CPEB Research Articles

Does polyadenylation of the human mos mRNA play a role in human oocyte maturation?

The mechanisms regulating human oocyte maturation in vivo or in vitro are not well characterized. Previous studies employing inhibition of protein synthesis or targeted ablation of the endogenous Mos mRNA, have suggested a role for regulated Mos RNA translational control during human oocyte maturation. However, while a cytoplasmic polyadenylation element binding protein (CPEB1) has been characterized and shown to be expressed in human oocytes, it has not been determined if regulated cytoplasmic polyadenylation of the maternal Mos mRNA occurs during human oocyte maturation.

Cytoplasmic polyadenylation element binding protein-dependent protein synthesis is regulated by calcium/calmodulin-dependent protein kinase II.

Phosphorylation of cytoplasmic polyadenylation element binding protein (CPEB) regulates protein synthesis in hippocampal dendrites. CPEB binds the 3' untranslated region (UTR) of cytoplasmic mRNAs and, when phosphorylated, initiates mRNA polyadenylation and translation. We report that, of the protein kinases activated in the hippocampus during synaptic plasticity, calcium/calmodulin-dependent protein kinase II (CaMKII) robustly phosphorylated the regulatory site (threonine 171) in CPEB in vitro. In postsynaptic density fractions or hippocampal neurons, CPEB phosphorylation increased when CaMKII was activated. These increases in CPEB phosphorylation were attenuated by a specific peptide inhibitor of CaMKII and by the general CaM-kinase inhibitor KN-93. Inhibitors of protein phosphatase 1 increased basal CPEB phosphorylation in neurons; this was also attenuated by a CaM-kinase inhibitor. To determine whether CaM-kinase activity regulates CPEB-dependent mRNA translation, hippocampal neurons were transfected with luciferase fused to a 3' UTR containing CPE-binding elements. Depolarization of neurons stimulated synthesis of luciferase; this was abrogated by inhibitors of protein synthesis, mRNA polyadenylation, and CaMKII. These results demonstrate that CPEB phosphorylation and translation are regulated by CaMKII activity and provide a possible mechanism for how dendritic protein synthesis in the hippocampus may be stimulated during synaptic plasticity.

Insulin and prolactin synergistically stimulate beta-casein messenger ribonucleic acid translation by cytoplasmic polyadenylation.

Previous studies have shown that the synthesis and stability of milk protein mRNAs are regulated by lactogenic hormones. We demonstrate here in cultured mouse mammary epithelial cells (CID 9) that insulin plus prolactin also synergistically increases the rate of milk protein mRNA translation. Insulin alone stimulates synthesis of both milk and nonmilk proteins, whereas prolactin alone has no effect, but insulin plus prolactin selectively stimulate synthesis of milk proteins more than insulin alone. The increase in beta-casein mRNA translation is also reflected in a shift to larger polysomes, indicating an effect on translational initiation. Inhibitors of the phosphatidylinositol 3-kinase, mammalian target of rapamycin, and MAPK pathways block insulin-stimulated total protein and beta-casein synthesis but not the synergistic stimulation. Conversely, cordycepin abolishes synergistic stimulation of protein synthesis without affecting insulin-stimulated translation. The poly(A) tract of beta-casein mRNA progressively increases from approximately 20 to about 200 A residues over 30 min of treatment with insulin plus prolactin. The 3'-untranslated region of beta-casein mRNA containing an unaltered cytoplasmic polyadenylation element is sufficient for the translational enhancement and mRNA-specific polyadenylation, based on transient transfection of cells with a reporter construct. Insulin and prolactin stimulate cytoplasmic polyadenylation element binding protein phosphorylation with no increase of cytoplasmic poly(A) polymerase activity.

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.

The active form of Xp54 RNA helicase in translational repression is an RNA-mediated oligomer.

Previously, we reported that in clam oocytes, cytoplasmic polyadenylation element-binding protein (CPEB) co-immunoprecipitates with p47, a member of the highly conserved RCK family of RNA helicases which includes Drosophila Me31B and Saccharomyces cerevisiae Dhh1. Xp54, the Xenopus homologue, with helicase activity, is a component of stored mRNP. In tethered function assays in Xenopus oocytes, we showed that MS2-Xp54 represses the translation of non-adenylated firefly luciferase mRNAs and that mutations in two core helicase motifs, DEAD and HRIGR, surprisingly, activated translation. Here we show that wild-type MS2-Xp54 tethered to the reporter mRNA 3'-untranslated region (UTR) represses translation in both oocytes and eggs in an RNA-dependent complex with endogenous Xp54. Injection of mutant helicases or adenylated reporter mRNA abrogates this association. Thus Xp54 oligomerization is a hallmark of translational repression. Xp54 complexes, which also contain CPEB and eIF4E in oocytes, change during meiotic maturation. In eggs, CPEB is degraded and, while eIF4E still interacts with Xp54, this interaction becomes RNA dependent. Supporting evidence for RNA-mediated oligomerization of endogenous Xp54, and RNA-independent association with CPEB and eIF4E in oocytes was obtained by gel filtration. Altogether, our data are consistent with a model in which the active form of the Xp54 RNA helicase is an oligomer in vivo which, when tethered, via either MS2 or CPEB to the 3'UTR, represses mRNA translation, possibly by sequestering eIF4E from the translational machinery.

Is a Prion the Memory Switch?

Prions are proteins that promote the conversion of proteins of the same type to an alternate conformational state; typically, there is a soluble conformation and an insoluble, aggregating conformation. Furthermore, the state of the protein is heritable in an epigenetic manner. In humans, a prion protein is responsible for transmissible spongiform encephalopathies; however, in yeast, prions are not pathogenic and represent a mechanism of epigenetic regulation of protein activity. Si et al. noted that the model organism Aplysia , which is used for studying neurobiological questions such as the mechanisms underlying synaptic plasticity, expressed a neuronal form of the translational regulatory protein cytoplasmic polyadenylation element binding protein (CPEB) that had an N-terminal domain enriched in glutamine and asparagine residues, like that of other prion proteins. When tested in several yeast assays for prion activity (the ability to promote heritable conformational changes in the heterologously expressed protein or proteins to which the N-terminal domain was fused), the Aplysia CPEB behaved as though it were a prion. Surprisingly, the aggregated state of CPEB appeared to be the active form of the protein, at least in the yeast assays, which is in contrast to yeast prion proteins that are inactivated when they aggregate. Analysis of the role of Aplysia CPEB in response to prolonged exposure to or a short pulse of serotonin indicated that CPEB protein increases in a posttranscriptional manner. Furthermore, serotonin-stimulated synthesis of CPEB occurred in isolated neurites that had been detached from the cell bodies. Treatment of cells or processes with antisense oligonucleotides to disrupt CPEB expression resulted in no loss of the initial facilitation response, but impairment of the maintenance of the enhanced synaptic efficacy. Pharmacologic analysis indicated that CPEB stimulation required phosphoinositide 3-kinase but did not require protein kinase A or protein kinase C. What remains to be determined is whether CPEB behaves as a prion in Aplysia and whether changes in the protein's conformation occur in response to synaptic activity associated with long-term potentiation (see Darnell for discussion). K. Si, S. Lindquist, E. R. Kandel, A neuronal isoform of the Aplysia CPEB has prion-like properties. Cell 115 , 879-891 (2003). [Online Journal] K. Si, M. Giustetto, A. Etkin, R. Hsu, A. M. Janisiewicz, M. C. Miniaci, J.-H. Kim, H. Zhu, E. R. Kandel, A neuronal isoform of CPEB regulates local protein synthesis and stabilizes synapse-specific long-term facilitation in Aplysia . Cell 115 , 893-904 (2003). [Online Journal] R. B. Darnell, Memory, synaptic transmission, and ... prions? Cell 115 , 767-768 (2003). [Online Journal]

A Neuronal Isoform of the Aplysia CPEB Has Prion-Like Properties

Prion proteins have the unusual capacity to fold into two functionally distinct conformations, one of which is self-perpetuating. When yeast prion proteins switch state, they produce heritable phenotypes. We report prion-like properties in a neuronal member of the CPEB family (cytoplasmic polyadenylation element binding protein), which regulates mRNA translation. Compared to other CPEB family members, the neuronal protein has an N-terminal extension that shares characteristics of yeast prion-determinants: a high glutamine content and predicted conformational flexibility. When fused to a reporter protein in yeast, this region confers upon it the epigenetic changes in state that characterize yeast prions. Full-length CPEB undergoes similar changes, but surprisingly it is the dominant, self-perpetuating prion-like form that has the greatest capacity to stimulate translation of CPEB-regulated mRNA. We hypothesize that conversion of CPEB to a prion-like state in stimulated synapses helps to maintain long-term synaptic changes associated with memory storage.

A Neuronal Isoform of CPEB Regulates Local Protein Synthesis and Stabilizes Synapse-Specific Long-Term Facilitation in Aplysia

Synapse-specific facilitation requires rapamycin-dependent local protein synthesis at the activated synapse. In Aplysia, rapamycin-dependent local protein synthesis serves two functions: (1) it provides a component of the mark at the activated synapse and thereby confers synapse specificity and (2) it stabilizes the synaptic growth associated with long-term facilitation. Here we report that a neuron-specific isoform of cytoplasmic polyadenylation element binding protein (CPEB) regulates this synaptic protein synthesis in an activity-dependent manner. Aplysia CPEB protein is upregulated locally at activated synapses, and it is needed not for the initiation but for the stable maintenance of long-term facilitation. We suggest that Aplysia CPEB is one of the stabilizing components of the synaptic mark.

Two previously undescribed members of the mouse CPEB family of genes and their inducible expression in the principal cell layers of the hippocampus.

Cytoplasmic polyadenylation element-binding (CPEB) proteins control polyadenylation-induced translation in early development. Studies in oocytes led to the delineation of Xenopus CPEB, the first member of the family to be identified, and its mouse homologue mCPEB-1. Recently, a second mouse family member, mCPEB-2, has been described in germ cells. Increasing evidence also implicates CPEB proteins as being important in the hippocampus, where these proteins are thought to regulate local protein synthesis and synaptic plasticity. We therefore carried out a systematic screen for CPEB genes in the mouse brain and report two previously undescribed gene family members: mCPEB-3 and -4. We next examined the expression of all four genes in the hippocampus and found that mCPEB-1, -2, and -4 transcripts are expressed in the principal cell layer in the CA3 and CA1 region and in the dentate gyrus of the hippocampus. mCPEB-3 was barely expressed in naïve animals but together with mCPEB-4 was strongly up-regulated after injection of kainate to initiate seizure activity. Whereas mCPEB-1 is regulated by the Aurora kinase, mCPEB-2, -3, and -4 do not contain Aurora kinase phosphorylation sites. However, alternative splice isoforms of mCPEB-2, -3, and -4 encode the so-called B region with phosphorylation sites for cAMP-dependent protein kinase, calcium/calmodulin-dependent protein kinase II, and S6 kinase. Only isoforms that encode the B region were expressed in the principal cell layer. Coexpression of mCPEB-1 and the B region-containing splice isoforms suggests that a variety of different signaling pathways can recruit CPEB activity in hippocampal neurons.

CPEB2, a novel putative translational regulator in mouse haploid germ cells.

Translational control of specific mRNAs by cytoplasmic polyadenylation has fundamental roles in gametogenesis. The cytoplasmic polyadenylation element binding (CPEB) protein regulates cytoplasmic polyadenylation of mRNAs as a trans factor in oogenesis and spermatogenesis. The CPEB protein contains two RNA recognition motifs and a Zn-finger structure. Proteins (KIAA0940 and KIAA1673) with similar structures are predicted from the genome database, but nothing is known about their expression and function. Here, we report another novel member of the CPEB protein family, CPEB2. Comparison of the amino acid sequences of CPEB family members suggests that the family can be divided structurally and, perhaps, functionally into two groups: the CPEB group, and the CPEB2-KIAA0940-KIAA1673 group. The CPEB2 maps to mouse chromosome distal 5B and is abundantly expressed in testis. However, it was detected by reverse transcription-polymerase chain reaction in all tissues that we examined. It preferentially binds to poly(U) and localizes to the cytoplasm in transfected HeLa cells. The CPEB2 is expressed postmeiotically in mouse spermatogenesis, suggesting a possible role in translational regulation of stored mRNAs in transcriptionally inactive haploid spermatids.

Orb and a long poly(A) tail are required for efficient oskar translation at the posterior pole of the Drosophila oocyte.

During Drosophila oogenesis, the posterior determinant, Oskar, is tightly localized at the posterior pole of the oocyte. The exclusive accumulation of Oskar at this site is ensured by localization-dependent translation of oskar mRNA: translation of oskar mRNA is repressed during transport and activated upon localization at the posterior cortex. Previous studies have suggested that oskar translation is poly(A)-independent. We show that a long poly(A) tail is required for efficient oskar translation, both in vivo and in vitro, but is not sufficient to overcome BRE-mediated repression. Moreover, we show that accumulation of Oskar activity requires the Drosophila homolog of Cytoplasmic Polyadenylation Element Binding protein (CPEB), Orb. As posterior localization of oskar mRNA is an essential prerequisite for its translation, it was critical to identify an allele of orb that does localize oskar mRNA to the posterior pole of the oocyte. We show that flies bearing the weak mutation orb(mel) localize oskar transcripts with a shortened poly(A) that fails to enhance oskar translation, resulting in reduced Oskar levels and posterior patterning defects. We conclude that Orb-mediated cytoplasmic polyadenylation stimulates oskar translation to achieve the high levels of Oskar protein necessary for posterior patterning and germline differentiation.

The cytoplasmic polyadenylation element binding protein and polyadenylation of messenger RNA in Aplysia neurons

Translation of some mRNAs in nerve terminals has been shown to be regulated by polyadenylation in an experience-dependent manner. The transcripts whose translation is controlled by regulated polyadenylation contain the cytoplasmic polyadenylation element (CPE), which binds to the highly conserved CPE-binding protein (CPEB). In Aplysia, neuron-specific actin mRNA, which has a CPE in its 3′ UTR, is located both in cell bodies and at nerve endings (synaptosomes). We found that actin mRNA from pleural ganglion sensory neurons becomes polyadenylated during long-term facilitation produced by treatment with serotonin or 8-bromo cAMP. We cloned two isoforms of CPEB (ApCPEB77 and ApCEPB49) from Aplysia nervous tissue. The larger form, which is predominant in nervous tissue, is similar to p82, the clam binding protein, as well as to vertebrate CPEBs. Moreover, synaptosomal actin mRNAs are polyadenylated following the treatment with 5-HT. Since both CPEB and polyadenylated actin mRNA are present in synaptosomes and synaptosomal actin protein increases during long-term facilitation, we suggest that the translation of actin message in nerve endings is up-regulated by polyadenylation to grow new synapses.

TACC3 expression and localization in the murine egg and ovary.

A protein spot cored from a silver-stained two dimensional (2D) gel of germinal vesicle stage immature mouse oocytes was identified as Transforming Acidic Coiled Coil containing protein (TACC3) by tandem mass spectrometry. PCR amplification revealed two alternatively spliced forms, Tacc3a and Tacc3b, in mouse ovarian cDNA libraries. TACC3a encoded a 630 aa protein with a predicted mass of 70 kDa. It contained seven 24 aa repeats at the N-terminus and two coiled-coil domains at the C-terminus. TACC3b encoded a 426 aa protein with a predicted mass of 49 kDa also containing two coiled coil domains, but lacking the 168 aa repeat region. In addition to homology to the TACC family members, murine TACC3 also showed 35.7% identity to the Xenopus protein, Maskin, a cytoplasmic polyadenylation element binding protein (CPEB)-associated factor. Northern blot analysis demonstrated that TACC3a is abundantly expressed in adult testis and spleen and is moderately expressed in the ovary, heart, and lung, suggesting a wide tissue distribution. Both myc-tagged TACC3a and TACC3b targeted to the cytoplasm of transiently transfected CV-1 cells. In situ hybridization of mouse ovarian tissue sections displayed abundant expression of TACC3 specifically in the cytoplasm of growing oocytes, but not in primordial or atretic follicles. This pattern of expression suggests that TACC3 is expressed in ovarian cells undergoing active growth and development.

N-methyl-D-aspartate receptor signaling results in Aurora kinase-catalyzed CPEB phosphorylation and alpha CaMKII mRNA polyadenylation at synapses.

Activity-dependent local translation of dendritic mRNAs is one process that underlies synaptic plasticity. Here, we demonstrate that several of the factors known to control polyadenylation-induced translation in early vertebrate development [cytoplasmic polyadenylation element-binding protein (CPEB), maskin, poly(A) polymerase, cleavage and polyadenylation specificity factor (CPSF) and Aurora] also reside at synaptic sites of rat hippocampal neurons. The induction of polyadenylation at synapses is mediated by the N-methyl-D-aspartate (NMDA) receptor, which transduces a signal that results in the activation of Aurora kinase. This kinase in turn phosphorylates CPEB, an essential RNA-binding protein, on a critical residue that is necessary for polyadenylation-induced translation. These data demonstrate a remarkable conservation of the regulatory machinery that controls signal-induced mRNA translation, and elucidates an axis connecting the NMDA receptor to localized protein synthesis at synapses.

In Caenorhabditis elegans, the RNA-binding domains of the cytoplasmic polyadenylation element binding protein FOG-1 are needed to regulate germ cell fates.

FOG-1 controls germ cell fates in the nematode Caenorhabditis elegans. Sequence analyses revealed that FOG-1 is a cytoplasmic polyadenylation element binding (CPEB) protein; similar proteins from other species have been shown to bind messenger RNAs and regulate their translation. Our analyses of fog-1 mutations indicate that each of the three RNA-binding domains of FOG-1 is essential for activity. In addition, biochemical tests show that FOG-1 is capable of binding RNA sequences in the 3'-untranslated region of its own message. Finally, genetic assays reveal that fog-1 functions zygotically, that the small fog-1 transcript has no detectable function, and that missense mutations in fog-1 cause a dominant negative phenotype. This last observation suggests that FOG-1 acts in a complex, or as a multimer, to regulate translation. On the basis of these data, we propose that FOG-1 binds RNA to regulate germ cell fates and that it does so by controlling the translation of its targets. One of these targets might be the fog-1 transcript itself.

A conserved role of a DEAD box helicase in mRNA masking.

Clam p82 is a member of the cytoplasmic polyadenylation element-binding protein (CPEB) family of RNA-binding proteins and serves dual functions in regulating gene expression in early development. In the oocyte, p82/CPEB is a translational repressor, whereas in the activated egg, it acts as a polyadenylation factor. Coimmunoprecipitations were performed with p82 antibodies in clam oocyte and egg lysates to identify stage-regulated accessory factors. p47 coprecipitates with p82 from oocyte lysates in an RNA-dependent manner and is absent from egg lysate p92-bound material. Clam p47 is a member of the RCK/p54 family of DEAD box RNA helicases. Xp54, the Xenopus homolog, with bona fide helicase activity, is an abundant and integral component of stored mRNP in oocytes (Ladomery et al., 1997). In oocytes, clam p47 and p82/CPEB are found in large cytoplasmic mRNP complexes. Whereas the helicase level is constant during embryogenesis, in contrast to CPEB, clam p47 translocates to nuclei at the two-cell stage. To address the role of this class of helicase in masking, Xp54 was tethered via 3' UTR MS2-binding sites to firefly luciferase, following microinjection of fusion protein and nonadenylated reporter mRNAs into Xenopus oocytes. Tethered helicase repressed luciferase translation three- to fivefold and, strikingly, mutations in two helicase motifs (DEAD--> DQAD and HRIGR-->HRIGQ), activated translation three- to fourfold, relative to MS2. These data suggest that this helicase family represses translation of maternal mRNA in early development, and that its activity may be attenuated during meiotic maturation, prior to cytoplasmic polyadenylation.

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.

Functioning of theDrosophila orbgene ingurkenmRNA localization and translation

The orb gene encodes an RNA recognition motif (RRM)-type RNA-binding protein that is a member of the cytoplasmic polyadenylation element binding protein (CPEB) family of translational regulators. Early in oogenesis, orb is required for the formation and initial differentiation of the egg chamber, while later in oogenesis it functions in the determination of the dorsoventral (DV) and anteroposterior axes of egg and embryo. In the studies reported here, we have examined the role of the orb gene in the gurken (grk)-Drosophila epidermal growth factor receptor (DER) signaling pathway. During the previtellogenic stages of oogenesis, the grk-DER signaling pathway defines the posterior pole of the oocyte by specifying posterior follicle cell identity. This is accomplished through the localized expression of Grk at the very posterior of the oocyte. Later in oogenesis, the grk-DER pathway is used to establish the DV axis. Grk protein synthesized at the dorsal anterior corner of the oocyte signals dorsal fate to the overlying follicle cell epithelium. We show that orb functions in both the early and late grk-DER signaling pathways, and in each case is required for the localized expression of Grk protein. We have found that orb is also required to promote the synthesis of a key component of the DV polarity pathway, K(10). Finally, we present evidence that Orb protein expression during the mid- to late stages of oogenesis is, in turn, negatively regulated by K(10).

Think globally, translate locally: what mitotic spindles and neuronal synapses have in common.

Early metazoan development is programmed by maternal mRNAs inherited by the egg at the time of fertilization. These mRNAs are not translated en masse at any one time or at any one place, but instead their expression is regulated both temporally and spatially. Recent evidence has shown that one maternal mRNA, cyclin B1, is concentrated on mitotic spindles in the early Xenopus embryo, where its translation is controlled by CPEB (cytoplasmic polyadenylation element binding protein), a sequence-specific RNA binding protein. Disruption of the spindle-associated translation of this mRNA results in a morphologically abnormal mitotic apparatus and inhibited cell division. Mammalian neurons, particularly in the synapto-dendritic compartment, also contain localized mRNAs such as that encoding alpha-CaMKII. Here, synaptic activation drives local translation, an event that is involved in synaptic plasticity and possibly long-term memory storage. Synaptic translation of alpha-CaMKII mRNA also appears to be controlled by CPEB, which is enriched in the postsynaptic density. Therefore, CPEB-controlled local translation may influence such seemingly disparate processes as the cell cycle and synaptic plasticity.

Biochemical Identification of Xenopus Pumilio as a Sequence-specific Cyclin B1 mRNA-binding Protein That Physically Interacts with a Nanos Homolog, Xcat-2, and a Cytoplasmic Polyadenylation Element-binding Protein

Translational activation of dormant cyclin B1 mRNA stored in oocytes is a prerequisite for the initiation or promotion of oocyte maturation in many vertebrates. Using a monoclonal antibody against the domain highly homologous to that of Drosophila Pumilio, we have shown for the first time in any vertebrate that a homolog of Pumilio is expressed in Xenopus oocytes. This 137-kDa protein binds to the region including the sequence UGUA at nucleotides 1335-1338 in the 3'-untranslated region of cyclin B1 mRNA, which is close to but does not overlap the cytoplasmic polyadenylation elements (CPEs). Physical in vitro association of Xenopus Pumilio with a Xenopus homolog of Nanos (Xcat-2) was demonstrated by a protein pull-down assay. The results of immunoprecipitation experiments showed in vivo interaction between Xenopus Pumilio and CPE-binding protein (CPEB), a key regulator of translational repression and activation of mRNAs stored in oocytes. This evidence provides a new insight into the mechanism of translational regulation through the 3'-end of mRNA during oocyte maturation. These results also suggest the generality of the function of Pumilio as a translational regulator of dormant mRNAs in both invertebrates and vertebrates.