induction of Mos-dependent polyadenylation, it is not required for Mos-independent polyadenylation. We characterized the elements conferring Mos dependence (Mos response elements) in the histone B4 and cyclin B1 mRNAs by mutational analysis. For histone B4 mRNA, the Mos response elements were in the coding region or 5' UTR. For cyclin B1 mRNA, the main Mos response element was a CPE that overlaps with the AAUAAA hexanucleotide. This indicates that the position of the CPE can have a profound influence on the timing of cytoplasmic polyadenylation.     

Redundant pathways for Cdc2 activation in Xenopus oocyte: either cyclin B or Mos synthesis

Xenopus oocytes are arrested in meiotic prophase I. Progesterone induces the resumption of meiotic maturation, which requires continuous protein synthesis to bring about Cdc2 activation. The identification of the newly synthesized proteins has long been a goal. Two plausible candidates have received extensive study. The synthesis of cyclin B and of c-Mos, a kinase that activates the mitogen-activated protein kinase pathway in oocytes, is clearly upregulated by translational control in response to progesterone. Recent studies suggest that ablation of either c-Mos or cyclin B synthesis by antisense oligonucleotides does not block meiotic maturation. Here, however, we show that when both pathways are simultaneously inhibited, progesterone no longer triggers maturation; adding back either c-Mos or cyclin B restores meiotic maturation. We conclude that the specific synthesis of either B-type cyclins or c-Mos, induced by progesterone, is required to induce meiotic maturation. The two pathways seem to be functionally redundant.     

Poly(A) polymerase and the regulation of cytoplasmic polyadenylation

Translational activation in oocytes and embryos is often regulated via increases in poly(A) length. Cleavage and polyadenylation specificity factor (CPSF), cytoplasmic polyadenylation element binding protein (CPEB), and poly(A) polymerase (PAP) have each been implicated in cytoplasmic polyadenylation in Xenopus laevis oocytes. Cytoplasmic polyadenylation activity first appears in vertebrate oocytes during meiotic maturation. Data presented here shows that complexes containing both CPSF and CPEB are present in extracts of X. laevis oocytes prepared before or after meiotic maturation. Assessment of a variety of RNA sequences as polyadenylation substrates indicates that the sequence specificity of polyadenylation in egg extracts is comparable to that observed with highly purified mammalian CPSF and recombinant PAP. The two in vitro systems exhibit a sequence specificity that is similar, but not identical, to that observed in vivo, as assessed by injection of the same RNAs into the oocyte. These findings imply that CPSFs intrinsic RNA sequence preferences are sufficient to account for the specificity of cytoplasmic polyadenylation of some mRNAs. We discuss the hypothesis that CPSF is required for all polyadenylation reactions, but that the polyadenylation of some mRNAs may require additional factors such as CPEB. To test the consequences of PAP binding to mRNAs in vivo, PAP was tethered to a reporter mRNA in resting oocytes using MS2 coat protein. Tethered PAP catalyzed polyadenylation and stimulated translation approximately 40-fold; stimulation was exclusively cis-acting, but was independent of a CPE and AAUAAA. Both polyadenylation and translational stimulation required PAPs catalytic core, but did not require the putative CPSF interaction domain of PAP. These results demonstrate that premature recruitment of PAP can cause precocious polyadenylation and translational stimulation in the resting oocyte, and can be interpreted to suggest that the role of other factors is to deliver PAP to the mRNA.     

Unmasking the role of the 3′ UTR in the cytoplasmic polyadenylation and translational regulation of maternal mRNAs

The poly(A)-dependent translational regulation of maternal mRNAs is an important mechanism to execute stage-specific programs of protein synthesis during early development. This control underlies many crucial developmental events including the meiotic maturation of oocytes and activation of the mitotic cell cycle at fertilization. A recent report⁽¹⁾ demonstrates that the 3′ untranslated region of the cyclin A1, B1, B2 and c-mos mRNAs determines the timing and extent of their cytoplasmic polyadenylation and translational activation during Xenopus oocyte maturation. These studies further establish that protein synthesis can be temporally and quantitatively controlled by developmentally regulated changes in the polyadenylation of maternal mRNAs.     

Modifications of the 5′ Cap of mRNAs during Xenopus Oocyte Maturation: Independence from Changes in Poly(A) Length and Impact on Translation

The translation of specific maternal mRNAs is regulated during early development. For some mRNAs, an increase in translational activity is correlated with cytoplasmic extension of their poly(A) tails; for others, translational inactivation is correlated with removal of their poly(A) tails. Recent results in several systems suggest that events at the 3′ end of the mRNA can affect the state of the 5′ cap structure, m⁷G(5′)ppp(5′)G. We focus here on the potential role of cap modifications on translation during early development and on the question of whether any such modifications are dependent on cytoplasmic poly(A) addition or removal. To do so, we injected synthetic RNAs into Xenopus oocytes and examined their cap structures and translational activities during meiotic maturation. We draw four main conclusions. First, the activity of a cytoplasmic guanine-7-methyltransferase increases during oocyte maturation and stimulates translation of an injected mRNA bearing a nonmethylated GpppG cap. The importance of the cap for translation in oocytes is corroborated by the sensitivity of protein synthesis to cap analogs and by the inefficient translation of mRNAs bearing nonphysiologically capped 5′ termini. Second, deadenylation during oocyte maturation does not cause decapping, in contrast to deadenylation-triggered decapping in Saccharomyces cerevisiae. Third, the poly(A) tail and the N-7 methyl group of the cap stimulate translation synergistically during oocyte maturation. Fourth, cap ribose methylation of certain mRNAs is very inefficient and is not required for their translational recruitment by poly(A). These results demonstrate that polyadenylation can cause translational recruitment independent of ribose methylation. We propose that polyadenylation enhances translation through at least two mechanisms that are distinguished by their dependence on ribose modification.     

Maturation of Xenopus laevis oocyte by progesterone requires poly(A) tail elongation of mRNA.

Meiotic maturation of Xenopus laevis oocytes by progesterone requires translation of stored maternal mRNAs. We investigated the role of poly(A) tail elongation of mRNAs during this process using cordycepin, which inhibits poly(A) tail elongation of mRNAs. When oocytes were treated with the buffer containing 10 mM cordycepin for 12 h, concentration of 3'-dATP in cytosol of oocytes increased to 0.7 mM, while that of ATP remained constant at around 1.2 mM. Incorporation of [32P]AMP into poly(A) mRNA was inhibited almost completely by this treatment. Progesterone-induced germinal vesicle breakdown (GVBD) was also abolished. Dose dependence of inhibition of progesterone-induced GVBD on cordycepin was similar to that of [32P]AMP incorporation into poly(A) mRNA. However, maturation-promoting factor-induced GVBD was unaffected by treatment of oocytes with cordycepin. Furthermore, the inhibition of GVBD by cordycepin was rescued by removal of cordycepin even in the presence of actinomycin D. Therefore, we concluded that poly(A) tail elongation of mRNA is required for induction of meiotic maturation of X. laevis oocytes. In addition, progesterone induced a 2.7-fold activation of [32P]AMP incorporation into the poly(A) tail of mRNA after a lag period of 3 h whereas GVBD was induced after 6-8 h from the progesterone treatment. Syntheses of most of the proteins were unaffected by treatment of oocytes with progesterone or cordycepin. However, syntheses of several proteins were increased or decreased by progesterone and cordycepin treatment.     

Aurora Kinase A Is Not Involved in CPEB1 Phosphorylation and cyclin B1 mRNA Polyadenylation during Meiotic Maturation of Porcine Oocytes

Regulation of mRNA translation by cytoplasmic polyadenylation is known to be important for oocyte maturation and further development. This process is generally controlled by phosphorylation of cytoplasmic polyadenylation element binding protein 1 (CPEB1). The aim of this study is to determine the role of Aurora kinase A in CPEB1 phosphorylation and the consequent CPEB1-dependent polyadenylation of maternal mRNAs during mammalian oocyte meiosis. For this purpose, we specifically inhibited Aurora kinase A with MLN8237 during meiotic maturation of porcine oocytes. Using poly(A)-test PCR method, we monitored the effect of Aurora kinase A inhibition on poly(A)-tail extension of long and short cyclin B1 encoding mRNAs as markers of CPEB1-dependent cytoplasmic polyadenylation. Our results show that inhibition of Aurora kinase A activity impairs neither cyclin B1 mRNA polyadenylation nor its translation and that Aurora kinase A is unlikely to be involved in CPEB1 activating phosphorylation.     

CPEB controls the cytoplasmic polyadenylation of cyclin, Cdk2 and c-mos mRNAs and is necessary for oocyte maturation in Xenopus.

Cytoplasmic polyadenylation is a key mechanism controlling maternal mRNA translation in early development. In most cases, mRNAs that undergo poly(A) elongation are translationally activated; those that undergo poly(A) shortening are deactivated. Poly(A) elongation is regulated by two cis-acting sequences in the 3'-untranslated region (UTR) of responding mRNAs, the polyadenylation hexanucleotide AAUAAA and the U-rich cytoplasmic polyadenylation element (CPE). Previously, we cloned and characterized the Xenopus oocyte CPE binding protein (CPEB), showing that it was essential for the cytoplasmic polyadenylation of B4 RNA. Here, we show that CPEB also binds the CPEs of G10, c-mos, cdk2, cyclins A1, B1 and B2 mRNAs. We find that CPEB is necessary for polyadenylation of these RNAs in egg extracts, suggesting that this protein is required for polyadenylation of most RNAs during oocyte maturation. Our data demonstrate that the complex timing and extent of polyadenylation are partially controlled by CPEB binding to multiple target sites in the 3' UTRs of responsive mRNAs. Finally, injection of CPEB antibody into oocytes not only inhibits polyadenylation in vivo, but also blocks progesterone-induced maturation. This is due to inhibition of polyadenylation and translation of c-mos mRNA, suggesting that CPEB is critical for early development.     

Differential phosphorylation controls Maskin association with eukaryotic translation initiation factor 4E and localization on the mitotic apparatus

Several cytoplasmic polyadenylation element (CPE)-containing mRNAs that are repressed in Xenopus oocytes become active during meiotic maturation. A group of factors that are anchored to the CPE are responsible for this repression and activation. Two of the most important are CPEB, which binds directly to the CPE, and Maskin, which associates with CPEB. In oocytes, Maskin also binds eukaryotic translation initiation factor 4E (eIF4E), an interaction that excludes eIF4G and prevents formation of the eIF4F initiation complex. When the oocytes are stimulated to reenter the meiotic divisions (maturation), CPEB promotes cytoplasmic polyadenylation. The newly elongated poly(A) tail becomes bound by poly(A) binding protein (PABP), which in turn binds eIF4G and helps it displace Maskin from eIF4E, thereby inducing translation. Here we show that Maskin undergoes several phosphorylation events during oocyte maturation, some of which are important for its dissociation from eIF4E and translational activation of CPE-containing mRNA. These sites are T58, S152, S311, S343, S453, and S638 and are phosphorylated by cdk1. Mutation of these sites to alanine alleviates the cdk1-induced dissociation of Maskin from eIF4E. Prior to maturation, Maskin is phosphorylated on S626 by protein kinase A. While this modification has no detectable effect on translation during oocyte maturation, it is critical for this protein to localize on the mitotic apparatus in somatic cells. These results show that Maskin activity and localization is controlled by differential phosphorylation.     

Xenopus Rbm9 is a novel interactor of XGld2 in the cytoplasmic polyadenylation complex

During early development, control of the poly(A) tail length by cytoplasmic polyadenylation is critical for the regulation of specific mRNA expression. Gld2, an atypical poly(A) polymerase, is involved in cytoplasmic polyadenylation in Xenopus oocytes. In this study, a new XGld2-interacting protein was identified: Xenopus RNA-binding motif protein 9 (XRbm9). This RNA-binding protein is exclusively expressed in the cytoplasm of Xenopus oocytes and interacts directly with XGld2. It is shown that XRbm9 belongs to the cytoplasmic polyadenylation complex, together with cytoplasmic polyadenylation element-binding protein (CPEB), cleavage and polyadenylation specificity factor (CPSF) and XGld2. In addition, tethered XRbm9 stimulates the translation of a reporter mRNA. The function of XGld2 in stage VI oocytes was also analysed. The injection of XGld2 antibody into oocytes inhibited polyadenylation, showing that endogenous XGld2 is required for cytoplasmic polyadenylation. Unexpectedly, XGld2 and CPEB antibody injections also led to an acceleration of meiotic maturation, suggesting that XGld2 is part of a masking complex with CPEB and is associated with repressed mRNAs in oocytes.     

Multiple sequence elements and a maternal mRNA product control cdk2 RNA polyadenylation and translation during early Xenopus development.

Cytoplasmic poly(A) elongation is one mechanism that regulates translational recruitment of maternal mRNA in early development. In Xenopus laevis, poly(A) elongation is controlled by two cis elements in the 3' untranslated regions of responsive mRNAs: the hexanucleotide AAUAAA and a U-rich structure with the general sequence UUUUUAAU, which is referred to as the cytoplasmic polyadenylation element (CPE). B4 RNA, which contains these sequences, is polyadenylated during oocyte maturation and maintains a poly(A) tail in early embryos. However, cdk2 RNA, which also contains these sequences, is polyadenylated during maturation but deadenylated after fertilization. This suggests that cis-acting elements in cdk2 RNA signal the removal of the poly(A) tail at this time. By using poly(A) RNA-injected eggs, we showed that two elements which reside 5' of the CPE and 3' of the hexanucleotide act synergistically to promote embryonic deadenylation of this RNA. When an identical RNA lacking a poly(A) tail was injected, these sequences also prevented poly(A) addition. When fused to CAT RNA, the cdk2 3' untranslated region, which contains these elements, as well as the CPE and the hexanucleotide, promoted poly(A) addition and enhanced chloramphenicol acetyltransferase activity during maturation, as well as repression of these events after fertilization. Incubation of fertilized eggs with cycloheximide prevented the embryonic inhibition of cdk2 RNA polyadenylation but did not affect the robust polyadenylation of B4 RNA. This suggests that a maternal mRNA, whose translation occurs only after fertilization, is necessary for the cdk2 deadenylation or inhibition of RNA polyadenylation. This was further suggested when poly(A)+ RNA isolated from two-cell embryos was injected into oocytes that were then allowed to mature. Such oocytes became deficient for cdk2 RNA polyadenylation but remained proficient for B4 RNA polyadenylation. These data show that CPE function is developmentally regulated by multiple sequences and factors.     

The clam 3' UTR masking element-binding protein p82 is a member of the CPEB family.

During early development gene expression is controlled principally at the translational level. Oocytes of the surf clam Spisula solidissima contain large stockpiles of maternal mRNAs that are translationally dormant or masked until meiotic maturation. Activation of the oocyte by fertilization leads to translational activation of the abundant cyclin and ribonucleotide reductase mRNAs at a time when they undergo cytoplasmic polyadenylation. In vitro unmasking assays have defined U-rich regions located approximately centrally in the 3' UTRs of these mRNAs as translational masking elements. A clam oocyte protein of 82 kDa, p82, which selectively binds the masking elements, has been proposed to act as a translational repressor. Importantly, mRNA-specific unmasking in vitro occurs in the absence of poly(A) extension. Here we show that clam p82 is related to Xenopus CPEB, an RNA-binding protein that interacts with the U-rich cytoplasmic polyadenylation elements (CPEs) of maternal mRNAs and promotes their polyadenylation. Cloned clam p82/CPEB shows extensive homology to Xenopus CPEB and related polypeptides from mouse, goldfish, Drosophila and Caenorhabditis elegans, particularly in their RNA-binding C-terminal halves. Two short N-terminal islands of sequence, of unknown function, are common to vertebrate CPEBs and clam p82. p82 undergoes rapid phosphorylation either directly or indirectly by cdc2 kinase after fertilization in meiotically maturing clam oocytes, prior to its degradation during the first cell cleavage. Phosphorylation precedes and, according to inhibitor studies, may be required for translational activation of maternal mRNA. These data suggest that clam p82 may be a functional homolog of Xenopus CPEB.     

Autoregulation of Musashi1 mRNA translation during Xenopus oocyte maturation

The mRNA translational control protein, Musashi, plays a critical role in cell fate determination through sequence‐specific interactions with select target mRNAs. In proliferating stem cells, Musashi exerts repression of target mRNAs to promote cell cycle progression. During stem cell differentiation, Musashi target mRNAs are de‐repressed and translated. Recently, we have reported an obligatory requirement for Musashi to direct translational activation of target mRNAs during Xenopus oocyte meiotic cell cycle progression. Despite the importance of Musashi in cell cycle regulation, only a few target mRNAs have been fully characterized. In this study, we report the identification and characterization of a new Musashi target mRNA in Xenopus oocytes. We demonstrate that progesterone‐stimulated translational activation of the Xenopus Musashi1 mRNA is regulated through a functional Musashi binding element (MBE) in the Musashi1 mRNA 3′ untranslated region (3′ UTR). Mutational disruption of the MBE prevented translational activation of Musashi1 mRNA and its interaction with Musashi protein. Further, elimination of Musashi function through microinjection of inhibitory antisense oligonucleotides prevented progesterone‐induced polyadenylation and translation of the endogenous Musashi1 mRNA. Thus, Xenopus Musashi proteins regulate translation of the Musashi1 mRNA during oocyte maturation. Our results indicate that the hierarchy of sequential and dependent mRNA translational control programs involved in directing progression through meiosis are reinforced by an intricate series of nested, positive feedback loops, including Musashi mRNA translational autoregulation. These autoregulatory positive feedback loops serve to amplify a weak initiating signal into a robust commitment for the oocyte to progress through the cell cycle and become competent for fertilization.Mol. Reprod. Dev. 79: 553‐563, 2012. © 2012 Wiley Periodicals, Inc.