CPEB3 low-complexity motif regulates local protein synthesis via protein-protein interactions in neuronal ribonucleoprotein granules.

Biomolecular condensates, membraneless organelles found throughout the cell, play critical roles in many aspects of cellular function. Ribonucleoprotein granules (RNPs) are a type of biomolecular condensate necessary for local protein synthesis and are involved in synaptic plasticity and long-term memory. Most of the proteins in RNPs possess low-complexity motifs (LCM), allowing for increased promiscuity of protein-protein interactions. Here, we describe the importance of protein-protein interactions mediated by the LCM of RNA-binding protein cytoplasmic polyadenylation element binding protein 3 (CPEB3). CPEB3 is necessary for long-term synaptic plasticity and memory persistence, but the mechanisms involved are still not completely elucidated. We now present key mechanisms involved in its regulation of synaptic plasticity. We find that CPEB3-LCM plays a role in appropriate local protein synthesis of messenger ribonucleic acid (mRNA) targets, through crucial protein-protein interactions that drive localization to neuronal Decapping protein 1 (DCP1)-bodies. Translation-promoting CPEB3 and translation-inhibiting CPEB1 are packaged into neuronal RNP granules immediately after chemical long-term potentiation is induced, but only translation-promoting CPEB3 is repackaged to these organelles at later time points. This localization to neuronal RNP granules is critical for functional influence on translation as well as overall local protein synthesis (measured as α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) insertion into the membrane and localization to the synapse). We therefore conclude that protein-protein interaction between the LCM of CPEB3 plays a critical role in local protein synthesis by utilizing neuronal RNP granules.

Experimental Resurrection of Ancestral Mammalian CPEB3 Ribozymes Reveals Deep Functional Conservation.

Self-cleaving ribozymes are genetic elements found in all domains of life, but their evolution remains poorly understood. A ribozyme located in the second intron of the cytoplasmic polyadenylation binding protein 3 gene (CPEB3) shows high sequence conservation in mammals, but little is known about the functional conservation of self-cleaving ribozyme activity across the mammalian tree of life or during the course of mammalian evolution. Here, we use a phylogenetic approach to design a mutational library and a deep sequencing assay to evaluate the in vitro self-cleavage activity of numerous extant and resurrected CPEB3 ribozymes that span over 100 My of mammalian evolution. We found that the predicted sequence at the divergence of placentals and marsupials is highly active, and this activity has been conserved in most lineages. A reduction in ribozyme activity appears to have occurred multiple different times throughout the mammalian tree of life. The in vitro activity data allow an evaluation of the predicted mutational pathways leading to extant ribozyme as well as the mutational landscape surrounding these ribozymes. The results demonstrate that in addition to sequence conservation, the self-cleavage activity of the CPEB3 ribozyme has persisted over millions of years of mammalian evolution.

Emotional Stress Induces Structural Plasticity in Bergmann Glial Cells via an AC5-CPEB3-GluA1 Pathway.

Stress alters brain function by modifying the structure and function of neurons and astrocytes. The fine processes of astrocytes are critical for the clearance of neurotransmitters during synaptic transmission. Thus, experience-dependent remodeling of glial processes is anticipated to alter the output of neural circuits. However, the molecular mechanisms that underlie glial structural plasticity are not known. Here we show that a single exposure of male and female mice to an acute stress produced a long-lasting retraction of the lateral processes of cerebellar Bergmann glial cells. These cells express the GluA1 subunit of AMPA-type glutamate receptors, and GluA1 knockdown is known to shorten the length of glial processes. We found that stress reduced the level of GluA1 protein and AMPA receptor-mediated currents in Bergmann glial cells, and these effects were absent in mice devoid of CPEB3, a protein that binds to GluA1 mRNA and regulates GluA1 protein synthesis. Administration of a ß-adrenergic receptor blocker attenuated the reduction in GluA1, and deletion of adenylate cyclase 5 prevented GluA1 suppression. Therefore, stress suppresses GluA1 protein synthesis via an adrenergic/adenylyl cyclase/CPEB3 pathway, and reduces the length of astrocyte lateral processes. Our results identify a novel mechanism for GluA1 subunit plasticity in non-neuronal cells and suggest a previously unappreciated role for AMPA receptors in stress-induced astrocytic remodeling.SIGNIFICANCE STATEMENT Astrocytes play important roles in synaptic transmission by extending fine processes around synapses. In this study, we showed that a single exposure to an acute stress triggered a retraction of lateral/fine processes in mouse cerebellar astrocytes. These astrocytes express GluA1, a glutamate receptor subunit known to lengthen astrocyte processes. We showed that astrocytic structural changes are associated with a reduction of GluA1 protein levels. This requires activation of ß-adrenergic receptors and is triggered by noradrenaline released during stress. We identified adenylyl cyclase 5, an enzyme that elevates cAMP levels, as a downstream effector and found that lowering GluA1 levels depends on CPEB3 proteins that bind to GluA1 mRNA. Therefore, stress regulates GluA1 protein synthesis via an adrenergic/adenylyl cyclase/CPEB3 pathway in astrocytes and remodels their fine processes.

Regulation of microRNA-221, -222, -21 and -27 in articular cartilage subjected to abnormal compressive forces.

KEY POINTS: microRNAs (miRs) are small non-coding molecules that regulate post-transcriptional target gene expression. miRs are involved in regulating cellular activities in response to mechanical loading in all physiological systems, although it is largely unknown whether this response differs with increasing magnitudes of load. miR-221, miR-222, miR-21-5p and miR-27a-5p were significantly increased in ex vivo cartilage explants subjected to increasing load magnitude and in in vivo joint cartilage exposed to abnormal loading. TIMP3 and CPEB3 are putative miR targets in chondrocytes Identification of mechanically regulated miRs that have potential to impact on tissue homeostasis provides a mechanism by which load-induced tissue behaviour is regulated, in both health and pathology, in all physiological systems. ABSTRACT: MicroRNAs (miRs) are small non-coding molecules that regulate post-transcriptional target gene expression and are involved in mechano-regulation of cellular activities in all physiological systems. It is unknown whether such epigenetic mechanisms are regulated in response to increasing magnitudes of load. The present study investigated mechano-regulation of miRs in articular cartilage subjected to 'physiological' and 'non-physiological' compressive loads in vitro as a model system and validated findings in an in vivo model of abnormal joint loading. Bovine full-depth articular cartilage explants were loaded to 2.5 MPa (physiological) or 7 MPa (non-physiological) (1 Hz, 15 min) and mechanically-regulated miRs identified using next generation sequencing and verified using a quantitative PCR. Downstream targets were verified using miR-specific mimics or inhibitors in conjunction with 3'-UTR luciferase activity assays. A subset of miRs were mechanically-regulated in ex vivo cartilage explants and in vivo joint cartilage. miR-221, miR-222, miR-21-5p and miR-27a-5p were increased and miR-483 levels decreased with increasing load magnitude. Tissue inhibitor of metalloproteinase 3 (TIMP3) and cytoplasmic polyadenylation element binding protein 3 (CPEB3) were identified as putative downstream targets. Our data confirm miR-221 and -222 mechano-regulation and demonstrates novel mechano-regulation of miR-21-5p and miR-27a-5p in ex vivo and in vivo cartilage loading models. TIMP3 and CPEB3 are putative miR targets in chondrocytes. Identification of specific miRs that are regulated by increasing load magnitude, as well as their potential to impact on tissue homeostasis, has direct relevance to other mechano-sensitive physiological systems and provides a mechanism by which load-induced tissue behaviour is regulated, in both health and pathology.

Lidocaine Inhibits Hepatocellular Carcinoma Development by Modulating circ_ITCH/miR-421/CPEB3 Axis.

BACKGROUND: Lidocaine plays an anticancer role in hepatocellular carcinoma. Nevertheless, the mechanism of lidocaine in hepatocellular carcinoma remains largely unclear. AIMS: This study aims to assess the function of lidocaine and explore the potential regulatory mechanism. METHODS: Hepatocellular carcinoma cells were challenged via lidocaine. Cell proliferation, apoptosis, migration, and invasion were detected via colony formation, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, flow cytometry, Western blot, and transwell analyses. Circular RNA itchy E3 ubiquitin protein ligase (circ_ITCH), microRNA-421 (miR-421), and cytoplasmic polyadenylation element-binding protein 3 (CPEB3) abundances were detected via quantitative reverse transcription polymerase chain reaction or Western blot. The relationship between miR-421 and circ_ITCH or CPEB3 was tested via dual-luciferase reporter analysis. The role of circ_ITCH in lidocaine-challenged cell growth in vivo was assessed via xenograft model. RESULTS: Lidocaine inhibited hepatocellular carcinoma cell proliferation by decreasing colony formation and cell viability. Lidocaine suppressed hepatocellular carcinoma cell migration and invasion and promoted apoptosis. circ_ITCH and CPEB3 levels were decreased in hepatocellular carcinoma tissues and cells, and were restored in cells via lidocaine treatment. circ_ITCH knockdown weakened the suppressive effect of lidocaine on hepatocellular carcinoma development, which was abolished via CPEB3 overexpression. circ_ITCH could modulate CPEB3 by competitively binding with miR-421. miR-421 knockdown mitigated the effect of circ_ITCH silence in lidocaine-challenged cells. circ_ITCH knockdown increased xenograft tumor growth. CONCLUSIONS: Lidocaine represses hepatocellular carcinoma cell proliferation, migration, and invasion and promotes apoptosis via regulating circ_ITCH/miR-421/CPEB3 axis, indicating a new insight into the mechanism of lidocaine in hepatocellular carcinoma.

MiR-20b-5p promotes hepatocellular carcinoma cell proliferation, migration and invasion by down-regulating CPEB3.

INTRODUCTION AND OBJECTIVES: This study aimed to explore the functional mechanism of the miRNA-20b-5p/cytoplasmic polyadenylation element binding protein 3 (miR-20b-5p/CPEB3) axis in hepatocellular carcinoma (HCC) so as to provide a new idea for targeted therapy of HCC. MATERIALS AND METHODS: Bioinformatics analysis was employed to obtain markedly differentially expressed miRNAs and mRNAs in The Cancer Genome Atlas-Liver Hepatocellular Carcinoma (TCGA-LIHC) dataset, so as to find target miRNA and its target mRNA. Real-time quantitative PCR was conducted to detect miR-20b-5p and CPEB3 mRNA expression. Western blot was performed to determine CPEB3 protein expression. Dual-luciferase reporter assay was carried out to verify the targeting relationship between miR-20b-5p and CPEB3. Cell counting kit-8 assay, wound healing assay, Transwell invasion assay and flow cytometry were conducted to evaluate the proliferation, migration, invasion and apoptosis of HCC cells. RESULTS: Bioinformatics analysis suggested that miR-20b-5p and CPEB3 were markedly highly and lowly expressed, respectively, in HCC tissue in TCGA-LIHC dataset. Over-expressing miR-20b-5p facilitated the proliferation, migration and invasion, and suppressed the apoptosis of HCC cells. Dual-luciferase reporter assay validated that there was a targeting relationship between miR-20b-5p and CPEB3. The inhibitory effect of CPEB3 over-expression on HCC cell proliferation, migration and invasion was reversed by over-expressing miR-20b-5p. CONCLUSIONS: The present study proved that miR-20b-5p promotes HCC cell proliferation, migration and invasion by inhibiting CPEB3 expression, which may provide a theoretical basis for the prognosis and treatment of HCC patients.

LINC00641 hinders the progression of cervical cancer by targeting miR-378a-3p/CPEB3.

BACKGROUND: LINC00641 was found to act in anti-tumor manner in several types of cancers. Nonetheless, the detailed functions of LINC00641 have not been determined in cervical cancer (CC). METHODS: The expression of LINC00641, miR-378a-3p and CPEB3 was examined using a quantitative reverse transcriptase-polymerase chain reaction. The relationships between LINC00641 and its downstream mechanism were illustrated by RNA pull-down and luciferase reporter experiments. RESULTS: LINC00641 was found to be under-expressed in CC cell lines. By overexpressing LINC00641, cell proliferative, migratory and invasive abilities, as well as epithelial mesenchymal transition (EMT) characteristics, were inhibited, whereas the rate of apoptosis was increased. Next, a starBase search (http://starbase.sysu.edu.cn) was applied to select microRNAs that had binding sequences with LINC00641. By up-regulating LINC00641 expression, miR-378a-3p expression displayed the strongest decline. Moreover, miR-378a-3p was found to be up-regulated in CC cell lines. In addition, LINC00641 hindered the progression of CC by decreasing miR-378a-3p expression. CPEB3 was discovered as a downstream target of miR-378a-3p and was under-expressed in CC cells. Furthermore, knockdown of CPEB3 could counter the influence of an overexpression of LINC00641 with respect to CC progression. CONCLUSIONS: LINC00641 suppressed the progression of CC by targeting miR-378a-3p/CPEB3, suggesting that LINC00641 may have positive therapeutic impact for treatment for CC.

LncRNA SUMO1P3 promotes proliferation and inhibits apoptosis in colorectal cancer by epigenetically silencing CPEB3.

Colorectal cancer (CRC) is a prevalent malignancy characterized with high morbidity and death rate. Due to late diagnosis, most CRC patients missed the proper timing for radical operation, which led to the high mortality in CRC. Therefore, identifying new prognostic and therapeutic targets is important. Long non-coding RNAs are reported as essential regulators for tumor progression, including in CRC. LncRNA SUMO1P3 has been documented as an oncogene promoting proliferation, cell cycle, and metastasis in several cancers, but its role in CRC has never been unveiled. The purpose of our study is to interrogate the functions and mechanism of SUMO1P3 in colorectal cancer. We validated the upregulation and the prognostic significance of SUMO1P3 in CRC. The loss-of-function assays suggested that SUMO1P3 provoked CRC cell proliferative ability, and retarded apoptotic ability. Cytoplasmic polyadenylation element binding protein 3 (CPEB3) has been newly acknowledged as a tumor suppressive gene in several cancers, and has been revealed to present low expression in CRC. We predicted through UCSC database and validated by ChIP assay that EZH2, a crucial regulator of trimethylation of histone H3 at lysine 27 (H3K27me3), bound to CPEB3 promoter. Further, we validated that SUMO1P3 epigenetically repressed CPEB3 through EZH2. Finally, rescue assays indicated that SUMO1P3 provoked proliferation, cell cycle, and retarded apoptosis through CPEB3. Consequently, current study showed that lncRNA SUMO1P3 promoted cell proliferative ability and inhibited apoptotic ability in CRC by epigenetically silencing CPEB3, providing a novel prognostic marker for CRC patients.

Secondary structure confirmation and localization of Mg2+ ions in the mammalian CPEB3 ribozyme.

Most of today's knowledge of the CPEB3 ribozyme, one of the few small self-cleaving ribozymes known to occur in humans, is based on comparative studies with the hepatitis delta virus (HDV) ribozyme, which is highly similar in cleavage mechanism and probably also in structure. Here we present detailed NMR studies of the CPEB3 ribozyme in order to verify the formation of the predicted nested double pseudoknot in solution. In particular, the influence of Mg(2+), the ribozyme's crucial cofactor, on the CPEB3 structure is investigated. NMR titrations, Tb(3+)-induced cleavage, as well as stoichiometry determination by hydroxyquinoline sulfonic acid fluorescence and equilibrium dialysis, are used to evaluate the number, location, and binding mode of Mg(2+)ions. Up to eight Mg(2+)ions interact site-specifically with the ribozyme, four of which are bound with high affinity. The global fold of the CPEB3 ribozyme, encompassing 80%-90% of the predicted base pairs, is formed in the presence of monovalent ions alone. Low millimolar concentrations of Mg(2+)promote a more compact fold and lead to the formation of additional structures in the core of the ribozyme, which contains the inner small pseudoknot and the active site. Several Mg(2+)binding sites, which are important for the functional fold, appear to be located in corresponding locations in the HDV and CPEB3 ribozyme, demonstrating the particular relevance of Mg(2+)for the nested double pseudoknot structure.

The 5'-AG5 CC-3' Fragment from the Human CPEB3 Ribozyme Forms an Ultrastable Parallel RNA G-Quadruplex.

An unusually thermostable G-quadruplex is formed by a sequence fragment of a naturally occurring ribozyme, the human CPEB3 ribozyme. Strong evidence is provided for the formation of a uniquely stable intermolecular G-quadruplex structure consisting of five tetrad layers, by using CD spectroscopy, UV melting curves, 2D NMR spectroscopy, and gel shift analysis. The cationic porphyrin TMPyP4 destabilizes the complex.

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.

Inhibition of Cpeb3 ribozyme elevates CPEB3 protein expression and polyadenylation of its target mRNAs and enhances object location memory.

A self-cleaving ribozyme that maps to an intron of the cytoplasmic polyadenylation element-binding protein 3 (Cpeb3) gene is thought to play a role in human episodic memory, but the underlying mechanisms mediating this effect are not known. We tested the activity of the murine sequence and found that the ribozyme's self-scission half-life matches the time it takes an RNA polymerase to reach the immediate downstream exon, suggesting that the ribozyme-dependent intron cleavage is tuned to co-transcriptional splicing of the Cpeb3 mRNA. Our studies also reveal that the murine ribozyme modulates maturation of its harboring mRNA in both cultured cortical neurons and the hippocampus: inhibition of the ribozyme using an antisense oligonucleotide leads to increased CPEB3 protein expression, which enhances polyadenylation and translation of localized plasticity-related target mRNAs, and subsequently strengthens hippocampal-dependent long-term memory. These findings reveal a previously unknown role for self-cleaving ribozyme activity in regulating experience-induced co-transcriptional and local translational processes required for learning and memory.

Stored within DNA are the instructions cells need to make proteins. In order for proteins to get made, the region of DNA that codes for the desired protein (known as the gene) must first be copied into a molecule called messenger RNA (or mRNA for short). Once transcribed, the mRNA undergoes further modifications, including removing redundant segments known as introns. It then travels to molecular machines that translate its genetic sequence into the building blocks of the protein. Following transcription, some RNAs can fold into catalytic segments known as self-cleaving ribozymes which promote the scission of their own genetic sequence. One such ribozyme resides in the intron of a gene for CPEB3, a protein which adds a poly(A) tail to various mRNAs, including some involved in learning and memory. Although this ribozyme is found in most mammals, its biological role is poorly understood. Previous studies suggested that the ribozyme cleaves itself at the same time as the mRNA for CPEB3 is transcribed. This led Chen et al. to hypothesize that the rate at which these two events occur impacts the amount of CPEB3 produced, resulting in changes in memory and learning. If the ribozyme cleaves quickly, the intron is disrupted and may not be properly removed, leading to less CPEB3 being made. However, if the ribozyme is inhibited, the intron remains intact and is efficiently excised, resulting in higher levels of CPEB3 protein. To test how the ribozyme impacts CPEB3 production, Chen et al. inhibited the enzyme from cutting itself with antisense oligonucleotides (ASOs). The ASOs were applied to in vitro transcription systems, neurons cultured in the laboratory and the brains of living mice in an area called the hippocampus. The in vitro and cell culture experiments led to higher levels of CPEB3 protein and the addition of more poly(A) tails to mRNAs involved in neuron communication. Injection of the ASOs into the brains of mice had the same effect, and also improved their memory and learning. The findings of Chen et al. show a new mechanism for controlling protein production, and suggest that ASOs could be used to increase the levels of CPEB3 and modulate neuronal activity. This is the first time a biological role for a self-cleaving ribozyme in mammals has been identified, and the approach used could be applied to investigate the function of two other self-cleaving ribozymes located in introns in humans.

Prenatal diesel exhaust exposure alters hippocampal synaptic plasticity in offspring.

Diesel exhaust particles (DEPs) are major air pollutants emitted from automobile engines. Prenatal exposure to DEPs has been linked to neurodevelopmental and neurodegenerative diseases associated with aging. However, the specific mechanism by DEPs impair the hippocampal synaptic plasticity in the offspring remains unclear. Pregnant C57BL/6 mice were administered DEPs solution via the tail vein every other day for a total of 10 injections, then the male offsprings were studied to assess learning and memory by the Morris water maze. Additionally, protein expression in the hippocampus, including CPEB3, NMDAR (NR1, NR2A, NR2B), PKA, SYP, PSD95, and p-CREB was analyzed using Western blotting and immunohistochemistry. The alterations in the histomorphology of the hippocampus were observed in male offspring on postnatal day 7 following prenatal exposure to DEPs. Furthermore, 8-week-old male offspring exposed to DEPs during prenatal development exhibited impairments in the Morris water maze test, indicating deficits in learning and memory. Mechanistically, the findings from our study indicate that exposure to DEPs during pregnancy may alter the expression of CPEB3, SYP, PSD95, NMDAR (NR1, NR2A, and NR2B), PKA, and p-CREB in the hippocampus of both immature and mature male offspring. The results offer evidence for the role of the NMDAR/PKA/CREB and CPEB3 signaling pathway in mediating the learning and memory toxicity of DEPs in male offspring mice. The alterations in signaling pathways may contribute to the observed damage to synaptic structure and transmission function plasticity caused by DEPs. The findings hold potential for informing future safety assessments of DEPs.

miR-351-5p regulation of CPEB3 affecting aluminium-induced learning and memory impairment in SD rats.

Aluminium exposure has been found to impair learning and memory abilities; however, the underlying molecular mechanisms remain unclear. In this study we conducted a double luciferase reporter assay to determine whether miR-351-5p regulates cytoplasmic polyadenylation element binding protein (CPEB) 3 mRNA. To this end, we overexpressed and inhibited miR-351-5p via stereotaxic microinjections of adeno-associated virus (AAV) into the hippocampus of Sprague Dawley rats in a sub-chronic aluminium exposure model to examine learning and memory ability using Morris water maze. Ultrastructural electron microscopy and Golgi staining were used to examine morphological changes in hippocampal neurons. In addition, we examined the levels of synaptic plasticity-related proteins (PRPs) and CPEB3 to determine the involvement of the miR-351-5P/CPEB3/PRPs pathway in aluminium neurotoxicity. Sub-chronic aluminium exposure reduced the spatial learning and memory ability of rats. Overexpression of AAV-miR-351-5P in the hippocampus aggravated the impairment of spatial learning and memory abilities of aluminium-treated rats, whereas inhibition of AAV-miR-351-5p expression alleviated it. Western blotting suggested that sub-chronic aluminium exposure increased miR-351-5p levels and reduced the expression of CPEB3 and PRPs in the hippocampus. Treatment with an AAV-miR-351-5p inhibitor partially recovered CPEB3 and PRPs. Double luciferase reporter assay results showed that CPEB3 was a direct target of miR-351-5p, while electron microscopy suggested that aluminium could damage mitochondria and synapses in the CA1 of the hippocampus. Golgi staining results indicated that aluminium could reduce the number of dendritic spines in hippocampal neurons. Inhibition of miR-351-5p restored the synaptic structure and growth of dendritic spines in the hippocampus. The involvement of the miR-351-5P/CPEB3/RPPs pathway in aluminium neurotoxicity was confirmed. Our findings suggest that inhibition of miR-351-5p can alleviate learning and memory impairments by increasing CPEB3 and PRPs.

Phase separation modulates the functional amyloid assembly of human CPEB3.

How functional amyloids are regulated to restrict their activity is poorly understood. The cytoplasmic polyadenylation element-binding protein 3 (CPEB3) is an RNA-binding protein that adopts an amyloid state key for memory persistence. Its monomer represses the translation of synaptic target mRNAs while phase separated, whereas its aggregated state acts as a translational activator. Here, we have explored the sequence-driven molecular determinants behind the functional aggregation of human CPEB3 (hCPEB3). We found that the intrinsically disordered region (IDR) of hCPEB3 encodes both an amyloidogenic and a phase separation domain, separated by a poly-A-rich region. The hCPEB3 amyloid core is composed by a hydrophobic region instead of the Q-rich stretch found in the Drosophila orthologue. The hCPEB3 phase separation domain relies on hydrophobic interactions with ionic strength dependence, and its droplet ageing process leads to a liquid-to-solid transition with the formation of a non-fibril-based hydrogel surrounded by starburst droplets. Furthermore, we demonstrate the differential behavior of the protein depending on its environment. Under physiological-like conditions, hCPEB3 can establish additional electrostatic interactions with ions, increasing the stability of its liquid droplets and driving a condensation-based amyloid pathway.

Neuralized1-Mediated CPEB3 Ubiquitination in the Spinal Dorsal Horn Contributes to the Pathogenesis of Neuropathic Pain in Rats.

Compelling evidence has shown that Neuralized1 (Neurl1) facilitates hippocampal-dependent memory storage by modulating cytoplasmic polyadenylation element-binding protein 3 (CPEB3)-dependent protein synthesis. In the current study, we investigated the role of Neurl1 in the pathogenesis of neuropathic pain and the underlying mechanisms. The neuropathic pain was evaluated by lumbar 5 spinal nerve ligation (SNL) in rats. Immunofluorescence staining, Western blotting, qRT-PCR, and coimmunoprecipitation (Co-IP) were performed to investigate the underlying mechanisms. Our results showed that SNL led to an increase of Neurl1 in the spinal dorsal horn. Spinal microinjection of AAV-EGFP-Neurl1 shRNA alleviated mechanical allodynia; decreased the level of CPEB3 ubiquitination; inhibited the production of GluA1, GluA2, and PSD95; and reduced GluA1-containing AMPA receptors in the membrane of the dorsal horn following SNL. Knockdown of spinal CPEB3 decreased the production of GluA1, GluA2, and PSD95 in the dorsal horn and attenuated abnormal pain after SNL. Overexpression of Neurl1 in the dorsal horn resulted in pain-related hypersensitivity in naïve rats; raised the level of CPEB3 ubiquitination; increased the production of GluA1, GluA2, and PSD95; and augmented GluA1-containing AMPA receptors in the membrane in the dorsal horn. Moreover, spinal Neurl1 overexpression-induced mechanical allodynia in naïve rats was partially reversed by repeated intrathecal injections of CPEB3 siRNA. Collectively, our results suggest that SNL-induced upregulation of Neurl1 through CPEB3 ubiquitination-dependent production of GluA1, GluA2, and PSD95 in the dorsal horn contributes to the pathogenesis of neuropathic pain in rats. Targeting spinal Neurl1 might be a promising therapeutic strategy for the treatment of neuropathic pain.

CPEB3, an RNA-Binding Protein, Modulates the Behavior of Endometriosis-Derived Stromal Cells via Regulating CXCL12.

Endometriosis is a benign gynecological disease sharing several features with malignant tumor. Cytoplasmic polyadenylation element-binding protein 3 (CPEB3), a potential target of miR-21-5p, is downregulated in endometriotic specimens. However, the function of CPEB3 in endometriosis is elusive. In this study, in cultured primary human endometrial stromal cells (ESCs), the overexpression and inhibition of CPEB3 were achieved by transduction of adenovirus-mediated CPEB3 overexpressed plasmid and shRNA, respectively. Functional analysis uncovered that upregulated CPEB3 reduced cell viability and arrested cell cycle entry. The expression of cyclin D1 and c-Myc was decreased after CPEB3 overexpression. Overexpression of CPEB3 facilitated ESC apoptotic potential, accompanied by increased Bax, cleaved-caspase 3 and cleaved-caspase 9, and reduced Bcl2. Moreover, elevated CPEB3 weakened migration and invasion abilities of ESCs. CPEB3 overexpression also reduced the expression of fibronectin and vimentin and the activities of matrix metalloproteinase (MMP)-9 and MMP-2. Interestingly, these effects were counteracted by CPEB3 inhibition. Furthermore, CPEB3 controlled the protein level of CXCL12, a homeostatic chemokine. CXCL12 elevation partially reversed the effects of CPEB3 on inhibiting ESC proliferation, migration and invasion, and promoting apoptosis. Based on these findings, it seems possible that CPEB3, as a critical player, attenuated the progression of endometriosis through repressing CXCL12 expression.

Circ_0007624 suppresses the development of esophageal squamous cell carcinoma via targeting miR-224-5p/CPEB3 to inactivate the EGFR/PI3K/AKT signaling.

Circular RNAs (circRNAs) have been confirmed to be involved in the regulation of esophageal squamous cell carcinoma (ESCC) progression. According to GEO datasets (GSE112496 and GSE150476), we identified that circ_0007624 was abnormally down-regulated in ESCC. However, there is still no reports regarding the function and mechanism of circ_0007624 in ESCC development. Here, we found that circ_0007624 was significantly underexpressed in ESCC tissues, and low expression of circ_0007624 was indicative of a poor prognosis. Overexpressing circ_0007624 or silencing miR-224-5p suppressed cell proliferation, metastasis, epithelial-mesenchymal transition (EMT), and promoted apoptosis in vitro. Also, circ_0007624 up-regulation slowed ESCC tumor growth in vivo. Mechanistically, circ_0007624 could serve as a competing endogenous RNA (ceRNA) by sponging miR-224-5p to antagonize its inhibitory effect on the target cytoplasmic polyadenylation element binding protein 3 (CPEB3). Rescue experiments showed that the anti-cancer properity role of circ_0007624 in ESCC is partly reversed by the restoration of miR-224-5p or down-regulation of CPEB3. Furthermore, EGFR/PI3K/AKT pathway was involved in the regulation of circ_0007624/miR-224-5p/CPEB3 axis in ESCC. Together, our findings demonstrate for the first time that circ_0007624/miR-224-5p/CPEB3 suppresses ESCC progression by inactivating EGFR/PI3K/AKT signaling, providing a basis for developing circ_0007624-targeted therapies for ESCC patients.

Mapping the Fibril Core of the Prion Subdomain of the Mammalian CPEB3 that is Involved in Long Term Memory Retention.

Long-term memory storage is modulated by the prion nature of CPEB3 forming the molecular basis for the maintenance of synaptic facilitation. Here we report that the first prion sub-domain PRD1 of mouse CPEB3 can autonomously form amyloid fibrils in vitro and punctate-like structures in vivo. A ninety-four amino acid sequence within the PRD1 domain, PRD1-core, displays high propensity towards aggregation and associated amyloid characteristics. PRD1-core is characterized using electron microscopy, X-ray diffraction, and solution-state NMR deuterium exchange experiments. Secondary structure elements deduced from solid-state NMR reveal a ß-rich core comprising of forty amino acids at the N-terminus of PRD1-core. The synthesized twenty-three amino acid long peptide containing the longest rigid segment (E124-H145) of the PRD1-core rapidly self-aggregates and forms fibrils, indicating a limited aggregation-prone region that could potentially activate the aggregation of the full-length protein. This study provides the first step in identifying the structural trigger for the CPEB3 aggregation process.

MiR-9-5p promotes cell proliferation and migration of hepatocellular carcinoma by targeting CPEB3.

Objective: This study aimed to observe the effect of miR-9-5p and CPEB3 on hepatocellular carcinoma (HCC) cells, and investigate the underlying targeting regulatory mechanism. Materials & methods: Various experiments like CCK-8, colony formation assay, wound healing assay and Transwell were performed for cancer cell activities detection, including cell proliferation, growth activity, migration and invasion. Results: MiR-9-5p was found to be highly expressed in HCC cells, while CPEB3 was poorly expressed (p < 0.05). The overexpression of miR-9-5p and the silencing of CPEB3 both could significantly promote cell proliferation, migration and invasion (p < 0.05). In addition, miR-9-5p could target to downregulate CPEB3 expression, thus accelerating cell proliferation, migration, invasion and epithelial-mesenchymal transition process in HCC. Conclusion: MiR-9-5p can target CPEB3, thereby promoting cell proliferation, migration and invasion in HCC. The axis of miR-9-5p/CPEB3 is expected to become a potential therapeutic target beneficial for HCC patients.