RESUMEN
The MUS81-EME1 endonuclease cleaves late replication intermediates at common fragile sites (CFSs) during early mitosis to trigger DNA-repair synthesis that ensures faithful chromosome segregation. Here, we show that these DNA transactions are promoted by RECQ5 DNA helicase in a manner dependent on its Ser727 phosphorylation by CDK1. Upon replication stress, RECQ5 associates with CFSs in early mitosis through its physical interaction with MUS81 and promotes MUS81-dependent mitotic DNA synthesis. RECQ5 depletion or mutational inactivation of its ATP-binding site, RAD51-interacting domain, or phosphorylation site causes excessive binding of RAD51 to CFS loci and impairs CFS expression. This leads to defective chromosome segregation and accumulation of CFS-associated DNA damage in G1 cells. Biochemically, RECQ5 alleviates the inhibitory effect of RAD51 on 3'-flap DNA cleavage by MUS81-EME1 through its RAD51 filament disruption activity. These data suggest that RECQ5 removes RAD51 filaments stabilizing stalled replication forks at CFSs and hence facilitates CFS cleavage by MUS81-EME1.
Asunto(s)
Sitios Frágiles del Cromosoma , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , ADN/biosíntesis , Endonucleasas/metabolismo , Mitosis , RecQ Helicasas/metabolismo , Origen de Réplica , Sitios de Unión , Proteína Quinasa CDC2 , Inestabilidad Cromosómica , Segregación Cromosómica , Quinasas Ciclina-Dependientes/metabolismo , ADN/genética , Daño del ADN , Proteínas de Unión al ADN/genética , Endodesoxirribonucleasas/metabolismo , Endonucleasas/genética , Células HEK293 , Células HeLa , Humanos , Fosforilación , Unión Proteica , Interferencia de ARN , Recombinasa Rad51/metabolismo , RecQ Helicasas/genética , Factores de Tiempo , TransfecciónRESUMEN
Motor neurons in the vertebrate spinal cord are stereotypically organized along the rostro-caudal axis in discrete columns that specifically innervate peripheral muscle domains. Originating from the same progenitor domain, the generation of spinal motor neurons is orchestrated by a spatially and temporally tightly regulated set of secreted molecules and transcription factors such as retinoic acid and the Lim homeodomain transcription factors Isl1 and Lhx1. However, the molecular interactions between these factors remained unclear. In this study we examined the role of the microRNA 9 (miR-9) in the specification of spinal motor neurons and identified Onecut1 (OC1) as one of its targets. miR-9 and OC1 are expressed in mutually exclusive patterns in the developing chick spinal cord, with high OC1 levels in early-born motor neurons and high miR-9 levels in late-born motor neurons. miR-9 efficiently represses OC1 expression in vitro and in vivo. Overexpression of miR-9 leads to an increase in late-born neurons, while miR-9 loss-of-function induces additional OC1(+) motor neurons that display a transcriptional profile typical of early-born neurons. These results demonstrate that regulation of OC1 by miR-9 is a crucial step in the specification of spinal motor neurons and support a model in which miR-9 expression in late-born LMCl neurons downregulates Isl1 expression through inhibition of OC1. In conclusion, our study contributes essential factors to the molecular network specifying spinal motor neurons and emphasizes the importance of microRNAs as key players in the generation of neuronal diversity.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/fisiología , MicroARNs/metabolismo , Neuronas Motoras/fisiología , Factores de Transcripción Onecut/metabolismo , Médula Espinal/embriología , Análisis de Varianza , Animales , Secuencia de Bases , Embrión de Pollo , Electroporación , Fluorescencia , Regulación del Desarrollo de la Expresión Génica/genética , Inmunohistoquímica , Hibridación in Situ , Luciferasas , MicroARNs/genética , Datos de Secuencia Molecular , Neuronas Motoras/metabolismo , Factores de Transcripción Onecut/genéticaRESUMEN
The Ca(2+)-stimulated adenylate cyclase 1 (AC1) is a key mediator of retinotopic map refinement and is required for the retraction response of retinal growth cones to the guidance cue ephrin-A5. We show here that AC1 is dynamically expressed in subpopulations of motor neurons in the spinal cord and sensory neurons of the dorsal root ganglia during development. AC1 was first detected around E12.5 in motoneurons of the medial aspect of the lateral motor column (LMCm) and the lateral region of the medial motor column (MMCl), which project to the ventral limb and body wall musculature, respectively. Expression levels gradually increased until they reached a maximum at a time when peripheral sensory and motor axons branch and establish connections with their targets. In barrelless mice, where a mutation inactivates the AC1 gene, sensory projections to the skin in the limbs and trunk region as well as innervations of the intercostal musculature provided by MMCl axons show increased branching. These results suggest a function of AC1 in the formation of peripheral nerve trajectories such as branching and pruning, after the initial projections have been laid down.