Maturation-specific polyadenylation and translational control: diversity of cytoplasmic polyadenylation elements, influence of poly(A) tail size, and formation of stable polyadenylation complexes.

Early embryonic development in Xenopus laevis is programmed in part by maternally derived mRNAs, many of which are translated at the completion of meiosis (oocyte maturation). Polysomal recruitment of at least one of these mRNAs, G10, is regulated by cytoplasmic poly(A) elongation which, in turn, is dependent upon the cytoplasmic polyadenylation element (CPE) UUUUUUAUAAAG and the hexanucleotide AAUAAA (L. L. McGrew, E. Dworkin-Rastl, M. B. Dworkin, and J. D. Richter, Genes Dev. 3:803-815, 1989). We have investigated whether sequences similar to the G10 RNA CPE that are present in other RNAs could also be responsible for maturation-specific polyadenylation. B4 RNA, which encodes a histone H1-like protein, requires a CPE of the sequence UUUUUAAU as well as the polyadenylation hexanucleotide. The 3' untranslated regions of Xenopus c-mos RNA and mouse HPRT RNA also contain U-rich CPEs since they confer maturation-specific polyadenylation when fused to Xenopus B-globin RNA. Polyadenylation of B4 RNA, which occurs very early during maturation, is limited to 150 residues, and it is this number that is required for polysomal recruitment. To investigate the possible diversity of factors and/or affinities that might control polyadenylation, egg extracts that faithfully adenylate exogenously added RNA were used in competition experiments. At least one factor is shared by B4 and G10 RNAs, although it has a much greater affinity for B4 RNA. Additional experiments demonstrate that an intact CPE and hexanucleotide are both required to compete for the polyadenylation apparatus. Gel mobility shift assays show that two polyadenylation complexes are formed on B4 RNA. Optimal complex formation requires an intact CPE and hexanucleotide but not ongoing adenylation. These data, plus additional RNA competition studies, suggest that stable complex formation is enhanced by an interaction of the trans-acting factors that bind the CPE and polyadenylation hexanucleotide.     

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.     

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 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.     

The 36-kilodalton embryonic-type cytoplasmic polyadenylation element-binding protein in Xenopus laevis is ElrA, a member of the ELAV family of RNA-binding proteins.

The translational activation of several maternal mRNAs in Xenopus laevis is dependent on cytoplasmic poly(A) elongation. Messages harboring the UUUUUAU-type cytoplasmic polyadenylation element (CPE) in their 3' untranslated regions (UTRs) undergo polyadenylation and translation during oocyte maturation. This CPE is bound by the protein CPEB, which is essential for polyadenylation. mRNAs that have the poly(U)12-27 embryonic-type CPE (eCPE) in their 3' UTRs undergo polyadenylation and translation during the early cleavage and blastula stages. A 36-kDa eCPE-binding protein in oocytes and embryos has been identified by UV cross-linking. We now report that this 36-kDa protein is ElrA, a member of the ELAV family of RNA-binding proteins. The proteins are identical in size, antibody directed against ElrA immunoprecipitates the 36-kDa protein, and the two proteins have the same RNA binding specificity in vitro. C12 and activin receptor mRNAs, both of which contain eCPEs, are detected in immunoprecipitated ElrA-mRNP complexes from eggs and embryos. In addition, this in vivo interaction requires the eCPE. Although a number of experiments failed to define a role for ElrA in cytoplasmic polyadenylation, the expression of a dominant negative ElrA protein in embryos results in an exogastrulation phenotype. The possible functions of ElrA in gastrulation are discussed.     

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.     

Translational control by cytoplasmic polyadenylation during Xenopus oocyte maturation: characterization of cis and trans elements and regulation by cyclin/MPF.

The expression of certain maternal mRNAs during oocyte maturation is regulated by cytoplasmic polyadenylation. To understand this process, we have focused on a maternal mRNA from Xenopus termed G10. This mRNA is stored in the cytoplasm of stage 6 oocytes until maturation when the process of poly(A) elongation stimulates its translation. Deletion analysis of the 3' untranslated region of G10 RNA has revealed that two sequence elements, UUUUUUAU and AAUAAA were both necessary and sufficient for polyadenylation and polysomal recruitment. In this communication, we have defined the U-rich region that is optimal for polyadenylation as UUUUUUAUAAAG, henceforth referred to as the cytoplasmic polyadenylation element (CPE). We have also identified unique sequence requirements in the 3' terminus of the RNA that can modulate polyadenylation even in the presence of wild-type cis elements. A time course of cytoplasmic polyadenylation in vivo shows that it is an early event of maturation and that it requires protein synthesis within the first 15 min of exposure to progesterone. MPF and cyclin can both induce polyadenylation but, at least with respect to MPF, cannot obviate the requirement for protein synthesis. To identify factors that may be responsible for maturation-specific polyadenylation, we employed extracts from oocytes and unfertilized eggs, the latter of which correctly polyadenylates exogenously added RNA. UV crosslinking demonstrated that an 82 kd protein binds to the U-rich CPE in egg, but not oocyte, extracts. The data suggest that progesterone, either in addition to or through MPF/cyclin, induces the synthesis of a factor during very early maturation that stimulates polyadenylation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Translational control by cytoplasmic polyadenylation in Xenopus oocytes

Elongation of the poly(A) tails of specific mRNAs in the cytoplasm is a crucial regulatory step in oogenesis and early development of many animal species. The best studied example is the regulation of translation by cytoplasmic polyadenylation elements (CPEs) in the 3' untranslated region of mRNAs involved in Xenopus oocyte maturation. In this review we discuss the mechanism of translational control by the CPE binding protein (CPEB) in Xenopus oocytes as follows: Finally we discuss some of the remaining questions regarding the mechanisms of translational regulation by cytoplasmic polyadenylation and give our view on where our knowledge is likely to be expanded in the near future.     

Dissolution of the maskin-eIF4E complex by cytoplasmic polyadenylation and poly(A)-binding protein controls cyclin B1 mRNA translation and oocyte maturation

Cytoplasmic polyadenylation stimulates the translation of several dormant mRNAs during oocyte maturation in XENOPUS: Polyadenylation is regulated by the cytoplasmic polyadenylation element (CPE), a cis-acting element in the 3'-untranslated region of responding mRNAs, and its associated factor CPEB. CPEB also binds maskin, a protein that in turn interacts with eIF4E, the cap-binding factor. Here, we report that based on antibody and mRNA reporter injection assays, maskin prevents oocyte maturation and the translation of the CPE-containing cyclin B1 mRNA by blocking the association of eIF4G with eIF4E. Dissociation of the maskin-eIF4E complex is essential for cyclin B1 mRNA translational activation, and requires not only cytoplasmic polyadenylation, but also the poly(A)-binding protein. These results suggest a molecular mechanism by which CPE- containing mRNA is activated in early development.     

Opposing polymerase-deadenylase activities regulate cytoplasmic polyadenylation

Cytoplasmic polyadenylation is one mechanism that regulates translation in early animal development. In Xenopus oocytes, polyadenylation of dormant mRNAs, including cyclin B1, is controlled by the cis-acting cytoplasmic polyadenylation element (CPE) and hexanucleotide AAUAAA through associations with CPEB and CPSF, respectively. Previously, we demonstrated that the scaffold protein symplekin contacts CPEB and CPSF and helps them interact with Gld2, a poly(A) polymerase. Here, we report the mechanism by which poly(A) tail length is regulated. Cyclin B1 pre-mRNA acquires a long poly(A) tail in the nucleus that is subsequently shortened in the cytoplasm. The shortening is controlled by CPEB and PARN, a poly(A)-specific ribonuclease. Gld2 and PARN both reside in the CPEB-containing complex. However, because PARN is more active than Gld2, the poly(A) tail is short. When oocytes mature, CPEB phosphorylation causes PARN to be expelled from the ribonucleoprotein complex, which allows Gld2 to elongate poly(A) by default.     

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.     

Symplekin and xGLD-2 are required for CPEB-mediated cytoplasmic polyadenylation

Cytoplasmic polyadenylation-induced mRNA translation is a hallmark of early animal development. In Xenopus oocytes, where the molecular mechanism has been defined, the core factors that control this process include CPEB, an RNA binding protein whose association with the CPE specifies which mRNAs undergo polyadenylation; CPSF, a multifactor complex that interacts with the near-ubiquitous polyadenylation hexanucleotide AAUAAA; and maskin, a CPEB and eIF4E binding protein whose regulation of initiation is governed by poly(A) tail length. Here, we define two new factors that are essential for polyadenylation. The first is symplekin, a CPEB and CPSF binding protein that serves as a scaffold upon which regulatory factors are assembled. The second is xGLD-2, an unusual poly(A) polymerase that is anchored to CPEB and CPSF even before polyadenylation begins. The identification of these factors has broad implications for biological process that employ polyadenylation-regulated translation, such as gametogenesis, cell cycle progression, and synaptic plasticity.     

CPEB degradation during Xenopus oocyte maturation requires a PEST domain and the 26S proteasome

Cytoplasmic poly(A) elongation is widely utilized during the early development of many organisms as a mechanism for translational activation. Targeting of mRNAs for this mechanism requires the presence of a U-rich element, the cytoplasmic polyadenylation element (CPE), and its binding protein, CPEB. Blocking cytoplasmic polyadenylation by interfering with the CPE or CPEB prevents the translational activation of mRNAs that are crucial for oocyte maturation. The CPE sequence and CPEB are also important for translational repression of mRNAs stored in the Xenopus oocyte during oogenesis. To understand the contribution of protein metabolism to these two roles for CPEB, we have examined the mechanisms influencing the expression of CPEB during oogenesis and oocyte maturation. Through a comparison of CPEB mRNA levels, protein synthesis, and accumulation, we find that CPEB is synthesized during oogenesis and stockpiled in the oocyte. Minimal synthesis of CPEB, <3.6%, occurs during oocyte maturation. In late oocyte maturation, 75% of CPEB is degraded coincident with germinal vesicle breakdown. Using proteasome and ubiquitination inhibitors, we demonstrate that CPEB degradation occurs via the proteasome pathway, most likely through ubiquitin-conjugated intermediates. In addition, we demonstrate that degradation requires a 14 amino acid PEST domain.     

Expression of cyclin B1 messenger RNA isoforms and initiation of cytoplasmic polyadenylation in the bovine oocyte

Oocytes can synthesize and store maternal mRNA in an inactive translational state until the start of in vitro maturation. Cytoplasmic polyadenylation, driven by 3'-untranslated region (UTR) cis-acting cytoplasmic polyadenylation element (CPE), is associated with translational activation of cyclin B1 mRNA during maturation. The main aim of the present study was to investigate if bovine oocyte cyclin B1 mRNA undergoes cytoplasmic polyadenylation/translation during in vitro maturation, as in other species. We have found that cyclin B1 mRNA is present in two isoforms, consisting of the same open reading frame but with different 3'-UTR lengths. Only the longest isoform (cyclin B1L) has a putative CPE sequence and other regulatory sequences, and its mRNA level decreases during early embryo development. The polyadenylation state of cyclin B1L during in vitro maturation was studied. Results demonstrated that cyclin B1L bears a relatively long poly(A) tail in germinal vesicle-stage oocytes, which is further lengthened at 10 h of maturation, before metaphase I. Interestingly, cyclin B1L bears a short poly(A) tail when the ovaries and the oocytes are transported and manipulated on ice to stop the polyadenylation process. Cytoplasmic polyadenylation most probably occurs during ovary transport in warm saline, when oocytes are still in their follicular environment. Our results also show a link between cytoplasmic polyadenylation of cyclin B1 and translation/appearance of cyclin B1 protein before in vitro maturation.     

Cytoplasmic polyadenylation element (CPE)- and CPE-binding protein (CPEB)-independent mechanisms regulate early class maternal mRNA translational activation in Xenopus oocytes

Meiotic cell cycle progression during vertebrate oocyte maturation requires the correct temporal translation of maternal mRNAs encoding key regulatory proteins. The mechanism by which specific mRNAs are temporally activated is unknown, although both cytoplasmic polyadenylation elements (CPE) within the 3'-untranslated region (3'-UTR) of mRNAs and the CPE-binding protein (CPEB) have been implicated. We report that in progesterone-stimulated Xenopus oocytes, the early cytoplasmic polyadenylation and translational activation of multiple maternal mRNAs occur in a CPE- and CPEB-independent manner. We demonstrate that polyadenylation response elements, originally identified in the 3'-UTR of the mRNA encoding the Mos proto-oncogene, direct CPE- and CPEB-independent polyadenylation of an early class of Xenopus maternal mRNAs. Our findings refute the hypothesis that CPE sequences alone account for the range of temporal inductions of maternal mRNAs observed during Xenopus oocyte maturation. Rather, our data indicate that the sequential action of distinct 3'-UTR-directed translational control mechanisms coordinates the complex temporal patterns and extent of protein synthesis during vertebrate meiotic cell cycle progression.     

Phosphorylation of CPEB by Eg2 mediates the recruitment of CPSF into an active cytoplasmic polyadenylation complex

The release of Xenopus oocytes from prophase I arrest is largely driven by the cytoplasmic polyadenylation-induced translation of dormant maternal mRNAs. Two cis elements, the CPE and the hexanucleotide AAUAAA, and their respective binding factors, CPEB and a cytoplasmic form of CPSF, control polyadenylation. The most proximal stimulus for polyadenylation is Eg2-catalyzed phosphorylation of CPEB serine 174. Here, we show that this phosphorylation event stimulates an interaction between CPEB and CPSF. This interaction is direct, does not require RNA tethering, and occurs through the 160 kDa subunit of CPSF. Eg2-stimulated and CPE-dependent polyadenylation is reconstituted in vitro using purified components. These results demonstrate that the molecular function of Eg2-phosphorylated CPEB is to recruit CPSF into an active cytoplasmic polyadenylation complex.     

Activity-dependent polyadenylation in neurons

Activity-dependent changes in protein synthesis modify synaptic efficacy. One mechanism that regulates mRNA translation in the synapto-dendritic compartment is cytoplasmic polyadenylation, a process controlled by CPEB, the cytoplasmic polyadenylation element (CPE)-specific RNA binding protein. In neurons, very few mRNAs are known CPEB substrates, and none appear to be responsible for the effects on plasticity that are found in the CPEB knockout mouse. These results suggest that the translation of other mRNAs is regulated by CPEB. To identify them, we have developed a functional assay based on the polyadenylation of brain-derived mRNAs injected into Xenopus oocytes, a surrogate system that carries out this 3' end processing event in an efficient manner. The polyadenylated RNAs were isolated by binding to and thermal elution from poly(U) agarose and identified by microarray analysis. Selected sequences that were positive for polyadenylation were cloned and retested for polyadenylation by injection into oocytes. These sequences were then examined for activity-dependent polyadenylation in cultured hippocampal neurons. Finally, the levels of two proteins encoded by polyadenylated mRNAs were examined in glutamate-stimulated synaptoneurosomes. These studies show that many mRNAs undergo activity-dependent polyadenylation in neurons and that this process coincides with increased translation in the synapto-dendritic compartment.