RESUMEN
In mammals, neurons in the peripheral nervous system (PNS) have regenerative capacity following injury, but it is generally absent in the CNS. This difference is attributed, at least in part, to the intrinsic ability of PNS neurons to activate a unique regenerative transcriptional program following injury. Here, we profiled gene expression following sciatic nerve crush in mice and identified long noncoding RNAs (lncRNAs) that act in the regenerating neurons and which are typically not expressed in other contexts. We show that two of these lncRNAs regulate the extent of neuronal outgrowth. We then focus on one of these, Silc1, and show that it regulates neuroregeneration in cultured cells and in vivo, through cis-acting activation of the transcription factor Sox11.
Asunto(s)
Regeneración Nerviosa/genética , ARN Largo no Codificante/fisiología , Animales , Línea Celular Tumoral , Ganglios Espinales , Regulación de la Expresión Génica/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuritas/metabolismo , Neuritas/fisiología , Neuronas/fisiología , Traumatismos de los Nervios Periféricos/genética , Traumatismos de los Nervios Periféricos/fisiopatología , ARN Largo no Codificante/genética , ARN Mensajero , Factores de Transcripción SOXC , Nervio Ciático/metabolismoRESUMEN
The number of known long noncoding RNA (lncRNA) functions is rapidly growing, but how those functions are encoded in their sequence and structure remains poorly understood. NORAD (noncoding RNA activated by DNA damage) is a recently characterized, abundant, and highly conserved lncRNA that is required for proper mitotic divisions in human cells. NORAD acts in the cytoplasm and antagonizes repressors from the Pumilio family that bind at least 17 sites spread through 12 repetitive units in NORAD sequence. Here we study conserved sequences in NORAD repeats, identify additional interacting partners, and characterize the interaction between NORAD and the RNA-binding protein SAM68 (KHDRBS1), which is required for NORAD function in antagonizing Pumilio. These interactions provide a paradigm for how repeated elements in a lncRNA facilitate function.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Unión al ADN/metabolismo , ARN Largo no Codificante/metabolismo , Proteínas de Unión al ARN/metabolismo , Regiones no Traducidas 3' , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/fisiología , Sitios de Unión , Línea Celular Tumoral , Segregación Cromosómica , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Regulación de la Expresión Génica , Humanos , ARN Largo no Codificante/química , ARN Largo no Codificante/genética , ARN Largo no Codificante/fisiología , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/fisiología , Proteínas Represoras/metabolismoRESUMEN
Long noncoding RNAs (lncRNAs) have been shown to play important roles in gene regulatory networks acting in early development. There has been rapid turnover of lncRNA loci during vertebrate evolution, with few human lncRNAs conserved beyond mammals. The sequences of these rare deeply conserved lncRNAs are typically not similar to each other. Here, we characterize HOXA-AS3 and HOXB-AS3, lncRNAs produced from the central regions of the HOXA and HOXB clusters. Sequence-similar orthologs of both lncRNAs are found in multiple vertebrate species and there is evident sequence similarity between their promoters, suggesting that the production of these lncRNAs predates the duplication of the HOX clusters at the root of the vertebrate lineage. This conservation extends to similar expression patterns of the two lncRNAs, in particular in cells transiently arising during early development or in the adult colon. Functionally, the RNA products of HOXA-AS3 and HOXB-AS3 regulate the expression of their overlapping HOX5-7 genes both in HT-29 cells and during differentiation of human embryonic stem cells. Beyond production of paralogous protein-coding and microRNA genes, the regulatory program in the HOX clusters therefore also relies on paralogous lncRNAs acting in restricted spatial and temporal windows of embryonic development and cell differentiation.
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Proteínas de Homeodominio/genética , Animales , Diferenciación Celular/genética , Proliferación Celular/genética , Células Madre Embrionarias/metabolismo , Endodermo/metabolismo , Enterocitos/metabolismo , Genes Homeobox , Proteínas de Homeodominio/metabolismo , Humanos , Familia de Multigenes/genética , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Homología de Secuencia , Vertebrados/genéticaRESUMEN
Social anxiety disorder is characterized by a persistent fear and avoidance of social situations, but available treatment options are rather unspecific. Using an established mouse social fear conditioning (SFC) paradigm, we profiled gene expression and chromatin alterations after the acquisition and extinction of social fear within the septum, a brain region important for social fear and social behaviors. Here, we particularly focused on the successful versus unsuccessful outcome of social fear extinction training, which corresponds to treatment responsive versus resistant patients in the clinics. Validation of coding and non-coding RNAs revealed specific isoforms of the long non-coding RNA (lncRNA) Meg3 regulated, depending on the success of social fear extinction. Moreover, PI3K/AKT was differentially activated with extinction success in SFC-mice. In vivo knockdown of specific Meg3 isoforms increased baseline activity of PI3K/AKT signaling, and mildly delayed social fear extinction. Using ATAC-Seq and CUT&RUN, we found alterations in the chromatin structure of specific genes, which might be direct targets of lncRNA Meg3.
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Extinción Psicológica , Miedo , ARN Largo no Codificante , Animales , Ratones , Cromatina , Fosfatidilinositol 3-Quinasas , Proteínas Proto-Oncogénicas c-akt , ARN Largo no Codificante/genética , TranscriptomaRESUMEN
Eukaryotic genomes are pervasively transcribed, with tens of thousands of RNAs emanating from uni- and bi-directional promoters and from active enhancers. In vertebrates, thousands of loci in each species produce a class of transcripts called long noncoding RNAs (lncRNAs) that are typically expressed at low levels and do not appear to give rise to functional proteins. Substantial numbers of lncRNAs are expressed at specific stages of embryonic development, in many cases from regions flanking key developmental regulators. Here, we review the known biological functions of such lncRNAs and the emerging paradigms of their modes of action. We also provide an overview of the growing arsenal of methods for lncRNA identification, perturbation and functional characterization.
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Desarrollo Embrionario/genética , ARN Largo no Codificante/fisiología , Células Madre/fisiología , Animales , Embrión de Mamíferos , Regulación del Desarrollo de la Expresión Génica , Humanos , Mamíferos/embriología , Mamíferos/genéticaRESUMEN
Downregulation of the urea cycle enzyme argininosuccinate synthase (ASS1) in multiple tumors is associated with a poor prognosis partly because of the metabolic diversion of cytosolic aspartate for pyrimidine synthesis, supporting proliferation and mutagenesis owing to nucleotide imbalance. Here, we find that prolonged loss of ASS1 promotes DNA damage in colon cancer cells and fibroblasts from subjects with citrullinemia type I. Following acute induction of DNA damage with doxorubicin, ASS1 expression is elevated in the cytosol and the nucleus with at least a partial dependency on p53; ASS1 metabolically restrains cell cycle progression in the cytosol by restricting nucleotide synthesis. In the nucleus, ASS1 and ASL generate fumarate for the succination of SMARCC1, destabilizing the chromatin-remodeling complex SMARCC1-SNF5 to decrease gene transcription, specifically in a subset of the p53-regulated cell cycle genes. Thus, following DNA damage, ASS1 is part of the p53 network that pauses cell cycle progression, enabling genome maintenance and survival. Loss of ASS1 contributes to DNA damage and promotes cell cycle progression, likely contributing to cancer mutagenesis and, hence, adaptability potential.
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Argininosuccinato Sintasa , Núcleo Celular , Citosol , Daño del ADN , Proteína p53 Supresora de Tumor , Humanos , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/genética , Citosol/metabolismo , Argininosuccinato Sintasa/metabolismo , Argininosuccinato Sintasa/genética , Núcleo Celular/metabolismo , Ciclo Celular/genéticaRESUMEN
Long non-coding RNAs (lncRNAs) are implicated in a plethora of cellular processes, but an in-depth understanding of their functional features or their mechanisms of action is currently lacking. Here we study Meteor, a lncRNA transcribed near the gene encoding EOMES, a pleiotropic transcription factor implicated in various processes throughout development and in adult tissues. Using a wide array of perturbation techniques, we show that transcription elongation through the Meteor locus is required for Eomes activation in mouse embryonic stem cells, with Meteor repression linked to a change in the subpopulation primed to differentiate to the mesoderm lineage. We further demonstrate that a distinct functional feature of the locus-namely, the underlying DNA element-is required for suppressing Eomes expression following neuronal differentiation. Our results demonstrate the complex regulation that can be conferred by a single locus and emphasize the importance of careful selection of perturbation techniques when studying lncRNA loci.
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ARN Largo no Codificante , Proteínas de Dominio T Box , Animales , Ratones , Diferenciación Celular/genética , Regulación de la Expresión Génica , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Active nucleocytoplasmic transport of macromolecules requires soluble transport carriers of the importin/karyopherin superfamily. Although the nuclear transport machinery is essential in all eukaryotic cells, neurons must also mobilise importins and associated proteins to overcome unique spatiotemporal challenges. These include switches in importin alpha subtype expression during neuronal differentiation, localized axonal synthesis of importin beta1 to coordinate a retrograde injury signaling complex on axonal dynein, and trafficking of regulatory and signaling molecules from synaptic terminals to cell bodies. Targeting of RNAs encoding critical components of the importins complex and the Ran system to axons allows sophisticated local regulation of the system for mobilization upon need. Finally, a number of importin family members have been associated with mental or neurodegenerative diseases. The extended roles recently discovered for importins in the nervous system might also be relevant in non-neuronal cells, and the localized modes of importin regulation in neurons offer new avenues to interrogate their cytoplasmic functions.
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Transporte Activo de Núcleo Celular , Neuronas/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Animales , Humanos , Carioferinas/metabolismo , Enfermedades del Sistema Nervioso/metabolismo , Plasticidad Neuronal , Neuronas/citologíaRESUMEN
Long noncoding RNAs (lncRNAs) are gathering increasing attention toward their roles in different biological systems. In mammals, the richest repertoires of lncRNAs are expressed in the brain and in the testis, and the diversity of lncRNAs in the nervous system is thought to be related to the diversity and the complexity of its cell types. Supporting this notion, many lncRNAs are differentially expressed between different regions of the brain or in particular cell types, and many lncRNAs are dynamically expressed during embryonic or postnatal neurogenesis. Less is known about the functions of these genes, if any, but they are increasingly implicated in diverse processes in health and disease. Here, we review the current knowledge about the roles and importance of lncRNAs in the central and peripheral nervous systems and discuss the specific niches within gene regulatory networks that might be preferentially occupied by lncRNAs.
RESUMEN
Chromodomain helicase DNA binding protein 2 (Chd2) is a chromatin remodeller implicated in neurological disease. Here we show that Chaserr, a highly conserved long noncoding RNA transcribed from a region near the transcription start site of Chd2 and on the same strand, acts in concert with the CHD2 protein to maintain proper Chd2 expression levels. Loss of Chaserr in mice leads to early postnatal lethality in homozygous mice, and severe growth retardation in heterozygotes. Mechanistically, loss of Chaserr leads to substantially increased Chd2 mRNA and protein levels, which in turn lead to transcriptional interference by inhibiting promoters found downstream of highly expressed genes. We further show that Chaserr production represses Chd2 expression solely in cis, and that the phenotypic consequences of Chaserr loss are rescued when Chd2 is perturbed as well. Targeting Chaserr is thus a potential strategy for increasing CHD2 levels in haploinsufficient individuals.
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Ensamble y Desensamble de Cromatina/genética , Proteínas de Unión al ADN/genética , Trastornos del Crecimiento/genética , ARN Largo no Codificante/genética , ARN Mensajero/metabolismo , Animales , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Genes Letales , Haploinsuficiencia , Heterocigoto , Homocigoto , Ratones , Ratones Noqueados , Regiones Promotoras GenéticasRESUMEN
How is protein synthesis initiated locally in neurons? We found that mTOR (mechanistic target of rapamycin) was activated and then up-regulated in injured axons, owing to local translation of mTOR messenger RNA (mRNA). This mRNA was transported into axons by the cell size-regulating RNA-binding protein nucleolin. Furthermore, mTOR controlled local translation in injured axons. This included regulation of its own translation and that of retrograde injury signaling molecules such as importin ß1 and STAT3 (signal transducer and activator of transcription 3). Deletion of the mTOR 3' untranslated region (3'UTR) in mice reduced mTOR in axons and decreased local translation after nerve injury. Both pharmacological inhibition of mTOR in axons and deletion of the mTOR 3'UTR decreased proprioceptive neuronal survival after nerve injury. Thus, mRNA localization enables spatiotemporal control of mTOR pathways regulating local translation and long-range intracellular signaling.
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Axones/metabolismo , Ganglios Espinales/lesiones , Biosíntesis de Proteínas , Nervio Ciático/lesiones , Serina-Treonina Quinasas TOR/biosíntesis , Regiones no Traducidas 3' , Animales , Tamaño de la Célula , Ratones , Ratones Endogámicos , Fosfoproteínas/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Ratas , Ratas Endogámicas BB , Ratas Sprague-Dawley , Transducción de Señal , Serina-Treonina Quinasas TOR/genética , NucleolinaRESUMEN
Reactive oxygen species (ROS) contribute to tissue damage and remodelling mediated by the inflammatory response after injury. Here we show that ROS, which promote axonal dieback and degeneration after injury, are also required for axonal regeneration and functional recovery after spinal injury. We find that ROS production in the injured sciatic nerve and dorsal root ganglia requires CX3CR1-dependent recruitment of inflammatory cells. Next, exosomes containing functional NADPH oxidase 2 complexes are released from macrophages and incorporated into injured axons via endocytosis. Once in axonal endosomes, active NOX2 is retrogradely transported to the cell body through an importin-ß1-dynein-dependent mechanism. Endosomal NOX2 oxidizes PTEN, which leads to its inactivation, thus stimulating PI3K-phosporylated (p-)Akt signalling and regenerative outgrowth. Challenging the view that ROS are exclusively involved in nerve degeneration, we propose a previously unrecognized role of ROS in mammalian axonal regeneration through a NOX2-PI3K-p-Akt signalling pathway.
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Axones/enzimología , Exosomas/enzimología , Ganglios Espinales/enzimología , NADPH Oxidasa 2/metabolismo , Degeneración Nerviosa , Regeneración Nerviosa , Traumatismos de los Nervios Periféricos/enzimología , Especies Reactivas de Oxígeno/metabolismo , Nervio Ciático/enzimología , Traumatismos de la Médula Espinal/enzimología , Animales , Axones/patología , Receptor 1 de Quimiocinas CX3C/metabolismo , Línea Celular , Modelos Animales de Enfermedad , Dineínas/metabolismo , Endocitosis , Endosomas/enzimología , Endosomas/patología , Exosomas/patología , Ganglios Espinales/lesiones , Ganglios Espinales/patología , Macrófagos/enzimología , Macrófagos/patología , Ratones Endogámicos C57BL , Ratones Noqueados , NADPH Oxidasa 2/deficiencia , NADPH Oxidasa 2/genética , Proteínas Nucleares/metabolismo , Fosfohidrolasa PTEN/metabolismo , Traumatismos de los Nervios Periféricos/genética , Traumatismos de los Nervios Periféricos/patología , Traumatismos de los Nervios Periféricos/fisiopatología , Fosfatidilinositol 3-Quinasa/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Nervio Ciático/lesiones , Nervio Ciático/patología , Nervio Ciático/fisiopatología , Transducción de Señal , Traumatismos de la Médula Espinal/genética , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/fisiopatología , beta CarioferinasRESUMEN
In the version of this Article originally published, the affiliations for Roland A. Fleck and José Antonio Del Río were incorrect due to a technical error that resulted in affiliations 8 and 9 being switched. The correct affiliations are: Roland A. Fleck: 8Centre for Ultrastructural Imaging, Kings College London, London, UK. José Antonio Del Río: 2Cellular and Molecular Neurobiotechnology, Institute for Bioengineering of Catalonia, Barcelona, Spain; 9Department of Cell Biology, Physiology and Immunology, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain; 10Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Barcelona, Spain. This has now been amended in all online versions of the Article.
RESUMEN
How can cells sense their own size to coordinate biosynthesis and metabolism with their growth needs? We recently proposed a motor-dependent bidirectional transport mechanism for axon length and cell size sensing, but the nature of the motor-transported size signals remained elusive. Here, we show that motor-dependent mRNA localization regulates neuronal growth and cycling cell size. We found that the RNA-binding protein nucleolin is associated with importin ß1 mRNA in axons. Perturbation of nucleolin association with kinesins reduces its levels in axons, with a concomitant reduction in axonal importin ß1 mRNA and protein levels. Strikingly, subcellular sequestration of nucleolin or importin ß1 enhances axonal growth and causes a subcellular shift in protein synthesis. Similar findings were obtained in fibroblasts. Thus, subcellular mRNA localization regulates size and growth in both neurons and cycling cells.
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Tamaño de la Célula , Neuronas Motoras/metabolismo , Fosfoproteínas/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Axones/metabolismo , Ratones Transgénicos , Neurogénesis , Biosíntesis de Proteínas/fisiología , NucleolinaRESUMEN
Size homeostasis is fundamental in cell biology, but it is not clear how large cells such as neurons can assess their own size or length. We examined a role for molecular motors in intracellular length sensing.Computational simulations suggest that spatial information can be encoded by the frequency of an oscillating retrograde signal arising from a composite negative feedback loop between bidirectional motor-dependent signals. The model predicts that decreasing either or both anterograde or retrograde signals should increase cell length, and this prediction was confirmed upon application of siRNAs for specific kinesin and/or dynein heavy chains in adult sensory neurons. Heterozygous dynein heavy chain 1 mutant sensory neurons also exhibited increased lengths both in vitro and during embryonic development.Moreover, similar length increases were observed in mouse embryonic fibroblasts upon partial downregulation of dynein heavy chain 1.Thus, molecular motors critically influence cell length sensing and growth control.
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Tamaño de la Célula , Fibroblastos/citología , Fibroblastos/metabolismo , Proteínas Motoras Moleculares/metabolismo , Animales , Transporte Biológico , Simulación por Computador , Citoesqueleto/metabolismo , Regulación hacia Abajo , Dineínas/ultraestructura , Citometría de Flujo , Heterocigoto , Cinesinas/metabolismo , Cinética , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Modelos Biológicos , Mutación/genética , Células 3T3 NIH , Neuritas/metabolismo , ARN Interferente Pequeño/metabolismo , Nervio Ciático/citología , Nervio Ciático/ultraestructura , Células Receptoras Sensoriales/citología , Células Receptoras Sensoriales/metabolismoRESUMEN
Subcellular localization of mRNA enables compartmentalized regulation within large cells. Neurons are the longest known cells; however, so far, evidence is lacking for an essential role of endogenous mRNA localization in axons. Localized upregulation of Importin ß1 in lesioned axons coordinates a retrograde injury-signaling complex transported to the neuronal cell body. Here we show that a long 3' untranslated region (3' UTR) directs axonal localization of Importin ß1. Conditional targeting of this 3' UTR region in mice causes subcellular loss of Importin ß1 mRNA and protein in axons, without affecting cell body levels or nuclear functions in sensory neurons. Strikingly, axonal knockout of Importin ß1 attenuates cell body transcriptional responses to nerve injury and delays functional recovery in vivo. Thus, localized translation of Importin ß1 mRNA enables separation of cytoplasmic and nuclear transport functions of importins and is required for efficient retrograde signaling in injured axons.