RESUMEN
Longitudinal bone growth in children is sustained by growth plates, narrow discs of cartilage that provide a continuous supply of chondrocytes for endochondral ossification1. However, it remains unknown how this supply is maintained throughout childhood growth. Chondroprogenitors in the resting zone are thought to be gradually consumed as they supply cells for longitudinal growth1,2, but this model has never been proved. Here, using clonal genetic tracing with multicolour reporters and functional perturbations, we demonstrate that longitudinal growth during the fetal and neonatal periods involves depletion of chondroprogenitors, whereas later in life, coinciding with the formation of the secondary ossification centre, chondroprogenitors acquire the capacity for self-renewal, resulting in the formation of large, stable monoclonal columns of chondrocytes. Simultaneously, chondroprogenitors begin to express stem cell markers and undergo symmetric cell division. Regulation of the pool of self-renewing progenitors involves the hedgehog and mammalian target of rapamycin complex 1 (mTORC1) signalling pathways. Our findings indicate that a stem cell niche develops postnatally in the epiphyseal growth plate, which provides a continuous supply of chondrocytes over a prolonged period.
Asunto(s)
Condrocitos/citología , Células Clonales/citología , Placa de Crecimiento/citología , Nicho de Células Madre/fisiología , Envejecimiento , Animales , Cartílago/citología , Autorrenovación de las Células , Células Clonales/metabolismo , Femenino , Placa de Crecimiento/metabolismo , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , RatonesRESUMEN
Somatic motor neurons are selectively vulnerable in spinal muscular atrophy (SMA), which is caused by a deficiency of the ubiquitously expressed survival of motor neuron protein. However, some motor neuron groups, including oculomotor and trochlear (ocular), which innervate eye muscles, are for unknown reasons spared. To reveal mechanisms of vulnerability and resistance in SMA, we investigate the transcriptional dynamics in discrete neuronal populations using laser capture microdissection coupled with RNA sequencing (LCM-seq). Using gene correlation network analysis, we reveal a TRP53-mediated stress response that is intrinsic to all somatic motor neurons independent of their vulnerability, but absent in relatively resistant red nucleus and visceral motor neurons. However, the temporal and spatial expression analysis across neuron types shows that the majority of SMA-induced modulations are cell type-specific. Using Gene Ontology and protein network analyses, we show that ocular motor neurons present unique disease-adaptation mechanisms that could explain their resilience. Specifically, ocular motor neurons up-regulate (1) Syt1, Syt5, and Cplx2, which modulate neurotransmitter release; (2) the neuronal survival factors Gdf15, Chl1, and Lif; (3) Aldh4, that protects cells from oxidative stress; and (4) the caspase inhibitor Pak4. Finally, we show that GDF15 can rescue vulnerable human spinal motor neurons from degeneration. This confirms that adaptation mechanisms identified in resilient neurons can be used to reduce susceptibility of vulnerable neurons. In conclusion, this in-depth longitudinal transcriptomics analysis in SMA reveals novel cell type-specific changes that, alone and combined, present compelling targets, including Gdf15, for future gene therapy studies aimed toward preserving vulnerable motor neurons.
Asunto(s)
Adaptación Fisiológica/fisiología , Neuronas Motoras/metabolismo , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/patología , Neuroprotección/genética , Adaptación Fisiológica/genética , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Ojo/inervación , Predisposición Genética a la Enfermedad/genética , Factor 15 de Diferenciación de Crecimiento/genética , Factor 15 de Diferenciación de Crecimiento/metabolismo , Captura por Microdisección con Láser , Ratones , Ratones Noqueados , Corteza Motora/patología , Análisis de Secuencia de ARN , Proteína 1 para la Supervivencia de la Neurona Motora/genética , Proteína 1 para la Supervivencia de la Neurona Motora/metabolismo , Proteína 2 para la Supervivencia de la Neurona Motora/genética , Proteína 2 para la Supervivencia de la Neurona Motora/metabolismo , Activación Transcripcional/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Fused in sarcoma (FUS) is a ubiquitously expressed RNA-binding protein proposed to function in various RNA metabolic pathways, including transcription regulation, pre-mRNA splicing, RNA transport and microRNA processing. Mutations in the FUS gene were identified in patients with amyotrophic lateral sclerosis (ALS), but the pathomechanisms by which these mutations cause ALS are not known. Here, we show that FUS interacts with the minor spliceosome constituent U11 snRNP, binds preferentially to minor introns and directly regulates their removal. Furthermore, a FUS knockout in neuroblastoma cells strongly disturbs the splicing of minor intron-containing mRNAs, among them mRNAs required for action potential transmission and for functional spinal motor units. Moreover, an ALS-associated FUS mutant that forms cytoplasmic aggregates inhibits splicing of minor introns by trapping U11 and U12 snRNAs in these aggregates. Collectively, our findings suggest a possible pathomechanism for ALS in which mutated FUS inhibits correct splicing of minor introns in mRNAs encoding proteins required for motor neuron survival.
Asunto(s)
Esclerosis Amiotrófica Lateral/patología , Intrones , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Empalme del ARN , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/metabolismo , Línea Celular , HumanosRESUMEN
The nonsense-mediated mRNA decay (NMD) pathway is well known as a translation-coupled quality control system that recognizes and degrades aberrant mRNAs with truncated open reading frames (ORF) due to the presence of a premature termination codon (PTC). However, a more general role of NMD in posttranscriptional regulation of gene expression is indicated by transcriptome-wide mRNA profilings that identified a plethora of physiological mRNAs as NMD targets. In this review, we focus on mechanistic aspects of target mRNA identification and degradation in mammalian cells, based on the available biochemical and genetic data, and point out knowledge gaps. Translation termination in a messenger ribonucleoprotein particle (mRNP) environment lacking necessary factors for proper translation termination emerges as a key determinant for subjecting an mRNA to NMD, and we therefore review recent structural and mechanistic insight into translation termination. In addition, the central role of UPF1, its crucial phosphorylation/dephosphorylation cycle and dynamic interactions with other NMD factors are discussed. Moreover, we address the role of exon junction complexes (EJCs) in NMD and summarize the functions of SMG5, SMG6 and SMG7 in promoting mRNA decay through different routes. This article is part of a Special Issue entitled: RNA Decay mechanisms.
Asunto(s)
Degradación de ARNm Mediada por Codón sin Sentido/genética , Estabilidad del ARN/genética , Ribonucleoproteínas/genética , Transactivadores/genética , Regiones no Traducidas 3' , Animales , Mamíferos , Terminación de la Cadena Péptídica Traduccional , Fosforilación , Biosíntesis de Proteínas , ARN HelicasasRESUMEN
Single-cell mRNA sequencing can dissect heterogeneous cell populations as it can identify cell types and cellular states based on their unique transcriptional signatures. We use fluorescence-activated cell sorting (FACS) to isolate individual cultured neurons derived from human-induced pluripotent stem cells (hiPSCs) followed by polyA-based Smart-Seq2 RNA sequencing to analyze the single-cell transcriptional profiles. We provide protocols and guidelines on dissociation, cell selection, and library preparation that can be readily adapted to other cell types or tissue samples.
Asunto(s)
Neuronas , Análisis de la Célula Individual , Humanos , Análisis de la Célula Individual/métodos , Análisis de Secuencia de ARN/métodos , Biblioteca de Genes , Neuronas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Perfilación de la Expresión Génica/métodosRESUMEN
Amyotrophic lateral sclerosis (ALS) is defined by the loss of upper motor neurons (MNs) that project from the cerebral cortex to the brain stem and spinal cord and of lower MNs in the brain stem and spinal cord which innervate skeletal muscles, leading to spasticity, muscle atrophy, and paralysis. ALS involves several disease stages, and multiple cell types show dysfunction and play important roles during distinct phases of disease initiation and progression, subsequently leading to selective MN loss. Why MNs are particularly vulnerable in this lethal disease is still not entirely clear. Neither is it fully understood why certain MNs are more resilient to degeneration in ALS than others. Brain stem MNs of cranial nerves III, IV, and VI, which innervate our eye muscles, are highly resistant and persist until the end-stage of the disease, enabling paralyzed patients to communicate through ocular tracking devices. MNs of the Onuf's nucleus in the sacral spinal cord, that innervate sphincter muscles and control urogenital functions, are also spared throughout the disease. There is also a differential vulnerability among MNs that are intermingled throughout the spinal cord, that directly relate to their physiological properties. Here, fast-twitch fatigable (FF) MNs, which innervate type IIb muscle fibers, are affected early, before onset of clinical symptoms, while slow-twitch (S) MNs, that innervate type I muscle fibers, remain longer throughout the disease progression. The resilience of particular MN subpopulations has been attributed to intrinsic determinants and multiple studies have demonstrated their unique gene regulation and protein content in health and in response to disease. Identified factors within resilient MNs have been utilized to protect more vulnerable cells. Selective vulnerability may also, in part, be driven by non-cell autonomous processes and the unique surroundings and constantly changing environment close to particular MN groups. In this article, we review in detail the cell intrinsic properties of resilient and vulnerable MN groups, as well as multiple additional cell types involved in disease initiation and progression and explain how these may contribute to the selective MN resilience and vulnerability in ALS.
RESUMEN
Endurance exercise promotes skeletal muscle vascularization, oxidative metabolism, fiber-type switching, and neuromuscular junction integrity. Importantly, the metabolic and contractile properties of the muscle fiber must be coupled to the identity of the innervating motor neuron (MN). Here, we show that muscle-derived neurturin (NRTN) acts on muscle fibers and MNs to couple their characteristics. Using a muscle-specific NRTN transgenic mouse (HSA-NRTN) and RNA sequencing of MN somas, we observed that retrograde NRTN signaling promotes a shift toward a slow MN identity. In muscle, NRTN increased capillary density and oxidative capacity and induced a transcriptional reprograming favoring fatty acid metabolism over glycolysis. This combination of effects on muscle and MNs makes HSA-NRTN mice lean with remarkable exercise performance and motor coordination. Interestingly, HSA-NRTN mice largely recapitulate the phenotype of mice with muscle-specific expression of its upstream regulator PGC-1É1. This work identifies NRTN as a myokine that couples muscle oxidative capacity to slow MN identity.
Asunto(s)
Neuronas Motoras , Neurturina , Animales , Ratones , Ratones Transgénicos , Neuronas Motoras/metabolismo , Músculo Esquelético/metabolismo , Neurturina/genética , Neurturina/metabolismo , Neurturina/farmacología , Estrés OxidativoRESUMEN
Mutations in the RNA-binding protein Fused in Sarcoma (FUS) cause early-onset amyotrophic lateral sclerosis (ALS). However, a detailed understanding of central RNA targets of FUS and their implications for disease remain elusive. Here, we use a unique blend of crosslinking and immunoprecipitation (CLIP) and NMR spectroscopy to identify and characterise physiological and pathological RNA targets of FUS. We find that U1 snRNA is the primary RNA target of FUS via its interaction with stem-loop 3 and provide atomic details of this RNA-mediated mode of interaction with the U1 snRNP. Furthermore, we show that ALS-associated FUS aberrantly contacts U1 snRNA at the Sm site with its zinc finger and traps snRNP biogenesis intermediates in human and murine motor neurons. Altogether, we present molecular insights into a FUS toxic gain-of-function involving direct and aberrant RNA-binding and strengthen the link between two motor neuron diseases, ALS and spinal muscular atrophy (SMA).
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , ARN Nuclear Pequeño/metabolismo , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/metabolismo , Ribonucleoproteína Nuclear Pequeña U1/metabolismo , Esclerosis Amiotrófica Lateral/genética , Animales , Línea Celular , Predisposición Genética a la Enfermedad/genética , Humanos , Ratones , Ratones Noqueados , Modelos Moleculares , Neuronas Motoras/metabolismo , Mutación , Dominios y Motivos de Interacción de Proteínas , ARN Nuclear Pequeño/química , Proteína FUS de Unión a ARN/química , Ribonucleoproteína Nuclear Pequeña U1/químicaRESUMEN
Oculomotor neurons, which regulate eye movement, are resilient to degeneration in the lethal motor neuron disease amyotrophic lateral sclerosis (ALS). It would be highly advantageous if motor neuron resilience could be modeled in vitro. Toward this goal, we generated a high proportion of oculomotor neurons from mouse embryonic stem cells through temporal overexpression of PHOX2A in neuronal progenitors. We demonstrate, using electrophysiology, immunocytochemistry, and RNA sequencing, that in vitro-generated neurons are bona fide oculomotor neurons based on their cellular properties and similarity to their in vivo counterpart in rodent and man. We also show that in vitro-generated oculomotor neurons display a robust activation of survival-promoting Akt signaling and are more resilient to the ALS-like toxicity of kainic acid than spinal motor neurons. Thus, we can generate bona fide oculomotor neurons in vitro that display a resilience similar to that seen in vivo.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Diferenciación Celular , Neuronas Motoras/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Transducción de Señal , Esclerosis Amiotrófica Lateral/patología , Animales , Supervivencia Celular , Proteínas de Homeodominio/metabolismo , Humanos , Ratones , Neuronas Motoras/patología , Células Madre Embrionarias de Ratones/patología , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Proximity-dependent trans-biotinylation by the Escherichia coli biotin ligase BirA mutant R118G (BirA*) allows stringent streptavidin affinity purification of proximal proteins. This so-called BioID method provides an alternative to the widely used co-immunoprecipitation (co-IP) to identify protein-protein interactions. Here, we used BioID, on its own and combined with co-IP, to identify proteins involved in nonsense-mediated mRNA decay (NMD), a post-transcriptional mRNA turnover pathway that targets mRNAs that fail to terminate translation properly. In particular, we expressed BirA* fused to the well characterized NMD factors UPF1, UPF2 and SMG5 and detected by liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) the streptavidin-purified biotinylated proteins. While the identified already known interactors confirmed the usefulness of BioID, we also found new potentially important interactors that have escaped previous detection by co-IP, presumably because they associate only weakly and/or very transiently with the NMD machinery. Our results suggest that SMG5 only transiently contacts the UPF1-UPF2-UPF3 complex and that it provides a physical link to the decapping complex. In addition, BioID revealed among others CRKL and EIF4A2 as putative novel transient interactors with NMD factors, but whether or not they have a function in NMD remains to be elucidated.
Asunto(s)
Proteínas Portadoras/metabolismo , Degradación de ARNm Mediada por Codón sin Sentido , Mapeo de Interacción de Proteínas/métodos , Mapas de Interacción de Proteínas , ARN Mensajero/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Biotinilación , Ligasas de Carbono-Nitrógeno/genética , Ligasas de Carbono-Nitrógeno/aislamiento & purificación , Ligasas de Carbono-Nitrógeno/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/aislamiento & purificación , Línea Celular , Cromatografía Liquida , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/aislamiento & purificación , Proteínas de Escherichia coli/metabolismo , Células HeLa , Humanos , Inmunoprecipitación/métodos , ARN Helicasas , Proteínas de Unión al ARN , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/aislamiento & purificación , Proteínas Represoras/metabolismo , Espectrometría de Masas en Tándem , Transactivadores/genética , Transactivadores/aislamiento & purificación , Factores de Transcripción/genética , Factores de Transcripción/aislamiento & purificaciónRESUMEN
In addition to classically defined immune mechanisms, cell-intrinsic processes can restrict virus infection and have shaped virus evolution. The details of this virus-host interaction are still emerging. Following a genome-wide siRNA screen for host factors affecting replication of Semliki Forest virus (SFV), a positive-strand RNA (+RNA) virus, we found that depletion of nonsense-mediated mRNA decay (NMD) pathway components Upf1, Smg5, and Smg7 led to increased levels of viral proteins and RNA and higher titers of released virus. The inhibitory effect of NMD was stronger when virus replication efficiency was impaired by mutations or deletions in the replicase proteins. Consequently, depletion of NMD components resulted in a more than 20-fold increase in production of these attenuated viruses. These findings indicate that a cellular mRNA quality control mechanism serves as an intrinsic barrier to the translation of early viral proteins and the amplification of +RNA viruses in animal cells.
Asunto(s)
Infecciones por Alphavirus/virología , Proteínas Portadoras/metabolismo , Degradación de ARNm Mediada por Codón sin Sentido , Virus de los Bosques Semliki/fisiología , Transactivadores/metabolismo , Replicación Viral , Infecciones por Alphavirus/genética , Infecciones por Alphavirus/metabolismo , Proteínas Portadoras/genética , Interacciones Huésped-Patógeno , Humanos , ARN Helicasas , Virus de los Bosques Semliki/genética , Transactivadores/genética , Proteínas Virales/genética , Proteínas Virales/metabolismo , Liberación del VirusRESUMEN
Cleavage/polyadenylation of mRNAs and 3' processing of replication-dependent histone transcripts are both mediated by large complexes that share several protein components. Functional studies of these shared proteins are complicated by the cooperative binding of the individual subunits. For CstF-64, an additional difficulty is that symplekin and CstF-77 bind mutually exclusively to its hinge domain. Here we have identified CstF-64 and symplekin mutants that allowed us to distinguish between these interactions and to elucidate the role of CstF-64 in the two processing reactions. The interaction of CstF-64 with symplekin is limiting for histone RNA 3' processing but relatively unimportant for cleavage/polyadenylation. In contrast, the nuclear accumulation of CstF-64 depends on its binding to CstF-77 and not to symplekin. Moreover, the CstF-64 paralogue CstF-64Tau can compensate for the loss of CstF-64. As CstF-64Tau has a lower affinity for CstF-77 than CstF-64 and is relatively unstable, it is the minor form. However, it may become up-regulated when the CstF-64 level decreases, which has biological implications for spermatogenesis and probably also for other regulatory events. Thus, the interactions between CstF-64/CstF-64Tau and CstF-77 are important for the maintenance of stoichiometric nuclear levels of the CstF complex components and for their intracellular localization, stability, and function.