CPEB Research Articles

Identification of Dynamic RNA-Binding Proteins Uncovers a Cpeb4-Controlled Regulatory Cascade During Pathological Cell Growth of Cardiomyocytes

RNA binding proteins (RBPs) have the potential to affect most post-transcriptional steps in gene expression. RBPs control critical elements of cellular function, but their specific role in cardiomyocytes is largely unknown. Here we defined RBPs in healthy and diseased primary cardiomyocytes at a system-wide level by RNA Interactome Capture. This identified 67 novel cardiomyocyte specific RBPs including several contractile proteins. Furthermore, we identified Cytoplasmic polyadenylation element binding protein 4 (Cpeb4) as a dynamic mRBP in diseased cardiomyocytes, regulating cardiac growth both in vitro and in vivo. To study Cpeb4 in cardiomyocytes, we identified mRNAs bound to and regulated by Cpeb4. Cpeb4 regulates cardiac remodeling by differential expression of transcription factors. Among Cpeb4 target mRNAs, two Zinc finger transcription factors (Zeb1 and Zbtb20) were identified. We show that Cpeb4 regulates the translation of these mRNAs and that Cpeb4 depletion increases their expression. Thus, Cpeb4 emerges as critical regulator of cardiomyocyte function by differential binding of specific mRNAs in response to pathological growth stimulation.

Depletion of CPEB1 protects against oxidized LDL-induced endothelial apoptosis and inflammation though SIRT1/LOX–1 signalling pathway

Atherosclerosis (AS) is a chronic inflammatory disease that results from Oxidized low-density lipoprotein (Ox-LDL) induced endothelial dysfunction. Cytoplasmic polyadenylation element binding protein 1 (CPEB1) is closely related to the development of epithelial cells, but the role of CPEB1 in AS remains unknown. The RNA and protein levels of CPEB1 expression are increased by Ox-LDL exposure, which is abrogated by c-Jun amino-terminal kinase (JNK) inhibitor SP600125. CPEB1 small interfering RNA (siRNA) suppressed the oxidative stress, inflammation, and apoptosis. Furthermore, CPEB1 siRNA enhanced the sirtuin 1 (SIRT1) transcription levels in Ox-LDL-treated HUVECs. Co-Immunoprecipitation (Co-IP) assay showed that CPEB1 siRNA declined the ubiquitination of SIRT1, and SIRT1 siRNA enhanced the Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), which were decreased by CPEB1 siRNA. In addition, LOX-1 and SIRT1 attenuated the protection of SIRT1 siRNA on Ox-LDL-induced oxidative stress. Therefore, our study revealed that CPEB1 depletion might play an anti-inflammatory and antiapoptotic role in Ox-LDL-induced apoptosis and inflammation though SIRT1/LOX‐1 signalling pathway.

Serine/Arginine-Rich Splicing Factor 3 Modulates the Alternative Splicing of Cytoplasmic Polyadenylation Element Binding Protein 2.

Triple negative breast cancer (TNBC) has an unusually low 5-year survival rate linked to higher metastatic rates. Our laboratory recently delineated a role for the alternative RNA splicing (AS) of cytoplasmic polyadenylation element binding protein 2 (CPEB2), via inclusion/exclusion of exon 4, in the metastasis of TNBC. In these studies, the mechanism governing the inclusion/exclusion of exon 4 was examined. Specifically, the RNA trans-factor, SRSF3, was found to be explicitly associated with CPEB2 exon 4. A SRSF3 consensus sequence was identified in exon 4, and mutation of this sequence abolished the association of SRSF3. The expression of SRSF3 was upregulated in TNBC cells upon the acquisition of anoikis resistance correlating with a reduction in the CPEB2A/B ratio. Importantly, downregulation of SRSF3 in these cells by siRNA induced the exclusion of exon 4 in cells increasing the ratio of CPEB2A (exon 4 excluded) to CPEB2B (exon 4 included). Downregulation of SRSF3 also reversed the CPEB2A/B ratio of a wild-type CPEB2 exon 4 minigene and endogenous CPEB2 pre-mRNA, but not a mutant CPEB2 minigene with the SRSF3 RNA cis-element ablated. SRSF3 downregulation ablated the anoikis resistance of TNBC cells, which was "rescued" by ectopic expression of CPEB2B. Finally, analysis of The Cancer Genome Atlas database showed a positive relationship between SRSF3 expression and lower CPEB2A/B ratios in aggressive breast cancers. IMPLICATIONS: These findings demonstrate that SRSF3 modulates CPEB2 AS to induce the expression of the CPEB2B isoform that drives TNBC phenotypes correlating with aggressive human breast cancer. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/17/9/1920/F1.large.jpg.

CPEB3 inhibits translation of mRNA targets by localizing them to P bodies

Protein synthesis is crucial for the maintenance of long-term memory-related synaptic plasticity. The cytoplasmic polyadenylation element-binding protein 3 (CPEB3) regulates the translation of several mRNAs important for long-term synaptic plasticity in the hippocampus. In previous studies, we found that the oligomerization and activity of CPEB3 are controlled by small ubiquitin-like modifier (SUMO)ylation. In the basal state, CPEB3 is SUMOylated; it is soluble and acts as a repressor of translation. Following neuronal stimulation, CPEB3 is de-SUMOylated; it now forms oligomers that are converted into an active form that promotes the translation of target mRNAs. To better understand how CPEB3 regulates the translation of its mRNA targets, we have examined CPEB3 subcellular localization. We found that basal, repressive CPEB3 is localized to membraneless cytoplasmic processing bodies (P bodies), subcellular compartments that are enriched in translationally repressed mRNA. This basal state is affected by the SUMOylation state of CPEB3. After stimulation, CPEB3 is recruited into polysomes, thus promoting the translation of its target mRNAs. Interestingly, when we examined CPEB3 recombinant protein in vitro, we found that CPEB3 phase separates when SUMOylated and binds to a specific mRNA target. These findings suggest a model whereby SUMO regulates the distribution, oligomerization, and activity of oligomeric CPEB3, a critical player in the persistence of memory.

Long non-coding RNA HCG11 modulates glioma progression through cooperating with miR-496/CPEB3 axis.

ObjectivesIt has been widely reported that long non‐coding RNAs (lncRNAs) can participate in multiple biological processes of human cancers. lncRNA HLA complex group 11 (HCG11) has been reported in human cancers as a tumour suppressor. This study focused on investigating the function and mechanism of HCG11 in glioma.Materials and methodsBased on The Cancer Genome Atlas (TCGA) data set and qRT‐PCR analysis, the expression pattern of HCG11 was identified in glioma samples. The mechanism associated with HCG11 downregulation was determined by mechanism experiments. Gain‐of‐function assays were conducted for the identification of HCG11 function in glioma progression. Mechanism investigation based on the luciferase reporter assay, RIP assay and pull‐down assay was used to explore the downstream molecular mechanism of HCG11. The role of molecular pathway in the progression of glioma was analysed in accordance with the rescue assays.Results HCG11 was expressed at low level in glioma samples compared with normal samples. FOXP1 could bind with HCG11 and transcriptionally inactivated HCG11. Overexpression of HCG11 efficiently suppressed cell proliferation, induced cell cycle arrest and promoted cell apoptosis. HCG11 was predominantly enriched in the cytoplasm of glioma cells and acted as a competing endogenous RNAs (ceRNAs) by sponging micro‐496 to upregulate cytoplasmic polyadenylation element binding protein 3 (CPEB3). CEPB3 and miR‐496 involved in HCG11‐mediated glioma progression.Conclusions HCG11 inhibited glioma progression by regulating miR‐496/CPEB3 axis.

The mitochondrial calcium uniporter contributes to morphine tolerance through pCREB and CPEB1 in rat spinal cord dorsal horn

BackgroundThe long-term use of opioid analgesics is limited by the development of unwanted side-effects, such as tolerance. The molecular mechanisms of morphine anti-nociceptive tolerance are still unclear. The mitochondrial calcium uniporter (MCU) is involved in painful hyperalgesia, but the role of MCU in morphine tolerance has not been uncharacterised. MethodsRats received intrathecal injection of morphine for 7 days to induce morphine tolerance. The mechanical withdrawal threshold was measured using von Frey filaments, and thermal latency using the hotplate test. The effects of an MCU inhibitor, antisense oligodeoxynucleotide against cyclic adenosine monophosphate response element (CRE)-binding protein (CREB) or cytoplasmic polyadenylation element-binding protein 1 (CPEB1) in morphine tolerance were examined. ResultsSpinal morphine tolerance was associated with an increased expression of neuronal MCU, phospho-CREB (pCREB), and CPEB1 in the spinal cord dorsal horn. MCU inhibition increased the mechanical threshold and thermal latency, and reduced the accumulation of mitochondrial calcium in morphine tolerance. Intrathecal antisense oligodeoxynucleotide against CREB or CPEB1 restored the anti-nociceptive effects of morphine compared with mismatch oligodeoxynucleotide in von Frey test and hotplate test. Chromatin immunoprecipitation with quantitative PCR assay showed that CREB knockdown reduced the interaction of pCREB with the ccdc109a gene (encoding MCU expression) promoter and decreased the MCU mRNA transcription. RNA immunoprecipitation assay suggested that CPEB1 binds to the MCU mRNA 3′ untranslated region. CPEB1 knockdown decreased the expression of MCU protein. ConclusionsThese findings suggest that spinal MCU is regulated by pCREB and CPEB1 in morphine tolerance, and that inhibition of MCU, pCREB, or CPEB1 may be useful in preventing the development of opioid tolerance.

Tumor suppressor role of cytoplasmic polyadenylation element binding protein 2 (CPEB2) in human mammary epithelial cells

BackgroundOver-expression of cyclooxygenase (COX)-2 promotes breast cancer progression by multiple mechanisms, including induction of stem-like cells (SLC). Combined gene expression and microRNA microarray analyses of empty vector vs COX-2- transfected COX-2 low MCF7 breast cancer cell line identified two COX-2-upregulated microRNAs, miR-526b and miR-655, both found to be oncogenic and SLC-promoting. Cytoplasmic Polyadenylation Element-Binding Protein 2 (CPEB2) was the single common target of both microRNAs, the functions of which remain controversial. CPEB2 has multiple isoforms (A-F), and paradoxically, a high B/A ratio was reported to impart anoikis-resistance and metastatic phenotype in triple- negative breast cancer cells. We tested whether CPEB2 is a tumor suppressor in mammary epithelial cells.MethodsWe knocked-out CPEB2 in the non-tumorigenic mammary epithelial cell line MCF10A by CRISPR/Cas9-double nickase approach, and knocked-down CPEB2 with siRNAs in the poorly malignant MCF7 cell line, both lines being high CPEB2-expressing. The resultant phenotypes for oncogenity were tested in vitro for both lines and in vivo for CPEB2KO cells. Finally, CPEB2 expression was compared between human breast cancer and non-tumor breast tissues.ResultsCPEB2 (isoform A) expression was inversely correlated with COX-2 or the above microRNAs in COX-2-divergent breast cancer cell lines. CPEB2KO MCF10A cells exhibited oncogenic properties including increased proliferation, migration, invasion, EMT (decreased E-Cadherin, increased Vimentin, N-Cadherin, SNAI1, and ZEB1) and SLC phenotype (increased tumorsphere formation and SLC marker-expression). Tumor-suppressor p53 protein was shown to be a novel translationally-regulated target of CPEB2, validated with polysome profiling. CPEB2KO, but not wild-type cells produced lung colonies upon intravenous injection and subcutaneous tumors and spontaneous lung metastases upon implantation at mammary sites in NOD/SCID/IL2Rϒ-null mice, identified with HLA immunostaining. Similarly, siRNA-mediated CPEB2 knockdown in MCF7 cells promoted oncogenic properties in vitro. Human breast cancer tissues (n = 105) revealed a lower mRNA expression for CPEB2 isoform A and also a lower A/B isoform ratio than in non-tumour breast tissues (n = 20), suggesting that CPEB2A accounts for the tumor-suppressor functions of CPEB2.ConclusionsCPEB2, presumably the isoform A, plays a key role in suppressing tumorigenesis in mammary epithelial cells by repressing EMT, migration, invasion, proliferation and SLC phenotype, via multiple targets, including a newly-identified translational target p53.

Functional Amyloids.

When protein/peptides aggregate, they usually form the amyloid state consisting of cross β-sheet structure built by repetitively stacked β-strands forming long fibrils. Amyloids are usually associated with disease including Alzheimer's. However, amyloid has many useful features. It efficiently transforms protein from the soluble to the insoluble state in an essentially two-state process, while its repetitive structure provides high stability and a robust prion-like replication mechanism. Accordingly, amyloid is used by nature in multifaceted and ingenious ways of life, ranging from bacteria and fungi to mammals. These include (1) Structure: Templating for small chemical molecules (Pmel17), biofilm formation in bacteria (curli), assisting aerial hyphae formation in streptomycetes (chaplins) or monolayer formation at a surface (hydrophobins). (2) Reservoirs: A storage state for peptide/proteins to protect them from their surroundings or vice versa (storage of peptide hormones in mammalian secretory granules or major basic protein in eosinophils). (3) Information carriers: The fungal immune system (HET-s prion in Podospora anserina, yeast prions) or long-term memory (e.g., mnemons in yeast, cytoplasmic polyadenylation element-binding protein in aplysia). Aggregation is also used to (4) "suppress" the function of the soluble protein (e.g., Cdc19 in yeast stress granules), or (5) "signaling" through formation of oligomers (e.g., HET-s prion, necroptosis-related proteins RIP1/RIP3). This review summarizes current knowledge on functional amyloids with a focus on the amyloid systems curli in bacteria, HET-s prion in P. anserina, and peptide hormone storage in mammals together with an attempt to highlight differences between functional and disease-associated amyloids.

MiRNA-301b-3p accelerates migration and invasion of high-grade ovarian serous tumor via targeting CPEB3/EGFR axis.

High-grade ovarian serous carcinoma (HGS-OvCa), a type of ovarian cancer with poor prognosis due to distant metastasis, is urgently in need of new therapeutic targets. microRNAs (miRNAs), a class of small noncoding RNAs, perform significant roles in tumor progression. Mounting evidence has revealed the aberrant expression of miRNA in various cancers, one of which is HGS-OvCa. Present study planned to investigate that miRNA-301b-3p accelerates migration and invasion of high-grade ovarian serous tumor via targeting CPEB3/EGFR axis. Upregulation of miR-301b-3p was uncovered in HGS-OvCa tissues and cell lines, and was identified to be associated with metastasis. The Kaplan-Meier analysis confirmed the association of miR-301b-3p with poor prognosis of HGS-OvCa patients. Transwell assay validated the oncogenic effect of miR-301b-3p on migration and invasion of HGS-OvCa cells. Cytoplasmic polyadenylation element binding protein 3 (CPEB3) was then identified as a target of miR-301b-3p. It was also discovered that CPEB3 was downregulated in HGS-OvCa tissues and cell lines. The Spearman correlation curve presented the negative correlation of CPEB3 expression with miR-301b-3p. Furthermore, rescue assays proved that miRNA-301b-3p regulated the invasion and migration through CPEB3. Western blot and qRT-PCR analysis showed that miRNA-301b-3p induced epidermal growth factor receptor and downstream metastasis-related proteins, p38, and extracellular signal-regulated kinase 1/2 (ERK1/2), through CPEB3. To be concluded, these results indicated that miRNA-301b-3p accelerated migration and invasion of high-grade ovarian serous tumor via targeting CPEB3/EGFR axis.

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.

CPEB1 is overexpressed in neurons derived from Down syndrome IPSCs and in the hippocampus of the mouse model Ts1Cje

Trisomy 21, also known as Down syndrome (DS), is the most frequent genetic cause of intellectual impairment. In mouse models of DS, deficits in hippocampal synaptic plasticity have been observed, in conjunction with alterations to local dendritic translation that are likely to influence plasticity, learning and memory. Here we show that expression of a local translational regulator, the Cytoplasmic Polyadenylation Element Binding Protein 1 (CPEB1), is enhanced in hippocampal neurons from the Ts1Cje DS mouse model. Interestingly, this protein, which is also involved in dendritic mRNA transport, is overexpressed in dendrites of neurons derived from DS human induced pluripotent stem cells (hIPSCs). Moreover, there is an increase in the mRNA levels of α-Calmodulin Kinase II (α-CaMKII) and Microtubule-associated protein 1B (MAP1B), two dendritic mRNAs, in Ts1Cje synaptoneurosomes. Taking into account the fundamental role of CPEB1 protein and its target mRNAs in synaptic plasticity, these data could be relevant to the intellectual impairment in the context of DS.

Effects of CPEB1 in the anterior cingulate cortex on visceral pain in mice

Patients with irritable bowel syndrome suffer from chronic visceral pain, and in some of them, this is accompanied by anxiety comorbidity. Cytoplasmic polyadenylation element binding protein 1 (CPEB1) mediates the cytoplasmic polyadenylation of mRNAs and facilitates their translation. Our previous studies have shown that CPEB1 knockdown in the amygdala exerts anxiolytic but not analgesic effects in a mouse model of inflammatory pain. However, the roles of CPEB1 in the anterior cingulate cortex (ACC) in visceral pain modulation remain unclear. In this study, a visceral pain mouse model was established by injecting zymosan into the colon of mice. Zymosan injection significantly induced visceral pain- and anxiety-like behaviors in mice and increased the levels of GluA1, phosphorylated GluA1 at S845 and S831, and CPEB1 in the ACC. CPEB1 knockdown in the ACC by AAV-CPEB1-shRNA reduced zymosan-induced pain- and anxiety-like behaviors in mice. This observation was closely correlated with reduced AMPA receptor, synaptophysin, and PSD95 levels. These data suggest that CPEB1 in the ACC is a potential therapeutic target for visceral pain and anxiety comorbidity.

CPEB4-Dependent Neonate-Born Granule Cells Are Required for Olfactory Discrimination

The rodent olfactory bulb (OB) contains two distinct populations of postnatally born interneurons, mainly granule cells (GCs), to support local circuits throughout life. During the early postnatal period (i.e., 2 weeks after birth), GCs are mostly produced locally from progenitor cells in the OB with a proportion of them deriving from proliferating cells in the rostral migratory stream (RMS). Afterward, the replenishment of GCs involves differentiated neuroblasts from the subventricular zone (SVZ) in a process known as adult neurogenesis. Although numerous studies have addressed the role of SVZ-born GCs in olfactory behaviors, the function of GCs produced early postnatally in the OB remains elusive. Our previous study demonstrated that the translational regulator, cytoplasmic polyadenylation element-binding protein 4 (CPEB4), is a survival factor exclusively for neonate-born but not SVZ/adult-derived GCs, so CPEB4-knockout (KO) mice provide unique leverage to study early postnatal-born GC-regulated olfactory functions. CPEB4-KO mice with hypoplastic OBs showed normal olfactory sensitivity and short-term memory, but impaired ability to spontaneously discriminate two odors. Such olfactory dysfunction was recapitulated in specific ablation of Cpeb4 gene in inhibitory interneurons but not in excitatory projection neurons or SVZ-derived interneurons. The continuous supply of GCs from adult neurogenesis eventually restored the OB size but not the discrimination function in 6-month-old KO mice. Hence, in the early postnatal OB, whose function cannot be replaced by adult-born GCs, construct critical circuits for odor discrimination.

Expression of indoleamine 2, 3-dioxygenase and cytoplasmic polyadenylation element binding protein 4 in gastric carcinoma and their clinical significance

Objective To investigate the expression of indoleamine 2, 3-dioxygenase (IDO) and cytoplasmic polyadenylation element binding protein 4 (CPEB4) in gastric carcinoma and their clinical significance. Methods A total of 92 cases of gastric carcinoma from January 2015 to December 2017 were collected. The immunohistochemical method was used to detect and analyze the expression levels of IDO and CPEB4 in gastric carcinoma and adjacent gastric tissues. Results The expression rate of IDO was 65.22% (60/92) in gastric carcinoma tissues, and 38.04% (35/92) in non-cancerous adjacent gastric tissues (t= 13.60, P<0.01). The expressions levels of IDO were related gastric carcinoma to TNM stage and lymph node metastasis (t=7.64, 5.37 respectively, P<0.05). The expression rate of CPEB4 was 57.61% (53/92) in gastric carcinoma tissues, and 32.61% (30/92) in non-cancerous adjacent gastric tissues (t=11.61, P<0.01). The expression levels of CPEB4 were related gastric carcinoma to histology grade, TNM stage and lymph node metastasis (t= 5.58, 6.85, 7.35 respectively, P<0.05). Conclusion The expression levels of IDO and CPEB4 may be related to the carcinogenesis and progression of gastric carcinoma. Key words: Indoleamine 2, 3-dioxygenase; Cytoplasmic polyadenylation element binding protein 4; Gastric carcinoma; Immunohistochemical method

Cytoplasmic polyadenylation element binding protein 2 (CPEB2) is required for tight-junction assembly for establishment of porcine trophectoderm epithelium.

Cytoplasmic polyadenylation element binding protein (CPEB) is an RNA-binding protein that promotes elongation of poly(A) tails and regulates mRNA translation. CPEB depletion in mammary epithelium is known to disrupt tight-junction (TJ) assembly via mislocalisation of tight junction protein 1 (TJP1), but the role of CPEB in the biological functions associated with TJs has not yet been studied. The objective of this study was to investigate the roles of CPEB2 during porcine parthenote development. CPEB2 was detected in both the nuclei and apical cytoplasm at the 4- and 8-cell stages and was localised to cell-cell contact after the initiation of the morula stage. Its depletion led to retarded blastocyst formation caused by impaired TJ assembly. Moreover, transcription of TJ-associated genes, including TJP1, Coxsackie virus and adenovirus receptor (CXADR) and occludin (OCLN), was not affected, but the corresponding proteins were not properly localised at the apical cell membrane in morulae, suggesting that CPEB2 confers mRNA stability or determines subcellular localisation for translation. Remarkably reduced relative levels of TJP1 transcripts bearing the 3'-untranslated region were noted, indicating that CPEB2 mediates TJP1 mRNA stability. In conclusion, our findings demonstrate that because of its regulation of TJP1, CPEB2 is required for TJ assembly during porcine blastocyst development.

A continuous model of physiological prion aggregation suggests a role for Orb2 in gating long-term synaptic information.

The regulation of mRNA translation at the level of the synapse is believed to be fundamental in memory and learning at the cellular level. The family of cytoplasmic polyadenylation element binding (CPEB) proteins emerged as an important RNA-binding protein family during development and in adult neurons. Drosophila Orb2 (homologue of mouse CPEB3 protein and of the neural isoform of Aplysia CPEB) has been found to be involved in the translation of plasticity-dependent mRNAs and has been associated with long-term memory. Orb2 protein presents two main isoforms, Orb2A and Orb2B, which form an activity-induced amyloid-like functional aggregate, thought to be the translation-inducing state of the RNA-binding protein. Here we present a first two-states continuous differential model for Orb2A–Orb2B aggregation. This model provides new working hypotheses for studying the role of prion-like CPEB proteins in long-term synaptic plasticity. Moreover, this model can be used as a first step to integrate translation- and protein aggregation-dependent phenomena in synaptic facilitation rules.

Micro-RNAs, their target proteins, predispositions and the memory of filial imprinting

Visual imprinting is a learning process whereby young animals come to prefer a visual stimulus after exposure to it (training). The intermediate medial mesopallium (IMM) in the domestic chick forebrain is critical for visual imprinting and contributes to molecular regulation of memory formation. We investigated the role of micro-RNAs (miRNAs) in such regulation. Twenty-four hours after training, miRNA spectra in the left IMM were compared between chicks with high preference scores (strong memory for imprinting stimulus), and chicks with low preference scores (weak memory for imprinting stimulus). Using criteria of significance and expression level, we chose gga-miR-130b-3p for further study and found that down-regulation correlated with learning strength. No effect was detected in posterior nidopallium, a region not involved in imprinting. We studied two targets of gga-miR-130b-3p, cytoplasmic polyadenylation element binding proteins 1 (CPEB-1) and 3 (CPEB-3), in two subcellular fractions (P2 membrane-mitochondrial and cytoplasmic) of IMM and posterior nidopallium. Only in the left IMM was a learning-related effect observed, in membrane CPEB-3. Variances from the regression with preference score and untrained chicks suggest that, in the IMM, gga-miR-130b-3p level reflects a predisposition, i.e. capacity to learn, whereas P2 membrane-mitochondrial CPEB-3 is up-regulated in a learning-specific way.

CPEB1 Expression Correlates with Severity of Posttraumatic Ankle Osteoarthritis and Aggravates Catabolic Effect of IL-1β on Chondrocytes

Most cases of posttraumatic ankle osteoarthritis (PTAOA) represent a sequela of ankle fractures. The cytoplasmic polyadenylation element-binding protein 1 (CPEB1) is an RNA binding protein that controls protein expression. Here, we report the previously unappreciated association of CPEB1 with PTAOA. We found that CPEB1 was upregulated in articular cartilage from patients with PTAOA. Additionally, its expression level positively correlated with disease severity. In human primary chondrocytes cultured in vitro, CPEB1 was upregulated when treated with pro-inflammatory cytokines, i.e., IL-1β and TNF-α, suggesting that the observed CPEB1 upregulation in articular cartilage of PTAOA patients may be attributed to local inflammatory milieu. Functionally, CPEB1 overexpression aggravated the catabolic effect of IL-1β on chondrocytes in vitro, and vice versa, its knockdown reduced this effect, together implying a detrimental role of CPEB1 involved in OA progression. In sum, our study identifies CPEB1 as a potential regulator of disease progression of PTAOA.

A combinatorial code for mRNA 3'-UTR-mediated translational control in the mouse oocyte.

Meiotic maturation of mammalian oocytes depends on the temporally and spatially regulated cytoplasmic polyadenylation and translational activation of maternal mRNAs. Cytoplasmic polyadenylation is controlled by cis-elements in the 3′-UTRs of mRNAs including the polyadenylation signal (PAS), which is bound by the cleavage and polyadenylation specificity factor (CPSF) and the cytoplasmic polyadenylation element (CPE), which recruits CPE binding proteins. Using the 3′-UTRs of mouse Cpeb1, Btg4 and Cnot6l mRNAs, we deciphered the combinatorial code that controls developmental stage-specific translation during meiotic maturation: (i) translation of a maternal transcript at the germinal vesicle (GV) stage requires one or more PASs that locate far away from CPEs; (ii) PASs distal and proximal to the 3′-end of the transcripts are equally effective in mediating translation at the GV stage, as long as they are not close to the CPEs; (iii) Both translational repression at the GV stage and activation after germinal vesicle breakdown require at least one CPE adjacent to the PAS; (iv) The numbers and positions of CPEs in relation to PASs within the 3′-UTR of a given transcript determines its repression efficiency in GV oocytes. This study reveals a previously unrecognized non-canonical mechanism by which the proximal PASs mediate 3′-terminal polyadenylation and translation of maternal transcripts.

CPEB2 Is Necessary for Proper Porcine Meiotic Maturation and Embryonic Development.

Oocyte meiotic maturation and embryogenesis are some of the most important physiological processes that occur in organisms, playing crucial roles in the preservation of life in all species. The post-transcriptional regulation of maternal messenger ribonucleic acids (mRNAs) and the post-translational regulation of proteins are critical in the control of oocyte maturation and early embryogenesis. Translational control affects the basic mechanism of protein synthesis, thus, knowledge of the key components included in this machinery is required in order to understand its regulation. Cytoplasmic polyadenylation element binding proteins (CPEBs) bind to the 3′-end of mRNAs to regulate their localization and translation and are necessary for proper development. In this study we examined the expression pattern of cytoplasmic polyadenylation element binding protein 2 (CPEB2) both on the mRNA (by real-time quantitative reverse transcription polymerase chain reaction, qRT-PCR) and protein (by Western blotting, WB) level, as well as its localization during the meiotic maturation of porcine oocytes and early embryonic development by immunocytochemistry (ICC). For the elucidation of its functions, CPEB2 knockdown by double-strand RNA (dsRNA) was used. We discovered that CPEB2 is expressed during all stages of porcine meiotic maturation and embryonic development. Moreover, we found that it is necessary to enable a high percentage of oocytes to reach the metaphase II (MII) stage, as well as for the production of good-quality parthenogenetic blastocysts.