Amyloid precursor proteins anchor CPEB to membranes and promote polyadenylation-induced translation

The cytoplasmic polyadenylation element (CPE) binding factor, CPEB, is a sequence-specific RNA binding protein that controls polyadenylation-induced translation in germ cells and at postsynaptic sites of neurons. A yeast two-hybrid screen with a mouse brain cDNA library identified the transmembrane amyloid precursor-like protein 1 (APLP1) as a CPEB-interacting factor. CPEB binds the small intracellular domain (ICD) of APLP1 and the related proteins APLP2 and APP. These proteins promote polyadenylation and translation by stimulating Aurora A catalyzed CPEB serine 174 phosphorylation. Surprisingly, CPEB, Maskin, CPSF, and several other factors involved in polyadenylation and translation and CPE-containing RNA are all detected on membranes by cell fractionation and immunoelectron microscopy. Moreover, most of the RNA that undergoes polyadenylation does so in membrane-containing fractions. These data demonstrate a link between cytoplasmic polyadenylation and membrane association and implicate APP family member proteins as anchors for localized mRNA polyadenylation and translation.     

The nuclear experience of CPEB: Implications for RNA processing and translational control

CPEB is a sequence-specific RNA binding protein that promotes polyadenylation-induced translation in early development, during cell cycle progression and cellular senescence, and following neuronal synapse stimulation. It controls polyadenylation and translation through other interacting molecules, most notably the poly(A) polymerase Gld2, the deadenylating enzyme PARN, and the eIF4E-binding protein Maskin. Here, we report that CPEB shuttles between the nucleus and cytoplasm and that its export occurs via the CRM1-dependent pathway. In the nucleus of Xenopus oocytes, CPEB associates with lampbrush chromosomes and several proteins involved in nuclear RNA processing. CPEB also interacts with Maskin in the nucleus as well as with CPE-containing mRNAs. Although the CPE does not regulate mRNA export, it influences the degree to which mRNAs are translationally repressed in the cytoplasm. Moreover, CPEB directly or indirectly mediates the alternative splicing of at least one pre-mRNA in mouse embryo fibroblasts as well as certain mouse tissues. We propose that CPEB, together with Maskin, binds mRNA in the nucleus to ensure tight translational repression upon export to the cytoplasm. In addition, we propose that nuclear CPEB regulates specific pre-mRNA alternative splicing.     

CPEB3 and CPEB4 in neurons: analysis of RNA-binding specificity and translational control of AMPA receptor GluR2 mRNA

CPEB is a sequence-specific RNA-binding protein that promotes polyadenylation-induced translation in oocytes and neurons. Vertebrates contain three additional genes that encode CPEB-like proteins, all of which are expressed in the brain. Here, we use SELEX, RNA structure probing, and RNA footprinting to show that CPEB and the CPEB-like proteins interact with different RNA sequences and thus constitute different classes of RNA-binding proteins. In transfected neurons, CPEB3 represses the translation of a reporter RNA in tethered function assays; in response to NMDA receptor activation, translation is stimulated. In contrast to CPEB, CPEB3-mediated translation is unlikely to involve cytoplasmic polyadenylation, as it requires neither the cis-acting AAUAAA nor the trans-acting cleavage and polyadenylation specificity factor, both of which are necessary for CPEB-induced polyadenylation. One target of CPEB3-mediated translation is GluR2 mRNA; not only does CPEB3 bind this RNA in vitro and in vivo, but an RNAi knockdown of CPEB3 in neurons results in elevated levels of GluR2 protein. These results indicate that CPEB3 is a sequence-specific translational regulatory protein.     

CPEB interacts with an ovary-specific eIF4E and 4E-T in early Xenopus oocytes

CPEB (cytoplasmic polyadenylation element-binding protein) is an important regulator of translation in oocytes and neurons. Although previous studies of CPEB in late Xenopus oocytes involve the eIF4E-binding protein maskin as the key factor for the repression of maternal mRNA, a second mechanism must exist, since maskin is absent earlier in oogenesis. Using co-immunoprecipitation and gel filtration assays, we show that CPEB specifically interacts, via protein/protein interactions, with the RNA helicase Xp54, the RNA-binding proteins P100(Pat1) and RAP55, the eIF4E-binding protein 4E-T, and an eIF4E protein. Remarkably, these CPEB complex proteins have been characterized, in one or more organism, as P-body, maternal, or neuronal granule components. We do not detect interactions with eIF4E1a, the canonical cap-binding factor, eIF4G, or eIF4A or with proteins expressed late in oogenesis, including maskin, PARN, and 4E-BP1. The eIF4E protein was identified as eIF4E1b, a close homolog of eIF4E1a, whose expression is restricted to oocytes and early embryos. Although eIF4E1b possesses all residues required for cap and eIF4G binding, it binds m(7)GTP weakly, and in pull-down assays, rather than binding eIF4G, it binds 4E-T, in a manner independent of the consensus eIF4E-binding site, YSKEELL. Wild type and Y-A mutant 4E-T (which binds eIF4E1b but not eIF4E1a), when tethered to a reporter mRNA, represses its translation in a cap-dependent manner, and injection of eIF4E1b antibody accelerates meiotic maturation. Altogether, our data suggest that CPEB, partnered with several highly conserved RNA-binding partners, inhibits protein synthesis in oocytes using a novel pairing of 4E-T and eIF4E1b.     

Critical role of amyloid-like oligomers of Drosophila Orb2 in the persistence of memory

A long-standing question in the study of long-term memory is how a memory trace persists for years when the proteins that initiated the process turn over and disappear within days. Previously, we postulated that self-sustaining amyloidogenic oligomers of cytoplasmic polyadenylation element-binding protein (CPEB) provide a mechanism for the maintenance of activity-dependent synaptic changes and, thus, the persistence of memory. Here, we found that the Drosophila CPEB Orb2 forms amyloid-like oligomers, and oligomers are enriched in the synaptic membrane fraction. Of the two protein isoforms of Orb2, the amyloid-like oligomer formation is dependent on the Orb2A form. A point mutation in the prion-like domain of Orb2A, which reduced amyloid-like oligomerization of Orb2, did not interfere with learning or memory persisting up to 24?hr. However the mutant flies failed to stabilize memory beyond 48?hr. These results support the idea that amyloid-like oligomers of neuronal CPEB are critical for the persistence of long-term memory.     

The Drosophila CPEB protein Orb2 has a novel expression pattern and is important for asymmetric cell division and nervous system function

Cytoplasmic polyadenylation element binding (CPEB) proteins bind mRNAs to regulate their localization and translation. While the first CPEBs discovered were germline specific, subsequent studies indicate that CPEBs also function in many somatic tissues including the nervous system. Drosophila has two CPEB family members. One of these, orb, plays a key role in the establishment of polarity axes in the developing egg and early embryo, but has no known somatic functions or expression outside of the germline. Here we characterize the other Drosophila CPEB, orb2. Unlike orb, orb2 mRNA and protein are found throughout development in many different somatic tissues. While orb2 mRNA and protein of maternal origin are distributed uniformly in early embryos, this pattern changes as development proceeds and by midembryogenesis the highest levels are found in the CNS and PNS. In the embryonic CNS, Orb2 appears to be concentrated in cell bodies and mostly absent from the longitudinal and commissural axon tracts. In contrast, in the adult brain, the protein is seen in axonal and dendritic terminals. Lethal effects are observed for both RNAi knockdowns and orb2 mutant alleles while surviving adults display locomotion and behavioral defects. We also show that orb2 funtions in asymmetric division of stem cells and precursor cells during the development of the embryonic nervous system and mesoderm.     

The Role of Functional Prion-Like Proteins in the Persistence of Memory

Prions are a self-templating amyloidogenic state of normal cellular proteins, such as prion protein (PrP). They have been identified as the pathogenic agents, contributing to a number of diseases of the nervous system. However, the discovery that the neuronal RNA-binding protein, cytoplasmic polyadenylation element-binding protein (CPEB), has a prion-like state that is involved in the stabilization of memory raised the possibility that prion-like proteins can serve normal physiological functions in the nervous system. Here, we review recent experimental evidence of prion-like properties of neuronal CPEB in various organisms and propose a model of how the prion-like state may stabilize memory.Prions are proteinaceous infectious agents that were discovered in the 1980s by Stanley Prusiner while studying Creutzfeldt–Jakob disease (Prusiner 1982). Prusiner and colleagues showed them to be an amyloidogenic, self-perpetuating, forms of a normal cellular protein, termed prion protein or PrP. Prp in its self-perpetuating state kills cells. Prusiner and colleagues found that PrPs exist in at least two conformations: monomeric and aggregated (Fig. 1). The transition among these forms occurs spontaneously and only the aggregated conformation is pathogenic. Soon, PrPs were found to contribute to other neurodegenerative disorders in people, including kuru, transmissible spongiform encephalopathies, as well as bovine spongiform encephalopathy in cows (Prusiner 1994; Aguzzi and Weissmann 1998).Open in a separate windowFigure 1.Pathogenic prions exist in two states (soluble and aggregated and self-perpetuating). The conversion from the soluble to the aggregated form is spontaneous and the aggregated, self-perpetuating form is often toxic and kills the cell.There is now a growing consensus that similar prion-like, self-templating mechanisms underlie a variety of neurodegenerative disorders, including amyotrophic lateral sclerosis, Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease (Polymenidou and Cleveland 2012).Not all prions, however, appear to be disease causing. Fungal prions, for instance, are nontoxic, and some may even be beneficial to the cells that harbor them (Wickner 1994; Shorter and Lindquist 2005; Crow and Li 2011). In 2003, Si and Kandel serendipitously discovered a prion-like protein in multicellular eukaryotes—the nervous system of the marine snail Aplysia—whose aggregated and self-perpetuating form contributes to the maintenance of long-term changes in synaptic efficacy. This functional prion-like protein differs from pathogenic prions in two important ways: (1) The conversion to the prion-like state is regulated by a physiological signal, and (2) the aggregated form has an identified physiological function (Fig. 2). Recent identification of new functional prion-like proteins in various organisms, including human, supports the idea that nonpathogenic prions may perform a wide range of biologically meaningful roles (Coustou et al. 1997; Eaglestone et al. 1999; True and Lindquist 2000; Ishimaru et al. 2003; True et al. 2004; Hou et al. 2011; Jarosz et al. 2014).Open in a separate windowFigure 2.“Functional” prion: memory. “Functional” prions differ from conventional prions in two ways. First, the conversion is triggered by a physiological signal, and second, the aggregated, self-perpetrating forms have a physiological function. 5-HT, Serotonin; DA, dopamine.In this review, we focus on functional prion-like proteins in the brain and specifically on the prion-like properties of the cytoplasmic polyadenylation element-binding protein (CPEB), and examine how the prion-like state can control protein synthesis at the synapse and, thereby, synaptic plasticity and long-lasting memory. We anticipate the studies of CPEB would also provide some generalizable concepts as to how prion-based protein switches in multicellular eukaryotes may work.     

A Role of CPEB1 in the Modulation of Proliferation and Neuronal Maturation of Rat Primary Neural Progenitor Cells

Cytoplasmic polyadenylation binding protein 1 (CPEB1) is a RNA binding protein, which regulates translation of target mRNAs by regulating polyadenylation status. CPEB1 plays important roles in the regulation of germline cell development by modulating cell cycle progression through the polyadenylation of target mRNAs such as cyclin B1. Similar mechanism is reported in proliferating astrocytes by us, although CPEB1 is involved in the transport of target mRNAs as well as local translation at dendritic spines. In this study, we found the expression of CPEB1 in cultured rat primary neural progenitor cells (NPCs). EGF stimulation of cultured NPCs induced rapid phosphorylation of CPEB1, a hallmark of CPEB1-dependent translational control along with cyclin B1 polyadenylation and translation. EGF-induced activation of ERK1/2 and Aurora A kinase was responsible for CPEB1 phosphorylation. Pharmacological inhibition studies suggested that ERK1/2 is involved in the activation of Aurora A kinase and regulation of CPEB1 phosphorylation in cultured NPCs. Long-term incubation in EGF resulted in the down-regulation of CPEB1 expression, which further increased expression of cyclin B1 and cell cycle progression. When we down-regulated the expression of CPEB1 in NPCs by siRNA transfection, the proliferation of NPCs was increased. Increased NPCs proliferation by down-regulation of CPEB1 resulted in eventual up-regulation of neuronal differentiation with increase in both pre- and post-synaptic proteins. The results from the present study may suggest the importance of translational control in the regulation of neuronal development, an emerging concept in many neurodevelopmental and psychiatric disorders such as autism spectrum disorder.     

Maskin is a CPEB-associated factor that transiently interacts with elF-4E

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. Here, we report that CPEB, which binds the CPE and stimulates polyadenylation, interacts with a new factor we term 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, and 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-4E to elF-4G and the translational activation of CPE-containing mRNAs.     

Novel RNA recognition motif domain in the cytoplasmic polyadenylation element binding protein 3

The family of cytoplasmic polyadenylation element binding proteins CPEB1, CPEB2, CPEB3, and CPEB4 binds to the 3′‐untranslated region (3′‐UTR) of mRNA, and plays significant roles in mRNA metabolism and translation regulation. They have a common domain organization, involving two consecutive RNA recognition motif (RRM) domains followed by a zinc finger domain in the C‐terminal region. We solved the solution structure of the first RRM domain (RRM1) of human CPEB3, which revealed that CPEB3 RRM1 exhibits structural features distinct from those of the canonical RRM domain. Our structural data provide important information about the RNA binding ability of CPEB3 RRM1. Proteins 2014; 82:2879–2886. © 2014 Wiley Periodicals, Inc.     

Transient CPEB dimerization and translational control

During oocyte development, the cytoplasmic polyadenylation element-binding protein (CPEB) nucleates a set of factors on mRNA that controls cytoplasmic polyadenylation and translation. The regulation of polyadenylation is mediated in part through serial phosphorylations of CPEB, which control both the dynamic integrity of the cytoplasmic polyadenylation apparatus and CPEB stability, events necessary for meiotic progression. Because the precise stoichiometry between CPEB and CPE-containing RNA is responsible for the temporal order of mRNA polyadenylation during meiosis, we hypothesized that, if CPEB production exceeded the amount required to bind mRNA, the excess would be sequestered in an inactive form. One attractive possibility for the sequestration is protein dimerization. We demonstrate that not only does CPEB form a dimer, but dimerization requires its RNA-binding domains. Dimer formation prevents CPEB from being UV cross-linked to RNA, which establishes a second pool of CPEB that is inert for polyadenylation and translational control. During oocyte maturation, the dimers are degraded much more rapidly than the CPEB monomers, due to their greater affinity for polo-like kinase 1 (plx1) and the ubiquitin E3 ligase β-TrCP. Because dimeric CPEB also binds cytoplasmic polyadenylation factors with greater affinity than monomeric CPEB, it may act as a hub or reservoir for the polyadenylation machinery. We propose that the balance between CPEB and its target mRNAs is maintained by CPEB dimerization, which inactivates spare proteins and prevents them from inducing polyadenylation of RNAs with low affinity binding sites. In addition, the dimers might serve as molecular hubs that release polyadenylation factors for translational activation upon CPEB dimer destruction.     

Activity-dependent polyadenylation in neurons

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

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

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

Translational control by neuroguidin, a eukaryotic initiation factor 4E and CPEB binding protein

CPEB-mediated translation is important in early development and neuronal synaptic plasticity. Here, we describe a new eukaryotic initiation factor 4E (eIF4E) binding protein, Neuroguidin (Ngd), and its interaction with CPEB. In the mammalian nervous system, Ngd is detected as puncta in axons and dendrites and in growth cones and filopodia. Ngd contains three motifs that resemble those present in eIF4G, 4EBP, Cup, and Maskin, all of which are eIF4E binding proteins. Ngd binds eIF4E directly, and all three motifs must be deleted to abrogate the interaction between these two proteins. In injected Xenopus oocytes, Ngd binds CPEB and, most importantly, represses translation in a cytoplasmic polyadenylation element (CPE)-dependent manner. In Xenopus embryos, Ngd is found in both neural tube and neural crest cells. The injection of morpholino-containing antisense oligonucleotides directed against ngd mRNA disrupts neural tube closure and neural crest migration; however, the wild-type phenotype is restored by the injection of a rescuing ngd mRNA. These data suggest that Ngd guides neural development by regulating the translation of CPE-containing mRNAs.     

CPEB, maskin, and cyclin B1 mRNA at the mitotic apparatus: implications for local translational control of cell division

In Xenopus development, the expression of several maternal mRNAs is regulated by cytoplasmic polyadenylation. CPEB and 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.     

New Phosphospecific Antibody Reveals Isoform-Specific Phosphorylation of CPEB3 Protein

Cytoplasmic Polyadenylation Element Binding proteins (CPEBs) are a family of polyadenylation factors interacting with 3’UTRs of mRNA and thereby regulating gene expression. Various functions of CPEBs in development, synaptic plasticity, and cellular senescence have been reported. Four CPEB family members of partially overlapping functions have been described to date, each containing a distinct alternatively spliced region. This region is highly conserved between CPEBs-2-4 and contains a putative phosphorylation consensus, overlapping with the exon seven of CPEB3. We previously found CPEBs-2-4 splice isoforms containing exon seven to be predominantly present in neurons, and the isoform expression pattern to be cell type-specific. Here, focusing on the alternatively spliced region of CPEB3, we determined that putative neuronal isoforms of CPEB3 are phosphorylated. Using a new phosphospecific antibody directed to the phosphorylation consensus we found Protein Kinase A and Calcium/Calmodulin-dependent Protein Kinase II to robustly phosphorylate CPEB3 in vitro and in primary hippocampal neurons. Interestingly, status epilepticus induced by systemic kainate injection in mice led to specific upregulation of the CPEB3 isoforms containing exon seven. Extensive analysis of CPEB3 phosphorylation in vitro revealed two other phosphorylation sites. In addition, we found plethora of potential kinases that might be targeting the alternatively spliced kinase consensus site of CPEB3. As this site is highly conserved between the CPEB family members, we suggest the existence of a splicing-based regulatory mechanism of CPEB function, and describe a robust phosphospecific antibody to study it in future.     

CPEB Regulation of TAK1 Synthesis Mediates Cytokine Production and the Inflammatory Immune Response

The cytoplasmic-element-binding (CPEB) protein is a sequence-specific RNA-binding protein that regulates cytoplasmic polyadenylation-induced translation. In mouse embryo fibroblasts (MEFs) lacking CPEB, many mRNAs encoding proteins involved in inflammation are misregulated. Correlated with this aberrant translation in MEFs, a macrophage cell line depleted of CPEB and treated with lipopolysaccharide (LPS) to stimulate the inflammatory immune response expresses high levels of interleukin-6 (IL-6), which is due to prolonged nuclear retention of NF-κB. Two proteins involved in NF-κB nuclear localization and IL-6 expression, IκBα and transforming growth factor beta-activated kinase 1 (TAK1), are present at excessively low and high steady-state levels, respectively, in LPS-treated CPEB-depleted macrophages. However, only TAK1 has an altered synthesis rate that is CPEB dependent and CPEB/TAK1 double depletion alleviates high IL-6 production. Peritoneal macrophages isolated from CPEB knockout (KO) mice treated with LPS in vitro also have prolonged NF-κB nuclear retention and produce high IL-6 levels. LPS-injected CPEB KO mice secrete prodigious amounts of IL-6 and other proinflammatory cytokines and exhibit hypersensitivity to endotoxic shock; these effects are mitigated when the animals are also injected with (5Z)-7-oxozeaenol, a potent and specific inhibitor of TAK1. These data show that CPEB control of TAK1 mRNA translation mediates the inflammatory immune response.