RESUMEN
Cell type-specific transcriptomes are enabled by the action of multiple regulators, which are frequently expressed within restricted tissue regions. In the present study, we identify one such regulator, Quaking 5 (Qki5), as an RNA-binding protein (RNABP) that is expressed in early embryonic neural stem cells and subsequently down-regulated during neurogenesis. mRNA sequencing analysis in neural stem cell culture indicates that Qki proteins play supporting roles in the neural stem cell transcriptome and various forms of mRNA processing that may result from regionally restricted expression and subcellular localization. Also, our in utero electroporation gain-of-function study suggests that the nuclear-type Qki isoform Qki5 supports the neural stem cell state. We next performed in vivo transcriptome-wide protein-RNA interaction mapping to search for direct targets of Qki5 and elucidate how Qki5 regulates neural stem cell function. Combined with our transcriptome analysis, this mapping analysis yielded a bona fide map of Qki5-RNA interaction at single-nucleotide resolution, the identification of 892 Qki5 direct target genes, and an accurate Qki5-dependent alternative splicing rule in the developing brain. Last, our target gene list provides the first compelling evidence that Qki5 is associated with specific biological events; namely, cell-cell adhesion. This prediction was confirmed by histological analysis of mice in which Qki proteins were genetically ablated, which revealed disruption of the apical surface of the lateral wall in the developing brain. These data collectively indicate that Qki5 regulates communication between neural stem cells by mediating numerous RNA processing events and suggest new links between splicing regulation and neural stem cell states.
Asunto(s)
Encéfalo/embriología , Adhesión Celular/fisiología , Células Madre Embrionarias de Ratones/metabolismo , Células-Madre Neurales/metabolismo , Precursores del ARN/metabolismo , Proteínas de Unión al ARN/metabolismo , Empalme Alternativo/fisiología , Animales , Comunicación Celular , Regulación hacia Abajo , Perfilación de la Expresión Génica , Ratones , Ratones Noqueados , Neurogénesis/genética , Neurogénesis/fisiología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Transducción de SeñalRESUMEN
Microprocessor complex, including DiGeorge syndrome critical region gene 8 (DGCR8) and DROSHA, recognizes and cleaves primary transcripts of microRNAs (pri-miRNAs) in the maturation of canonical miRNAs. The study of DGCR8 haploinsufficiency reveals that the efficiency of this activity varies for different miRNA species. It is thought that this variation might be associated with the risk of schizophrenia with 22q11 deletion syndrome caused by disruption of the DGCR8 gene. However, the underlying mechanism for varying action of DGCR8 with each miRNA remains largely unknown. Here, we used in vivo monitoring to measure the efficiency of DGCR8-dependent microprocessor activity in cultured cells. We confirmed that this system recapitulates the microprocessor activity of endogenous pri-miRNA with expression of a ratiometric fluorescence reporter. Using this system, we detected mir-9-2 as one of the most efficient targets. We also identified a novel DGCR8-responsive RNA element, which is highly conserved among mammalian species and could be regulated at the epi-transcriptome (RNA modification) level. This unique feature between DGCR8 and pri-miR-9-2 processing may suggest a link to the risk of schizophrenia.
Asunto(s)
MicroARNs/genética , Proteínas de Unión al ARN/metabolismo , Línea Celular , Línea Celular Tumoral , Haploinsuficiencia/genética , Humanos , MicroARNs/metabolismo , Procesamiento Postranscripcional del ARN , Proteínas de Unión al ARN/genética , Esquizofrenia/genéticaRESUMEN
Pancreatic intraepithelial neoplasia is a pre-malignant lesion that can progress to pancreatic ductal adenocarcinoma, a highly lethal malignancy marked by its late stage at clinical presentation and profound drug resistance. The genomic alterations that commonly occur in pancreatic cancer include activation of KRAS2 and inactivation of p53 and SMAD4 (refs 2-4). So far, however, it has been challenging to target these pathways therapeutically; thus the search for other key mediators of pancreatic cancer growth remains an important endeavour. Here we show that the stem cell determinant Musashi (Msi) is a critical element of pancreatic cancer progression both in genetic models and in patient-derived xenografts. Specifically, we developed Msi reporter mice that allowed image-based tracking of stem cell signals within cancers, revealing that Msi expression rises as pancreatic intraepithelial neoplasia progresses to adenocarcinoma, and that Msi-expressing cells are key drivers of pancreatic cancer: they preferentially harbour the capacity to propagate adenocarcinoma, are enriched in circulating tumour cells, and are markedly drug resistant. This population could be effectively targeted by deletion of either Msi1 or Msi2, which led to a striking defect in the progression of pancreatic intraepithelial neoplasia to adenocarcinoma and an improvement in overall survival. Msi inhibition also blocked the growth of primary patient-derived tumours, suggesting that this signal is required for human disease. To define the translational potential of this work we developed antisense oligonucleotides against Msi; these showed reliable tumour penetration, uptake and target inhibition, and effectively blocked pancreatic cancer growth. Collectively, these studies highlight Msi reporters as a unique tool to identify therapy resistance, and define Msi signalling as a central regulator of pancreatic cancer.
Asunto(s)
Carcinoma Ductal Pancreático/tratamiento farmacológico , Resistencia a Antineoplásicos/efectos de los fármacos , Imagen Molecular , Proteínas del Tejido Nervioso/genética , Neoplasias Pancreáticas/tratamiento farmacológico , Proteínas de Unión al ARN/genética , Animales , Carcinoma in Situ/genética , Carcinoma in Situ/patología , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Transformación Celular Neoplásica/genética , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Resistencia a Antineoplásicos/genética , Femenino , Eliminación de Gen , Genes Reporteros/genética , Humanos , Masculino , Ratones , Modelos Genéticos , Células Neoplásicas Circulantes/metabolismo , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/metabolismo , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/farmacocinética , Oligonucleótidos Antisentido/uso terapéutico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Proteínas de Unión al ARN/metabolismo , Transducción de Señal/efectos de los fármacos , Tasa de Supervivencia , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Fused in sarcoma/translated in liposarcoma (FUS) is a causative gene of amyotrophic lateral sclerosis (ALS). Mutated FUS causes accumulation of DNA damage and cytosolic stress granule (SG) formation, thereby motor neuron (MN) death. However, key molecular aetiology remains unclear. Here, we applied a novel platform technology, iBRN, "Non- biased" Bayesian gene regulatory network analysis based on induced pluripotent stem cell (iPSC)-derived cell model, to elucidate the molecular aetiology using transcriptome of iPSC-derived MNs harboring FUSH517D. iBRN revealed "hub molecules", which strongly influenced transcriptome network, such as miR-125b-5p-TIMELESS axis and PRKDC for the molecular aetiology. Next, we confirmed miR-125b-5p-TIMELESS axis in FUSH517D MNs such that miR-125b-5p regulated several DNA repair-related genes including TIMELESS. In addition, we validated both introduction of miR-125b-5p and knocking down of TIMELESS caused DNA damage in the cell culture model. Furthermore, PRKDC was strongly associated with FUS mis-localization into SGs by DNA damage under impaired DNA-PK activity. Collectively, our iBRN strategy provides the first compelling evidence to elucidate molecular aetiology in neurodegenerative diseases.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Redes Reguladoras de Genes/fisiología , Células Madre Pluripotentes Inducidas/fisiología , MicroARNs/genética , Proteína FUS de Unión a ARN/genética , Esclerosis Amiotrófica Lateral/metabolismo , Teorema de Bayes , Línea Celular Tumoral , Daño del ADN/fisiología , Técnicas de Inactivación de Genes/métodos , Humanos , MicroARNs/biosíntesis , Proteína FUS de Unión a ARN/biosíntesisRESUMEN
Ferroptosis was recently defined as a novel type of programmed cell death depending on iron and lipid peroxidation. It is biologically different from other types of cell death such as apoptosis. While the involvement of ferroptosis in cancer, patient and animal model have been intensely studied, ferroptosis in human motor neuron model is still clearly unknown. Here we carefully assessed ferroptosis using human iPS cell-derived motor neuron (hiMNs). We found that almost all hiMNs died by the treatment of glutathione peroxidase 4 (GPX4) inhibitors. Importantly, the cell death was rescued by one antioxidant, vitamin E acetate, iron chelators and lipid peroxidase inhibitors with high dynamic ranges. Finally, these data clearly indicated that ferroptosis constitutively occurs in hiMNs, suggesting the possibility that it might play a biologically and pathologically important roles in motor neuron death such as motor neuron disease (MND)/Amyotrophic lateral sclerosis (ALS).
Asunto(s)
Muerte Celular , Ferroptosis , Neuronas Motoras/citología , Antioxidantes/farmacología , Muerte Celular/efectos de los fármacos , Línea Celular , Inhibidores Enzimáticos/farmacología , Ferroptosis/efectos de los fármacos , Humanos , Neuronas Motoras/efectos de los fármacos , Fosfolípido Hidroperóxido Glutatión Peroxidasa/antagonistas & inhibidoresRESUMEN
Two polypyrimidine tract RNA-binding proteins (PTBs), one near-ubiquitously expressed (Ptbp1) and another highly tissue-restricted (Ptbp2), regulate RNA in interrelated but incompletely understood ways. Ptbp1, a splicing regulator, is replaced in the brain and differentiated neuronal cell lines by Ptbp2. To define the roles of Ptbp2 in the nervous system, we generated two independent Ptbp2-null strains, unexpectedly revealing that Ptbp2 is expressed in neuronal progenitors and is essential for postnatal survival. A HITS-CLIP (high-throughput sequencing cross-linking immunoprecipitation)-generated map of reproducible Ptbp2-RNA interactions in the developing mouse neocortex, combined with results from splicing-sensitive microarrays, demonstrated that the major action of Ptbp2 is to inhibit adult-specific alternative exons by binding pyrimidine-rich sequences upstream of and/or within them. These regulated exons are present in mRNAs encoding proteins associated with control of cell fate, proliferation, and the actin cytoskeleton, suggesting a role for Ptbp2 in neurogenesis. Indeed, neuronal progenitors in the Ptbp2-null brain exhibited an aberrant polarity and were associated with regions of premature neurogenesis and reduced progenitor pools. Thus, Ptbp2 inhibition of a discrete set of adult neuronal exons underlies early brain development prior to neuronal differentiation and is essential for postnatal survival.
Asunto(s)
Empalme Alternativo/fisiología , Encéfalo/embriología , Diferenciación Celular/fisiología , Proteínas del Tejido Nervioso/metabolismo , Células-Madre Neurales/metabolismo , Proteína de Unión al Tracto de Polipirimidina/metabolismo , ARN Mensajero/metabolismo , Animales , Encéfalo/metabolismo , Exones/fisiología , Ribonucleoproteínas Nucleares Heterogéneas/genética , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Ratones , Ratones Mutantes , Proteínas del Tejido Nervioso/genética , Células-Madre Neurales/citología , Proteína de Unión al Tracto de Polipirimidina/genética , ARN Mensajero/genéticaRESUMEN
A set of tissue-specific splicing factors are thought to govern alternative splicing events during neural progenitor cell (NPC)-to-neuron transition by regulating neuron-specific exons. Here, we propose one such factor, RNA-binding protein Quaking 5 (Qki5), which is specifically expressed in the early embryonic neural stem cells. We performed mRNA-SEQ (Sequence) analysis using mRNAs obtained by developing cerebral cortices in Qk (Quaking) conditional knockout (cKO) mice. As expected, we found a large number of alternative splicing changes between control and conditional knockouts relative to changes in transcript levels. DAVID (The Database for Annotation, Visualization and Integrated Discovery) and Metascape analyses suggested that the affected spliced genes are involved in axon development and microtubule-based processes. Among these, the mRNA coding for the Ninein protein is listed as one of Qki protein-dependent alternative splicing targets. Interestingly, this exon encodes a very long polypeptide (2121 nt), and has been previously defined as a dynamic RNA switch during the NPC-to-neuron transition. Additionally, we validated that the regulation of this large exon is consistent with the Qki5-dependent alternative exon inclusion mode suggested by our previous Qki5 HITS-CLIP (high throughput sequencing-cross linking immunoprecipitation) analysis. Taken together, these data suggest that Qki5 is an important factor for alternative splicing in the NPC-to-neuron transition.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Exones/genética , Regulación de la Expresión Génica , Células-Madre Neurales/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Unión al ARN/metabolismo , ARN/genética , Empalme Alternativo/genética , Animales , Citoesqueleto/metabolismo , Ontología de Genes , Ratones Transgénicos , ARN/metabolismo , Precursores del ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de Secuencia de ARNRESUMEN
Adaptation to hypoxia depends on a conserved α/ß heterodimeric transcription factor called Hypoxia Inducible Factor (HIF), whose α-subunit is regulated by oxygen through different concurrent mechanisms. In this study, we have identified the RNA binding protein dMusashi, as a negative regulator of the fly HIF homologue Sima. Genetic interaction assays suggested that dMusashi participates of the HIF pathway, and molecular studies carried out in Drosophila cell cultures showed that dMusashi recognizes a Musashi Binding Element in the 3' UTR of the HIFα transcript, thereby mediating its translational repression in normoxia. In hypoxic conditions dMusashi is downregulated, lifting HIFα repression and contributing to trigger HIF-dependent gene expression. Analysis performed in mouse brains revealed that murine Msi1 protein physically interacts with HIF-1α transcript, suggesting that the regulation of HIF by Msi might be conserved in mammalian systems. Thus, Musashi is a novel regulator of HIF that inhibits responses to hypoxia specifically when oxygen is available.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Biosíntesis de Proteínas , Proteínas de Unión al ARN/metabolismo , Animales , Secuencia de Bases , Proteínas de Unión al ADN/genética , Regulación hacia Abajo/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/crecimiento & desarrollo , Sitios Genéticos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Mamíferos , Modelos Biológicos , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal/genética , Tráquea/crecimiento & desarrollo , Transcripción GenéticaRESUMEN
Mitochondria have many functions, including ATP generation. The electron transport chain (ETC) and the coupled ATP synthase generate ATP by consuming oxygen. Reactive oxygen species (ROS) are also produced by ETC, and ROS damage deoxyribonucleic acids, membrane lipids and proteins. Recent analysis indicate that mitochondrial unfolded protein response (UPRmt), which enhances expression of mitochondrial chaperones and proteases to remove damaged proteins, is activated when damaged proteins accumulate in the mitochondria. In Caenorhabditis elegans, HAF-1, a putative ortholog of human ABCB10, plays an essential role in signal transduction from mitochondria to nuclei to enhance UPRmt. Therefore, it is possible that ABCB10 has a role similar to that of HAF-1. However, it has not been reported whether ABCB10 is a factor in the signal transduction pathway to enhance UPRmt. In this study, ABCB10 was depleted in HepG2 cells using small interfering RNA (siRNA), and the effect was examined. ABCB10 depletion upregulated ROS and the expression of ROS-detoxifying enzymes (SOD2, GSTA1, and GSTA2), and SESN3, a protein induced by ROS to protect the cell from oxidative stress. In addition, ABCB10 depletion significantly decreased expression of UPRmt-related mitochondrial chaperones (HSPD1 and DNAJA3), and a mitochondrial protease (LONP1). However, the putative activity of ABCB10 to export peptides from mitochondria was not lost by ABCB10 depletion. Altogether, these data suggest that ABCB10 is involved in UPRmt signaling pathway similar to that of HAF-1, although ABCB10 probably does not participate in peptide export from mitochondria.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Mitocondrias/metabolismo , Transducción de Señal/genética , Respuesta de Proteína Desplegada , Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteasas ATP-Dependientes/genética , Proteasas ATP-Dependientes/metabolismo , Chaperonina 60/genética , Chaperonina 60/metabolismo , Perfilación de la Expresión Génica , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Proteínas del Choque Térmico HSP40/genética , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Células Hep G2 , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Oxidación-Reducción , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismoRESUMEN
Osteoblasts, osteoclasts, chondrocytes, and macrophages that participate in the bone repair process are derived from hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). However, the roles of these stem cells during the repair of injured bone tissue are still unclear. In the present study, we examined the effects of bone defect on HSCs and MSCs in bone marrow and spleen in 75 mice and its mechanism. We analyzed the HSC and MSC populations in these tissues of a mouse with femoral bone damage by using flow cytometry. The number of HSCs in the bone marrow of mice with damaged femurs was significantly lower than the number of these cells in the bone marrow of the contralateral intact femurs on day 2 after injury. Meanwhile, the number of MSCs in the bone marrow of mice with damaged femurs was significantly higher than that of the contralateral femurs. Both intraperitoneal administration of AMD3100, a C-X-C chemokine receptor 4 (CXCR4) antagonist, and local treatment with an anti-stromal cell-derived factor-1 (SDF-1) antibody blunted the observed decrease in HSC and increase in MSC populations within the bone marrow of injured femurs. In conclusion, the present study revealed that there is a concurrent decrease and increase in the numbers of HSCs and MSCs, respectively, in the bone marrow during repair of mouse femoral bone damage. Furthermore, the SDF-1/CXCR4 system was implicated as contributing to the changes in these stem cell populations upon bone injury.
Asunto(s)
Células de la Médula Ósea/fisiología , Regeneración Ósea/fisiología , Quimiocina CXCL12/fisiología , Células Madre Hematopoyéticas/fisiología , Células Madre Mesenquimatosas/fisiología , Animales , Anticuerpos/farmacología , Bencilaminas , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Regeneración Ósea/efectos de los fármacos , Huesos/citología , Huesos/lesiones , Recuento de Células , Quimiocina CXCL12/antagonistas & inhibidores , Ciclamas , Fémur/citología , Fémur/lesiones , Fémur/fisiología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Compuestos Heterocíclicos/farmacología , Masculino , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores CXCR4/antagonistas & inhibidoresRESUMEN
Fibrodysplasia ossificans progressiva (FOP) is a disorder of skeletal malformations and progressive heterotopic ossification. The constitutively activating mutation (R206H) of the bone morphogenetic protein type 1 receptor, activin-like kinase 2 (ALK2), is responsible for the pathogenesis of FOP. Although transfection of the causal mutation of FOP into myoblasts enhances osteoclast formation by transforming growth factor-ß (TGF-ß), the role of osteoclasts in heterotopic ossification is unknown. We therefore examined the effects of alendronate, SB431542 and SB203580 on heterotopic ossification induced by the causal mutation of FOP. Total bone mineral content as well as numbers of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated and alkaline phosphatase (ALP)-positive cells in heterotopic bone were significantly higher in muscle tissues implanted with ALK2 (R206H)-transfected mouse myoblastic C2C12 cells than in the tissues implanted with empty vector-transfected cells in nude mice. Alendronate, an aminobisphosphonate, did not affect total mineral content or numbers of TRAP-positive multinucleated and ALP-positive cells in heterotopic bone, which were enhanced by the implantation of ALK2 (R206H)-transfected C2C12 cells, although it significantly decreased serum levels of cross-linked C-telopeptide of type I collagen, a bone resorption index. Moreover, neither SB431542, an inhibitor of TGF-ß receptor type I kinase, nor SB203580, an inhibitor of p38 mitogen-activated protein kinase, affected the increase in heterotopic ossification due to the implantation of ALK2 (R206H)-transfected C2C12 cells. In conclusion, the present study indicates that osteoclast inhibition does not affect heterotopic ossification enhanced by FOP-related mutation.
Asunto(s)
Receptores de Activinas Tipo I/genética , Miositis Osificante/genética , Osificación Heterotópica/etiología , Osteoclastos/fisiología , Alendronato/farmacología , Fosfatasa Alcalina/análisis , Fosfatasa Alcalina/sangre , Animales , Benzamidas/farmacología , Calcio/sangre , Línea Celular , Colágeno Tipo I/sangre , Dioxoles/farmacología , Imidazoles/farmacología , Masculino , Ratones , Ratones Desnudos , Mutación , Mioblastos/trasplante , Osteoclastos/efectos de los fármacos , Péptidos/sangre , Fósforo/sangre , Piridinas/farmacologíaRESUMEN
Fibrodysplasia ossificans progressiva is characterized by extensive ossification within muscle tissues, and its molecular pathogenesis is responsible for the constitutively activating mutation (R206H) of the bone morphogenetic protein type 1 receptor, activin-like kinase 2 (ALK2). In this study, we investigated the effects of implanting ALK2 (R206H)-transfected myoblastic C2C12 cells into nude mice on osteoclast formation during heterotopic ossification in muscle and subcutaneous tissues. The implantation of ALK2 (R206H)-transfected C2C12 cells with BMP-2 in nude mice induced robust heterotopic ossification with an increase in the formation of osteoclasts in muscle tissues but not in subcutaneous tissues. The implantation of ALK2 (R206H)-transfected C2C12 cells in muscle induced heterotopic ossification more effectively than that of empty vector-transfected cells. A co-culture of ALK2 (R206H)-transfected C2C12 cells as well as the conditioned medium from ALK2 (R206H)-transfected C2C12 cells enhanced osteoclast formation in Raw264.7 cells more effectively than those with empty vector-transfected cells. The transfection of ALK2 (R206H) into C2C12 cells elevated the expression of transforming growth factor (TGF)-ß, whereas the inhibition of TGF-ß signaling suppressed the enhanced formation of osteoclasts in the co-culture with ALK2 (R206H)-transfected C2C12 cells and their conditioned medium. In conclusion, this study demonstrated that the causal mutation transfection of fibrodysplasia ossificans progressiva in myoblasts enhanced the formation of osteoclasts from its precursor through TGF-ß in muscle tissues.
Asunto(s)
Receptores de Activinas Tipo I/metabolismo , Músculo Esquelético/metabolismo , Miositis Osificante/genética , Osificación Heterotópica/metabolismo , Osteoclastos/metabolismo , Osteogénesis , Receptores de Activinas Tipo I/genética , Animales , Proteína Morfogenética Ósea 2/genética , Proteína Morfogenética Ósea 2/metabolismo , Células Cultivadas , Medios de Cultivo Condicionados/farmacología , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Músculo Esquelético/patología , Mutación Missense , Mioblastos/citología , Mioblastos/efectos de los fármacos , Mioblastos/metabolismo , Mioblastos/trasplante , Células 3T3 NIH , Osificación Heterotópica/genética , Osteoclastos/citología , Ratas , Transducción de Señal , Tejido Subcutáneo/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Activity-dependent gene regulation in neurons has been hypothesized to be under transcriptional control and to include dramatic increases in immediate early genes (IEGs) after neuronal activity. In addition, several reports have focused on post-transcriptional regulation, which could be mediated by neuronal post-transcriptional regulators, including RNA binding proteins (RNABPs). One such protein family is the neuronal Elavls (nElavls; Elavl2, Elavl3, and Elavl4), whose members are widely expressed in peripheral and central nervous system. Previous reports showed that Elavl3 and 4 are up-regulated following repeated stimulation such as during cocaine administration, a seizure, or a spatial discrimination task. In this study, we focused on Elavl2, a candidate gene for schizophrenia and studied its role in neuronal activity. First we found that Elavl2 has a cell-type specific expression pattern that is highly expressed in hippocampal CA3 pyramidal neurons and hilar interneurons using Elavl2 specific antibody. Second, unexpectedly, we discovered that the Elavl2 protein level in the hippocampus was acutely down-regulated for 3 h after a kainic acid (KA)-induced seizure in the hippocampal CA3 region. In addition, level of Gap43 mRNA, a target mRNA of Elavl2 is decreased 12 h after KA treatment, thus suggesting the involvement of Elavl2 in activity-dependent RNA regulation.
Asunto(s)
Región CA3 Hipocampal/efectos de los fármacos , Proteína 2 Similar a ELAV/metabolismo , Agonistas de Aminoácidos Excitadores/farmacología , Proteína GAP-43/genética , Ácido Kaínico/farmacología , ARN Mensajero/genética , Animales , Región CA3 Hipocampal/citología , Región CA3 Hipocampal/metabolismo , Proteína 2 Similar a ELAV/análisis , Regulación de la Expresión Génica/efectos de los fármacos , Interneuronas/citología , Interneuronas/metabolismo , Masculino , Ratones , Células Piramidales/citología , Células Piramidales/efectos de los fármacos , Células Piramidales/metabolismo , ARN Mensajero/metabolismo , Convulsiones/inducido químicamenteRESUMEN
HuD is a neuronal RNA-binding protein that plays an important role in neuronal differentiation of the nervous system. HuD has been reported to have three RNA recognition motifs (RRMs) and three splice variants (SVs) that differ in their amino acid sequences between RRM2 and RRM3. This study investigates whether these SVs have specific roles in neuronal differentiation. In primary neural epithelial cells under differentiating conditions, HuD splice variant 1 (HuD-sv1), which is a general form, and HuD-sv2 were expressed at all tested times, whereas HuD-sv4 was transiently expressed at the beginning of differentiation, indicating that HuD-sv4 might play a role compared different from that of HuD-sv1. Indeed, HuD-sv4 did not promote neuronal differentiation in epithelial cells, whereas HuD-sv1 did promote neuronal differentiation. HuD-sv4 overexpression showed less neurite-inducing activity than HuD-sv1 in mouse neuroblastoma N1E-115 cells; however, HuD-sv4 showed stronger growth-arresting activity. HuD-sv1 was localized only in the cytoplasm, whereas HuD-sv4 was localized in both the cytoplasm and the nuclei. The Hu protein has been reported to be involved in translation and alternative splicing in the cytoplasm and nuclei, respectively. Consistent with this observation, HuD-sv1 showed translational activity on p21, which plays a role in growth arrest and neuronal differentiation, whereas HuD-sv4 did not. By contrast, HuD-sv4 showed stronger pre-mRNA splicing activity than did HuD-sv1 on Clasp2, which participates in cell division. Therefore, HuD SVs might play a role in controlling the timing of proliferation/differentiation switching by controlling the translation and alternative splicing of target genes.
Asunto(s)
Proteínas ELAV/metabolismo , Células Epiteliales/metabolismo , Neurogénesis/fisiología , Neuronas/metabolismo , Isoformas de Proteínas/metabolismo , Empalme Alternativo , Animales , Línea Celular Tumoral , Proliferación Celular , Proteínas ELAV/genética , Proteína 4 Similar a ELAV , Células Epiteliales/citología , Ratones , Neuritas/metabolismo , Neuronas/citología , Isoformas de Proteínas/genéticaRESUMEN
An increasing number of studies have demonstrated that neural RNA-binding proteins (nRNABPs) participate in several steps of neural development through post-transcriptional regulation of their RNA targets (Grabowski Curr Opin Genet Dev 21:388-394, 2011). Classical genetics and in vitro biochemical approaches have identified several important RNA targets of nRNABPs linked to cell-fate decision and neuronal functions. In recent years, new technologies, such as unbiased in vivo protein-RNA interaction approaches, high-throughput sequencing-cross-linked immunoprecipitation (HITS-CLIP), microarrays, RNAseq and others, have been developed. The use of these with genetics has succeeded in defining a dynamic range of RNA targets of RNABPs at the transcriptome-wide level. This new platform also provides the mechanistic insights into a specific biological function of nRNABPs. This review highlights the discoveries and challenges of the interplay between the nRNABPs and their biological functions in neural development.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Sistema Nervioso/crecimiento & desarrollo , Neurogénesis , Proteínas de Unión al ARN/metabolismo , Animales , Humanos , Neuronas/metabolismo , InvestigaciónRESUMEN
BACKGROUND: G-quadruplexes are four-stranded structures formed in guanine-rich nucleotide sequences. Several functional roles of DNA G-quadruplexes have so far been investigated, where their putative functional roles during DNA replication and transcription have been suggested. A necessary condition for G-quadruplex formation is the presence of four regions of tandem guanines called G-runs and three nucleotide subsequences called loops that connect G-runs. A simple computational way to detect potential G-quadruplex regions in a given genomic sequence is pattern matching with regular expression. Although many putative G-quadruplex motifs can be found in most genomes by the regular expression-based approach, the majority of these sequences are unlikely to form G-quadruplexes because they are unstable as compared with canonical double helix structures. RESULTS: Here we present elaborate computational models for representing DNA G-quadruplex motifs using hidden Markov models (HMMs). Use of HMMs enables us to evaluate G-quadruplex motifs quantitatively by a probabilistic measure. In addition, the parameters of HMMs can be trained by using experimentally verified data. Computational experiments in discriminating between positive and negative G-quadruplex sequences as well as reducing putative G-quadruplexes in the human genome were carried out, indicating that HMM-based models can discern bona fide G-quadruplex structures well and one of them has the possibility of reducing false positive G-quadruplexes predicted by existing regular expression-based methods. Furthermore, our results show that one of our models can be specialized to detect G-quadruplex sequences whose functional roles are expected to be involved in DNA transcription. CONCLUSIONS: The HMM-based method along with the conventional pattern matching approach can contribute to reducing costly and laborious wet-lab experiments to perform functional analysis on a given set of potential G-quadruplexes of interest. The C++ and Perl programs are available at http://tcs.cira.kyoto-u.ac.jp/~ykato/program/g4hmm/.
Asunto(s)
G-Cuádruplex , Genómica/métodos , Cadenas de Markov , Motivos de Nucleótidos , Bases de Datos Genéticas , HumanosRESUMEN
Further development in research of bone regeneration is necessary to meet the clinical demand for bone reconstruction. Recently, we reported that plasminogen is crucial for bone repair through enhancement of vessel formation. However, the details of the role of tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) in the bone repair process still remain unknown. Herein, we examined the effects of plasminogen activators on bone repair after a femoral bone defect using tPA-deficient (tPA(-/-)) and uPA-deficient (uPA(-/-)) mice. Bone repair of the femur was delayed in tPA(-/-) mice, unlike that in wild-type (tPA(+/+)) mice. Conversely, the bone repair was comparable between wild-type (uPA(+/+)) and uPA(-/-) mice. The number of proliferative osteoblasts was decreased at the site of bone damage in tPA(-/-) mice. Moreover, the proliferation of primary calvarial osteoblasts was reduced in tPA(-/-) mice. Recombinant tPA facilitated the proliferation of mouse osteoblastic MC3T3-E1 cells. The proliferation enhanced by tPA was antagonized by the inhibition of endogenous annexin 2 by siRNA and by the inhibition of extracellular signal-regulated kinase (ERK)1/2 phosphorylation in MC3T3-E1 cells. Vessel formation as well as the levels of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) were decreased at the damaged site in tPA(-/-) mice. Our results provide novel evidence that tPA is crucial for bone repair through the facilitation of osteoblast proliferation related to annexin 2 and ERK1/2 as well as enhancement of vessel formation related to VEGF and HIF-1α at the site of bone damage.
Asunto(s)
Regeneración Ósea , Osteoblastos/metabolismo , Activador de Tejido Plasminógeno/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Animales Recién Nacidos , Anexina A2/antagonistas & inhibidores , Anexina A2/genética , Anexina A2/metabolismo , Huesos/irrigación sanguínea , Huesos/citología , Huesos/metabolismo , Huesos/patología , Línea Celular , Proliferación Celular , Células Cultivadas , Cruzamientos Genéticos , Femenino , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neovascularización Fisiológica , Osteoblastos/citología , Osteoblastos/patología , Interferencia de ARN , Proteínas Recombinantes/metabolismo , Activador de Tejido Plasminógeno/deficiencia , Activador de Tejido Plasminógeno/genética , Activador de Plasminógeno de Tipo Uroquinasa/deficiencia , Activador de Plasminógeno de Tipo Uroquinasa/genética , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
Membrane microdomains consisting of sphingomyelin (SM) and cholesterol appear to be important for signal transduction in T-cell activation. The present study was designed to elucidate the role of membrane SM in vivo and in vitro using sphingomyelin synthase 1 (SMS1) knock out (SMS1(-/-)) mice and Concanavalin A (ConA)-induced hepatitis. After establishing SMS1(-/-) mice, we investigated CD4+ T-cell functions including proliferation, cytokine production and signal transduction in vivo. We also examined severity of hepatitis, cytokine production in serum and liver after ConA injection at a dose of 20 mg kg(-1). CD4+ T cells from SMS1(-/-) mice showed severe deficiency of membrane SM and several profound defects compared with wild-type controls as follows: (i) cellular proliferation and production of IL-2 and IFN-γ by co-cross-linking of CD3 and CD4; (ii) tyrosine phosphorylation of LAT and its association with ZAP-70; (iii) clustering and co-localization of TCR with lipid rafts. Consistent with these impaired CD4+ T-cell functions in vitro, SMS1(-/-) mice showed decreased serum levels of IL-6 and IFN-γ by ConA injection, which renders SMS1(-/-) mice less sensitive to ConA-induced hepatitis. These results indicated that the deficiency of membrane SM caused the CD4+ T-cell dysfunction through impaired lipid raft function contributed to protection of ConA-induced liver injury, suggesting that the membrane SM is critical for full T-cell activation both in vitro and in vivo.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/patología , Concanavalina A/inmunología , Hepatitis/inmunología , Microdominios de Membrana/patología , Transferasas (Grupos de Otros Fosfatos Sustitutos)/deficiencia , Animales , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transferasas (Grupos de Otros Fosfatos Sustitutos)/inmunologíaRESUMEN
Fibrodysplasia ossificans progressiva (FOP) is a skeletal disorder with progressive heterotopic ossification in skeletal muscle. A mutation causing constitutive activation in a bone morphogenetic protein (BMP) type 1 receptor [ALK2(R206H)] is found in most patients with FOP. However, the details in the heterotopic ossification of muscle in FOP and the role of matrix metalloproteinase-10 (MMP-10) in bone remain to be fully elucidated. In the present study, we investigated the role of MMP-10 in the differentiation of mouse myoblastic C2C12 cells into osteoblasts. MMP-10 was extracted as a factor, whose expression was most extensively enhanced by ALK2 (R206H) transfection in C2C12 cells. MMP-10 significantly augmented the levels of Osterix, type 1 collagen, alkaline phosphatase (ALP) and osteocalcin mRNA as well as ALP activity enhanced by BMP-2 in C2C12 cells. Moreover, a reduction in endogenous MMP-10 levels by siRNA significantly decreased the levels of Runx2, Osterix, type 1 collagen, ALP and osteocalcin mRNA enhanced by BMP-2 in these cells. In addition, MMP-10 increased the phosphorylation of Smad1/5/8 as well as enhanced the levels of Smad6 and Smad7 mRNA induced by BMP-2. In conclusion, the present study first demonstrated that MMP-10 promotes the differentiation of myoblasts into osteoblasts by interacting with the BMP signaling pathway. MMP-10 may play some important role in the heterotopic ossification of muscle in FOP.
Asunto(s)
Proteína Morfogenética Ósea 2/metabolismo , Diferenciación Celular , Regulación Enzimológica de la Expresión Génica , Metaloproteinasa 10 de la Matriz/metabolismo , Osteoblastos/citología , Transducción de Señal , Receptores de Activinas Tipo I/genética , Receptores de Activinas Tipo I/metabolismo , Sustitución de Aminoácidos , Animales , Proteína Morfogenética Ósea 2/agonistas , Proteína Morfogenética Ósea 2/genética , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/genética , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/metabolismo , Línea Celular , Colágeno Tipo I/agonistas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Humanos , Metaloproteinasa 10 de la Matriz/química , Metaloproteinasa 10 de la Matriz/genética , Ratones , Proteínas Mutantes/agonistas , Proteínas Mutantes/metabolismo , Mioblastos/citología , Mioblastos/enzimología , Mioblastos/metabolismo , Osteoblastos/enzimología , Osteoblastos/metabolismo , Osteocalcina/agonistas , Osteocalcina/genética , Osteocalcina/metabolismo , Interferencia de ARN , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteína smad6/agonistas , Proteína smad6/genética , Proteína smad6/metabolismo , Proteína smad7/agonistas , Proteína smad7/genética , Proteína smad7/metabolismo , Factor de Transcripción Sp7 , Factores de Transcripción/agonistas , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Phosphoinositides (PIPs) act as intracellular signaling molecules that regulate various cellular processes. Abnormalities in PIP metabolism cause various pathological conditions, including neurodegenerative diseases, cancer and immune disorders. Several neurological diseases with diverse phenotypes, such as ataxia with cerebellar atrophy or intellectual disability without brain malformation, are caused by mutations in INPP4A, which encodes a phosphoinositide phosphatase. We examined two strains of Inpp4a mutant mice with distinct cerebellar phenotypes: the Inpp4aΔEx1,2 mutant exhibited striatal degeneration without cerebellar atrophy, and the Inpp4aΔEx23 mutant exhibited a severe striatal phenotype with cerebellar atrophy. Both strains exhibited reduced expression of Inpp4a mutant proteins in the cerebellum. N-terminal-truncated Inpp4a proteins were expressed from the Inpp4aΔEx1,2 allele by alternative translation initiation and had phosphatase activity for PI(3,4)P2, whereas the Inpp4a mutant protein encoded by Inpp4aΔEx23 completely lacked phosphatase activity. Our results indicate that the diverse phenotypes observed in Inpp4a-related neurological diseases could be due to the varying protein expression levels and retained phosphatase activity in different Inpp4a variants. These findings provide insights into the role of INPP4A mutations in disease pathogenesis and may help to develop personalized therapy.