CPEB Protein Research Articles

Developmental expression of FOG-1/CPEB protein and its control in the Caenorhabditis elegans hermaphrodite germ line.

The specification of a germ cell as sperm or oocyte and determination of cell number remain unsolved questions in developmental biology. This paper examines Caenorhabditis elegans FOG-1, a CPEB-related RNA-binding protein that controls the sperm fate. We find that abundant FOG-1 protein is observed transiently in germ cells just prior to their expression of an early sperm-differentiation marker. As the germline tissue elongates, abundant FOG-1 appears more and more distally as sperm become specified, but disappears when the germ line switches to oogenesis. This dynamic pattern is controlled by both globally acting and germline-specific sex-determining regulators. Importantly, the extent of FOG-1 expression corresponds roughly to sperm number in wild-type and mutants, altering sperm number. By contrast, three other key regulators of the sperm/oocyte decision do not similarly correspond to sperm number. We suggest that FOG-1 is precisely modulated in both time and space to specify sperm fate and control sperm number.

The fragile X mental retardation protein–RNP granules show an mGluR-dependent localization in the post-synaptic spines

The localization of RNA/mRNA in dendrites plays a role in both local and temporal regulation of protein synthesis, which is required for certain forms of synaptic plasticity. A key molecule in these processes is the fragile X mental retardation protein (FMRP). Using in situ hybridization coupled to immunofluorescence confocal microscopy, we find that the FMRP–RNP particle contains αCaMKII and BC1 RNAs as well as Staufen and CPEB proteins. Furthermore, following mGluR activation, the FMRP–mRNP complex moves into spines as shown by co-localization with the PSD-95 and Shank proteins. This study shows, for the first time, that the translationally inactive FMRP–mRNP complex relocates into neuronal spines after stimulation and that de novo protein synthesis mainly contributes to the pool of FMRP at synapses.

Autoregulation of GLD-2 cytoplasmic poly(A) polymerase

Cytoplasmic polyadenylation regulates mRNA stability and translation and is required for early development and synaptic plasticity. The GLD-2 poly(A) polymerase catalyzes cytoplasmic polyadenylation in the germline of metazoa. Among vertebrates, the enzyme is encoded by two isoforms of mRNA that differ only in the length of their 3'-UTRs. Here we focus on regulation of vertebrate GLD-2 mRNA. We show that the 3'-UTR of GLD-2 mRNA elicits its own polyadenylation and translational activation during frog oocyte maturation. We identify the sequence elements responsible for repression and activation, and demonstrate that CPEB and PUF proteins likely mediate repression in the resting oocyte. Regulated polyadenylation of GLD-2 mRNA is conserved, as are the key regulatory elements. Poly(A) tails of GLD-2 mRNA increase in length in the brain in response to neuronal stimulation, suggesting that a comparable system exists in that tissue. We propose a positive feedback circuit in which translation of GLD-2 mRNA is stimulated by its polyadenylation, thereby reinforcing the switch to polyadenylate and activate batteries of mRNAs.

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.

Cytoplasmic CstF-77 Protein Belongs to a Masking Complex with Cytoplasmic Polyadenylation Element-binding Protein in Xenopus Oocytes

Regulated mRNA translation is a hallmark of oocytes and early embryos, of which cytoplasmic polyadenylation is a major mechanism. This process involves multiple protein components, including the CPSF (cleavage and polyadenylation specificity factor), which is also required for nuclear polyadenylation. The CstF (cleavage stimulatory factor), with CPSF, is required for the pre-mRNA cleavage before nuclear polyadenylation. However, some evidence suggests that the CstF-77 subunit might have a function independent of nuclear polyadenylation, which could be related to the cell cycle. As such, we addressed the question whether CstF-77 might have a role in cytoplasmic polyadenylation. We investigated the function of the CstF-77 protein in Xenopus oocytes, and show that CstF-77 has indeed a role in the cytoplasm. The Xenopus CstF-77 protein (X77K) localizes mainly to the nucleus, but also in punctuate cytoplasmic foci. We show that X77K resides in a cytoplasmic complex with eIF4E, CPEB (cytoplasmic polyadenylation element-binding protein), CPSF-100 and XGLD2, but is not required for cytoplasmic polyadenylation per se. Impairment of X77K function in ovo leads to an acceleration of the G(2)/M transition, with a premature synthesis of Mos and AuroraA proteins. However, the kinetic of Mos mRNA polyadenylation is not modified. Furthermore, X77K represses mRNA translation in vitro. These results suggest that X77K could be involved in masking of mRNA prior to polyadenylation.

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.

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 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.

Role of cdc2 kinase phosphorylation and conserved N-terminal proteolysis motifs in cytoplasmic polyadenylation-element-binding protein (CPEB) complex dissociation and degradation.

Cytoplasmic polyadenylation-element-binding protein (CPEB) is a well-characterized and important regulator of translation of maternal mRNA in early development in organisms ranging from worms, flies and clams to frogs and mice. Previous studies provided evidence that clam and Xenopus CPEB are hyperphosphorylated at germinal vesicle breakdown (GVBD) by cdc2 kinase, and degraded shortly after. To examine the conserved features of CPEB that mediate its modification during meiotic maturation, we microinjected mRNA encoding wild-type and mutated clam CPEB into Xenopus oocytes that were subsequently allowed to mature with progesterone. We observed that (i) ectopically expressed clam CPEB is phosphorylated at GVBD and subsequently degraded, mirroring the fate of the endogenous Xenopus CPEB protein, (ii) mutation of nine Ser/Thr Pro-directed kinase sites prevents phosphorylation and degradation and (iii) deletion of the PEST box, and to a lesser extent of the putative cyclin destruction box, generates a stable and phosphorylated version of CPEB. We conclude that phosphorylation of both consensus and non-consensus sites by cdc2 kinase targets clam CPEB for PEST-mediated destruction. We also show that phosphorylation of CPEB mediates its dissociation from ribonucleoprotein complexes, prior to degradation. Our findings reinforce results obtained in Xenopus, and have implications for CPEB from other invertebrates including Drosophila, Caenorhabditis elegans and Aplysia, which lack PEST boxes.

CPEB phosphorylation and cytoplasmic polyadenylation are catalyzed by the kinase IAK1/Eg2 in maturing mouse oocytes.

In both vertebrates and invertebrates, the expression of several maternal mRNAs is regulated by cytoplasmic polyadenylation. In Xenopus oocytes, where most of the biochemical details of this process have been examined, polyadenylation is controlled by CPEB, a sequence-specific RNA binding protein. The activity of CPEB, which is to recruit cleavage and polyadenylation specificity factor (CPSF) and poly(A) polymerase (PAP) into an active cytoplasmic polyadenylation complex, is controlled by Eg2-catalyzed phosphorylation. Soon after CPEB phosphorylation and resulting polyadenylation take place, the interaction between maskin, a CPEB-associated factor, and eIF4E, the cap-binding protein, is destroyed, which results in the recruitment of mRNA into polysomes. Polyadenylation also occurs in maturing mouse oocytes, although the biochemical events that govern the reaction in these cells are not known. In this study, we have examined the phosphorylation of CPEB and have assessed the necessity of this protein for polyadenylation in maturing mouse oocytes. Immunohistochemistry has revealed that all the factors that control polyadenylation and translation in Xenopus oocytes (CPEB, CPSF, PAP, maskin, and IAK1, the murine homologue of Eg2) are also present in the cytoplasm of mouse oocytes. After the induction of maturation, a kinase is activated that phosphorylates CPEB on a critical regulatory residue, an event that is essential for CPEB activity. A peptide that competitively inhibits the activity of IAK1/Eg2 blocks the progression of meiosis in injected oocytes. Finally, a CPEB protein that acts as a dominant negative mutation because it cannot be phosphorylated by IAK1/Eg2, prevents cytoplasmic polyadenylation. These data indicate that cytoplasmic polyadenylation in mouse oocytes is mediated by IAK1/Eg2-catalyzed phosphorylation of CPEB.

An autoregulatory feedback loop directs the localized expression of the Drosophila CPEB protein Orb in the developing oocyte.

The RRM-type RNA binding protein Orb plays a central role in the establishment of polarity in the Drosophila egg and embryo. In addition to its role in the formation and initial differentiation of the egg chamber, orb is required later in oogenesis for the determination of the dorsoventral (DV) and anteroposterior (AP) axes. In DV axis formation, Orb protein is required to localize and translate gurken mRNA at the dorsoanterior part of the oocyte. In AP axis formation, Orb is required for the translation of oskar mRNA. In each case, Orb protein is already localized at the appropriate sites within the oocyte before the arrival of the mRNAs encoding axis determinants. We present evidence that an autoregulatory mechanism is responsible for directing the on site accumulation of Orb protein in the Drosophila oocyte. This orb autoregulatory activity ensures the accumulation of high levels of Orb protein at sites in the oocyte that contain localized orb message.

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.

Regulation of Cell Fate in Caenorhabditis elegans by a Novel Cytoplasmic Polyadenylation Element Binding Protein

The fog-1 gene of Caenorhabditis elegans specifies that germ cells differentiate as sperm rather than as oocytes. We cloned fog-1 through a combination of transformation rescue experiments, RNA-mediated inactivation, and mutant analyses. Our results show that fog-1 produces two transcripts, both of which are found in germ cells but not in the soma. Furthermore, two deletion mutants alter these transcripts and are likely to eliminate fog-1 activity. The larger transcript is expressed under the control of sex-determination genes, is necessary for fog-1 activity, and is sufficient to rescue a fog-1 mutant. This transcript encodes a novel member of the CPEB family of RNA-binding proteins. Because CPEB proteins in Xenopus and Drosophila regulate gene expression at the level of translation, we propose that FOG-1 controls germ cell fates by regulating the translation of specific messenger RNAs.

The Drosophila CPEB Homolog, Orb, Is Required for Oskar Protein Expression in Oocytes

The establishment of polarity axes in the Drosophila egg and embryo depends upon the localization and on-site expression of maternal mRNAs. The critical step in the targeting of posterior determinants is the localization of oskar (osk) mRNA to the pole and its on-site translation. Osk protein then recruits other posterior group gene products involved in the formation of pole plasm and in the localization and regulation of the posterior determinant, nanos. Here we have investigated the role of the Drosophila CPEB homolog, the orb gene, in the osk mRNA localization pathway. We demonstrate that the expression of Osk protein is dependent upon the orb gene. In strong orb mutants, Osk protein expression is undetectable, while in the hypomorphic mutant, orbmel, little or no on-site expression of Osk protein at the posterior pole is observed. The defects in Osk protein accumulation inorb mutant ovaries are correlated with a reduction in the length of the osk poly(A) tails. We show that osk mRNA is in immunoprecipitable complexes with Orb protein in ovaries and that the osk 3′ UTR can be UV cross-linked to Orb protein in ovarian extracts. These data suggest that Orb is required to activate the translation of osk mRNA and at that this may be accomplished by a mechanism similar to that used by the Xenopus CPEB protein to control translation of “masked” mRNAs.

CPEB is a specificity factor that mediates cytoplasmic polyadenylation during Xenopus oocyte maturation

The translational activation of several maternal mRNAs during Xenopus oocyte maturation is stimulated by cytoplasmic poly(A) elongation, which requires the uridine-rich cytoplasmic polyadenylation element (CPE) and the hexanucleotide AAUAAA. Here, we have enriched a CPE-binding protein (CPEB) by single-step RNA affinity chromatography, have obtained a CPEB cDNA, and have assessed the role of CPEB in cytoplasmic polyadenylation. The 62 kDa CPEB contains two RNA recognition motifs, and within this region, it is 62% identical to orb, an oocyte-specific RNA-binding protein from Drosophila. CPEB mRNA and protein are abundant in oocytes and are not detected in embryos beyond the gastrula stage. During oocyte maturation, CPEB is phosphorylated at a time that corresponds with the induction of polyadenylation. Immunodepletion of CPEB from polyadenylation-proficient egg extracts renders them incapable of adenylating exogenous RNA. Partial restoration of polyadenylation in depleted extracts is achieved by the addition of CPEB, thus demonstrating that this protein is required for cytoplasmic polyadenylation.