RESUMO
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.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Células-Tronco Embrionárias/fisiologia , MicroRNAs/fisiologia , Proteínas de Ligação a RNA/fisiologia , Processamento Alternativo , Animais , Apoptose/genética , Linhagem Celular , Proliferação de Células/genética , Transição Epitelial-Mesenquimal/genética , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , MicroRNAs/genéticaRESUMO
Enhanced glycolysis is a main feature of pluripotent stem cells (PSCs) and is proposed to be important for the maintenance and induction of pluripotency. The molecular mechanism underlying enhanced glycolysis in PSCs is not clear. Using Dgcr8-/- mouse embryonic stem cells (ESCs) that lack mature miRNAs, we found that miR-290 cluster of miRNAs stimulates glycolysis by upregulating glycolytic enzymes Pkm2 and Ldha, which are also essential for the induction of pluripotency during reprogramming. Mechanistically, we identified Mbd2, a reader for methylated CpGs, as the target of miR-290 cluster that represses glycolysis and reprogramming. Furthermore, we discovered Myc as a key target of Mbd2 that controls metabolic switch in ESCs. Importantly, we demonstrated that miR-371 cluster, a human homolog of miR-290 cluster, stimulates glycolysis to promote the reprogramming of human fibroblasts. Hence, we identified a previously unappreciated mechanism by which miR-290/371 miRNAs orchestrate epigenetic, transcriptional and metabolic networks to promote pluripotency in PSCs and during reprogramming.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Glicólise/fisiologia , Redes e Vias Metabólicas/fisiologia , MicroRNAs/metabolismo , Células-Tronco Pluripotentes/enzimologia , Células-Tronco Pluripotentes/fisiologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Imunoprecipitação da Cromatina , Primers do DNA/genética , Proteínas de Ligação a DNA/genética , Técnicas de Inativação de Genes , Glicólise/genética , Redes e Vias Metabólicas/genética , Camundongos , Camundongos Knockout , MicroRNAs/genética , Proteínas Proto-Oncogênicas c-myc/genética , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
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.
Assuntos
Fatores de Crescimento de Fibroblastos/genética , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Células-Tronco Embrionárias Murinas/metabolismo , RNA Longo não Codificante/genética , Fatores de Transcrição/genética , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Embrião de Mamíferos , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Sistema de Sinalização das MAP Quinases , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Fosforilação , Ligação Proteica , RNA Longo não Codificante/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Adaptadoras da Sinalização Shc/genética , Proteínas Adaptadoras da Sinalização Shc/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismoRESUMO
Serum- and 2i-cultured embryonic stem cells (ESCs) show different epigenetic landscapes and transcriptomic profiles. The difference in the function and expression of microRNAs (miRNAs) between these two states remains unclear. Here, we showed that 2i- and serum-cultured ESCs exhibited distinctive miRNA expression profiles with >100 miRNAs differentially expressed, and the expression changes were largely due to transcriptional regulation. We further characterized the function of miRNAs differentially expressed under two conditions and found that ESCs exhibited higher degree of dependency on miRNAs for rapid proliferation; since Dgcr8-/- or Dicer1-/- but not wild-type ESCs showed slower growth rate and more accumulation in the G1 phase under 2i than serum condition. More interestingly, introduction of various self-renewal-silencing miRNAs in wild-type or Dgcr8-/- ESCs failed to silence the self-renewal in 2i medium, but regained the ability to silence the self-renewal upon the addition of serum. Our findings reveal significant differences in the expression and function of miRNAs between serum- and 2i-cultured ESCs.
Assuntos
Regulação da Expressão Gênica , MicroRNAs/genética , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Soro/citologia , Animais , Diferenciação Celular/genética , Proliferação de Células/genética , Autorrenovação Celular/genética , Células Cultivadas , Fase G1/genética , Camundongos , Fase S/genéticaRESUMO
The molecular mechanism controlling the dismantling of naive pluripotency is poorly understood. Here we show that microRNAs (miRNAs) have important roles during naive to primed pluripotency transition. Dgcr8(-/-) embryonic stem cells (ESCs) failed to completely silence the naive pluripotency program, as well as to establish the primed pluripotency program during differentiation. miRNA profiling revealed that expression levels of a large number of miRNAs changed dynamically and rapidly during naive to primed pluripotency transition. Furthermore, a miRNA screen identified numerous miRNAs promoting naive to primed pluripotency transition. Unexpectedly, multiple miRNAs from miR-290 and miR-302 clusters, previously shown as pluripotency-promoting miRNAs, demonstrated the strongest effects in silencing naive pluripotency. Knockout of both miR-290 and miR-302 clusters but not either alone blocked the silencing of naive pluripotency program. Mechanistically, the miR-290/302 family of miRNAs may facilitate the exit of naive pluripotency in part by promoting the activity of MEK pathway and through directly repressing Akt1. Our study reveals miRNAs as an important class of regulators potentiating ESCs to transition from naive to primed pluripotency, and uncovers context-dependent functions of the miR-290/302 family of miRNAs at different developmental stages.