RESUMEN
The DiGeorge syndrome critical region gene 8 (Dgcr8) knockout strategy has been widely used to study the function of canonical microRNAs (miRNAs) in vitro and in vivo. However, primary miRNA (pri-miRNA) transcripts are accumulated in Dgcr8 knockout cells due to interrupted processing. Whether abnormally accumulated pri-miRNAs have any function is unknown. Here, using clustered regularly interspaced short palindromic repeats system/CRISPR-associated protein 9 (CRISPR/Cas9), we successfully knocked out the primary microRNA-290~295 (pri-miR-290~295) cluster, the most highly expressed miRNA cluster in mouse embryonic stem cells (ESCs), in Dgcr8 knockout background. We found that the major defects associated with Dgcr8 knockout in mouse ESCs, including higher expression of epithelial-to-mesenchymal transition (EMT) markers, slower proliferation, G1 accumulation, and defects in silencing self-renewal, were not affected by the deletion of pri-miR-290~290 cluster. Interestingly, the transcription of neighboring gene nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing 12(Nlrp12) was upregulated upon the deletion of the pri-miR-290~295 cluster. Together, our results suggested that the major defects in Dgcr8 knockout ESCs were not due to the accumulation of pri-miR-290~295, and the deletion of miRNA genes could affect the transcription of neighboring DNA elements.
Asunto(s)
MicroARNs/genética , Células Madre Embrionarias de Ratones/metabolismo , Interferencia de ARN , Proteínas de Unión al ARN/genética , Animales , Biomarcadores , Ciclo Celular/genética , Diferenciación Celular/genética , Proliferación Celular , Autorrenovación de las Células/genética , Transición Epitelial-Mesenquimal , Regulación del Desarrollo de la Expresión Génica , Silenciador del Gen , Ratones , Ratones Noqueados , FenotipoRESUMEN
Long noncoding RNAs (lncRNAs) have emerged as important components of gene regulatory network in embryonic stem cells (ESCs). However, the function and molecular mechanism of lncRNAs are still largely unknown. Here we identifies Trincr1 (TRIM71 interacting long noncoding RNA 1) lncRNA that regulates the FGF/ERK signaling and self-renewal of ESCs. Trincr1 is exported by THOC complex to cytoplasm where it binds and represses TRIM71, leading to the downregulation of SHCBP1 protein. Knocking out Trincr1 leads to the upregulation of phosphorylated ERK and ERK pathway target genes and the decrease of ESC self-renewal, while knocking down Trim71 completely rescues the defects of Trincr1 knockout. Furthermore, ectopic expression of Trincr1 represses FGF/ERK signaling and the self-renewal of neural progenitor cells (NPCs). Together, this study highlights lncRNA as an important player in cell signaling network to coordinate cell fate specification.
Asunto(s)
Factores de Crecimiento de Fibroblastos/genética , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/genética , Células Madre Embrionarias de Ratones/metabolismo , ARN Largo no Codificante/genética , Factores de Transcripción/genética , Animales , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Embrión de Mamíferos , Factores de Crecimiento de Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Sistema de Señalización de MAP Quinasas , Ratones , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Células Madre Embrionarias de Ratones/citología , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Fosforilación , Unión Proteica , ARN Largo no Codificante/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Adaptadoras de la Señalización Shc/genética , Proteínas Adaptadoras de la Señalización Shc/metabolismo , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismoRESUMEN
microRNAs (miRNAs) are small noncoding RNAs that play important regulatory roles in plants, animals and viruses. Measuring miRNA activity in vivo remains a big challenge. Here, using an miRNA-mediated single guide RNA (sgRNA)-releasing strategy and dCas9-VPR to drive a transgene red fluorescent protein, we create an miRNA sensor that can faithfully measure miRNA activity at cellular levels and use it to monitor differentiation status of stem cells. Furthermore, by designing sgRNAs to target endogenous loci, we adapted this system to control the expression of endogenous genes or mutate specific DNA bases upon induction by cell-type-specific miRNAs. Finally, by miRNA sensor library screening, we discover a previously undefined layer of heterogeneity associated with miR-21a activity in mouse embryonic stem cells. Together, these results highlight the utility of an miRNA-induced CRISPR-Cas9 system as miRNA sensors and cell-type-specific genome regulation tools.
Asunto(s)
Sistemas CRISPR-Cas , Animales , Proteína 9 Asociada a CRISPR , Diferenciación Celular/genética , Células Cultivadas , Células Madre Embrionarias/metabolismo , Genoma , Células HeLa , Humanos , Ratones , MicroARNs , ARN Interferente Pequeño/metabolismo , Activación Transcripcional , TransgenesRESUMEN
Alternative pre-mRNA splicing plays important roles in regulating self-renewal and differentiation of embryonic stem cells (ESCs). However, how specific alternative splicing programs are established in ESCs remains elusive. Here, we show that a subset of alternative splicing events in ESCs is dependent on miR-294 expression. Remarkably, roughly 60% of these splicing events are affected by the depletion of Muscleblind-Like Splicing Regulator 1 and 2 (Mbnl1/2). Distinct from canonical miRNA function, miR-294 represses Mbnl1/2 through both posttranscriptional and epigenetic mechanisms. Furthermore, we uncover non-canonical functions of MBNL proteins that bind and promote the expression of miR-294 targets, including Cdkn1a and Tgfbr2, thereby opposing the role of miR-294 in regulating cell proliferation, apoptosis, and epithelial-mesenchymal transition (EMT). Our study reveals extensive interactions between miRNAs and splicing factors, highlighting their roles in regulating cell type-specific alternative splicing and defining gene expression programs during development and cellular differentiation.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Células Madre Embrionarias/fisiología , MicroARNs/fisiología , Proteínas de Unión al ARN/fisiología , Empalme Alternativo , Animales , Apoptosis/genética , Línea Celular , Proliferación Celular/genética , Transición Epitelial-Mesenquimal/genética , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , MicroARNs/genéticaRESUMEN
Dgcr8 knockout cells provide a great means to understand the function of microRNAs (miRNAs) in vitro and in vivo. Current strategies to study miRNA function in Dgcr8 knockout cells depend on transient transfection of chemically synthesized miRNA mimics, which is costly and not suitable for long-term study and genetic selection of miRNA function. Here, we developed a cost-effective DGCR8-independent stable miRNA expression (DISME) strategy based on a short hairpin RNA vector that can be precisely processed by DICER. Using DISME, we found that miR-294 promoted the formation of meso-endoderm lineages during embryonic stem cell differentiation. Furthermore, DISME allowed for a pooled screen of miRNA function and identified an miR-183-182 cluster of miRNAs promoting self-renewal and pluripotency in mouse embryonic stem cells. Altogether, our study demonstrates that DISME is a robust and cost-effective strategy that allows for long-term study and genetic selection of miRNA function in a Dgcr8 knockout background.
