RESUMEN
MicroRNAs (miRNAs) are short (â¼22â nt) single-stranded non-coding RNAs that regulate gene expression at the post-transcriptional level. Over recent years, many studies have extensively characterized the involvement of miRNA-mediated regulation in neurogenesis and brain development. However, a comprehensive catalog of cortical miRNAs expressed in a cell-specific manner in progenitor types of the developing mammalian cortex is still missing. Overcoming this limitation, here we exploited a double reporter mouse line previously validated by our group to allow the identification of the transcriptional signature of neurogenic commitment and provide the field with the complete atlas of miRNA expression in proliferating neural stem cells, neurogenic progenitors and newborn neurons during corticogenesis. By extending the currently known list of miRNAs expressed in the mouse brain by over twofold, our study highlights the power of cell type-specific analyses for the detection of transcripts that would otherwise be diluted out when studying bulk tissues. We further exploited our data by predicting putative miRNAs and validated the power of our approach by providing evidence for the involvement of miR-486 in brain development.
Asunto(s)
MicroARNs/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Neuronas/citología , Neuronas/metabolismo , Animales , Northern Blotting , Biología Computacional/métodos , Electroporación , Femenino , Perfilación de la Expresión Génica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Inmunohistoquímica , Hibridación in Situ , Ratones , Ratones Endogámicos C57BL , MicroARNs/genética , Neurogénesis/genética , Neurogénesis/fisiologíaRESUMEN
circSLC45A4 is the main RNA splice isoform produced from its genetic locus and one of the highest expressed circRNAs in the developing human frontal cortex. Knockdown of this highly conserved circRNA in a human neuroblastoma cell line is sufficient to induce spontaneous neuronal differentiation, measurable by increased expression of neuronal marker genes. Depletion of circSlc45a4 in the developing mouse cortex causes a significant reduction of the basal progenitor pool and increases the expression of neurogenic regulators. Furthermore, knockdown of circSlc45a4a induces a significant depletion of cells in the cortical plate. In addition, deconvolution of the bulk RNA-seq data with the help of single-cell RNA-seq data validates the depletion of basal progenitors and reveals an increase in Cajal-Retzius cells. In summary, we present a detailed study of a highly conserved circular RNA that is necessary to maintain the pool of neural progenitors in vitro and in vivo.
Asunto(s)
Encéfalo/fisiología , Perfilación de la Expresión Génica/métodos , Neuronas/metabolismo , ARN Circular/metabolismo , Animales , Diferenciación Celular , Femenino , Humanos , RatonesRESUMEN
Circular (circ) RNAs have recently emerged as a novel class of transcripts whose identification and function remain elusive. Among many tissues and species, the mammalian brain is the organ in which circRNAs are more abundant and first evidence of their functional significance started to emerge. Yet, even within this well-studied organ, annotation of circRNAs remains fragmentary, their sequence is unknown, and their expression in specific cell types was never investigated. Overcoming these limitations, here we provide the first comprehensive identification of circRNAs and assessment of their expression patterns in proliferating neural stem cells, neurogenic progenitors, and newborn neurons of the developing mouse cortex. Extending the current knowledge about the diversity of this class of transcripts by the identification of nearly 4,000 new circRNAs, our study is the first to provide the full sequence information and expression patterns of circRNAs in cell types representing the lineage of neurogenic commitment. We further exploited our data by evaluating the coding potential, evolutionary conservation, and biogenesis of circRNAs that we found to arise from a specific subclass of linear mRNAs. Our study provides the arising field of circRNA biology with a powerful new resource to address the complexity and potential biological significance of this new class of transcripts.
Asunto(s)
Secuencia de Bases/genética , Neurogénesis/genética , ARN Circular/genética , Células Madre/fisiología , Transcriptoma/genética , Empalme Alternativo/genética , Animales , Sitios de Unión , Corteza Cerebelosa/citología , Exones/genética , Femenino , Ratones , Ratones Transgénicos , MicroARNs/genética , MicroARNs/metabolismo , Embarazo , Biosíntesis de Proteínas/genética , ARN Circular/metabolismo , ARN Mensajero/genéticaRESUMEN
The transcription factor C/EBPα is required for granulocytic differentiation of normal myeloid progenitors and is frequently inactivated in acute myeloid leukemia (AML) cells. Ectopic expression of C/EBPα in AML cells suppresses proliferation and induces differentiation suggesting that restoring C/EBPα expression/activity in AML cells could be therapeutically useful. Unfortunately, current approaches of gene or protein delivery in leukemic cells are unsatisfactory. However, "drug repurposing" is becoming a very attractive strategy to identify potential new uses for existing drugs. In this study, we assessed the biological effects of candidate C/EBPα-mimetics identified by interrogation of the Connectivity Map database. We found that amantadine, an antiviral and anti-Parkinson agent, induced a monocyte-macrophage-like differentiation of HL60, U937, Kasumi-1 myeloid leukemia cell lines, as indicated by morphology and differentiation antigen expression, when used in combination with suboptimal concentration of all trans retinoic acid (ATRA) or Vit D3. The effect of amantadine depends, in part, on increased activity of the vitamin D receptor (VDR), since it induced VDR expression and amantadine-dependent monocyte-macrophage differentiation of HL60 cells was blocked by expression of dominant-negative VDR. These results reveal a new function for amantadine and support the concept that screening of the Connectivity Map database can identify small molecules that mimic the effect of transcription factors required for myelo-monocytic differentiation.