CPEB Protein Research Articles

Identification of New FG-Repeat Nucleoporins with Amyloid Properties.

Amyloids are fibrillar protein aggregates with a cross-β structure. More than two hundred different proteins with amyloid or amyloid-like properties are already known. Functional amyloids with conservative amyloidogenic regions were found in different organisms. Protein aggregation appears to be beneficial for the organism in these cases. Therefore, this property might be conservative for orthologous proteins. The amyloid aggregates of the CPEB protein were suggested to play an important role in the long-term memory formation in Aplysia californica, Drosophila melanogaster, and Mus musculus. Moreover, the FXR1 protein demonstrates amyloid properties among the Vertebrates. A few nucleoporins (e.g., yeast Nup49, Nup100, Nup116, and human Nup153 and Nup58), are supposed or proved to form amyloid fibrils. In this study, we performed wide-scale bioinformatic analysis of nucleoporins with FG-repeats (phenylalanine-glycine repeats). We demonstrated that most of the barrier nucleoporins possess potential amyloidogenic properties. Furthermore, the aggregation-prone properties of several Nsp1 and Nup100 orthologs in bacteria and yeast cells were analyzed. Only two new nucleoporins, Drosophila melanogaster Nup98 and Schizosaccharomyces pombe Nup98, aggregated in different experiments. At the same time, Taeniopygia guttata Nup58 only formed amyloids in bacterial cells. These results rather contradict the hypothesis about the functional aggregation of nucleoporins.

CPEB and translational control by cytoplasmic polyadenylation: impact on synaptic plasticity, learning, and memory.

The late 1990s were banner years in molecular neuroscience; seminal studies demonstrated that local protein synthesis, at or near synapses, was necessary for synaptic plasticity, the underlying cellular basis of learning and memory [1, 2]. The newly made proteins were proposed to "tag" the stimulated synapse, distinguishing it from naive synapses, thereby forming a cellular memory [3]. Subsequent studies demonstrated that the transport of mRNAs from soma to dendrite was linked with translational unmasking at synapses upon synaptic stimulation. It soon became apparent that one prevalent mechanism governing these events is cytoplasmic polyadenylation, and that among the proteins that control this process, CPEB, plays a central role in synaptic plasticity, and learning and memory. In vertebrates, CPEB is a family of four proteins, all of which regulate translation in the brain, that have partially overlapping functions, but also have unique characteristics and RNA binding properties that make them control different aspects of higher cognitive function. Biochemical analysis of the vertebrate CPEBs demonstrate them to respond to different signaling pathways whose output leads to specific cellular responses. In addition, the different CPEBs, when their functions go awry, result in pathophysiological phenotypes resembling specific human neurological disorders. In this essay, we review key aspects of the vertebrate CPEB proteins and cytoplasmic polyadenylation within the context of brain function.

Mining of Small Interacting Ligands with Cytoplasmic Polyadenylation Element-binding Protein 4

Cytoplasmic polyadenylation element-binding protein 4 (CPEB 4) is a family member of CPEB proteins that are involved in regulating posttranscriptional expression of mRNAs. These proteins act by binding to cytoplasmic polyadenylation element of genes at their untranslated region with the conservation site of UUUUUAU. CPEB 4 proteins are responsible for modulating gene expression by the process of translational activation or repression by polyadenylation event. Overexpression of CPEB 4 gene has been reported in several classes of cancer such as gastric cancer, neuronal cancer, and pancreatic cancer. CPEB 4 sometimes acts like oncogene and can cause different types of cancer. CPEB 4 protein has been studied in detail in the current research. CPEB 4 protein structure was retrieved from the PDB database with PDB ID: 5DIF and total of 25 ligands were selected through a literature survey, chemical databases, etc., to perform molecular interaction study, docking method was used. Glide docking was done using Schrodinger software, and results were analyzed. Result shows that 2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxy-chromen-4-one (PubChem ID: 5281670) ligand has stable interaction with a Glide score of −8.26 and shows bonds with CPEB 4 protein at positions Lys(A: 924), Gly(B: 791), Asn(A: 879), and Thr(A: 878). These findings can help in modeling suitable ligands against the CPEB 4 protein responsible for several types of cancers.

Long-Term Memory Formation in Drosophila Depends on the 3'UTR of CPEB Gene orb2.

Activation of local translation in neurites in response to stimulation is an important step in the formation of long-term memory (LTM). CPEB proteins are a family of translation factors involved in LTM formation. The Drosophila CPEB protein Orb2 plays an important role in the development and function of the nervous system. Mutations of the coding region of the orb2 gene have previously been shown to impair LTM formation. We found that a deletion of the 3'UTR of the orb2 gene similarly results in loss of LTM in Drosophila. As a result of the deletion, the content of the Orb2 protein remained the same in the neuron soma, but significantly decreased in synapses. Using RNA immunoprecipitation followed by high-throughput sequencing, we detected more than 6000 potential Orb2 mRNA targets expressed in the Drosophila brain. Importantly, deletion of the 3'UTR of orb2 mRNA also affected the localization of the Csp, Pyd, and Eya proteins, which are encoded by putative mRNA targets of Orb2. Therefore, the 3'UTR of the orb2 mRNA is important for the proper localization of Orb2 and other proteins in synapses of neurons and the brain as a whole, providing a molecular basis for LTM formation.

Comparative analyses of vertebrate CPEB proteins define two subfamilies with coordinated yet distinct functions in post-transcriptional gene regulation

BackgroundVertebrate CPEB proteins bind mRNAs at cytoplasmic polyadenylation elements (CPEs) in their 3′ UTRs, leading to cytoplasmic changes in their poly(A) tail lengths; this can promote translational repression or activation of the mRNA. However, neither the regulation nor the mechanisms of action of the CPEB family per se have been systematically addressed to date.ResultsBased on a comparative analysis of the four vertebrate CPEBs, we determine their differential regulation by phosphorylation, the composition and properties of their supramolecular assemblies, and their target mRNAs. We show that all four CPEBs are able to recruit the CCR4-NOT deadenylation complex to repress the translation. However, their regulation, mechanism of action, and target mRNAs define two subfamilies. Thus, CPEB1 forms ribonucleoprotein complexes that are remodeled upon a single phosphorylation event and are associated with mRNAs containing canonical CPEs. CPEB2–4 are regulated by multiple proline-directed phosphorylations that control their liquid–liquid phase separation. CPEB2–4 mRNA targets include CPEB1-bound transcripts, with canonical CPEs, but also a specific subset of mRNAs with non-canonical CPEs.ConclusionsAltogether, these results show how, globally, the CPEB family of proteins is able to integrate cellular cues to generate a fine-tuned adaptive response in gene expression regulation through the coordinated actions of all four members.

Communicating with people with hearing loss: COVID-19 and beyond.

To maintain a balance of self-renewal versus neurogenesis, neural stem cells (NSCs) undergo asymmetric cell division along an invariant polarity axis instructed by centrosomes. In the NSCs of the third instar <i>Drosophila</i> larval brain, the interphase centrosomes are defined by marked asymmetries in protein composition and functional activity as microtubule-organizing centers. Here we show that a conserved RNA-binding protein, Orb2, supports NSC centrosome asymmetry by localizing to the cytoplasm, where it promotes robust apical centrosome maturation and transient basal centrosome inactivation, required for centrosome segregation and spindle morphogenesis. Orb2 is required cell autonomously within NSCs to support centrosome asymmetry and maintenance of the stem cell pool. We suggest Orb2 plays opposing roles in centrosome activation and inactivation at the apical versus basal centrosomes respectively, possibly through the translational regulation of multiple mRNAs. Conversely, loss of <i>orb2</i> manifests in microcephaly independent of Orb2 function in NSCs. Bioinformatics uncovers a significant overlap among RNA targets between <i>Drosophila</i> Orb2 and human CPEB4, consistent with a conserved role for CPEB proteins in centrosome regulation and neurodevelopment.

The Drosophila CPEB Protein Orb Specifies Oocyte Fate by a 3'UTR-Dependent Autoregulatory Loop.

orb encodes one of the two fly CPEB proteins. These widely conserved proteins bind to the 3'UTRs of target messenger RNAs (mRNAs) and activate or repress their translation. We show here that a positive autoregulatory loop driven by the orb gene propels the specification of oocyte identity in Drosophila egg chambers. Oocyte fate specification is mediated by a 3'UTR-dependent mechanism that concentrates orb mRNAs and proteins in one of the two pro-oocytes in the 16-cell germline cyst. When the orb 3'UTR is deleted, orb mRNA and protein fail to localize and all 16 cells become nurse cells. In wild type, the oocyte is specified when orb and other gene products concentrate in a single cell in region 2b of the germarium. A partially functional orb 3'UTR replacement delays oocyte specification until the egg chambers reach stage 2 of oogenesis. Before this point, orb mRNA and protein are unlocalized, as are other markers of oocyte identity, and the oocyte is not specified. After stage 2, ∼50% of the chambers successfully localize orb in a single cell, and this cell assumes oocyte identity. In the remaining chambers, the orb autoregulatory loop is not activated and no oocyte is formed. Finally, maintenance of oocyte identity requires continuous orb activity.

Amyloid-like Assembly Activates a Phosphatase in the Developing Drosophila Embryo.

Prion-like proteins can assume distinct conformational and physical states in the same cell. Sequence analysis suggests that prion-like proteins are prevalent in various species; however, it remains unclear what functional space they occupy in multicellular organisms. Here, we report the identification of a prion-like protein, Herzog (CG5830), through a multimodal screen in Drosophila melanogaster. Herzog functions as a membrane-associated phosphatase and controls embryonic patterning, likely being involved in TGF-β/BMP and FGF/EGF signaling pathways. Remarkably, monomeric Herzog is enzymatically inactive and becomes active upon amyloid-like assembly. The prion-like domain of Herzog is necessary for both its assembly and membrane targeting. Removal of the prion-like domain impairs activity, while restoring assembly on the membrane using a heterologous prion-like domain and membrane-targeting motif can restore phosphatase activity. This study provides an example of a prion-like domain that allows an enzyme to gain essential functionality via amyloid-like assembly to control animal development.

Elucidating the role of CPEB proteins in translational control of maternal transcripts during zebrafish development

Elucidating the role of CPEB proteins in translational control of maternal transcripts during zebrafish development

Altered splicing leads to reduced activation of CPEB3 in high-grade gliomas.

Cytoplasmic polyadenylation element binding proteins (CPEBs) are auxiliary translational factors that associate with consensus sequences present in 3′UTRs of mRNAs, thereby activating or repressing their translation. Knowing that CPEBs are players in cell cycle regulation and cellular senescence prompted us to investigate their contribution to the molecular pathology of gliomas–most frequent of intracranial tumors found in humans. To this end, we performed methylation analyses in the promoter regions of CPEB1-4 and identified the CPEB1 gene to be hypermethylated in tumor samples. Decreased expression of CPEB1 protein in gliomas correlated with the rising grade of tumor malignancy. Abundant expression of CPEBs2-4 was observed in several glioma specimens. Interestingly, expression of CPEB3 positively correlated with tumor progression and malignancy but negatively correlated with protein phosphorylation in the alternatively spliced region. Our data suggest that loss of CPEB3 activity in high-grade gliomas is caused by expression of alternatively spliced variants lacking the B-region that overlaps with the kinase recognition site. We conclude that deregulation of CPEB proteins may be a frequent phenomenon in gliomas and occurs on the level of transcription involving epigenetic mechanism as well as on the level of mRNA splicing, which generates isoforms with compromised biological properties.

Wispy and Orb cooperate in the cytoplasmic polyadenylation of localized gurken mRNA.

In Drosophila, the dorsal-ventral (D-V) axis of the oocyte is dependent on Gurken (Grk) protein distribution. This is achieved through the cytoplasmic localization of grk mRNA and regulation of its translation. During mid-late stages of oogenesis, grk mRNA and protein are localized to the dorsal-anterior of the oocyte, while unlocalized grk transcripts are translationally silenced. As females carrying mutations in the gene encoding the CPEB protein Orb lay ventralized eggs due to insufficient Grk levels, it seemed likely that cytoplasmic polyadenylation of grk transcripts may play a role in their translational regulation. We have found that grk is polyadenylated throughout oogenesis, with poly(A) tails of approximately 30-50 A residues. Hyperadenylated grk transcripts, with poly(A) tails of 50-90 As, are detected in late stage egg chambers, but they fail to accumulate in oocytes deficient in Orb or the poly(A) polymerase Wispy (Wisp). wisp females also lay weakly ventralized eggs, demonstrating that they produce inadequate amounts of Grk. Finally, unlocalized grk transcripts are also not appropriately hyperadenylated. Localized cytoplasmic polyadenylation of grk mRNA by Wisp and Orb is necessary to achieve appropriate Grk protein accumulation in the D/A corner of the oocyte during mid to late oogenesis.

Persistence of Memory and Prion Mechanisms: A Perspective

Long‐term memory for behavioral sensitization is synapse specific and requires local protein synthesis at the activated synapse. Kausik Si next found that local protein synthesis is regulated by a neuron‐specific isoform of cytoplasmic polyadenylation element binding protein (CPEB). Aplysia CPEB protein is a regulator of protein synthesis that upregulated locally at activated synapses, and it is needed not for the initiation but for the stable maintenance of long‐term facilitation.Recently we have extended this work in two directions:1) We found CPEB‐3 as a homolog of ApCPEB in the mammalian brain and found that CPEB‐3 is activated by Neuralized, an ubiquitin hydrolase.2) We next found for a new class of prions in the mammalian brain, and found a completely new candidate – TIA (T‐cell intracellular antigen). TIA has classic prion properties in yeast and serves as part of the cellular response to systemic stress. TIA serves as a sex‐specific protective factor in PTSD and does so in female mice only.In my talk I will to provide an overall view of the emerging biology of functional prions and the various roles of these prions in brain and behavior.

Spermatid cyst polarization in Drosophila depends upon apkc and the CPEB family translational regulator orb2.

Mature Drosophila sperm are highly polarized cells—on one side is a nearly 2 mm long flagellar tail that comprises most of the cell, while on the other is the sperm head, which carries the gamete's genetic information. The polarization of the sperm cells commences after meiosis is complete and the 64-cell spermatid cyst begins the process of differentiation. The spermatid nuclei cluster to one side of the cyst, while the flagellar axonemes grows from the other. The elongating spermatid bundles are also polarized with respect to the main axis of the testis; the sperm heads are always oriented basally, while the growing tails extend apically. This orientation within the testes is important for transferring the mature sperm into the seminal vesicles. We show here that orienting cyst polarization with respect to the main axis of the testis depends upon atypical Protein Kinase C (aPKC), a factor implicated in polarity decisions in many different biological contexts. When apkc activity is compromised in the male germline, the direction of cyst polarization within this organ is randomized. Significantly, the mechanisms used to spatially restrict apkc activity to the apical side of the spermatid cyst are different from the canonical cross-regulatory interactions between this kinase and other cell polarity proteins that normally orchestrate polarization. We show that the asymmetric accumulation of aPKC protein in the cyst depends on an mRNA localization pathway that is regulated by the Drosophila CPEB protein Orb2. orb2 is required to properly localize and activate the translation of apkc mRNAs in polarizing spermatid cysts. We also show that orb2 functions not only in orienting cyst polarization with respect to the apical-basal axis of the testis, but also in the process of polarization itself. One of the orb2 targets in this process is its own mRNA. Moreover, the proper execution of this orb2 autoregulatory pathway depends upon apkc.

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. In zebrafish, 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 is the cytoplasmic polyadenylation element (CPE)-binding proteins (CPEBs), which bind to a sequence in the 3'-untranslated regions 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 we now provide 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, ElrA protein is then produced and available to regulate the translation of additional mRNAs during embryogenesis. We have also identified a potential target of ElrA, the maternal mRNA for hnRNPab, which 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.

The Functioning of the Drosophila CPEB Protein Orb Is Regulated by Phosphorylation and Requires Casein Kinase 2 Activity

The Orb CPEB protein regulates translation of localized mRNAs in Drosophila ovaries. While there are multiple hypo- and hyperphosphorylated Orb isoforms in wild type ovaries, most are missing in orbF303, which has an amino acid substitution in a buried region of the second RRM domain. Using a proteomics approach we identified a candidate Orb kinase, Casein Kinase 2 (CK2). In addition to being associated with Orb in vivo, we show that ck2 is required for orb functioning in gurken signaling and in the autoregulation of orb mRNA localization and translation. Supporting a role for ck2 in Orb phosphorylation, we find that the phosphorylation pattern is altered when ck2 activity is partially compromised. Finally, we show that the Orb hypophosphorylated isoforms are in slowly sedimenting complexes that contain the translational repressor Bruno, while the hyperphosphorylated isoforms assemble into large complexes that co-sediment with polysomes and contain the Wisp poly(A) polymerase.

Yeast prions as a model of neurodegenerative infectious amyloidoses in humans

Several neurodegenerative diseases (so-called age-related diseases) in humans are associated with development of protein aggregates--amyloids. Prion diseases--kuru, Kreutzfeldt-Jakob and Gerstmann-Straussler-Sheinker diseases, fatal familial insomnia, etc.--are examples of infectious amyloidoses. A model system for investigation of mechanisms of amyloidogenesis and of its infectious nature had been developed as a result of yeast prion discovery. The existence of a prion network as an interaction of different prions identified in yeast is being confirmed recently as an interaction of different anyloids in humans. The potential danger of amyloidoses is conditioned by the very structure of almost all proteins containing fragments capable to be organized as beta-sheets, which lead to their aggregation being exposed. Meanwhile, there are several well-defined examples of the adaptive value of amyloid aggregates: cytoplasmic incompatibility factor in Podospora anserina, spider silk, cytoplasmic stress granules in mammals, prion form of CPEB protein responsible for the neuron activity in Aplisia, etc. These facts should be taken into consideration when seeking antiamyloid drugs. Discovery of protein inheritance in lower eukaryotes modifies our knowledge of the template principle significance in biology and adds a concept of conformational templates (II order templates) involved in reproduction of the three-dimensional structure of the supramolecular complexes in the cell.

An Interview with Eric Kandel

An Interview with Eric Kandel

Comparative in Silico Analyses of Cpeb1–4 with Functional Predictions

Cytoplasmic polyadenylation element binding proteins (Cpebs) are a family of proteins that bind to defined groups of mRNAs and regulate their translation. While Cpebs were originally identified as important features of oocyte maturation, recent interest is due to their prospective roles in neural system plasticity. In this study we made use of bioinformatic tools and methods including NCBI Blast, UCSC Blat, and Invitrogen Vector NTI to comprehensively analyze all known isoforms of four mouse Cpeb paralogs extracted from the national UniGene, UniProt, and NCBI protein databases. We identified multiple alternative splicing variants for each Cpeb. Regions of commonality and distinctiveness were evident when comparing Cpeb2, 3, and 4. In addition, we performed cross-ortholog comparisons among multiple species. The exon patterns were generally conserved across vertebrates. Mouse and human isoforms were compared in greater detail as they are the most represented in the current databases. The homologous and distinct regions are strictly conserved in mouse Cpeb and human CPEB proteins. Novel variants were proposed based on cross-ortholog comparisons and validated using biological methods. The functions of the alternatively spliced regions were predicted using the Eukaryotic Linear Motif resource. Together, the large number of transcripts and proteins indicate the presence of a hitherto unappreciated complexity in the regulation and functions of Cpebs. The evolutionary retention of variable regions as described here is most likely an indication of their functional significance.

Enforcing temporal control of maternal mRNA translation during oocyte cell-cycle progression

Meiotic cell-cycle progression in progesterone-stimulated Xenopus oocytes requires that the translation of pre-existing maternal mRNAs occur in a strict temporal order. Timing of translation is regulated through elements within the mRNA 3' untranslated region (3' UTR), which respond to cell cycle-dependant signalling. One element that has been previously implicated in the temporal control of mRNA translation is the cytoplasmic polyadenylation element (CPE). In this study, we show that the CPE does not direct early mRNA translation. Rather, early translation is directed through specific early factors, including the Musashi-binding element (MBE) and the MBE-binding protein, Musashi. Our findings indicate that although the cyclin B5 3' UTR contains both CPEs and an MBE, the MBE is the critical regulator of early translation. The cyclin B2 3' UTR contains CPEs, but lacks an MBE and is translationally activated late in maturation. Finally, utilizing antisense oligonucleotides to attenuate endogenous Musashi synthesis, we show that Musashi is critical for the initiation of early class mRNA translation and for the subsequent activation of CPE-dependant mRNA translation.

Translational Unmasking of Emi2 Directs Cytostatic Factor Arrest in Meiosis II

Cytostatic factor (CSF) arrests unfertilized vertebrate eggs in metaphase of meiosis II by inhibiting the anaphase-promoting complex/cyclosome (APC/C) from mediating cyclin destruction. The APC/C inhibitor Emi2/XErp1 satisfies a number of historical criteria for the molecular identification of CSF, but the mechanism by which CSF is activated selectively in meiosis II is the remaining unexplained criterion. Here we provide an explanation by showing that Emi2 is expressed specifically in meiosis II through translational de-repression or “unmasking” of its mRNA. We find that Emi2 protein is undetectable in immature, G2/prophase-arrested Xenopus oocytes and accumulates ~90 minutes after germinal vesicle breakdown. The 3’ untranslated region of Emi2 mRNA contains cytoplasmic polyadenylation elements that directly bind the CPEB protein and confer temporal regulation of Emi2 polyadenylation and translation. Our results demonstrate that cytoplasmic polyadenylation and translational unmasking of Emi2 directs meiosis II-specific CSF arrest.