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OBJECTIVE: Brown adipose tissue (BAT) plays an important role in mammalian thermogenesis through the expression of uncoupling protein 1 (UCP1). Our previous study identified cytoplasmic polyadenylation element binding protein 2 (CPEB2) as a key regulator that activates the translation of Ucp1 with a long 3'-untranslated region (Ucp1L) in response to adrenergic signaling. Mice lacking CPEB2 or Ucp1L exhibited reduced UCP1 expression and impaired thermogenesis; however, only CPEB2-null mice displayed obesogenic phenotypes. Hence, this study aims to investigate how CPEB2-controlled translation impacts body weight. METHODS: Body weight measurements were conducted on mice with global knockout (KO) of CPEB2, UCP1 or Ucp1L, as well as those with conditional knockout of CPEB2 in neurons or adipose tissues. RNA sequencing coupled with bioinformatics analysis was used to identify dysregulated gene expression in CPEB2-deficient BAT. The role of CPEB2 in regulating PRD1-BF1-RIZ1 homologous-domain containing 16 (PRDM16) expression was subsequently confirmed by RT-qPCR, Western blotting, polysomal profiling and luciferase reporter assays. Adeno-associated viruses (AAV) expressing CPEB2 or PRDM16 were delivered into BAT to assess their efficacy in mitigating weight gain in CPEB2-KO mice. RESULTS: We validated that defective BAT function contributed to the increased weight gain in CPEB2-KO mice. Transcriptomic profiling revealed upregulated expression of genes associated with muscle development in CPEB2-KO BAT. Given that both brown adipocytes and myocytes stem from myogenic factor 5-expressing precursors, with their cell-fate differentiation regulated by PRDM16, we identified that Prdm16 was translationally upregulated by CPEB2. Ectopic expression of PRDM16 in CPEB2-deprived BAT restored gene expression profiles and decreased weight gain in CPEB2-KO mice. CONCLUSIONS: In addition to Ucp1L, activation of Prdm16 translation by CPEB2 is critical for sustaining brown adipocyte function. These findings unveil a new layer of post-transcriptional regulation governed by CPEB2, fine-tuning thermogenic and metabolic activities of brown adipocytes to control body weight.

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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.

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INTRODUCTION: Cisplatin-based chemotherapy is one of the fundamental therapeutic modalities for gastric cancer (GC). Chemoresistance to cisplatin is a great clinical challenge, and its underlying mechanisms remain poorly understood. Circular RNAs (circRNAs) are involved in the pathophysiology of multiple human malignancies. METHODS: High-throughput sequencing was performed to determine the differentially expressed profile of circRNA in GC tissues and cisplatin-resistant GC cells. Quantitative real-time polymerase chain reaction and Fluorescence in situ hybridization was utilized to confirm the dysregulation of circ_0008315 in GC tissues. To evaluate the prognostic significance of circ_0008315 in GC, we used Kaplan-Meier plot. The self-renewal ability of drug-resistant GC cell was verified through tumor sphere formation assay. GC organoids were constructed to simulate the tumor microenvironment and verified the function of circ_0008315 in cisplatin resistance of gastric cancer. In vivo evaluation was conducted using patient-derived xenograft models. Dual-luciferase reporter gene, RNA immunoprecipitation and miRNA pull-down assays were employed to investigate the molecular mechanisms of circ_0008315 in GC. RESULTS: We revealed that a novel circRNA hsa_circ_0008315 was upregulated in GC and cisplatin-resistant GC cells. Elevated circ_0008315 was also observed in cisplatin-resistant GC organoid model. High circ_0008315 expression predicted unfavorable survival outcome in GC patients. Downregulation of circ_0008315 expression inhibited proliferation, mobility, and epithelial-mesenchymal transition of GC cells in vitro and in vivo. Reducing circ_0008315 expression in cisplatin-resistant GC organoid model reversed cisplatin resistance. Mechanistically, circ_0008315 modulated the stem cell properties of GC through the miR-3666/CPEB4 signaling pathway, thereby promoting cisplatin resistance and GC malignant progression. Furthermore, we developed PLGA-PEG nanoparticles targeting circ_0008315, and the nanoparticles could effectively inhibit GC proliferation and cisplatin resistance. CONCLUSION: Circ_0008315 exacerbates GC progression and cisplatin resistance, and can be used as a prognostic predictor. Circ_0008315 may function as a promising nanotherapeutic target for GC treatment.

Asunto(s)

Cisplatino , Resistencia a Antineoplásicos , ARN Circular , Neoplasias Gástricas , Neoplasias Gástricas/genética , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/patología , Cisplatino/farmacología , Cisplatino/uso terapéutico , Humanos , ARN Circular/genética , ARN Circular/metabolismo , Resistencia a Antineoplásicos/genética , Línea Celular Tumoral , Animales , Ratones , Carcinogénesis/genética , Ratones Desnudos , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Proliferación Celular/efectos de los fármacos , Femenino , Masculino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Microambiente Tumoral/genética , Transición Epitelial-Mesenquimal/genética , MicroARNs/genética , MicroARNs/metabolismo , Ratones Endogámicos BALB C

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BACKGROUND: Local translation at synapses is important for rapidly remodeling the synaptic proteome to sustain long-term plasticity and memory. While the regulatory mechanisms underlying memory-associated local translation have been widely elucidated in the postsynaptic/dendritic region, there is no direct evidence for which RNA-binding protein (RBP) in axons controls target-specific mRNA translation to promote long-term potentiation (LTP) and memory. We previously reported that translation controlled by cytoplasmic polyadenylation element binding protein 2 (CPEB2) is important for postsynaptic plasticity and memory. Here, we investigated whether CPEB2 regulates axonal translation to support presynaptic plasticity. METHODS: Behavioral and electrophysiological assessments were conducted in mice with pan neuron/glia- or glutamatergic neuron-specific knockout of CPEB2. Hippocampal Schaffer collateral (SC)-CA1 and temporoammonic (TA)-CA1 pathways were electro-recorded to monitor synaptic transmission and LTP evoked by 4 trains of high-frequency stimulation. RNA immunoprecipitation, coupled with bioinformatics analysis, were used to unveil CPEB2-binding axonal RNA candidates associated with learning, which were further validated by Western blotting and luciferase reporter assays. Adeno-associated viruses expressing Cre recombinase were stereotaxically delivered to the pre- or post-synaptic region of the TA circuit to ablate Cpeb2 for further electrophysiological investigation. Biochemically isolated synaptosomes and axotomized neurons cultured on a microfluidic platform were applied to measure axonal protein synthesis and FM4-64FX-loaded synaptic vesicles. RESULTS: Electrophysiological analysis of hippocampal CA1 neurons detected abnormal excitability and vesicle release probability in CPEB2-depleted SC and TA afferents, so we cross-compared the CPEB2-immunoprecipitated transcriptome with a learning-induced axonal translatome in the adult cortex to identify axonal targets possibly regulated by CPEB2. We validated that Slc17a6, encoding vesicular glutamate transporter 2 (VGLUT2), is translationally upregulated by CPEB2. Conditional knockout of CPEB2 in VGLUT2-expressing glutamatergic neurons impaired consolidation of hippocampus-dependent memory in mice. Presynaptic-specific ablation of Cpeb2 in VGLUT2-dominated TA afferents was sufficient to attenuate protein synthesis-dependent LTP. Moreover, blocking activity-induced axonal Slc17a6 translation by CPEB2 deficiency or cycloheximide diminished the releasable pool of VGLUT2-containing synaptic vesicles. CONCLUSIONS: We identified 272 CPEB2-binding transcripts with altered axonal translation post-learning and established a causal link between CPEB2-driven axonal synthesis of VGLUT2 and presynaptic translation-dependent LTP. These findings extend our understanding of memory-related translational control mechanisms in the presynaptic compartment.

Asunto(s)

Plasticidad Neuronal , Proteínas de Unión al ARN , Transmisión Sináptica , Proteína 2 de Transporte Vesicular de Glutamato , Animales , Ratones , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Plasticidad Neuronal/fisiología , Transmisión Sináptica/fisiología , Proteína 2 de Transporte Vesicular de Glutamato/metabolismo , Proteína 2 de Transporte Vesicular de Glutamato/genética , Ratones Noqueados , Axones/metabolismo , Axones/fisiología , ARN Mensajero/metabolismo , ARN Mensajero/genética , Masculino , Biosíntesis de Proteínas

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Background: Uterine Corpus Endometrial Carcinoma (UCEC) stands as one of the prevalent malignancies impacting women globally. Given its heterogeneous nature, personalized therapeutic approaches are increasingly significant for optimizing patient outcomes. This study investigated the prognostic potential of cellular senescence genes(CSGs) in UCEC, utilizing machine learning techniques integrated with large-scale genomic data. Methods: A comprehensive analysis was conducted using transcriptomic and clinical data from 579 endometrial cancer patients sourced from the Cancer Genome Atlas (TCGA). A subset of 503 CSGs was assessed through weighted gene co-expression network analysis (WGCNA) alongside machine learning algorithms, including Gaussian Mixture Model (GMM), support vector machine - recursive feature elimination (SVM-RFE), Random Forest, and eXtreme Gradient Boosting (XGBoost), to identify key differentially expressed cellular senescence genes. These genes underwent further analysis to construct a prognostic model. Results: Our analysis revealed two distinct molecular clusters of UCEC with significant differences in tumor microenvironment and survival outcomes. Utilizing cellular senescence genes, a prognostic model effectively stratified patients into high-risk and low-risk categories. Patients in the high-risk group exhibited compromised overall survival and presented distinct molecular and immune profiles indicative of tumor progression. Crucially, the prognostic model demonstrated robust predictive performance and underwent validation in an independent patient cohort. Conclusion: The study emphasized the significance of cellular senescence genes in UCEC progression and underscored the efficacy of machine learning in developing reliable prognostic models. Our findings suggested that targeting cellular senescence holds promise as a strategy in personalized UCEC treatment, thus warranting further clinical investigation.

Asunto(s)

Senescencia Celular , Neoplasias Endometriales , Aprendizaje Automático , Humanos , Femenino , Senescencia Celular/genética , Neoplasias Endometriales/genética , Neoplasias Endometriales/mortalidad , Neoplasias Endometriales/patología , Pronóstico , Biomarcadores de Tumor/genética , Regulación Neoplásica de la Expresión Génica , Microambiente Tumoral/genética , Transcriptoma , Perfilación de la Expresión Génica , Persona de Mediana Edad

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Pancreatic cancer is the deadliest malignancy with a poor response to chemotherapy but is potentially indicated for ferroptosis therapy. Here we identified that cytoplasmic polyadenylation element binding protein 1 (CPEB1) regulates NRF2 proteostasis and susceptibility to ferroptosis in pancreatic ductal adenocarcinoma (PDAC). We found that CPEB1 deficiency in cancer cells promotes the translation of p62/SQSTM1 by facilitating mRNA polyadenylation. Consequently, upregulated p62 enhances NRF2 stability by sequestering KEAP1, an E3 ligase for proteasomal degradation of NRF2, leading to the transcriptional activation of anti-ferroptosis genes. In support of the critical role of this signaling cascade in cancer therapy, CPEB1-deficient pancreatic cancer cells display higher resistance to ferroptosis-inducing agents than their CPEB1-normal counterparts in vitro and in vivo. Furthermore, based on the pathological evaluation of tissue specimens from 90 PDAC patients, we established that CPEB1 is an independent prognosticator whose expression level is closely associated with clinical therapeutic outcomes in PDAC. These findings identify the role of CPEB1 as a key ferroptosis regulator and a potential prognosticator in pancreatic cancer.

Asunto(s)

Ferroptosis , Factor 2 Relacionado con NF-E2 , Neoplasias Pancreáticas , Humanos , Ferroptosis/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Factor 2 Relacionado con NF-E2/metabolismo , Factor 2 Relacionado con NF-E2/genética , Línea Celular Tumoral , Animales , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Factores de Escisión y Poliadenilación de ARNm/genética , Ratones , Proteostasis , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Carcinoma Ductal Pancreático/genética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch/genética , Ratones Desnudos

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BACKGROUND: CPEB1 is an alternative polyadenylation binding protein that promotes or suppresses the expression of related mRNAs and proteins by binding to a highly conserved Cytoplasmic Polyadenylation Element (CPE) in the mRNAs 3'UTR. It is found to express abnormally in multiple tumors and affect tumorigenesis through many pathways. This review summarizes the functions and mechanisms of CPEB1 in a variety of cancers and suggests new directions for future related treatments. METHODS: A total of 95 articles were eligible for inclusion based on the year, quality of the research, and the strength of association with CPEB1. In this review, current research about how CPEB1 affects the initiation and progression of glioblastoma, breast cancer, hepatocellular carcinoma, gastric cancer, colorectal cancer, non-small cell lung cancer, prostate cancer, and melanoma are dissected, and the biomedical functions and mechanisms are summarized. RESULTS: CPEB1 mostly presents as a tumor suppressor for breast cancer, endometrial carcinoma, hepatocellular carcinoma, non-small cell lung cancer, prostate cancer, and melanoma. However, glioblastoma, gastric cancer, and colorectal cancer it exhibit two opposing properties of tumorigenesis, either promoting or inhibiting it. CONCLUSION: CPEB1 is likely to serve as a target and dynamic detection index or prognostic indicator for its function of apoptosis, activity, proliferation, migration, invasion, stemness, drug resistance, and even ferroptosis in various cancers.

RESUMEN

The RNA-binding protein cytoplasmic polyadenylation element binding 1 (CPEB1) plays a fundamental role in regulating mRNA translation in oocytes. However, the specifics of how and which protein kinase cascades modulate CPEB1 activity are still controversial. Using genetic and pharmacological tools, and detailed time courses, we have re-evaluated the relationship between CPEB1 phosphorylation and translation activation during mouse oocyte maturation. We show that both the CDK1/MAPK and AURKA/PLK1 pathways converge on CPEB1 phosphorylation during prometaphase of meiosis I. Only inactivation of the CDK1/MAPK pathway disrupts translation, whereas inactivation of either pathway alone leads to CPEB1 stabilization. However, CPEB1 stabilization induced by inactivation of the AURKA/PLK1 pathway does not affect translation, indicating that destabilization and/or degradation is not linked to translational activation. The accumulation of endogenous CCNB1 protein closely recapitulates the translation data that use an exogenous template. These findings support the overarching hypothesis that the activation of translation during prometaphase in mouse oocytes relies on a CDK1/MAPK-dependent CPEB1 phosphorylation, and that translational activation precedes CPEB1 destabilization.

Asunto(s)

Meiosis , Oocitos , Biosíntesis de Proteínas , Factores de Transcripción , Factores de Escisión y Poliadenilación de ARNm , Animales , Femenino , Ratones , Aurora Quinasa A/metabolismo , Aurora Quinasa A/genética , Proteína Quinasa CDC2/metabolismo , Proteína Quinasa CDC2/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Ciclina B1/metabolismo , Ciclina B1/genética , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Factores de Escisión y Poliadenilación de ARNm/genética , Oocitos/metabolismo , Oocitos/citología , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/genética , Transducción de Señal , Factores de Transcripción/metabolismo , Factores de Transcripción/genética

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Background: This study aimed to characterize the clinical phenotype and genetic variations in patients with Kallmann syndrome (KS). Methods: This study involved the collection and analysis of clinical data from an individual with sporadic KS. Following this, peripheral blood samples were obtained from the patient and his parents. Genomic deoxyribonucleic acid was extracted and subjected to whole-exome sequencing and genomic copy number variation (CNV) detection. Finally, Sanger sequencing was performed to validate the suspected pathogenic variants. Results: Whole-exome sequencing confirmed that the child carried both the IL17RD variant (c.2101G>A, p.Gly701Ser) inherited from the mother and the new CPEB4 variant (c.1414C>T, p.Arg472*). No pathogenic CNVs were identified in CNV testing. Conclusion: Bioinformatics analysis shows that the IL17RD protein undergoing Gly701Ser mutation and is speculated to be phosphorylated and modified, thereby disrupting fibroblast growth factor signaling. This study also suggested that the CPEB4 might play a crucial role in the key signaling process affecting olfactory bulb morphogenesis. Overall, the findings of this study broaden the gene expression profile of KS-related pathogenic genes. This offers a new avenue for exploring the pathogenic mechanism of KS and provides valuable insights for precise clinical diagnosis and treatment strategies for this condition.

Asunto(s)

Síndrome de Kallmann , Femenino , Niño , Humanos , Síndrome de Kallmann/genética , Variaciones en el Número de Copia de ADN , Mutación , Fenotipo , Madres , Receptores de Interleucina/genética , Proteínas de Unión al ARN/genética

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AIMS: Trophoblast cell dysfunction is one of the important factors leading to preeclampsia (PE). Cytoplasmic polyadenylation element-binding 2 (CPEB2) has been found to be differentially expressed in PE patients, but whether it mediates PE process by regulating trophoblast cell function is unclear. METHODS: The expression of CPEB2 and somatostatin receptor 3 (SSTR3) was detected by quantitative real-time PCR, Western blot (WB) and immunofluorescence staining. Cell functions were analyzed by CCK-8 assay, EdU assay, flow cytometry and transwell assay. Epithelial-mesenchymal transition (EMT)-related protein levels were detected by WB. The interaction of CPEB2 and SSTR3 was confirmed by RIP assay, dual-luciferase reporter assay and PCR poly(A) tail assay. Animal experiments were performed to explore the effect of CPEB2 on PE progression in vivo, and the placental tissues of rat were used for H&E staining, immunohistochemical staining and TUNEL staining. RESULTS: CPEB2 was lowly expressed in PE patients. CPEB2 upregulation accelerated trophoblast cell proliferation, migration, invasion and EMT, while its knockdown had an opposite effect. CPEB2 bound to the CPE site in the 3'-UTR of SSTR3 mRNA to suppress SSTR3 translation through reducing poly(A) tails. Besides, SSTR3 overexpression suppressed trophoblast cell proliferation, migration, invasion and EMT, while its silencing accelerated trophoblast cell functions. However, these effects could be reversed by CPEB2 upregulation and knockdown, respectively. In vivo experiments, CPEB2 overexpression relieved histopathologic changes, inhibited apoptosis, promoted proliferation and enhanced EMT in the placenta of PE rat by decreasing SSTR3 expression. CONCLUSION: CPEB2 inhibited PE progression, which promoted trophoblast cell functions by inhibiting SSTR3 translation through polyadenylation.

Asunto(s)

Poliadenilación , Preeclampsia , Proteínas de Unión al ARN , Receptores de Somatostatina , Trofoblastos , Embarazo , Humanos , Preeclampsia/metabolismo , Preeclampsia/genética , Preeclampsia/patología , Femenino , Animales , Receptores de Somatostatina/metabolismo , Receptores de Somatostatina/genética , Ratas , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Trofoblastos/metabolismo , Trofoblastos/patología , Proliferación Celular , Transición Epitelial-Mesenquimal/genética , Ratas Sprague-Dawley , Adulto , Progresión de la Enfermedad , Movimiento Celular/genética , Biosíntesis de Proteínas , Placenta/metabolismo , Placenta/patología

RESUMEN

Chronic cerebral ischemia (CCI) is a clinical syndrome characterised by brain dysfunction due to decreased chronic cerebral perfusion. CCI initiates several inflammatory pathways, including pyroptosis. RNA-binding proteins (RBPs) play important roles in CCI. This study aimed to explore whether the interaction between RBP-Cpeb4 and Dclk2 affected Ehf phosphorylation to regulate neuronal pyroptosis. HT22 cells and mice were used to construct oxygen glucose deprivation (OGD)/CCI models. We found that Cpeb4 and Dclk2 were upregulated in OGD-treated HT22 cells and CCI-induced hippocampal CA1 tissues. Cpeb4 upregulated Dclk2 expression by increasing Dclk2 mRNA stability. Knockdown of Cpeb4 or Dclk2 inhibited neuronal pyroptosis in OGD-treated HT22 cells and CCI-induced hippocampal CA1 tissues. By binding to the promoter regions of Caspase1 and Caspase3, the transcription factor Ehf reduced their promoter activities and inhibited the transcription. Dclk2 phosphorylated Ehf and changed its nucleoplasmic distribution, resulting in the exit of p-Ehf from the nucleus and decreased Ehf levels. It promoted the expression of Caspase1 and Caspase3 and stimulated neuronal pyroptosis of HT22 cells induced by OGD. Cpeb4/Dclk2/Ehf pathway plays an important role in the regulation of cerebral ischemia-induced neuronal pyroptosis.

Asunto(s)

Isquemia Encefálica , Neuronas , Piroptosis , Animales , Masculino , Ratones , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Línea Celular , Enfermedad Crónica , Quinasas Similares a Doblecortina , Ratones Endogámicos C57BL , Neuronas/metabolismo , Neuronas/patología , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Piroptosis/fisiología , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética

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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.

Asunto(s)

Efectos Tardíos de la Exposición Prenatal , Emisiones de Vehículos , Femenino , Embarazo , Humanos , Ratones , Animales , Masculino , Emisiones de Vehículos/toxicidad , Aprendizaje por Laberinto , Efectos Tardíos de la Exposición Prenatal/metabolismo , Ratones Endogámicos C57BL , Receptores de N-Metil-D-Aspartato/metabolismo , Hipocampo/metabolismo , Plasticidad Neuronal , Proteínas de Unión al ARN/metabolismo

RESUMEN

During oocyte maturation and early embryogenesis, changes in mRNA poly(A)-tail lengths strongly influence translation, but how these tail-length changes are orchestrated has been unclear. Here, we performed tail-length and translational profiling of mRNA reporter libraries (each with millions of 3' UTR sequence variants) in frog oocytes and embryos and in fish embryos. Contrasting to previously proposed cytoplasmic polyadenylation elements (CPEs), we found that a shorter element, UUUUA, together with the polyadenylation signal (PAS), specify cytoplasmic polyadenylation, and we identified contextual features that modulate the activity of both elements. In maturing oocytes, this tail lengthening occurs against a backdrop of global deadenylation and the action of C-rich elements that specify tail-length-independent translational repression. In embryos, cytoplasmic polyadenylation becomes more permissive, and additional elements specify waves of stage-specific deadenylation. Together, these findings largely explain the complex tapestry of tail-length changes observed in early frog and fish development, with strong evidence of conservation in both mice and humans.

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Regiones no Traducidas 3' , Oocitos , Poli A , Poliadenilación , Biosíntesis de Proteínas , ARN Mensajero , Animales , Oocitos/metabolismo , Oocitos/citología , Poli A/metabolismo , Poli A/genética , Regiones no Traducidas 3'/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Ratones , Humanos , Embrión no Mamífero/metabolismo , Desarrollo Embrionario/genética , Femenino , Xenopus laevis/metabolismo , Xenopus laevis/embriología , Xenopus laevis/genética , Citoplasma/metabolismo

RESUMEN

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.

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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.

Asunto(s)

ARN Catalítico , Ratones , Humanos , Animales , ARN Catalítico/genética , ARN Catalítico/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Poliadenilación , Memoria a Largo Plazo , Neuronas/metabolismo , Proteínas de Unión al ARN/metabolismo

RESUMEN

The ribonucleic acid (RNA)-binding protein Cytoplasmic Polyadenylation Element Binding Protein 1 (CPEB1), a key member of the CPEB family, is essential in controlling gene expression involved in both healthy physiological and pathological processes. CPEB1 can bind to the 3'- untranslated regions (UTR) of substrate messenger ribonucleic acid (mRNA) and regulate its translation. There is increasing evidence that CPEB1 is closely related to the pathological basis of atherosclerosis. According to recent investigations, many pathological processes, including inflammation, lipid metabolism, endothelial dysfunction, angiogenesis, oxidative stress, cellular senescence, apoptosis, and insulin resistance, are regulated by CPEB1. This review considers the prevention and treatment of atherosclerotic heart disease in relation to the evolution of the physiological function of CPEB1, recent research breakthroughs, and the potential participation of CPEB1 in atherosclerosis.

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Aterosclerosis , Factores de Escisión y Poliadenilación de ARNm , Humanos , Aterosclerosis/metabolismo , Aterosclerosis/genética , Aterosclerosis/patología , Animales , Factores de Escisión y Poliadenilación de ARNm/genética , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Regulación de la Expresión Génica , Transducción de Señal

RESUMEN

Glioblastoma is the most frequent form of malignant brain tumor. Cytoplasmic polyadenylation element binding protein 4 (CPEB4) is overexpressed and involved in the tumorigenesis and metastasis of glioblastoma. miR-130a-3p has been revealed to be aberrantly expressed in tumors and has aroused wide attention. In present study, we would like to investigate the effect and potential mechanism of miR-130a-3p on the proliferation and migration in glioblastoma. The relative expression levels of miR-130a-3p and CPEB4 in glioblastoma cell lines were detected by real-time quantitative polymerase chain reaction. Cell viability and migration were detected by methylthiazolyl tetrazolium assay and transwell assay, and cell cycle analysis was detected by flow cytometry. The expression of CPEB4 protein and epithelial-mesenchymal transition associated markers were detected by western blot. Bioinformatics and luciferase activity analysis were used to verify the targeting relationship between miR-130a-3p and CPEB4. We observed that the expression of CPEB4 was upregulated while that of miR-130a-3p was downregulated in glioblastoma cell lines. CPEB4 was validated as a target of miR-130a-3p by luciferase activity assay. Increased levels of miR-130a-3p inhibited the proliferation and migration of the glioblastoma cells and the overexpression of miR-130a-3p inhibited epithelial-mesenchymal transition. However, CPEB4 overexpression resisted the inhibitory effects of miR-130a-3p. Our study elucidates CPEB4 is upregulated because of the downregulated miR-130a-3p in glioblastoma, which enhances the glioblastoma growth and migration, suggesting a potential therapeutic target for the disease.

Asunto(s)

Glioblastoma , MicroARNs , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Línea Celular Tumoral , Glioblastoma/genética , Proliferación Celular/genética , Luciferasas/metabolismo , Movimiento Celular/genética , Proteínas de Unión al ARN/genética

RESUMEN

Several array-based microRNA (miRNA) expression studies independently showed increased expression of miRNAs hsa-miR-130a-3p, -142-3p, -144-3p, -144-5p, -223-3p, -17-5p, and -30e-5p in gingiva affected by periodontal inflammation. We aimed to determine direct target genes and signaling pathways regulated by these miRNAs to identify processes relevant to gingival inflammatory responses and tissue homeostasis. We transfected miRNA mimics (mirVana) for each of the 7 miRNAs separately into human primary gingival fibroblasts cultured from 3 different donors. Following RNA sequencing, differential gene expression and second-generation gene set enrichment analyses were performed. miRNA inhibition and upregulation was validated at the transcript and protein levels using quantitative reverse transcriptase polymerase chain reaction, Western blotting, and reporter gene assays. All 7 miRNAs significantly increased expression of the gene MET proto-oncogene, receptor tyrosine kinase (MET). Expression of known periodontitis risk genes CPEB1, ABCA1, and ATP6V1C1 was significantly repressed by hsa-miR-130a-3p, -144-3p, and -144-5p, respectively. The genes WASL, ENPP5, ARL6IP1, and IDH1 showed the most significant and strongest downregulation after hsa-miR-142-3p, -17-5p, -223-3p, and -30e-5p transfection, respectively. The most significantly regulated gene set of each miRNA related to cell cycle (hsa-miRNA-144-3p and -5p [Padj = 4 × 10-40 and Padj = 4 × 10-6], -miR-17-5p [Padj = 9.5 × 10-23], -miR-30e-5p [Padj = 8.2 × 10-18], -miR-130a-3p [Padj = 5 × 10-15]), integrin cell surface interaction (-miR-223-3p [Padj = 2.4 × 10-7]), and interferon signaling (-miR-142-3p [Padj = 5 × 10-11]). At the end of acute inflammation, gingival miRNAs bring together complex regulatory networks that lead to increased expression of the gene MET. This underscores the importance of mesenchymal cell migration and invasion during gingival tissue remodeling and proliferation in restoring periodontal tissue homeostasis after active inflammation. MET, a receptor of the mitogenic hepatocyte growth factor fibroblast secreted, is a core gene of this process.

Asunto(s)

Encía , MicroARNs , Humanos , Encía/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Transducción de Señal/genética , Regulación hacia Arriba , Inflamación , Perfilación de la Expresión Génica

RESUMEN

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.

Asunto(s)

Proteínas de Unión al ARN , Humanos , Amiloide/química , Amiloide/metabolismo , Proteínas de Unión al ARN/metabolismo , Separación de Fases

RESUMEN

During early embryonic development, the RNA-binding protein CPEB mediates cytoplasmic polyadenylation and translational activation through a combinatorial code defined by the cy-toplasmic polyadenylation element (CPE) present in maternal mRNAs. However, in non-neuronal somatic cells, CPEB accelerates deadenylation to repress translation of the target, including c-myc mRNA, through an ill-defined cis-regulatory mechanism. Using RNA mutagenesis and electrophoretic mobility shift assays, we demonstrated that a combination of tandemly arranged consensus (cCPE) and non-consensus (ncCPE) cytoplasmic polyadenylation elements (CPEs) constituted a combinatorial code for CPEB-mediated c-myc mRNA decay. CPEB binds to cCPEs with high affinity (Kd = ~250 nM), whereas it binds to ncCPEs with low affinity (Kd > ~900 nM). CPEB binding to a cCPE enhances CPEB binding to the proximal ncCPE. In contrast, while a cCPE did not activate mRNA degradation, an ncCPE was essential for the induction of degradation, and a combination of a cCPE and ncCPEs further promoted degradation. Based on these findings, we propose a model in which the high-affinity binding of CPEB to the cCPE accelerates the binding of the second CPEB to the ncCPEs, resulting in the recruitment of deadenylases, acceleration of deadenylation, and repression of c-myc mRNAs.

Asunto(s)

Oocitos , Factores de Escisión y Poliadenilación de ARNm , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Oocitos/metabolismo , ARN/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Factores de Transcripción/metabolismo , Humanos

RESUMEN

The critical importance of circular RNAs (circRNAs) in human cancers, including cervical cancer (CC), has been discovered in recent years. However, the function and mechanism of hsa_circ_0000069 (circ_0000069) in CC have been fully understood. The expression levels of circ_0000069, microRNAs (miR-1270, miR-1276 and miR-620) and cytoplasmic polyadenylation element binding protein 4 (CPEB4) mRNA were detected by quantitative real time polymerase chain reaction (qRT-PCR). Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, wound healing, transwell and tube formation assays were used to clarify the effects of circ_0000069 on the functional behaviors of CC cells. The binding relationships among miR-1270, circ_0000069 and CPEB4 were detected by dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. A xenograft tumor model was established to explore the effect of circ_0000069 on tumor growth in vivo. Circ_0000069 was upregulated in CC clinical samples and cell lines, and its expression was associated with the clinical stage of CC patients. Circ_0000069 knockdown significantly decreased cell proliferation, invasion, migration, and tube formation and increased cell apoptosis in vitro. Moreover, miR-1270 was a direct target of circ_0000069, and CPEB4 was the downstream target of miR-1270. Knockdown of miR-1270 reversed the inhibitory effect of circ_0000069 knockdown on CC progression, and CPEB4 overexpression overturned the effect of miR-1270 on CC progression. In xenograft experiments, the oncogenic effect of circ_0000069 on tumor growth was verified. Altogether, circ_0000069 adsorbed miR-1270 to upregulate CPEB4 expression, thereby promoting the malignant phenotypes of CC cells. Circ_0000069 might be a potential target for treatment of CC.