CPEB binding to RNA and polyadenylation

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). The Xenopus oocyte CPE binding protein (CPEB) is essential for the cytoplasmic polyadenylation of B4 RNA. CPEB also binds the CPEs of G10, c-mos, cdk2, cyclins A1, B1 and B2 mRNAs. 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. These 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 (Stebbins-Boaz, 1996).

CPEB is an RNA binding protein that interacts with the maturation-type cytoplasmic polyadenylation element (CPE) (consensus UUUUUAU) to promote polyadenylation and translational activation of maternal mRNAs in Xenopus laevis. CPEB, which is conserved from mammals to invertebrates, is composed of three regions: an amino-terminal portion with no obvious functional motif; two RNA recognition motifs (RRMs), and a cysteine-histidine region that is reminiscent of a zinc finger. In this study, the physical properties of CPEB required for RNA binding have been examined. CPEB can interact with RNA as a monomer; phosphorylation, which modifies the protein during oocyte maturation, has little effect on RNA binding. Deletion mutations of CPEB have been overexpressed in Escherichia coli and used in a series of RNA gel shift experiments. Although a full-length and a truncated CPEB that lacks 139 amino-terminal amino acids avidly bind CPE-containing RNA , proteins that have either RRM deleted bind RNA much less efficiently. CPEB that has had the cysteine-histidine region deleted has no detectable capacity to bind RNA. Single alanine substitutions of specific cysteine or histidine residues within this region also abolish RNA binding, pointing to the importance of this highly conserved domain of the protein. Chelation of metal ions by 1,10-phenanthroline inhibits the ability of CPEB to bind RNA; however, RNA binding is restored if the reaction is supplemented with zinc. CPEB also binds other metals such as cobalt and cadmium, but these destroy RNA binding. These data indicate that the RRMs and a zinc finger region of CPEB are essential for RNA binding (Hake, 1999).

Cytoplasmic polyadenylylation is an essential process that controls the translation of maternal mRNAs during early development and depends on two cis elements in the 3' untranslated region: the polyadenylylation hexanucleotide AAUAAA and a U-rich cytoplasmic polyadenylylation element (CPE). In searching for factors that could mediate cytoplasmic polyadenylylation of mouse c-mos mRNA, which encodes a serine/threonine kinase necessary for oocyte maturation, the mouse homolog of CPEB was isolated. This protein binds to the CPEs of a number of mRNAs in Xenopus oocytes and is required for their polyadenylylation. Mouse CPEB (mCPEB) is a 62-kDa protein that binds to the CPEs of c-mos mRNA. mCPEB mRNA is present in the ovary, testis, and kidney; within the ovary, this RNA is restricted to oocytes. mCPEB shows 80% overall identity with its Xenopus counterpart, with a higher homology in the carboxyl-terminal portion, which contains two RNA recognition motifs and a cysteine/histidine repeat. Proteins from arthropods and nematodes are also similar to this region, suggesting an ancient and widely used mechanism to control polyadenylylation and translation (Gebauer, 1996).

CPEB homologs: Protein interactions

In Xenopus, the CPE is a bifunctional 3' UTR sequence that maintains maternal mRNA in a dormant state in oocytes and activates polyadenylation-induced translation during oocyte maturation. CPEB, which binds the CPE and stimulates polyadenylation, interacts with a new factor termed maskin. Maskin contains a peptide sequence that is conserved among elF-4E-binding proteins. Affinity chromatography demonstrates that CPEB, maskin, and elF-4E reside in a complex in oocytes. Yeast two-hybrid analyses indicate that CPEB and maskin bind directly, as do maskin and elF-4E. While CPEB and maskin remain together during oocyte maturation, the maskin-elF-4E interaction is substantially reduced. The dissolution of this complex may result in the binding of elF-4 to elF-4G and the translational activation of CPE-containing mRNAs (Stebbins-Boaz, 2000).

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, it has been 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 have shown in vivo interaction between Xenopus Pumilio and CPE-binding protein (CPEB: Drosophila homolog Orb), 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 (Nakahata, 2001).

The actual biological roles of XPum are completely unknown at present, but it can be speculated that XPum plays an important role in translational control of cyclin B1 mRNA, as in Drosophila. CPEB directly binds to maskin, a protein that can also bind directly to the cap-binding translation initiation factor elF-4E, which leads to translational repression. The dissociation of maskin from elF-4E allows elF-4G to bind to elF-4E, which brings elF-3 and the 40 S ribosomal subunit to the mRNA to initiate translation via cap-ribose methylation. Recent studies have also shown that a progesterone-induced early phosphorylation of CPEB at serine 174 is catalyzed by Eg2 and that this phosphorylation recruits cleavage and polyadenylation specificity factor into an active cytoplasmic polyadenylation complex. Thus, CPEB plays a key role in both translational repression and activation of mRNAs stored in oocytes. XPum is physically associated with CPEB in oocytes. In cooperation with CPEB, XPum may control the CPEB/maskin-mediated translational masking and unmasking to assure the highly coordinated successive translational activation of masked mRNAs during oocyte maturation. Further studies are required to understand the biological significance of the interactions among XPum, CPEB, and cyclin B1 mRNA, as well as to elucidate the functions of XPum in oocytes (Nakahata, 2001).

Activity-dependent local translation of dendritic mRNAs is one process that underlies synaptic plasticity. Several of the factors known to control polyadenylation-induced translation in early vertebrate development [cytoplasmic polyadenylation element-binding protein (CPEB), maskin, poly(A) polymerase (see hiiragi), 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 elucidate an axis connecting the NMDA receptor to localized protein synthesis at synapses (Huang, 2002).

XGef was isolated in a screen for proteins interacting with CPEB, a regulator of mRNA translation in early Xenopus development. XGef is a Rho-family guanine nucleotide exchange factor and activates Cdc42 in mammalian cells. Endogenous XGef (58 kDa) interacts with recombinant CPEB, and recombinant XGef interacts with endogenous CPEB in Xenopus oocytes. Injection of XGef antibodies into stage VI Xenopus oocytes blocks progesterone-induced oocyte maturation and prevents the polyadenylation and translation of c-mos mRNA; injection of XGef rescues these events. Overexpression of XGef in oocytes accelerates progesterone-induced oocyte maturation and the polyadenylation and translation of c-mos mRNA. Overexpression of a nucleotide exchange deficient version of XGef, which retains the ability to interact with CPEB, no longer accelerates oocyte maturation or Mos synthesis, suggesting that XGef exchange factor activity is required for the influence of overexpressed XGef on oocyte maturation. XGef overexpression continues to accelerate c-mos polyadenylation in the absence of Mos protein, but does not stimulate MAPK phosphorylation, MPF activation, or oocyte maturation, indicating that XGef may function through the Mos pathway to influence oocyte maturation. These results suggest that XGef may be an early acting component of the progesterone-induced oocyte maturation pathway (Reverte, 2003).

CPEB homologs in invertebrates: translational control

Cytoplasmic polyadenylation element binding (CPEB) proteins bind to and regulate the translation of specific mRNAs. CPEBs from Xenopus, Drosophila, and Spisula participate in oogenesis. The biological roles of all identifiable CPEB homologs in a single organism, Caenorhabditis elegans, have been examined. Four homologs are apparent in the C. elegans genome: cbp-1, cpb-2, cpb-3, and fog-1. Surprisingly, two homologs, CPB-1 and FOG-1, have key functions in spermatogenesis and are dispensable for oogenesis. CPB-2 and CPB-3 also appear not to be required for oogenesis. CPB-1 is essential for progression through meiosis: cpb-1(RNAi) spermatocytes fail to undergo the meiotic cell divisions. CPB-1 protein is present in the germ line just prior to overt spermatogenesis; once sperm differentiation begins, CPB-1 disappears. CPB-1 physically interacts with FBF, another RNA-binding protein and 3' UTR regulator. In addition to its role in controlling the sperm/oocyte switch, FBF also appears to be required for spermatogenesis, consistent with its interaction with CPEB. A second CPEB homolog, FOG-1, is required for specification of the sperm fate. The fog-1 gene produces fog-1(L) and fog-1(S) transcripts. The fog-1(L) RNA is enriched in animals making sperm and is predicted to encode a larger protein; fog-1(S) RNA is enriched in animals making oocytes and is predicted to encode a smaller protein. The relative abundance of the two mRNAs is controlled temporally during germ-line development and by the sex determination pathway in a fashion that suggests that the fog-1(L) species encodes the active form. In sum, these results demonstrate that, in C. elegans, two CPEB proteins have distinct functions in the germ line, both in spermatogenesis: FOG-1 specifies the sperm cell fate and CPB-1 executes that decision (Luitjens, 2000).

Xenopus CPEB and C. elegans CPB-1 both control progression through meiosis. In Xenopus, CPEB is required for meiotic maturation during oogenesis. Specifically, CPEB activates translation of c-mos mRNA, which encodes a MAP kinase kinase kinase. Elevation of c-MOS kinase activity triggers a MAPK cascade that leads to activation of MPF and meiotic maturation. Drosophila orb is also required for oogenesis, but its role in the meiotic cell cycle is less clear. Like Xenopus CPEB, the C. elegans cpb-1 gene controls progression through meiosis, but it does so in spermatogenesis rather than oogenesis. Animals deficient for cpb-1 activity possess spermatocytes that have failed to execute either meiosis I or meiosis II. The CPB-1 protein is restricted to a region in which germ cells have entered meiotic prophase I, but have not yet begun to express an early sperm differentiation marker. This arrest of cpb-1(RNAi) animals in early spermatogenesis and the localization of CPB-1 to early meiotic cells together suggest that CPB-1 is critical for initiating meiotic maturation during spermatogenesis. Furthermore, CPB-1 is localized to the cytoplasm, consistent with a role in post-transcriptional regulation. It is suggested that CPB-1 protein regulates the translation of mRNAs that are required for progression through spermatogenesis. The target of this regulation is unlikely to be c-mos per se, since no c-mos homolog has been detected in C. elegans, but could be another component of the MAP kinase cascade (Luitjens, 2000).

Like frog CPEB and clam p82, CPB-1 abundance decreases as meiosis proceeds, suggesting a conserved mode of control. In C. elegans spermatocytes, CPB-1 protein is abundant during first meiotic prophase, but drops precipitously once meiosis resumes. In frogs and clams, the level of CPEB similarly decreases upon oocyte maturation, as the translation of several mRNAs increases. Three possible models are envisioned for how CPB-1 controls progression of meiosis in spermatogenesis. First, CPB-1 might repress one or more critical mRNAs (e.g., functional analogs of c-mos) that promote the completion of meiosis; relief of that repression allows meiosis to proceed. Indeed, recent studies suggest that specific phosphorylation of CPEB relieves repression of c-mos in Xenopus. Second, CPB-1 may activate one or more mRNAs that initiate meiosis. Finally, CPB-1 might initially repress mRNAs required for meiotic maturation and subsequently activate them. These scenarios all are consistent with destruction of CPB-1 once maturation has begun; having completed its role in launching meiotic maturation, its persistence might interfere with subsequent events in development (Luitjens, 2000 and references therein).

The fog-1 gene is essential for specification of germ cells to the sperm fate; in fog-1 null mutants, germ cells that normally would differentiate as sperm are instead sexually transformed into oocytes. Thus, fog-1 is a germ line-specific sex-determining gene. The identification of FOG-1 as a CPEB homolog suggests that a translational regulator can specify a specific cell fate. By genetic criteria, fog-1 acts at the end of the sex-determination pathway to specify germ cells as sperm rather than oocytes. It is suggested that FOG-1 controls gene expression and cell fate at the translational level, by analogy to CPEB functions in other species. Although other translational regulators can influence many cell fates broadly the cell fate transformation observed in animals lacking FOG-1 is highly specific, suggesting a simple binary switch that determines whether a cell becomes a sperm or an oocyte. Direct tests of this hypothesis will require ectopic expression of FOG-1 to determine whether it is sufficient to drive cells to become sperm (Luitjens, 2000).

The fog-1 gene produces at least three transcripts: a longer fog-1(L) transcript and two shorter fog-1(S) transcripts. Both fog-1(L) and fog-1(S) mRNAs encode the conserved RRM and C/H motifs typical of CPEBs, but compared with the fog-1(S) transcripts, fog-1(L) encodes an additional 214 or 269 amino acids in the N-terminal region of the protein. The two fog-1(S) mRNAs contain in-frame methionines and are predicted to encode N-terminally truncated forms of FOG-1. Two experiments suggest that fog-1(L) encodes the active form of FOG-1 that is required for the specification of spermatogenesis. (1) In wild-type animals, the fog-1(L) mRNA is abundant during midlarval development when fog-1 activity is required to promote spermatogenesis and is much reduced in later development once that activity is no longer required; in contrast, fog-1(S) is not detectable during midlarval development and increases later when fog-1 activity is no longer required. (2) In a mutant that produces only sperm in an otherwise female animal, fog-1(L) predominates, whereas in a mutant that produces only oocytes, fog-1(S) is more abundant. One simple interpretation is that the longer mRNA produces active FOG-1 protein and the shorter one produces either nonfunctional FOG-1 or a protein that antagonizes the active form (Luitjens, 2000).

In frogs, flies, and clams, CPEBs are essential during oogenesis. By contrast, in C. elegans, cpb-1 and fog-1 control spermatogenesis and have no essential role in oogenesis: oocytes that lack fog-1, cpb-1, or both are functional. Intriguingly, cpb-3 mRNA is enriched during oogenesis and its predicted protein, CPB-3, is most closely related by sequence to Xenopus CPEB and Drosophila Orb. Nonetheless, no effect on oogenesis was observed by cpb-3(RNAi), either alone or in combination with the other homologs. Furthermore, RNAi directed against the remaining CPEB homolog cpb-2, which is actually enriched during spermatogenesis, has no dramatic effect on any tissue, and RNAi directed against cpb-1, cpb-2, cpb-3 into a fog-1 mutant, which is predicted to remove all CPEB activity from the animal, results in only the fog-1 defect. Thus, no effect on oogenesis could be detected by removing one or more CPEBs singly or in combination. Because these are negative results and rely on RNAi to reduce expression, they are inconclusive. However, RNAi against other CPEB homologs (fog-1 and cpb-1) is effective, because fog-1(RNAi) mimics a fog-1 null mutant, and cpb-1(RNAi) removes CPB-1 protein and yields a completely penetrant and dramatic effect on spermatogenesis (Luitjens, 2000).

With this caveat, it is suggested that C. elegans CPEBs are not essential for oogenesis, and meiosis of oocytes in particular. In light of CPEB functions in the oocytes of other organisms, what does this mean? Two possible explanations are envisioned. One possibility is that the regulatory machinery controlling meiosis during oogenesis in flies, frogs, and clams has been switched to control meiosis during spermatogenesis in C. elegans. This view gains credibility from the finding that the role of the DAZ RNA-binding protein is reversed in C. elegans compared with flies and humans: In C. elegans, DAZ-1 is required for oogenesis but not spermatogenesis, whereas Drosophila DAZ, called boule, is essential for spermatogenesis but not oogenesis, and the human DAZ gene cluster is implicated in spermatogenesis. If this idea is right, a sex reversal in the regulatory machinery may have occurred during evolution of C. elegans. An alternative and perhaps more likely explanation is that CPEB and DAZ RNA-binding proteins were used in a common metazoan ancestor to regulate common germ-line processes (such as meiosis) during both oogenesis and spermatogenesis. During evolution, the functions of these genes may have become more specialized. Because much work to date has focused on the role of CPEB in oogenesis, sperm functions may have been missed in other organisms. In particular, Orb protein is present in Drosophila males. Although orb mutant males are fertile, a second CPEB homolog has been identified in the fly genome and could play a critical role in spermatogenesis. Similarly, CPEB mRNAs are found in mouse testes. These findings suggest that spermatogenic functions of CPEB homologs may be common (Luitjens, 2000 and references therein).

A working model depicting the role of CPEBs during C. elegans spermatogenesis has been presented. FOG-1 is essential for the initial specification of a germ cell to the sperm fate. Once that fate has been established, CPB-1 is required to initiate maturation of the primary spermatocyte. FOG-1 and CPB-1 therefore act at two nodal decision points during spermatogenesis. Because CPEB can act as both a repressor and activator, it is suggested that this RNA-binding protein may be particularly well-suited to the role of a molecular switch, consistent with its being used twice in that fashion during C. elegans spermatogenesis (Luitjens, 2000).

CPB-1 may play a role in spermatogenesis beyond its function in promoting progression through meiosis. This hypothesis is based on the physical interaction of CPB-1 with FBF. The CPB-1/FBF interaction occurs independently of RNA binding, and it is mediated by the first 80 amino acids of CPB-1. FBF is an RNA-binding protein that controls the switch from spermatogenesis to oogenesis and is a homolog of Drosophila Pumilio. It binds to a regulatory element in the fem-3 3' UTR and represses its activity. For this process, FBF interacts with NOS-3 protein. FBF is likely to be required for some step in spermatogenesis in addition to its previously identified role in the sperm/oocyte switch. Because male gametes produced in the absence of CPB-1 arrest early in spermatogenesis, it cannot be assessed whether cpb-1 is also required for subsequent steps. However, one simple model is suggested -- that cpb-1 functions in an fbf-independent fashion for progression through meiosis but then binds to and cooperates with fbf to promote a later step in spermatogenesis. Similarly, orb has multiple roles during oogenesis in Drosophila. The interaction of FBF with both CPB-1 and NOS-3 for apparently distinct functions underscores the possibility of a combinatorial network of translational regulators in germ-line development (Luitjens, 2000).

In other organisms, CPEB homologs bind to and regulate multiple mRNAs. The identification of mRNA targets for both FOG-1 and CPB-1 should help illuminate the mechanism by which the CPEB family of regulators control spermatogenesis. Furthermore, analysis of the targets may also reveal how a family of translational regulators diverged during evolution to adopt novel biological roles (Luitjens, 2000).

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. Fertilization of the oocyte leads to rapid polysomal recruitment of the abundant cyclin and ribonucleotide reductase mRNAs at about the time they undergo cytoplasmic polyadenylation. Clam p82, a 3' UTR RNA-binding protein, and a member of the CPEB (cytoplasmic polyadenylation element binding protein) family, functions as a translational masking factor in oocytes and as a polyadenylation factor in fertilized eggs. In meiotically maturing clam oocytes, p82/CPEB is rapidly phosphorylated on multiple residues to a 92-kDa apparent size, prior to its degradation during the first cell cleavage. The protein kinase(s) that phosphorylates clam p82/CPEB have been examined using a clam oocyte activation cell-free system that responds to elevated pH, mirroring the pH rise that accompanies fertilisation. p82/CPEB phosphorylation requires Ca2+ (<100 microM), in addition to raised pH. Examination of the calcium dependency combined with the use of specific inhibitors implicates the combined and independent actions of cdc2 and MAP kinases in p82/CPEB phosphorylation. Calcium is necessary for both the activation and the maintenance of MAP kinase, whose activity is transient in vitro, as in vivo. While cdc2 kinase plays a role in the maintenance of MAP kinase activity, it is not required for the activation of MAP kinase. A model of clam p82/CPEB phosphorylation is proposed in which MAP kinase initially phosphorylates clam p82/CPEB, at a minor subset of sites that does not alter its migration, and cdc2 kinase is necessary for the second wave of phosphorylation that results in the large mobility size shift of clam p82/CPEB. The possible roles of phosphorylation for the function and regulation of p82/CPEB are discussed (Katsu, 1999).

In the transcriptionally inert maturing oocyte and early embryo, control of gene expression is largely mediated by regulated changes in translational activity of maternal mRNAs. Some mRNAs are activated in response to poly(A) tail lengthening; in other cases activation results from de-repression of the inactive or masked mRNA. The 3' UTR cis-acting elements that direct these changes are defined, principally in Xenopus and mouse, and the study of their trans-acting binding factors is just beginning to shed light on the mechanism and regulation of cytoplasmic polyadenylation and translational masking. In the marine invertebrate, Spisula solidissima, the timing of activation of three abundant mRNAs (encoding cyclin A and B and the small subunit of ribonucleotide reductase, RR) in fertilized oocytes correlates with their cytoplasmic polyadenylation. However, in vitro, mRNA-specific unmasking occurs in the absence of polyadenylation. p82, a protein defined as selectively binding the 3' UTR masking elements, is a homolog of Xenopus CPEB (cytoplasmic polyadenylation element binding protein). The elements that support polyadenylation in clam egg lysates include multiple U-rich CPE-like motifs as well as the nuclear polyadenylation signal AAUAAA. This represents the first detailed analysis of invertebrate cis-acting cytoplasmic polyadenylation signals. Polyadenylation activity correlates with p82 binding in wild-type and CPE-mutant RR 3' UTR RNAs. Moreover, since anti-p82 antibodies specifically neutralize polyadenylation in egg lysates, it is concluded that clam p82 is a functional homolog of Xenopus CPEB, and plays a positive role in polyadenylation. Anti-p82 antibodies also result in specific translational activation of masked mRNAs in oocyte lysates, lending support to the model of clam p82 as a translational repressor. It is proposed therefore that clam p82/CPEB has dual functions in masking and cytoplasmic polyadenylation (Minshall, 1999).

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 as translational masking elements located in the approximate centers in the 3' UTRs of these mRNAs. 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. 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 (Walker, 1999).

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. A CPE-binding protein (CPEB) has been enriched by single-step RNA affinity chromatography; a CPEB cDNA has been obtained, and the role of CPEB in cytoplasmic polyadenylation has been assessed. The 62 kDa CPEB contains two RNA recognition motifs, and within this region, CPEB 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 (Hake, 1994).

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). Actin mRNA from pleural ganglion sensory neurons becomes polyadenylated during long-term facilitation produced by treatment with serotonin or 8-bromo cAMP. Two isoforms of CPEB (ApCPEB77 and ApCEPB49) were cloned 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, it is suggested that the translation of actin message in nerve endings is up-regulated by polyadenylation to grow new synapses (Liu, 2003).

The expression of CPEB proteins is sequentially regulated during zebrafish oogenesis and embryogenesis

In many species there is little transcription in the mature oocyte, and zygotic transcription does not begin immediately after fertilization; this is the case in zebrafish, where zygotic transcription is not initiated until the mid-blastula transition. Thus the production of new proteins during oogenesis and early embryogenesis is dependent on the translation of maternal mRNAs. In a growing number of species, the translation of key maternal transcripts is coupled to their cytoplasmic polyadenylation. One family of RNA-binding proteins implicated in this process are the cytoplasmic polyadenylation element (CPE)-binding proteins (CPEBs), which bind to a sequence in the 3'- untranslated regions (3'-UTRs) of regulated transcripts and mediate their storage/repression or translation. In several species, there is evidence for two classes of CPEBs, a larger oocyte-type and a smaller CPEB that functions during embryogenesis. This appears to be the case in zebrafish as well, and this study provides evidence suggesting that the oocyte-type CPEB (zorba) regulates the translation of the embryonic-type (ElrA) by keeping the ElrA transcript in storage until fertilization. When zorba levels fall, the ElrA protein is then produced and available to regulate the translation of additional mRNAs during embryogenesis. A potential target of ElrA, the maternal mRNA for hnRNPab, was identified that is a potential homolog of the Drosophila gene squid, whose product plays a role in patterning the Drosophila oocyte and embryo. These data suggest that during zebrafish embryogenesis, cytoplasmic polyadenylation mediates a cascade of translational control whose final targets play central patterning roles during embryogenesis (O'Connell, 2014).

A fly trap mechanism provides sequence-specific RNA recognition by CPEB proteins

Cytoplasmic changes in polyA tail length is a key mechanism of translational control and is implicated in germline development, synaptic plasticity, cellular proliferation, senescence, and cancer progression. The presence of a U-rich cytoplasmic polyadenylation element (CPE) in the 3' untranslated regions (UTRs) of the responding mRNAs gives them the selectivity to be regulated by the CPE-binding (CPEB) family of protein, which recognizes RNA via the tandem RNA recognition motifs (RRMs). This study reports the solution structures of the tandem RRMs of two human paralogs (CPEB1 and CPEB4) in their free and RNA-bound states. The structures reveal an unprecedented arrangement of RRMs in the free state that undergo an original closure motion upon RNA binding that ensures high fidelity. Structural and functional characterization of the ZZ domain (zinc-binding domain) of CPEB1 suggests a role in both protein-protein and protein-RNA interactions. Together with functional studies, the structures reveal how RNA binding by CPEB proteins leads to an optimal positioning of the N-terminal and ZZ domains at the 3' UTR, which favors the nucleation of the functional ribonucleoprotein complexes for translation regulation (Afroz, 2014).

CPEB homologs in vertebrates: oocyte maturation and translational control

During Xenopus oocyte maturation, poly(A) elongation controls the translational recruitment of specific mRNAs that possess a CPE (cytoplasmic polyadenylation element). To investigate the activation of polyadenylation, oocyte extracts that are not normally competent for polyadenylation were used. Addition of cell lysates containing baculovirus-expressed cyclin to these extracts induces the polyadenylation of exogenous B4 RNA. The involvement of p34cdc2 kinase in cyclin-mediated polyadenylation was demonstrated by p13-Sepharose depletion; removal of the kinase from oocyte extracts with this affinity matrix abolishes polyadenylation activation. Reintroduction of cell lysates containing baculovirus-expressed p34cdc2, however, completely restores this activity. To identify factors of the polyadenylation apparatus that might be responsible for the activation, UV cross-linking was employed and a 58-kD protein was identified that binds the B4 CPE in oocyte extracts. In polyadenylation-proficient egg extracts, this protein has a slower electrophoretic mobility, which suggests a post-translational modification. A similar size shift of the protein is evident in oocyte extracts supplemented with lysates containing baculovirus-expressed cyclin and p34cdc2. This size shift, which is reversed by treatment with acid phosphatase, coincides temporally with cyclin-induced polyadenylation activation. It is proposed that p34cdc2 kinase activity leads to the phosphorylation of the 58-kD CPE-binding protein and that this event is crucial for the cytoplasmic polyadenylation that occurs during oocyte maturation (Paris, 1991).

The animal/vegetal axis of the zebrafish egg is established during oogenesis, but the molecular factors responsible for its specification are unknown. As a first step towards the identification of such factors, the first demonstration of asymmetrically distributed maternal mRNAs in the zebrafish oocyte is presented here. To date, three classes of mRNAs have been identifed, characterized by the stage of oocyte maturation at which they concentrate to the future animal pole. One of these mRNAs has been characterized: zorba. Zorba encodes a homolog of the Drosophila Orb and Xenopus CPEB RNA-binding proteins; it belongs to the group of earliest mRNAs to localize at the animal pole, where it becomes restricted to a tight subcortical crescent at stage III of oogenesis. This localization is independent of microtubules and microfilaments; the distribution of Zorba protein parallels that of its mRNA (Bally-Cuif, 1998).

During Xenopus oocyte maturation, cyclin B1 mRNA is translationally activated by cytoplasmic polyadenylation. This process is dependent on cytoplasmic polyadenylation elements (CPEs) in the 3' untranslated region (UTR) of the mRNA. To determine whether a titratable factor might be involved in the initial translational repression (masking) of this mRNA, high levels of cyclin B1 3' UTR were injected into oocytes. While this treatment has no effect on the poly(A) tail length of endogenous cyclin B1 mRNA, it induces cyclin B1 synthesis. A mutational analysis reveals that the most efficient unmasking element in the cyclin 3' UTR is the CPE. However, other U-rich sequences that resemble the CPE in structure, but that do not bind the CPE-binding polyadenylation factor CPEB, fail to induce unmasking. When fused to the chloramphenical acetyl transferase (CAT) coding region, the cyclin B1 3' UTR inhibits CAT translation in injected oocytes. In addition, a synthetic 3' UTR containing multiple copies of the CPE also inhibits translation, and does so in a dose-dependent manner. Furthermore, efficient CPE-mediated masking requires cap-dependent translation. During the normal course of progesterone-induced maturation, cytoplasmic polyadenylation is necessary for mRNA unmasking. A model to explain how cyclin B1 mRNA masking and unmasking could be regulated by the CPE is presented (de Moor, 1999).

Full-grown Xenopus oocytes arrest at the G2/M border of meiosis I. Progesterone breaks this arrest, leading to the resumption of the meiotic cell cycles and maturation of the oocyte into a fertilizable egg. In these oocytes, progesterone interacts with an unidentified surface-associated receptor, which induces a non-transcriptional signaling pathway that stimulates the translation of dormant c-mos messenger RNA. Mos, a mitogen-activated protein (MAP) kinase kinase kinase, indirectly activates MAP kinase, which in turn leads to oocyte maturation. The translational recruitment of c-mos and several other mRNAs is regulated by cytoplasmic polyadenylation, a process that requires two 3' untranslated regions, the cytoplasmic polyadenylation element (CPE) and the polyadenylation hexanucleotide AAUAAA. Although the signaling events that trigger c-mos mRNA polyadenylation and translation are unclear, they probably involve the activation of CPEB, the CPE binding factor. An early site-specific phosphorylation of CPEB is essential for the polyadenylation of c-mos mRNA and its subsequent translation, and for oocyte maturation. This selective, early phosphorylation of CPEB is catalysed by Eg2, a member of the Aurora family of serine/threonine protein kinases (Mendez, 2000a).

The release of Xenopus oocytes from prophase I arrest is largely driven by the cytoplasmic polyadenylation-induced translation of dormant maternal mRNAs. Cytoplasmic polyadenylation requires two cis-acting sequences and three core polyadenylation factors. The cis elements are the hexanucleotide AAUAAA, which is also essential for nuclear pre-mRNA cleavage and polyadenylation, and an upstream U-rich sequence called the cytoplasmic polyadenylation element (CPE). The core factors include CPEB, an RNA recognition motif (RRM), and zinc finger-containing protein that has a strong binding preference for the CPE, and a cytoplasmic form of cleavage and polyadenylation specificity factor (CPSF), which interacts with AAUAAA. In contrast to the nuclear form of CPSF, which consists of four subunits of 160 kDa, 100 kDa, 70 kDa, and 30 kDa, the cytoplasmic form contains the 160 kDa (or a functional equivalent thereof), the 100 kDa, and the 30 kDa subunits, but lacks the 70 kDa species. The third core factor is poly(A) polymerase (PAP). Based on the events associated with nuclear polyadenylation, the function of CPSF is probably to recruit PAP to the end of the mRNA. The molecular function of CPEB in cytoplasmic polyadenylation has not been elucidated. Although all the factors required for cytoplasmic polyadenylation are present in oocytes, they become active only at the onset of maturation. In response to progesterone-stimulated maturation, CPEB undergoes an early phosphorylation at a single residue that is essential for polyadenylation. This phosphorylation event is catalyzed by Eg2, a member of the Aurora family of Ser/Thr protein kinases. In this study the molecular function of this CPEB phosphorylation has been investigated (Mendez, 2000b and references therein).

The most proximal stimulus for polyadenylation is Eg2-catalyzed phosphorylation of CPEB serine 174. 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 (Mendez, 2000b).

There are some key similarities between cytoplasmic and nuclear polyadenylation. In the nucleus, the affinity of CPSF for the AAUAAA is very low and must be stabilized by other RNA binding proteins. These other factors include CstF, which binds the GU-rich element downstream of the AAUAAA element, and U1A protein, which binds an upstream U-rich element present in at least one viral mRNA. While cytoplasmic polyadenylation has no downstream element, the upstream element is certainly equivalent to the CPE, and the function of CPEB is analogous to that shown for U1A, to stabilize the binding of CPSF to the AAUAAA. However, while there is no evidence that the CPSF-stabilizing activity of U1A or CstF can be modulated by phosphorylation, that of CPEB is regulated by Eg2-catalyzed phosphorylation (Mendez, 2000b and references therein).

In spite of these similarities, there are also some key differences that distinguish nuclear from cytoplasmic polyadenylation. (1) While both processes require CPSF, the cytoplasmic form of this multiprotein complex lacks the 70 kDa subunit. While the function of the 70 kDa protein in nuclear polyadenylation is unclear, it is possible that cytoplasmic CPSF contains a functional substitute, or that cytoplasmic polyadenylation has dispensed altogether with any need for a 70 kDa protein-like activity. (2) While nuclear polyadenylation is strongly coupled to pre-mRNA cleavage, the cytoplasmic process is not associated with any cleavage event. (3) Nuclear pre-mRNA polyadenylation occurs on virtually all mRNAs whereas cytoplasmic polyadenylation takes place on just those few species that contain a CPE. (4) cdc2-catalyzed phosphorylations of PAP inhibit nuclear pre-mRNA polyadenylation as cells enter M phase; cytoplasmic polyadenylation, in contrast, is induced as cells enter M phase (i.e., maturation) (Mendez, 2000b and references therein).

While Eg2-catalyzed phosphorylation of CPEB probably occurs on all CPE-containing mRNAs, there are additional processes that control the timing of polyadenylation. That is, the mRNA encoding the serine/threonine kinase Mos is the first mRNA known to undergo polyadenylation-induced translation following progesterone stimulation. Mos synthesis activates the M phase promoting factor (cyclinB/cdc2), which in turn induces the polyadenylation of other mRNAs later during maturation. What distinguishes early from late adenylating mRNAs? While additional mRNA-specific regulatory proteins could certainly affect the timing of polyadenylation, the number of CPEs appears to have an important influence. For example, the early adenylating c-mos mRNA has a single CPE while the late adenylating cyclin B1, histone B4, and cyclin A1 mRNAs have two CPEs. If one of the cyclin B1 CPEs is removed, the message adenylates early and is Mos (and cdc2) independent. It is possible that two CPEs, and thus two CPEBs, could actually inhibit the formation of an active polyadenylation complex by, for example, dimerizing or interacting with another factor that bridges the two CPEBs. While such a configuration of factors could preclude a CPEB-CPSF interaction early during maturation, a late cdc2-mediated partial destruction of CPEB could liberate one of the molecules to interact with CPSF and promote polyadenylation (Mendez, 2000b).

The polyadenylation complex includes other factors, which, while not directly influencing polyadenylation, mediate translational regulatory events associated with polyadenylation. The translational repression (masking) of CPE-containing mRNAs prior to progesterone stimulation appears to be mediated by maskin, a CPEB-associated protein that also binds the cap binding protein eIF4E. The maskin-eIF4E interaction prevents the association of eIF4G with eIF4E, thereby inhibiting translation. The dissociation of the maskin-eIF4E complex, which presumably allows eIF4G to bind eIF4E, is coincident with, and possibly the result of, cytoplasmic polyadenylation (Mendez, 2000b and references therein).

Translational activation of several dormant mRNAs in vertebrate oocytes is mediated by cytoplasmic polyadenylation, a process controlled by the cytoplasmic polyadenylation element (CPE) and its binding protein CPEB. The translation of CPE-containing mRNAs does not occur en masse at any one time, but instead is temporally regulated. In Xenopus, partial destruction of CPEB controls the temporal translation of CPE-containing mRNAs. While some mRNAs, such as the one encoding Mos, are polyadenylated at prophase I, the polyadenylation of cyclin B1 mRNA requires the partial destruction of CPEB that occurs at metaphase I. CPEB destruction is mediated by a PEST box and Cdc2-catalyzed phosphorylation, and is essential for meiotic progression to metaphase II. CPEB destruction is also necessary for mitosis in the early embryo. These data indicate that a change in the CPEB:CPE ratio is necessary to activate mRNAs at metaphase I and drive the cells' entry into metaphase II (Mendez, 2002).

In maturing mouse oocytes, protein synthesis is required for meiotic maturation subsequent to germinal vesicle breakdown (GVBD). While the number of different proteins that must be synthesized for this progression to occur is unknown, at least one of them appears to be cyclin B1, the regulatory subunit of M-phase-promoting factor. The mechanism of cyclin B1 mRNA translational control during mouse oocyte maturation has been investigated. TU-rich cytoplasmic polyadenylation element (CPE), a cis element in the 3' UTR of cyclin B1 mRNA, mediates translational repression in GV-stage oocytes. The CPE is also necessary for cytoplasmic polyadenylation, which stimulates translation during oocyte maturation. The injection of oocytes with a cyclin B1 antisense RNA, which probably precludes the binding of a factor to the CPE, delays cytoplasmic polyadenylation as well as the transition from GVBD to metaphase II. CPEB (related to Drosophila Orb) is an RNA recognition motif and zinc finger-containing protein that has a strong specificity for the CPE. CPEB, which interacts with the cyclin B1 CPE and is present throughout meiotic maturation, becomes phosphorylated at metaphase I. These data indicate that CPEB is involved in both the repression and the stimulation of cyclin B1 mRNA and suggest that the phosphorylation of this protein could be involved in regulating its activity (Tay, 2000).

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, the mechanisms influencing the expression of CPEB during oogenesis and oocyte maturation have been examined. Through a comparison of CPEB mRNA levels, protein synthesis, and accumulation, it has been found 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, it has been demonstrated that CPEB degradation occurs via the proteasome pathway, most likely through ubiquitin-conjugated intermediates. In addition, degradation requires a 14 amino acid PEST domain (Reverte, 2001).

CPEB is an mRNA-binding protein that stimulates polyadenylation-induced translation of maternal mRNA once it is phosphorylated on Ser 174 or Thr 171 (species-dependent). Disruption of the CPEB gene in mice causes an arrest of oogenesis at embryonic day 16.5 (E16.5), when most oocytes are in pachytene of prophase I. CPEB undergoes Thr 171 phosphorylation at E16.5, but dephosphorylation at the E18.5, when most oocytes are entering diplotene. Although phosphorylation is mediated by the kinase aurora, the dephosphorylation is due to the phosphatase PP1. The temporal control of CPEB phosphorylation suggests a mechanism in which mRNA translation of CPE-containing messages is stimulated at pachytene and metaphase I (Tay, 2003).

The results presented here suggest a mechanism by which the translation of maternal mRNAs is differentially controlled during murine meiosis. As oogenesis progresses into pachytene, the kinase aurora is activated, perhaps by phosphorylation. The upstream kinase that phosphorylates aurora is unclear, although some evidence indicates that PKA is involved. Activated aurora then phosphorylates CPEB Thr 171, which stimulates the polyadenylation and translation of SCP1 and SCP3 mRNAs. The translation of other mRNAs might also be stimulated by CPEB at this time. SCP1 and SCP3 help form the synaptonemal complex, which is necessary for meiotic progression to diplotene. At diplotene, PP1 dephosphorylates and inactivates CPEB, an event that allows key CPE-containing mRNAs such as mos to accumulate but remain translationally dormant. As the fully grown (GV) oocytes begin to mature, aurora again becomes active and phosphorylates CPEB, which in turn induces the polyadenylation and translation of mos, and other mRNAs with encoded products that either stimulate maturation or lead to cytostatic factor (CSF)-mediated meta-phase II arrest (Tay, 2003).

Two additional points of upstream CPEB regulation should be considered. (1) Although aurora, in addition to CPEB, appears to be inactive in E18.5 diplotene oocytes (i.e., no T171 phosphorylation), it is plausible that the kinase is active at this time but its ability to phosphorylate CPEB is overcome by a very active PP1. To investigate this possibility, the phosphorylation experiments were perfomed except that E18.5 ovary extracts were supplemented with I-2, the PP1 inhibitor. Neither I-2-supplemented nor un-supplemented extracts supported T171 phosphorylation. Because I-2 inhibits dephosphorylation in the extracts, the lack of T171 phoshorylation can be attributed to inactive aurora rather than overriding PP1 activity. It is also interesting to note that PP1 has been suggested to inactivate aurora as well. Perhaps PP1 acts on a CPEB and aurora-containing complex to inactivate these proteins simultaneously at diplotene. (2) It is inferred that PP1, which is present in mouse oocytes, is also regulated; it is inactive during E16.5 (pachytene) when CPEB phosphorylation is robust but active at E18.5 (diplotene) to dephosphorylate CPEB. PP1 activity is regulated by a number of modulator proteins, some of which could function during oogenesis (Tay, 2003).

MAPK cascade couples maternal mRNA translation and degradation to meiotic cell cycle progression in mouse oocyte

Mammalian oocyte maturation depends on the translational activation of stored maternal mRNAs upon meiotic resumption. Cytoplasmic polyadenylation element binding protein-1 (CPEB1; see Drosophila Orb2) is a key oocyte factor that regulates maternal mRNA translation. However, the signal that triggers CPEB1 activation at the onset of mammalian oocyte maturation is not known. This study provides evidence that a mitogen-activated protein kinase (MAPK) cascade couples maternal mRNA translation to meiotic cell cycle progression in mouse oocytes, by triggering CPEB1 phosphorylation and degradation. Mutations of the phosphorylation sites or ubiquitin E3 ligase binding sites in CPEB1 have a dominant negative effect in oocytes, and mimic the phenotype of ERK1/2 (see Drosophila Rolled) knockout, by impairing spindle assembly and mRNA translation. Overexpression of the CPEB1-downstream translation activator DAZL (see Drosophila Boule) in ERK1/2-deficient oocytes partially rescued the meiotic defects, indicating that ERK1/2 is essential for spindle assembly, metaphase II arrest, and maternal-zygotic transition (MZT) primarily by triggering the translation of key maternal mRNAs. Taken together, ERK1/2-mediated CPEB1 phosphorylation/degradation is a major mechanism of maternal mRNA translational activation, and is crucial for mouse oocyte maturation and MZT (Sha, 2016).

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: (1) CPEB (Orb in Drosophila), an RNA binding protein whose association with the CPE specifies which mRNAs undergo polyadenylation; (2) cleavage and polyadenylation specificity factor (CPSF), a multifactor complex that interacts with the near-ubiquitous polyadenylation hexanucleotide AAUAAA, and (3) maskin, a CPEB and eIF4E binding protein whose regulation of initiation is governed by poly(A) tail length. Two new factors that are essential for polyadenylation are defined in this study. 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 (Bernard, 2004).

Maskin was initially identified as one of a few proteins that coimmunoprecipitated with CPEB. One of the other proteins was identified through a short peptide that had no match in the databases. A BLAST search, however, revealed this protein to be the Xenopus homolog of symplekin, whose counterpart in mammalian cells interacts with CPSF and cleavage stimulatory factor (CstF), components of the nuclear pre-mRNA cleavage and polyadenylation machinery. In yeast, the apparent symplekin homolog Pta1 is necessary for both cleavage and polyadenylation of nuclear pre-mRNA. In spite of these observations, the biochemical function of symplekin is unknown, although it has been suggested to act as a molecular platform upon which the nuclear polyadenylation machinery is assembled (Bernard, 2004 and references therein).

In this study, symplekin is demonstrated to reside in a cytoplasmic polyadenylation complex and directly contacts both CPSF and CPEB. The injection of symplekin antibody into oocytes or its addition to egg extracts inhibits polyadenylation. Immunodepletion of symplekin from egg extracts also disrupts polyadenylation; however, the introduction of recombinant symplekin to the depleted extracts fails to restore polyadenylation. Because symplekin immunodepletion also results in the loss of CPEB and CPSF from the extracts, this may explain why supplemental symplekin did not restore polyadenylation. Surprisingly, complementation of the depleted extract with symplekin, CPSF, and CPEB still did not stimulate polyadenylation. These observations suggested that yet another essential polyadenylation factor was codepleted with symplekin. An analysis of the factors associated with the symplekin-containing complex revealed the presence of the Xenopus homolog of GLD-2, a member of an unusual family of nucleotidyltransferases, identified in S. pombe, S. cerevisiae, and C. elegans, that has poly(A) polymerase activity. Most importantly, supplemental Xenopus GLD-2 (xGLD-2), together with symplekin, CPEB, and CPSF, restores polyadenylation to the symplekin-depleted extracts. Moreover, the immunodepleted symplekin-associated complex is able to polyadenylate RNA in an extraordinarily robust manner. Finally, xGLD-2 is anchored to the cytoplasmic polyadenylation complex through direct interactions with both CPEB and CPSF in polyadenylation-deficient oocyte extracts as well as in polyadenylation-proficient egg extracts (Bernard, 2004).

The results presented here necessitate a revision in previous models for cytoplasmic polyadenylation. Because CPEB, CPSF, symplekin, and xGLD-2 are all present in a complex prior to polyadenylation, the recruitment of CPSF by CPEB, and the recruitment of the polymerase by CPSF, to the RNA no longer appears tenable. In contrast it has been shown that aurora A-catalyzed CPEB serine 174 phosphorylation stimulates an association with CPSF that results in polyadenylation. Moreover, this CPEB phosphorylation stimulates polyadenylation in vitro using purified CPEB, CPSF, and conventional poly(A) polymerase. It is proposed that, even though symplekin bridges unphosphorylated CPEB and CPSF, phospho-CPEB undergoes a conformational change such that its increased affinity for CPSF activates xGLD-2, perhaps by helping juxtapose the polymerase and the 3' end of the mRNA. The apparent increased affinity of phospho-CPEB for xGLD-2 may help activate the polymerase as well. It is also possible that the activity of the polymerase is modified by covalent (e.g., phosphorylation) or noncovalent (e.g., associating with a factor) interactions. Indeed, canonical poly(A) polymerase activity is regulated by phosphorylation as well as through interactions with other proteins. Attempts are being made to identify additional components of the cytoplasmic polyadenylation complex that could modify xGLD-2 activity. It is noteworthy that PAP, like xGLD-2, binds to CPSF160, and this interaction plays a significant role in targeting PAP in nuclear AAUAAA-dependent polyadenylation. It is conceivable that CPSF160 plays a very similar role with xGLD-2 in cytoplasmic polyadenylation (Bernard, 2004).

Finally, CPEB-mediated polyadenylation-induced translation occurs not only during oocyte maturation but during cell cycle progression and neuronal synaptic stimulation as well. Based on this report, it is posited that symplekin and GLD-2, like CPEB, are likely to be important for these processes. Indeed, both Cid 1 and Cid 13 influence cell cycle progression in yeast, although these phenomena are not related to CPEB. In metazoans, the apparent assembly of factors on the symplekin scaffold suggests that this protein might be a useful hook to isolate entire complexes involved in unique forms of cytoplasmic polyadenylation, including specialized RNA substrates. For example, polyadenylation in mammalian neurons is stimulated by NMDA receptor activation, which is certainly not the case during cell cycle progression. Perhaps neuronal cytoplasmic polyadenylation complexes contain ancillary factors that uniquely allow them to respond to NMDA stimulation; such factors might be identified through symplekin coimmunoprecipitation and mass spectrometry. Factors involved in cytoplasmic polaydenylation during the cell cycle could be identified in a similar manner (Bernard, 2004).

CPEB homologs and regulation of early cell divisions during embryonic development

In Xenopus development, the expression of several maternal mRNAs is regulated by cytoplasmic polyadenylation. CPEB (Drosophila homolog: Orb) and CPEB associated factor maskin, two factors that control polyadenylation-induced translation are present on the mitotic apparatus of animal pole blastomeres in embryos. Cyclin B1 protein and mRNA, whose translation is regulated by polyadenylation, are colocalized with CPEB and maskin. CPEB interacts with microtubules and is involved in the localization of cyclin B1 mRNA to the mitotic apparatus. Agents that disrupt polyadenylation-induced translation inhibit cell division and promote spindle and centrosome defects in injected embryos. Two of these agents inhibit the synthesis of cyclin B1 protein, and one, which has little effect on this process, disrupts the localization of cyclin B1 mRNA and protein. These data suggest that CPEB-regulated mRNA translation is important for the integrity of the mitotic apparatus and for cell division (Groisman, 2000).

The observation that CPEB and maskin are localized on animal pole spindles and centrosomes was completely unexpected. Because the spindles of metaphase II-arrested Xenopus eggs are associated with the animal pole cortex, it seems probable that CPEB and maskin either move along a microtubule array to the mitotic apparatus formed in this region, or became passively incorporated with microtubules as the spindles are formed. In either case, these proteins form a gradient along the spindles, with the greatest concentration nearest the centrosomes. Because CPEB (especially) and maskin bind tubulin in vitro, they probably interact directly with the mitotic apparatus. This is almost certainly the case with CPEB because deletion of a region that includes a PEST domain abrogates both tubulin binding in vitro and centrosome localization in vivo. While PEST domains have been implicated in rapid protein degradation, they are also sites of protein-protein interaction. In contrast to CPEB and maskin, the other factors involved in polyadenylation-induced translation [eIF4E, poly(A) polymerase, and CPSF] are coincident with, but not particularly concentrated on, the mitotic apparatus. The transient nature of protein kinase Eg2 colocalization with CPEB on centrosomes could have a crucial impact on polyadenylation-induced translation. For example, poly(A) tail length is probably controlled by a dynamic equilibrium between elongation and contraction. One mechanism by which this equilibrium could be shifted in one direction or the other is to modulate CPEB activity; such activity could be regulated by the cell cycle-controlled localization of Eg2 on centrosomes. Irrespective of this possibility, the observation that general translation factors are associated with spindles and centrosomes indicates that regulated protein synthesis, in close proximity to the mitotic apparatus, could occur (Groisman, 2000).

The finding that CPE-containing mRNAs are concentrated on the mitotic apparatus in the animal pole brings up two obvious questions: how do they get there, and what purpose do they serve? While this paper does not address the mechanism of RNA localization per se, the sequence-specificity would almost certainly be dependent upon CPEB. Indeed, this is strongly suggested by the observation that CPEBdelta4 acts as a dominant negative mutation for cyclin B1 mRNA localization. However, maskin might also play an important role in this process. The carboxy half of maskin is composed of a coiled-coil domain that is 70% identical with a similar motif in Drosophila dTACC (Gergely, 2000), a member of the TACC (transforming acidic coiled-coil) family of proteins. dTACC, like maskin, is localized in the oocyte, in this case to the anterior pole; it is centrosomal in embryos, and it is essential for Bicoid mRNA localization (Gergely, 2000). Neither maskin nor dTACC contain obvious RNA binding motifs, suggesting that their involvement in RNA localization is mediated by other factors. Orb, the Drosophila homolog of CPEB, is also important for mRNA localization, but whether it interacts with dTACC is not known (Groisman, 2000).

The treatment of embryos with cycloheximide, blocks cell division in S-phase, which results in centrosome replication. This result has been confirmed by injecting a maskin-derived eIF4E blocking peptide that, like cycloheximide, inhibits protein synthesis in general. However, the results demonstrating that CPEB antibody, CPEB dominant negative mutant deltaN, and cordycepin each inhibit cell division, induce the formation of multiple centrosomes, and destroy the integrity of the mitotic apparatus, suggest that the synthesis of key factors is mediated by cytoplasmic polyadenylation. It is important to note that these agents, all of which prevent polyadenylation-induced translation in injected oocytes, specifically inhibit the expression of the relatively few (perhaps a dozen or so are known) mRNAs that undergo poly(A) elongation, one of which is cyclin B1. Because CPEB antibody and cordycepin also inhibit cyclin B1 synthesis in embryos, it follows that the translation of this message is regulated by polyadenylation at this stage of development as well. Therefore, while cell cycle progression is clearly governed by cyclin synthesis and destruction, these data demonstrate that regulated cyclin B1 mRNA translation is also important for cell division in early embryos. In the vegetal region, however, this may not be the case because the injection of CPEB antibody into a vegetal pole blastomere of a 16 cell embryo has no obvious deleterious effect on cell division (Groisman, 2000).

In addition to agents that affect CPEB activity, maskin antibody injection also results in spindle/centrosome defects in embryos. These defects are similar to those observed in Drosophila embryos that have mutations in dTACC. Because dTACC has no obvious eIF4E binding domain, it is difficult to determine whether this protein and maskin are functionally homologous. However, these results suggest that they have at least partially overlapping activities (Groisman, 2000 and references therein).

The observation that cyclin B1 mRNA and protein, together with CPEB and maskin, is concentrated on spindles and centrosomes might suggest that these structures are the sites of translational control. While consistent with this possibility, the relative amount of this message, or the amounts of these proteins for that matter, that actually resides on the mitotic apparatus is difficult to estimate, and therefore the functional importance of this concentration is not necessarily clear-cut. However, CPEBdelta4, which is fully capable of binding CPE-containing mRNA but is defective for microtubule binding, mostly destroys the concentration of cyclin B1 mRNA and protein on the mitotic apparatus while having little effect on overall cyclin levels. This result suggests that the inhibition of cell division, which results from CPEBdelta4 injection, is due to the loss of cyclin B1 synthesis on the mitotic apparatus. Therefore, it is proposed that in animal pole blastomeres of Xenopus embryos, the translation of cyclin B1 mRNA is regulated locally, on or near spindles and centrosomes (Groisman, 2000).

Local cyclin mRNA translation might be necessary to ensure that the protein product is delivered to the sight at which it is needed. This would appear to be the case in Drosophila embryos, where both cyclin mRNA and protein are detected on the mitotic apparatus. Given that Xbub3 mRNA is also associated with spindles, it is suspected that local synthesis of this checkpoint control protein is also an important regulatory event. Additional CPE-containing mRNAs may also be found on spindles, and experiments to address this possibility are presently underway (Groisman, 2000).

The synthesis and destruction of cyclin B drives mitosis in eukaryotic cells. Cell cycle progression is also regulated at the level of cyclin B translation. In cycling extracts from Xenopus embryos, progression into M phase requires the polyadenylation-induced translation of cyclin B1 mRNA. Polyadenylation is mediated by the phosphorylation of CPEB by Aurora, a kinase whose activity oscillates with the cell cycle. Exit from M phase seems to require deadenylation and subsequent translational silencing of cyclin B1 mRNA by Maskin, a CPEB and eIF4E binding factor, whose expression is cell cycle regulated. These observations suggest that regulated cyclin B1 mRNA translation is essential for the embryonic cell cycle. Mammalian cells also display a cell cycle-dependent cytoplasmic polyadenylation, suggesting that translational control by polyadenylation might be a general feature of mitosis in animal cells (Groisman, 2002).

CPEB homologs in vertebrates: developmental role

Long-term changes in synaptic efficacy may require the regulated translation of dendritic mRNAs. While the basis of such regulation is unknown, it seemed possible that some features of translational control in development could be recapitulated in neurons. Polyadenylation-induced translation of oocyte mRNAs requires the cis-acting CPE sequence and the CPE-binding protein CPEB. CPEB is also present in the dendritic layers of the hippocampus, at synapses in cultured neurons, and in postsynaptic densities of adult brain. alpha-CaMKII mRNA, which is localized in dendrites and is necessary for synaptic plasticity and LTP, contains two CPEs. These CPEs are bound by CPEB; they mediate polyadenylation-induced translation in injected Xenopus oocytes. In the intact brain, visual experience induces alpha-CaMKII mRNA polyadenylation and translation, suggesting that this process likely occurs at synapses (Wu, 1999).

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. This study reports prion-like properties in a neuronal member of the CPEB family (cytoplasmic polyadenylation element binding protein) that 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. It is hypothesized that conversion of CPEB to a prion-like state in stimulated synapses helps to maintain long-term synaptic changes associated with memory storage (Si, 2003a).

The term 'prion' was first applied to the proteinacious infectious agent in a group of mammalian neurodegenerative disorders (transmissible spongiform encephalopathies). Transmissibility is widely believed to stem from the ability of the prion form of PrP protein, PrPsc, to promote the conformational change of the normal cellular form, PrPc, to the PrPsc conformation. A similar mechanism might explain the unusual dominant, cytoplasmic inheritance of certain traits in the yeast Saccharomyces cerevisiae and a wide array of genetic and biochemical evidence supports this hypothesis (Si, 2003a and references therein).

Unlike PrP, yeast prions are generally not pathogenic. Rather, they produce changes in phenotype that mimic conventional loss-of-function mutations. The patterns of their inheritance, however, are very different. The loss-of-function phenotypes caused by yeast prions are dominant, rather than recessive. Moreover, they are inherited in a non-Mendelian fashion. After cells containing the prion form of the protein are mated to cells containing the nonprion form and then sporulated, most progeny contain the prion form. The dominant nonnuclear inheritance of prion phenotypes results from two basic factors: (1) proteins that are in the prion conformation promote conversion of other proteins of the same type to the prion state; (2) the prion form of the protein is transferred from a mother cell to her daughters and mating partners, creating a dominant change in phenotype that is perpetuated through subsequent generations. The study of fungal prions has established that stable self-perpetuating conformational changes in proteins can occur in diverse organisms, produce distinct phenotypes, involve molecules with very different physiological functions, and can sometimes be beneficial (Si, 2003a and references therein).

In the well-characterized yeast prions [SUP35], [URE3], and [RNQ1], a specific region is responsible for prion behavior. These regions of 65-250 amino acids show no sequence homology to each other but share four striking characteristics: (1) they are unusually rich in the polar residues glutamine (Q) or asparagine (N); (2) by structural-prediction algorithms they score as having a low propensity for any particular secondary structure, an indication of conformational flexibility that likely relates to their ability to switch states; (3) they can exist as soluble species or ordered self-perpetuating aggregates; and (4) they are dispensable for the normal function of their associated protein domains. While studying a neuron-specific isoform of cytoplasmic polyadenylation element binding protein (CPEB) in Aplysia californica, it was noted that its N terminus has features reminiscent of a prion-like domain in yeast (Si, 2003a).

CPEB was initially identified in Xenopus oocytes as a translational regulator that activates dormant mRNAs by elongating their poly (A) tails. CPEB serves not only as an activator, but in some cases it is also a repressor (deMoor and Richter, 1999). These dual functions are normally controlled by phosphorylation (Mendez, 2002). However, the neuronal isoform of Aplysia CPEB lacks these phosphorylation sites. Instead synaptic stimulation with serotonin increases the amount of the CPEB protein (Si, 2003b). Furthermore, in Aplysia, the neuronal CPEB is causally involved in maintaining long-term synaptic facilitation (Si, 2003a; Si, 2003b).

This study reports that N-terminal domain of the Aplysia neuronal CPEB can confer upon another protein, the glucocorticoid receptor, a self-perpetuating change in state with the hallmarks of a yeast prion. Moreover, full-length CPEB itself has prion-like properties, and surprisingly the prion form of the protein is the more active form of the protein. Although direct evidence is lacking that the protein exists in a prion-like state in Aplysia, these findings suggest a mechanism by which a prion-like state of CPEB could selectively sustain an altered rate of translation locally, at some synapses and not at others, and thereby contribute to the long-term maintenance of a self-sustaining synapse-specific plastic change (Si, 2003a).

The amino-terminal end of Aplysia CPEB has an unusual amino acid composition. The N-terminal 160 amino acids of 44 randomly selected Aplysia proteins have an average glutamine + asparagine (Q + N) content of 10%, typical of proteins in other species. In contrast, the N-terminal 160 amino acids of ApCPEB have a Q + N content of 48%. The Aplysia CPEB N-terminal domain also resembles yeast prion domains in that it lacks predictable secondary structure when analyzed by standard structural predication algorithms. A search of the protein sequence database has revealed putative homologs of the Aplysia neuronal CPEB in Drosophila, mouse, and humans, with N-terminal extensions of similar character. Moreover, the Drosophila (Si, 2003) and mouse isoforms have been found to be expressed in neurons (Theis, 2003). The conservation of such a CPEB neuronal isoform suggests that the N-terminal extension has an evolutionarily conserved function. Since many different rigorous tests for prions are available in yeast cells, they were employed for further characterization of the properties of Aplysia CPEB (Si, 2003a).

One test of a prion determinant is the ability to confer upon another protein, to which it is fused, a capacity to exist in distinct physical and functional states that are interconvertible and heritable. As has been described for the prion-determining region of yeast prions, the N-terminal 160 amino acids of Aplysia CPEB (CPEBQ) were fused to the green fluorescence protein (GFP). GFP is normally soluble in yeast, but the fusion to CPEBQ conferred for the GFP a capacity to exist in distinct states: a few large aggregates, many small aggregates, or soluble protein. Notably, these distinct states were heritable. Mother cells almost always gave rise to daughter cells in which the GFP fusion protein was in the same state: soluble small aggregates, or large aggregates (Si, 2003a).

Advantage has been taken of the fact that prion domains in yeast are modular and can transfer prion-like behaviors to heterologous proteins. As has been descibed the prion domain of Sup35, NM, the N-terminal domain of CPEB was fused to a constitutively active variant of the rat glucocorticoid receptor (GR526), a transcription factor. A ß-galactosidase gene under the control of a GR response element provided a convenient blue-white colony color assay for GR activity: blue when the protein is active and white when the protein is inactive. Most cells expressing CPEBQ-GR526 were blue and remained blue from generation to generation, indicating that the CPEBQ fusion did not impair the transcriptional activity of GR526. As is the case for most yeast prions and for the NM-GR fusion, the CPEBQ-GR activity had a metastable character. Blue colonies gave rise to white colonies with a frequency (10-5) that is higher than the typical rate of spontaneous mutation (10-6). These white colonies continued to give rise to white colonies upon restreaking for generation after generation, but occasionally gave rise to blue colonies again. In contrast, GR526 alone rarely (10-6) produced white colonies, and when it did, they never reverted to blue colonies again (Si, 2003a).

This interconversion between blue and white states could result from either a genetic or an epigenetic change. Analysis of plasmids extracted from blue and white CPEBQ-GR526 colonies, and of the cells themselves after loss of the plasmids, demonstrated that the changes in GR activity in blue and white cells were not due to mutations in either the plasmids or the cellular genomes. Furthermore, as shown by immunoblotting, the level of GR526 produced by white and blue colonies was similar. Thus, when CPEBQ is fused to GR526, the fusion protein acquires the ability to exist in two functionally distinct states that are heritable and interconvertible at low frequency, in an epigenetic manner (Si, 2003a).

Thus, Aplysia CPEB has properties consistent with it being a prion-like protein. As is the case with other well-characterized prion determinants in yeast, the N-terminal domain of Aplysia CPEB is modular and transferable. When fused to GR it produces distinct, heritable, functional states that are associated with distinct physical states; the prion state is inactive and this state is readily transmitted to cells with active protein in a dominant, heritable manner (Si, 2003a).

As is also the case with conventional prions, the full-length Aplysia CPEB has a self-perpetuating epigenetic state that is associated with a distinct physical state. However the full-length CPEB differs from other yeast prions in a number of features. In yeast prions that have been characterized to date, the self-perpetuating prion state is biochemically inactive. By contrast, the dominant self-perpetuating state of CPEB is the active state. This is not completely unprecedented: the dominant self-perpetuating form of the fungal prion protein [HetS] also is associated with a genetic gain of function although the biochemical activities associated with [HetS] are still unknown. Also, a self-activating protease exhibits prion-like genetic behavior in the active state (Roberts, 2003). The current findings also suggest that the CPEB protein forms small aggregates or multimers in its active state. This raises the question: how can an aggregated form of the protein be active? One possibility is that in the aggregated state the protein either retains or acquires certain biochemical activities. Surprisingly (but consistent with its prion-like properties), it was found that it is only the aggregated state of the protein that binds to CPE containing RNA. The soluble monomeric state of the protein does not bind RNA. However, it is not known if the large aggregates of the Aplysia CPEB are the functional state in vivo. The large aggregates seen in vitro and sometimes in vivo are likely to be an expansion of smaller multimeric forms of the protein and it may be the smaller multimeric forms that are the functional RNA binding unit. As is true for other prions, it is not yet known if the change in state is self-autonomous or requires other factors. In any case, once the active state is achieved, it is self-perpetuating and transmissible to other CPEB proteins (Si, 2003a).

Although direct evidence in neurons is lacking, it is speculated that CPEB has at least two conformational states: one is inactive or acts as a repressor (perhaps monomeric), the other is active (perhaps multimeric). Based on these considerations, a model is proposed for CPEB that represents a variant of the conventional prion mechanisms. This variant has features that are particularly relevant in the context of long-term memory storage. Long-term memory in both invertebrates and mice involves long-term synapse-specific modifications. In the invertebrate Aplysia, the synapse-specific modifications are mediated by serotonin, a modulatory neurotransmitter released during learning. This long-term synapse-specific modification requires independent mechanisms for both spatial restriction (synapse specificity) and for persistence (duration). Serotonin acts locally partly by increasing the amount of CPEB in a restricted synapse-specific way. If CPEB has prion-like properties in Aplysia, this single molecule can achieve both restriction and persistence (Si, 2003a).

How might this work? In a naive synapse, the basal level of CPEB is low. Unlike conventional prions in this state the protein might be less active, inactive, or may even inhibit translation of certain CPE-containing mRNAs. Indeed in Xenopus oocytes, CPEB acts both as a translation repressor and as an activator for cyclin B1 mRNA. Since synaptic stimulation by serotonin leads to an increase in the neuronal CPEB, serotonin might trigger the conversion to the prion-like state either by itself or in concert with other signals. It is noteworthy that a transient increase in the protein level acts as general trigger for the conversion of all yeast prion proteins to the prion state. The prion-like state of neuronal CPEB, resulting from synaptic stimulation by serotonin, might be more active, have altered substrate specificity, or be devoid of the inhibitory function of the basal state. Once the prion state is achieved in the activated synapse, dormant mRNAs that are made in the cell body and distributed globally to all synapses could be activated locally. The prion-like state would thereby contribute to the long-term maintenance of a self-sustaining, synapse-specific plastic change (Si, 2003a).

According to this model, this variant form of prion mechanism evident in CPEB requires the action of a neurotransmitter for switching the protein to its active self-perpetuating state. This is in a sense equivalent to a posttranslational modification induced by physiological signal. However, a prion-like mechanism introduces an additional feature into signal transduction; once the protein achieves its prion state it is self-perpetuating and no longer requires for maintenance continued signaling either by kinases or phosphatases. Moreover, its activity state is less easily reversed. In principle, mechanisms such as these, where a physiological signal activates a non-Mendelian epigenetic self-perpetuating state of protein, could work in many other biological contexts such as differentiation and transcription (Si, 2003a).

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. 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. It is suggested that Aplysia CPEB is one of the stabilizing components of the synaptic mark (Si, 2003b).

Synaptic plasticity, the ability of neurons to modulate the strength of their synapses, is thought to be a key mechanism contributing to learning and memory storage. Like behavioral memory, synaptic plasticity has at least two temporally distinct forms: a short-term form lasting minutes and a long-term form lasting days and weeks. These temporally distinct forms of synaptic plasticity have distinct molecular requirements: the short-term form depends on the covalent modifications of preexisting proteins and the strengthening of preexisting connections, whereas the long-term forms require the synthesis of new protein and the establishment of new synaptic connections. Because long-term synaptic plasticity requires transcription and therefore the nucleus, it raises a question: must all long-term changes necessarily be cell-wide or can long-term changes be restricted to some synapses and not others (Si, 2003b)?

To address this question, Martin developed a new culture system in Aplysia, where a single bifurcated sensory neuron of the gill withdrawal reflex was plated in contact with two spatially separated gill motor neurons (Martin, 1997). In this culture system, application to one of the two sets of synapses of a single pulse of serotonin (5-HT) (a neurotransmitter released in vivo by interneurons activated during learning) results in a synapse-specific short-term facilitation of preexisting connections that lasts for minutes. In contrast, application of five pulses of 5-HT, designed to simulate the spaced training that leads to long-term memory, elicits a synapse-specific long-term facilitation (LTF) that lasts for three or more days. Whereas the short-term form does not require new protein synthesis, the long-term form requires both CREB-dependent transcription and rapamycin-sensitive local protein synthesis at activated synapses, leading to the stabilization of new connections. Moreover, a synapse-specific long-term facilitation that is initiated in one branch can be captured at the other by application of a single pulse of 5-HT (which by itself is capable of producing only a short-term facilitation). Similar observations have been made for the synaptic capture of long-term potentiation (LTP) in slices of mammalian hippocampus. Interestingly, local protein synthesis is not needed for capture per se; it is however required for the stable maintenance, beyond 24 hr, of the synaptic growth initiated by capture (Si, 2003b and references therein).

How is such a synapse-specific facilitation achieved? The capturing of the synapse-specific long-term facilitation initiated in one branch by the other branch led Martin (1997) to postulate that the repeated pulses of 5-HT serve at least two functions: (1) they send a signal from the synapse to the nucleus that activates transcription and (2) they mark the activated synapse. The newly synthesized mRNAs and proteins necessary for long-term facilitation are then presumed to be transported to all the synapses of the neuron, but these gene products are only productively utilized by the marked synapse. Synaptic capture by a single pulse of serotonin further suggests that the signaling required for short-term facilitation can produce the mark (Martin, 1997; Si, 2003b).

What is the molecular nature of the synaptic mark? In their initial experiments, Casadio (1999) found that the mark has at least two components: (1) a PKA-dependent component needed for the initial capture of synapse-specific facilitation and for the growth of new synaptic connections and (2) a rapamycin-sensitive, local protein synthesis-dependent component needed for the long-term maintenance of facilitation and stabilization of growth beyond 24 hr (Casadio, 1999). Because mRNAs are made in the cell body, the need for the local translation of some mRNAs suggests that these mRNAs may be dormant before they reach the site of translation. If that is true, then the synaptic mark for stabilization might be a regulator of translation capable of activating translationally dormant mRNAs (Si, 2003b).

In searching for such a translational regulator, focus was placed on cytoplasmic polyadenylation element binding protein (CPEB), a molecule that activates dormant mRNAs in other biological contexts. Work on Xenopus oocytes has revealed that some translationally dormant mRNAs are activated following elongation of their poly (A) tail. This polyadenylation-dependent translational control requires two cis-acting elements at the 3' UTR of the mRNAs, a polyadenylation sequence AAUAAA and a cytoplasmic polyadenylation element (CPE) with a general structure of UUUUUAU. Cytoplasmic polyadenylation is regulated by a CPE binding protein, CPEB. Although initially discovered in developing oocytes, CPEB was subsequently also found in cultured hippocampal neurons and in the postsynaptic density fraction of mouse synaptosomes (Si, 2003b and references therein).

CPEB has four important features that make it an attractive candidate for a synapse-specific mark for stabilization: (1) it is activated through an extracellular signal; (2) it activates mRNAs that are translationally dormant; (3) it is spatially restricted; and (4) some of the mRNAs targeted by CPEB are involved in cellular growth (Si, 2003b).

A neuron-specific isoform of CPEB in Aplysia is shown to be regulated in a novel way. It is induced in the neurites of sensory neurons by a single pulse of 5-HT and this induction is dependent on rapamycin-sensitive protein synthesis. Depletion of CPEB locally at the activated synapse inhibits the long-term maintenance of synaptic facilitation but not its early expression. Thus, CPEB has the properties required of the local rapamycin-sensitive protein synthesis-dependent component of marking and supports the idea that there are separate mechanisms for the initiation of the long-term synaptic facilitation and for its stabilization. A similar isoform of CPEB was found in the mouse, in the human nervous system, and in Drosophila (Theis, 2003), which suggests that the mechanism for marking may be evolutionarily conserved (Si, 2003b).

The CPEB-like protein in the Aplysia CNS is an 82 kDa polypeptide, which is homologous to the CPEB of Xenopus oocytes and mice. However, unlike the CPEBs found in Xenopus and in the mouse, Aplysia CPEB (ApCPEB) lacks the consensus phosphorylation site for Eg2, a member of the Aurora family of serine/threonine protein kinases (Si, 2003b).

To determine how general this neuronal isoform of CPEB is, the genomic database of Drosophila was examined. In addition to Orb a second isoform was found, a putative open reading frame CG5735, which has an amino acid sequence similar to Aplysia neuronal CPEB. CG5735 has four putative isoforms, CG5735R-A to -D. Of these four isoforms, CG5735R-A has a domain organization that is similar to Aplysia neuronal CPEB. Indeed, unlike Orb mRNA, which is expressed throughout development, the mRNA for CG5735R-A is not detectable by RT-PCR in the embryonic stage but is detected in the larval stage. The highest level of expression of the CG5735R-A mRNA is in the adult, including in the adult brain. CG5735R-A encodes a 62 kDa protein. Immunoblotting of adult Drosophila brain extract with affinity-purified antibodies raised against recombinant CG5735R-A revealed a major protein around 62 kDa. The mRNAs for the other putative open reading frames CG5735RB-D are expressed throughout development. The functional significance of these four different isoforms is not immediately apparent (Si, 2003b).

Because the Aplysia and the Drosophila protein were isolated based on their sequence homology to known CPEBs, the biochemical properties of the CPEBs were examined. The CPEB in Xenopus has the ability to bind mRNA containing a CPE. The recombinant Aplysia neuronal CPEB also binds to CPE-containing mRNAs and does so as a multimer. Another property associated with CPEB is that it regulates translation of the bound RNA through its interaction with three protein factors: (1) maskin, (2) Xgef, the rho family guanine nucleotide exchange factor, and (3) CPSF160, the cleavage polyadenylation specificity factor (Si, 2003b and references therein).

None of the interacting partners of CPEB have yet been isolated from Aplysia. However, Aplysia neuronal CPEB does interact with heterologous CPSF160, such as the yeast CPSF160 homolog Cstf1. A Drosophila homolog (CG8606) of the second interacting partner, Xgef, has been recently identified (Reverte, 2003). Based on the Drosophila genomic sequence database, the Xgef homolog from Drosophila brain (Drogef) was isolated and it was expressed as a GST-tagged protein in E. coli. When total protein extract made from Drosophila was incubated with purified recombinant GST-tagged Drogef, it selectively pulled down the Drosophila CG5735 antibody reacting polypeptides. This interaction between GSTDrogef and CG5735 is specific because incubating the same cell extract with GST alone did not yield anything. Based on their ability to bind CPE-containing mRNAs and their interaction with two of the three interacting partners, it is concluded that these neuronal isoforms are indeed CPEBs (Si, 2003b).

In the search for the components of the rapamycin-sensitive protein synthesis-dependent synaptic mark required for stabilization of synapse-specific facilitation, the Aplysia homolog of the cytoplasmic polyadenylation element binding protein (CPEB), a protein capable of activating dormant mRNAs, was isolated. It has been suggested that the rapamycin-sensitive component of the synaptic mark should have the following properties: (1) it should be made or activated at the synapse by a signal for short-term facilitation; (2) it should be dependent on the activity of a rapamycin-sensitive signaling pathway; (3) it should be stable for 3-4 hr from the time of initiation; and (4) inhibition of the activity of the mark should influence selectively the late phase of long-term facilitation without interfering with the initiation of the long-term facilitation or its early expression (Si, 2003b).

Aplysia CPEB qualifies as a protein synthesis-dependent component of the mark based on all of these criteria. Moreover, the fact that inhibitors of PKA did not block induction of CPEB suggests that the PKA-dependent component of the mark and the local protein synthesis-dependent components of the mark are in parallel rather than in series. A parallel arrangement of these marking components might serve as a cellular gatekeeping mechanism that ensures that not every short-term change that activates PKA results in a long-term change in synaptic strength (Si, 2003b).

The neuronal isoform of Aplysia CPEB is translated locally following even a single pulse of 5-HT, and the newly synthesized CPEB is critical for stabilization of the LTF. How might ApCPEB stabilize this late phase? Long-term facilitation involves both insertion of new active zones into preexisting but empty presynaptic terminals as well as the growth of new synaptic terminals. The insertion of new active zones and the growth of new synaptic terminals requires both structural changes in the shape, size, and morphology of the synapse as well as regulatory controls that determine where and when to grow. The genes involved in both structural and regulatory aspects of synaptic growth might be potential targets of ApCPEB. The structural aspect of synapse formation are dynamically controlled through a reorganization of the cytoskeleton and can be achieved either by redistributing preexisting cytoskeleton components or by their local synthesis. The observation that Aplysia N-actin and Talpha1-tubulin mRNAs are present in the neurites of the sensory neuron and can be polyadenylated in response to 5-HT suggests that at least some of the structural components for synaptic growth can be controlled through CPEB-mediated local protein synthesis. In addition, CPEB has been found to be involved in the regulation of axonal synthesis of EphA2 (Brittis, 2002), a member of a family of receptor tyrosine kinases. Local synthesis of EphA2 is needed for axonal pathfinding. Mammalian Eph receptors are involved in the establishment of neuronal identity, neuronal pathfinding, formation of excitatory synapses, and changes in synaptic structure. Thus, CPEB might contribute to the stabilization of learning-related synaptic growth by controlling the synthesis of both structural molecules such as tubulin and N-actin and regulatory molecules such as members of the ephrin family (Si, 2003b).

The Aplysia homolog of CPEB is neuron specific and distinct from the known CPEBs. The neuronal isoform of CPEB is not unique to Aplysia. It is also present in human, mice, and flies. In Aplysia, the regulation of the neuronal isoform of CPEB is distinct from the regulation of CPEB in maturing Xenopus oocytes. In maturing oocytes, CPEB is regulated through phosphorylation by Eg2 at a canonical LDS/TR site. This phosphorylation induces the recruitment of the multisubunit cleavage and specificity factor CPSF to the polyadenylation signal AAUAAA. The recruitment of CPSF is thought to engage the poly (A) polymerase (PAP) leading to the elongation of the Poly A tail (Si, 2003b).

In contrast, the neuronal isoform of Aplysia CPEB lacks the canonical LDSR site or its variant LDSH. While the absence of a canonical phosphorylation site does not formally rule out the possibility that there might be other phosphorylation sites, no phosphorylation of Aplysia CPEB was observed in vivo despite repeated attempts to look for it. The failure to find evidence for phosphorylation raised the question of how an unphosphorylated form of CPEB might activate poly (A) elongation. In Xenopus oocytes, the phosphorylation of CPEB is not needed for mRNA binding. The unphosphorylated CPEB can also bind to CPSF160. Instead, the phosphorylation of CPEB by Eg2 increases by 4- to 5-fold the affinity of CPEB to CPSF160. Conceivably, a 4- to 5-fold increase in the amount of the Aplysia CPEB protein can achieve the same degree of activation as a 4- to 5-fold increase in affinity. Several CPEB-like molecules have been identfied from the genomic sequence of human, mice, and Drosophila, and some of these resemble the Aplysia CPEB in that they lack the Eg2 phosphorylation sites. It will be interesting to know whether these novel CPEB molecules that lack the Eg2 phosphorylation sites also represent neuron-specific or neuron-enriched isoforms of CPEB as they do in Aplysia and in flies. If that were so, it would imply that the regulation of some forms of CPEB activity at the synapse might utilize a mechanism that is distinct from that utilized for the activation of classical CPEB. However, it is also possible that both phosphorylation-dependent and -independent pathways can be operative in neuronal cells (Si, 2003b).

These several results suggest a model of synaptic marking in which a single pulse of 5-HT activates a rapamycin-sensitive signaling pathway and thereby results in the synthesis of ApCPEB. Newly made CPEB then recruits the polyadenylation machinery important for the activation of two types of molecules: (1) structural molecules important for synaptic growth, such as N-actin and Talphs-tubulin, and (2) regulatory molecules that determine where and how much to grow. These findings in turn raise further questions: is there a continuous need for the local synthesis of a set of molecules to maintain the learning-related synaptic changes over long periods of time? If so, how can it be achieved by a translational regulator such as CPEB in the face of a continuous turnover of the protein? What are the cellular mechanisms by which long-term maintenance and perpetuation of altered synaptic states are achieved? A possible solution to some of these questions has come from the analysis of novel properties of the ApCPEB protein (Si, 2003b).

CPEB regulation of human cellular senescence, energy metabolism, and p53 mRNA translation

Cytoplasmic polyadenylation element-binding protein (CPEB) stimulates polyadenylation and translation in germ cells and neurons. This study shows that CPEB-regulated translation is essential for the senescence of human diploid fibroblasts. Knockdown of CPEB causes skin and lung cells to bypass the M1 crisis stage of senescence; reintroduction of CPEB into the knockdown cells restores a senescence-like phenotype. Knockdown cells that have bypassed senescence undergo little telomere erosion. Surprisingly, knockdown of exogenous CPEB that induced a senescence-like phenotype results in the resumption of cell growth. CPEB knockdown cells have fewer mitochondria than wild-type cells and resemble transformed cells by having reduced respiration and reactive oxygen species (ROS), normal ATP levels, and enhanced rates of glycolysis. p53 mRNA contains cytoplasmic polyadenylation elements in its 3' untranslated region (UTR), which promote polyadenylation. In CPEB knockdown cells, p53 mRNA has an abnormally short poly(A) tail and a reduced translational efficiency, resulting in an ~50% decrease in p53 protein levels. An shRNA-directed reduction in p53 protein by about 50% also results in extended cellular life span, reduced respiration and ROS, and increased glycolysis. Together, these results suggest that CPEB controls senescence and bioenergetics in human cells at least in part by modulating p53 mRNA polyadenylation-induced translation (Burns, 2008).

orb: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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