RESUMEN
The regulation of miRNAs is critical to the definition of cell identity and behavior in normal physiology and disease. To date, the dynamics of miRNA degradation and the mechanisms involved in remain largely obscure, in particular, in higher organisms. Here, we developed a pulse-chase approach based on metabolic RNA labeling to calculate miRNA decay rates at genome-wide scale in mammalian cells. Our analysis revealed heterogeneous miRNA half-lives, with many species behaving as stable molecules (T1/2> 24 h), while others, including passenger miRNAs and a number (25/129) of guide miRNAs, are quickly turned over (T1/2= 4-14 h). Decay rates were coupled with other features, including genomic organization, transcription rates, structural heterogeneity (isomiRs), and target abundance, measured through quantitative experimental approaches. This comprehensive analysis highlighted functional mechanisms that mediate miRNA degradation, as well as the importance of decay dynamics in the regulation of the miRNA pool under both steady-state conditions and during cell transitions.
Asunto(s)
MicroARNs/genética , Animales , Proteínas Argonautas/metabolismo , Fibroblastos , Regulación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Ratones , MicroARNs/metabolismo , Interferencia de ARN , Estabilidad del ARN , Ribonucleasa III/metabolismo , Factores de Tiempo , Transcripción GenéticaRESUMEN
Recent evidence indicates that the miRNA biogenesis factors DROSHA, DGCR8, and DICER exert non-overlapping functions, and have also roles in miRNA-independent regulatory mechanisms. However, it is currently unknown whether miRNA-independent functions of DGCR8 play any role in the maintenance of neuronal progenitors and during corticogenesis. Here, by phenotypic comparison of cortices from conditional Dgcr8 and Dicer knockout mice, we show that Dgcr8 deletion, in contrast to Dicer depletion, leads to premature differentiation of neural progenitor cells and overproduction of TBR1-positive neurons. Remarkably, depletion of miRNAs upon DCGR8 loss is reduced compared to DICER loss, indicating that these phenotypic differences are mediated by miRNA-independent functions of DGCR8. We show that Dgcr8 mutations induce an earlier and stronger phenotype in the developing nervous system compared to Dicer mutants and that miRNA-independent functions of DGCR8 are critical for corticogenesis. Finally, our data also suggest that the Microprocessor complex, with DROSHA and DGCR8 as core components, directly regulates the Tbr1 transcript, containing evolutionarily conserved hairpins that resemble miRNA precursors, independently of miRNAs.
Asunto(s)
Proteínas de Unión al ADN/genética , Regulación del Desarrollo de la Expresión Génica , MicroARNs/genética , Neocórtex/embriología , Neocórtex/metabolismo , Proteínas de Unión al ARN/genética , Animales , Apoptosis/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Línea Celular , Proliferación Celular , Proteínas de Unión al ADN/metabolismo , Eliminación de Gen , Proteínas de Homeodominio/metabolismo , Ratones , Ratones Noqueados , Ratones Transgénicos , Neocórtex/patología , Proteínas del Tejido Nervioso , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Neurogénesis/genética , Neuronas/metabolismo , Interferencia de ARN , Proteínas de Unión al ARN/metabolismo , Proteínas de Dominio T Box , Factores de Transcripción/metabolismoRESUMEN
Long noncoding RNAs (lncRNAs) are emerging as regulators of fundamental biological processes. Here we report on the characterization of an intergenic lncRNA expressed in epithelial tissues which we termed EPR (Epithelial cell Program Regulator). EPR is rapidly downregulated by TGF-ß and its sustained expression largely reshapes the transcriptome, favors the acquisition of epithelial traits, and reduces cell proliferation in cultured mammary gland cells as well as in an animal model of orthotopic transplantation. EPR generates a small peptide that localizes at epithelial cell junctions but the RNA molecule per se accounts for the vast majority of EPR-induced gene expression changes. Mechanistically, EPR interacts with chromatin and regulates Cdkn1a gene expression by affecting both its transcription and mRNA decay through its association with SMAD3 and the mRNA decay-promoting factor KHSRP, respectively. We propose that EPR enables epithelial cells to control proliferation by modulating waves of gene expression in response to TGF-ß.
Asunto(s)
Estabilidad del ARN/genética , ARN Largo no Codificante/genética , Proteína smad3/metabolismo , Transcriptoma/genética , Factor de Crecimiento Transformador beta/farmacología , Animales , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Células Cultivadas , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Humanos , ARN Largo no Codificante/efectos de los fármacosRESUMEN
Physical exercise stimulates adult hippocampal neurogenesis and is considered a relevant strategy for preventing age-related cognitive decline in humans. The underlying mechanisms remains controversial. Here, we show that exercise increases proliferation of neural precursor cells (NPCs) of the mouse dentate gyrus (DG) via downregulation of microRNA 135a-5p (miR-135a). MiR-135a inhibition stimulates NPC proliferation leading to increased neurogenesis, but not astrogliogenesis, in DG of resting mice, and intriguingly it re-activates NPC proliferation in aged mice. We identify 17 proteins (11 putative targets) modulated by miR-135 in NPCs. Of note, inositol 1,4,5-trisphosphate (IP3) receptor 1 and inositol polyphosphate-4-phosphatase type I are among the modulated proteins, suggesting that IP3 signaling may act downstream miR-135. miR-135 is the first noncoding RNA essential modulator of the brain's response to physical exercise. Prospectively, the miR-135-IP3 axis might represent a novel target of therapeutic intervention to prevent pathological brain aging.
Asunto(s)
Células Madre Adultas/metabolismo , Envejecimiento/metabolismo , MicroARNs/biosíntesis , Células-Madre Neurales/metabolismo , Neurogénesis , Condicionamiento Físico Animal , Animales , Proliferación Celular , Regulación de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Péptidos y Proteínas de Señalización Intracelular/biosíntesis , Ventrículos Laterales/citología , Ventrículos Laterales/metabolismo , Ratones , Ratones Noqueados , Nicho de Células Madre , Proteínas Quinasas p38 Activadas por Mitógenos/biosíntesisRESUMEN
MicroRNAs (miRNAs) are an evolutionary conserved class of short, single-stranded noncoding RNAs (<18-22 nt in length) that act in posttranscriptional regulation of gene expression in higher eukaryotes. The abundance of a miRNA is a key feature in control of its activity and, therefore, a number of mechanisms finely regulate miRNA levels, acting at both transcriptional and posttranscriptional level. Recent evidences, including our research, highlighted the role of miRNA decay as a mechanism controlling the miRNA pool. We describe in this chapter an optimized methodology to determine miRNA degradation rates in mammalian cells. Our approach is based on metabolic pulse labeling with 4-thiouridine (4sU), a uridine analog that is incorporated in nascent RNA and allows thiol-specific biotinylation and selective pull-down of labeled RNA. In particular, given the long average half-life and the complex biogenetic process of miRNAs, we developed a "pulse-chase" protocol where 4sU is removed from the medium after a long labeling period (2-3 h pulse), and labeled RNA is purified at different time points to measure the decay of labeled molecules. By combining the 4sU-based "pulse-chase" approach with high-throughput small RNA sequencing (sRNAseq), it is possible to quantify at genome-wide level miRNA degradation rates.
Asunto(s)
MicroARNs , Procesamiento Postranscripcional del ARN , Estabilidad del ARN , Coloración y Etiquetado/métodos , Tiouridina , Células HeLa , Humanos , MicroARNs/biosíntesis , MicroARNs/química , MicroARNs/genética , Tiouridina/química , Tiouridina/metabolismoRESUMEN
Little is known about miRNA decay. A target-directed miRNA degradation mechanism (TDMD) has been suggested, but further investigation on endogenous targets is necessary. Here, we identify hundreds of targets eligible for TDMD and show that an endogenous RNA (Serpine1) controls the degradation of two miRNAs (miR-30b-5p and miR-30c-5p) in mouse fibroblasts. In our study, TDMD occurs when the target is expressed at relatively low levels, similar in range to those of its miRNAs (100-200 copies per cell), and becomes more effective at high target:miRNA ratios (>10:1). We employ CRISPR/Cas9 to delete the miR-30 responsive element within Serpine1 3'UTR and interfere with TDMD. TDMD suppression increases miR-30b/c levels and boosts their activity towards other targets, modulating gene expression and cellular phenotypes (i.e., cell cycle re-entry and apoptosis). In conclusion, a sophisticated regulatory layer of miRNA and gene expression mediated by specific endogenous targets exists in mammalian cells.
Asunto(s)
Fibroblastos/metabolismo , MicroARNs/genética , Serpina E2/metabolismo , Regiones no Traducidas 3' , Animales , Apoptosis , Sistemas CRISPR-Cas , Ciclo Celular , Eliminación de Gen , Expresión Génica , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Ratones , MicroARNs/metabolismo , Mutación , Fenotipo , Estabilidad del ARN , Análisis de Secuencia de ARN , Serpina E2/genéticaRESUMEN
The cancer-associated loss of microRNA (miRNA) expression leads to a proliferative advantage and aggressive behavior through largely unknown mechanisms. Here, we exploit a model system that recapitulates physiological terminal differentiation and its reversal upon oncogene expression to analyze coordinated mRNA/miRNA responses. The cell cycle reentry of myotubes, forced by the E1A oncogene, was associated with a pattern of mRNA/miRNA modulation that was largely reciprocal to that induced during the differentiation of myoblasts into myotubes. The E1A-induced mRNA response was preponderantly Retinoblastoma protein (Rb)-dependent. Conversely, the miRNA response was mostly Rb-independent and exerted through tissue-specific factors and Myc. A subset of these miRNAs (miR-1, miR-34, miR-22, miR-365, miR-29, miR-145, and Let-7) was shown to coordinately target Rb-dependent cell cycle and DNA replication mRNAs. Thus, a dual level of regulation-transcriptional regulation via Rb-E2F and posttranscriptional regulation via miRNAs-confers robustness to cell cycle control and provides a molecular basis to understand the role of miRNA subversion in cancer.