RESUMEN
Local mRNA translation mediates the adaptive responses of axons to extrinsic signals, but direct evidence that it occurs in mammalian CNS axons in vivo is scant. We developed an axon-TRAP-RiboTag approach in mouse that allows deep-sequencing analysis of ribosome-bound mRNAs in the retinal ganglion cell axons of the developing and adult retinotectal projection in vivo. The embryonic-to-postnatal axonal translatome comprises an evolving subset of enriched genes with axon-specific roles, suggesting distinct steps in axon wiring, such as elongation, pruning, and synaptogenesis. Adult axons, remarkably, have a complex translatome with strong links to axon survival, neurotransmission, and neurodegenerative disease. Translationally co-regulated mRNA subsets share common upstream regulators, and sequence elements generated by alternative splicing promote axonal mRNA translation. Our results indicate that intricate regulation of compartment-specific mRNA translation in mammalian CNS axons supports the formation and maintenance of neural circuits in vivo.
Asunto(s)
Axones/metabolismo , Biosíntesis de Proteínas , Proteoma/metabolismo , Células Ganglionares de la Retina/metabolismo , Empalme Alternativo , Animales , Regulación del Desarrollo de la Expresión Génica , Ratones , Proteoma/análisis , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Ribosomas/metabolismo , Colículos Superiores/metabolismo , Transmisión SinápticaRESUMEN
Protocadherin19 (PCDH19)-related epilepsy syndrome is a rare disorder characterized by early-onset epilepsy, intellectual disability, and autistic behaviors. PCDH19 is located on the X chromosome and encodes a calcium-dependent single-pass transmembrane protein, which regulates cell-to-cell adhesion through homophilic binding. In human, 90% of heterozygous females, containing PCDH19 wild-type and mutant cells due to random X inactivation, are affected, whereas mutant males, containing only mutant cells, are typically not. The current view, the cellular interference, is that the altered interactions between wild-type and mutant cells during development, rather than loss of function itself, are responsible. However, studies using Pcdh19 knockout mice showed that the complete loss of function also causes autism-like behaviors both in males and females, suggesting that other functions of PCDH19 may also contribute to pathogenesis. To address whether mosaicism is required for PCDH19-related epilepsy, we generated Xenopus tropicalis tadpoles with complete or mosaic loss of function by injecting antisense morpholino oligonucleotides into the blastomeres of neural lineage at different stages of development. We found that either mosaic or complete knockdown results in seizure-like behaviors, which could be rescued by antiseizure medication, and repetitive behaviors. Our results suggest that the loss of PCDH19 function itself, in addition to cellular interference, may also contribute to PCDH19-related epilepsy.
Asunto(s)
Cadherinas , Epilepsia , Protocadherinas , Animales , Femenino , Masculino , Conducta Animal , Cadherinas/genética , Cadherinas/metabolismo , Epilepsia/genética , Epilepsia/metabolismo , Mosaicismo , XenopusRESUMEN
During the maternal-to-zygotic transition (MZT), maternal RNAs are actively degraded and replaced by newly synthesized zygotic transcripts in a highly coordinated manner. However, it remains largely unknown how maternal mRNA decay is triggered in early vertebrate embryos. Here, through genome-wide profiling of RNA abundance and 3' modification, we show that uridylation is induced at the onset of maternal mRNA clearance. The temporal control of uridylation is conserved in vertebrates. When the homologs of terminal uridylyltransferases TUT4 and TUT7 (TUT4/7) are depleted in zebrafish and Xenopus, maternal mRNA clearance is significantly delayed, leading to developmental defects during gastrulation. Short-tailed mRNAs are selectively uridylated by TUT4/7, with the highly uridylated transcripts degraded faster during the MZT than those with unmodified poly(A) tails. Our study demonstrates that uridylation plays a crucial role in timely mRNA degradation, thereby allowing the progression of early development.
Asunto(s)
Embrión de Mamíferos/enzimología , Embrión no Mamífero/enzimología , Nucleotidiltransferasas/metabolismo , Estabilidad del ARN , ARN Mensajero/metabolismo , Transcriptoma , Xenopus laevis/genética , Pez Cebra/genética , Animales , Animales Modificados Genéticamente , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Drosophila melanogaster/embriología , Drosophila melanogaster/enzimología , Drosophila melanogaster/genética , Gastrulación , Regulación del Desarrollo de la Expresión Génica , Edad Gestacional , Ratones Endogámicos ICR , Nucleotidiltransferasas/genética , Procesamiento Postranscripcional del ARN , ARN Mensajero/genética , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriología , Xenopus laevis/metabolismo , Pez Cebra/embriología , Pez Cebra/metabolismoRESUMEN
OBJECTIVE: Angiogenesis, the process of building complex vascular structures, begins with sprout formation on preexisting blood vessels, followed by extension of the vessels through proliferation and migration of endothelial cells. Based on the potential therapeutic benefits of preventing angiogenesis in pathological conditions, many studies have focused on the mechanisms of its initiation as well as control. However, how the extension of vessels is terminated remains obscure. Thus, we investigated the negative regulation mechanism. APPROACH AND RESULTS: We report that increased intracellular calcium can induce dephosphorylation of the endothelial receptor tyrosine kinase Tie2. The calcium-mediated dephosphorylation was found to be dependent on Tie2-calmodulin interaction. The Tyr1113 residue in the C-terminal end loop of the Tie2 kinase domain was mapped and found to be required for this interaction. Moreover, mutation of this residue into Phe impaired both the Tie2-calmodulin interaction and calcium-mediated Tie2 dephosphorylation. Furthermore, expressing a mutant Tie2 incapable of binding to calmodulin or inhibiting calmodulin function in vivo causes unchecked growth of the vasculature in Xenopus. Specifically, knockdown of Tie2 in Xenopus embryo retarded the sprouting and extension of intersomitic veins. Although human Tie2 expression in the Tie2-deficient animals almost completely rescued the retardation, the Tie2(Y1113F) mutant caused overgrowth of intersomitic veins with strikingly complex and excessive branching patterns. CONCLUSIONS: We propose that the calcium/calmodulin-dependent negative regulation of Tie2 can be used as an inhibitory signal for vessel growth and branching to build proper vessel architecture during embryonic development.
Asunto(s)
Señalización del Calcio , Calmodulina/metabolismo , Embrión no Mamífero/irrigación sanguínea , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Neovascularización Fisiológica , Receptor TIE-2/metabolismo , Xenopus/embriología , Animales , Células CHO , Cricetulus , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Genotipo , Células HEK293 , Humanos , Mutación , Fenotipo , Fosforilación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Receptor TIE-2/genética , Transfección , Tirosina , Xenopus/genética , Xenopus/metabolismoRESUMEN
Localized mRNA translation regulates synapse function and axon maintenance, but how compartment-specific mRNA repertoires are regulated is largely unknown. We developed an axonal transcriptome capture method that allows deep sequencing of metabolically labeled mRNAs from retinal ganglion cell axon terminals in mouse. Comparing axonal-to-somal transcriptomes and axonal translatome-to-transcriptome enables genome-wide visualization of mRNA transport and translation and unveils potential regulators tuned to each process. FMRP and TDP-43 stand out as key regulators of transport, and experiments in Fmr1 knockout mice validate FMRP's role in the axonal transportation of synapse-related mRNAs. Pulse-and-chase experiments enable genome-wide assessment of mRNA stability in axons and reveal a strong coupling between mRNA translation and decay. Measuring the absolute mRNA abundance per axon terminal shows that the adult axonal transcriptome is stably maintained by persistent transport. Our datasets provide a rich resource for unique insights into RNA-based mechanisms in maintaining presynaptic structure and function in vivo.
Asunto(s)
Axones , Sistema Nervioso Central , Animales , Ratones , Axones/metabolismo , Sistema Nervioso Central/metabolismo , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Mamíferos/genética , Mamíferos/metabolismo , ARN/metabolismo , Transporte de ARN , ARN Mensajero/metabolismoRESUMEN
Paulownia coreana has traditionally been used as the medicine and health food in the treatment of cancer and infectious diseases. In the present study, a new antiproliferation agent, isoatriplicolide tiglate (PCAC) was isolated from the chloroform soluble fraction of the leaves of Paulownia coreana. The antiproliferation activities of PCAC plant extract was examined in breast and cervical cancer cell lines in a time-and dose-dependent manners. Our in vitro experiments showed that PCAC suppresses the cell growth and proliferation of cancer cells at a relatively low concentration (< 10 µg/mL) and induces apoptosis at a high concentration (> 50 µg/mL). Western blot analysis showed that concentration higher than 50 µg/mL induces a time-dependent increase in the percentage of apoptotic cells. In this case, PCAC uses both extrinsic and intrinsic pathways for the apoptosis. PCAC treatment decreased the expression of pro-caspase 8, 9, and 3, the main regulators of apoptotic cell death, in MDA-MB-231 cells, accompanied by the activation of caspase 8, 9, and 3. More importantly, PCAC inhibited the in vitro proliferation of six other human breast and cervical cancer cell lines. In conclusion, our data strongly suggest that PCAC acts as an antiproliferation agents particularly against breast and cervical cancers by inducing cell cycle arrest in the S/G2 phase and caspase dependent apoptosis at relatively low (< 10 µg/mL) and high (> 50 µg/mL) concentrations, respectively.
Asunto(s)
Helechos/química , Extractos Vegetales/farmacología , Sesquiterpenos/farmacología , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Extractos Vegetales/química , Sesquiterpenos/químicaRESUMEN
p62/SQSTM1 is known to act as a key mediator in the selective autophagy of protein aggregates, or aggrephagy, by steering ubiquitinated protein aggregates towards the autophagy pathway. Here, we use a yeast two-hybrid screen to identify the prefoldin-like chaperone UXT as an interacting protein of p62. We show that UXT can bind to protein aggregates as well as the LB domain of p62, and, possibly by forming an oligomer, increase p62 clustering for its efficient targeting to protein aggregates, thereby promoting the formation of the p62 body and clearance of its cargo via autophagy. We also find that ectopic expression of human UXT delays SOD1(A4V)-induced degeneration of motor neurons in a Xenopus model system, and that specific disruption of the interaction between UXT and p62 suppresses UXT-mediated protection. Together, these results indicate that UXT functions as an autophagy adaptor of p62-dependent aggrephagy. Furthermore, our study illustrates a cooperative relationship between molecular chaperones and the aggrephagy machinery that efficiently removes misfolded protein aggregates.
Asunto(s)
Autofagia/genética , Proteínas de Ciclo Celular/genética , Chaperonas Moleculares/genética , Agregado de Proteínas , Proteína Sequestosoma-1/genética , Superóxido Dismutasa-1/genética , Animales , Autofagia/efectos de los fármacos , Proteínas de Ciclo Celular/metabolismo , Ganglios Espinales/citología , Ganglios Espinales/metabolismo , Regulación de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Células HeLa , Humanos , Leupeptinas/farmacología , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Chaperonas Moleculares/metabolismo , Neuronas Motoras/citología , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/metabolismo , Cultivo Primario de Células , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/metabolismo , Agregado de Proteínas/efectos de los fármacos , Pliegue de Proteína/efectos de los fármacos , Proteína Sequestosoma-1/metabolismo , Transducción de Señal , Superóxido Dismutasa-1/metabolismo , Transgenes , Xenopus laevis , Proteína Fluorescente RojaRESUMEN
Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The efficient in vivo visualization of blood vessels and the reliable quantification of their complexity are key to understanding the biology and disease of the vascular network. Here, we provide a detailed method to visualize blood vessels with a commercially available fluorescent dye, human plasma acetylated low density lipoprotein DiI complex (DiI-AcLDL), and to quantify their complexity in Xenopus tropicalis. Blood vessels can be labeled by a simple injection of DiI-AcLDL into the beating heart of an embryo, and blood vessels in the entire embryo can be imaged in live or fixed embryos. Combined with gene perturbation by the targeted microinjection of nucleic acids and/or the bath application of pharmacological reagents, the roles of a gene or of a signaling pathway on vascular development can be investigated within one week without resorting to sophisticated genetically engineered animals. Because of the well-defined venous system of Xenopus and its stereotypic angiogenesis, the sprouting of pre-existing vessels, vessel complexity can be quantified efficiently after perturbation experiments. This relatively simple protocol should serve as an easily accessible tool in diverse fields of cardiovascular research.
Asunto(s)
Neovascularización Fisiológica , Xenopus/embriología , Xenopus/fisiología , Animales , Colorantes Fluorescentes , Microscopía FluorescenteRESUMEN
The p34(SEI-1) oncoprotein is involved in a transcriptional regulation, cell cycle regulation, apoptosis, development and many other important cellular functions. Our present study suggests that p34(SEI-1) can promote metastasis by enhancing migration and invasion of cancer cells. Consistently, p34(SEI-1) expression was found to be increased as the tumor invasiveness progressed in human breast tissues. p34(SEI-1) may promote cancer metastasis by activating the PI3K/AKT signaling pathway. In this process, p34(SEI-1) activates two different serine/threonine kinases, AKT or ILK, depending on the expression status of HER2/neu oncogene. In HER2/neu suppressed cancer cells, p34(SEI-1) promoted metastasis mainly by activating AKT via phosphorylation of the 473 serine residue. In HER2/neu expressing cancer cells, p34(SEI-1) overexpression downregulates HER2/neu expression, leading to the activation of another crucial serine/threonine kinase ILK due to phosphorylation of the 178 threonine residue instead of AKT. These results suggest that p34(SEI-1) affects cancer metastasis by regulating two different signaling pathways depending on the HER2/neu expression level, in which AKT and ILK modulation can be stimulated by p34(SEI-1) overexpression.
Asunto(s)
Neoplasias de la Mama/patología , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor ErbB-2/genética , Transactivadores/metabolismo , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Movimiento Celular , Femenino , Células HEK293 , Humanos , Sistema de Señalización de MAP Quinasas , Células MCF-7 , Metástasis de la Neoplasia/patología , Proteínas Nucleares/genética , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Transactivadores/genética , Factores de TranscripciónRESUMEN
A 34-KD protein encoded by the SEI-1 gene (p34(SEI1)), is a relatively recently discovered oncoprotein that has multiple important biological functions. Our data show that p34(SEI-1) enhances cancer cell survival and promotes tumorigenesis by downregulating the tumor suppressor PTEN, a negative regulator of the PI3K/AKT signaling pathway, and therefore activating the PI3K/AKT signaling pathway. In this process, p34(SEI-1) positively affects NEDD4-1 gene expression both at the transcriptional and protein levels. Furthermore, the expression levels of p34(SEI-1) and NEDD4-1 were found to be coordinated in tumor tissues obtained from patients with breast cancer. We also show that p34(SEI-1) affects the subcellular localization of PTEN.
Asunto(s)
Neoplasias de la Mama/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Proteínas Nucleares/metabolismo , Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transactivadores/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Apoptosis , Western Blotting , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Proliferación Celular , Complejos de Clasificación Endosomal Requeridos para el Transporte/antagonistas & inhibidores , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Femenino , Humanos , Técnicas para Inmunoenzimas , Ubiquitina-Proteína Ligasas Nedd4 , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , Fosfohidrolasa PTEN/genética , Fosfatidilinositol 3-Quinasas/genética , Proteolisis , Proteínas Proto-Oncogénicas c-akt/genética , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transactivadores/antagonistas & inhibidores , Transactivadores/genética , Factores de Transcripción , Células Tumorales Cultivadas , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
Angelica genus (umbelliferae) has traditionally been used as the medicine and health food considered alleviating several used in the treatment of cancer as well as non-cancerous diseases. Angelica purpuraefolia Chung is an endemic species and a folk medicine in Korea. We have previously reported that two natural khellactone isolated from the rhizomes of A. purpuraefolia has significant antiplasmodial activity against Plasmodium falciparum. In the present study, we scientifically evaluated the effect of (+)-4'-decanoyl-cis-khellactone and (+)-3'-decanoyl-cis-khellactone from A. purpuraefolia on cell proliferation and apoptotic cell death. The anti-proliferative and apoptotic effects of (+)-4'-decanoyl-cis-khellactone and (+)-3'-decanoyl-cis-khellactone (10-50 µg/ml) were subjected to in vitro evaluation using four breast (MDA-MB-231, MCF-7, HS578T, and T47D), three cervical (HeLa, SiHa, and C33A) cancer cell lines, and NIH 3T3 normal cells using FACS and western analyses. Our in vitro experiments showed that (+)-4'-decanoyl-cis-khellactone and (+)-3'-decanoyl-cis-khellactone suppress the growth and proliferation of cancer cells at a relatively low concentration (<10 µg/ml) and induce apoptosis at a high concentration (>50 µg/ml). FACS analysis showed that the cell cycle arrest in the S/G2 phase was induced by the treatment with 10 µg/ml of (+)-4'-decanoyl-cis-khellactone and (+)-3'-decanoyl-cis-khellactone in MDA-MB-231 cells. Western blot analysis also showed that concentration higher than 50 µg/ml of (+)-4'-decanoyl-cis-khellactone induced a time-dependent increase in the percentage of apoptotic cells. However, our results showed that (+)-4'-decanoyl-cis-khellactone uses both extrinsic and intrinsic pathways but (+)-3'-decanoyl-cis-khellactone uses only an intrinsic pathway for the apoptosis. More importantly, (+)-4'-decanoyl-cis-khellactone and (+)-3'-decanoyl-cis-khellactone inhibited the in vitro proliferation of six other human breast and cervical cancer cell lines. Our data strongly suggest that (+)-4'-decanoyl-cis-khellactone and (+)-3'-decanoyl-cis-khellactone act as an anti-cancer supplement particularly against breast and cervical cancers by inducing cell cycle arrest in the S/G2 phase and caspase-dependent apoptosis at relatively low and high concentrations, respectively.
Asunto(s)
Angelica/química , Proliferación Celular/efectos de los fármacos , Cumarinas/farmacología , Medicina de Hierbas , Western Blotting , Neoplasias de la Mama/patología , Ciclo Celular , Línea Celular Tumoral , Separación Celular , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Citometría de Flujo , Humanos , Neoplasias del Cuello Uterino/patologíaRESUMEN
Breast cancer is one of the most common types of cancer in women and is highly treatable by radiotherapy. However, repeated exposure to radiation results in tumor cell resistance. Understanding the molecular mechanisms involved in the response of tumors to γ-irradiation is important for improving radiotherapy. For this reason, we aimed to identify radiation-responsive genes at the protein level. In the present study, we observed differentially expressed proteins using 2D-PAGE and MALDI-TOF-MS for the global analysis of protein expression patterns in response to ionizing radiation (IR). When the expression patterns of proteins were compared to a control gel, numerous spots were found that differed greatly. Among them, 11 spots were found to be significantly different. One set of proteins (GH2, RGS17, BAK1, CCNH, TSG6, RAD51B, IGFBP1 and CASP14) was upregulated and another set of proteins (C1QRF, PLSCR2 and p34(SE1-1)) was downregulated after exposure to γ-rays. These proteins are known to be related to cell cycle control, apoptosis, DNA repair, cell proliferation and other signaling pathways.