RESUMEN
The activities of RNA polymerase and the spliceosome are responsible for the heterogeneity in the abundance and isoform composition of mRNA in human cells. However, the dynamics of these megadalton enzymatic complexes working in concert on endogenous genes have not been described. Here, we establish a quasi-genome-scale platform for observing synthesis and processing kinetics of single nascent RNA molecules in real time. We find that all observed genes show transcriptional bursting. We also observe large kinetic variation in intron removal for single introns in single cells, which is inconsistent with deterministic splice site selection. Transcriptome-wide footprinting of the U2AF complex, nascent RNA profiling, long-read sequencing, and lariat sequencing further reveal widespread stochastic recursive splicing within introns. We propose and validate a unified theoretical model to explain the general features of transcription and pervasive stochastic splice site selection.
Asunto(s)
Precursores del ARN/genética , Sitios de Empalme de ARN/fisiología , Transcripción Genética , Exones/genética , Humanos , Intrones/genética , Precursores del ARN/metabolismo , Sitios de Empalme de ARN/genética , Empalme del ARN/genética , Empalme del ARN/fisiología , ARN Mensajero/metabolismo , Empalmosomas/metabolismo , TranscriptomaRESUMEN
MicroRNAs are important regulators of immune responses. Here, we show miR-221 and miR-222 modulate the intestinal Th17 cell response. Expression of miR-221 and miR-222 was induced by proinflammatory cytokines and repressed by the cytokine TGF-ß. Molecular targets of miR-221 and miR-222 included Maf and Il23r, and loss of miR-221 and miR-222 expression shifted the transcriptomic spectrum of intestinal Th17 cells to a proinflammatory signature. Although the loss of miR-221 and miR-222 was tolerated for maintaining intestinal Th17 cell homeostasis in healthy mice, Th17 cells lacking miR-221 and miR-222 expanded more efficiently in response to IL-23. Both global and T cell-specific deletion of miR-221 and miR-222 rendered mice prone to mucosal barrier damage. Collectively, these findings demonstrate that miR-221 and miR-222 are an integral part of intestinal Th17 cell response that are induced after IL-23 stimulation to constrain the magnitude of proinflammatory response.
Asunto(s)
Inflamación/inmunología , Interleucina-23/metabolismo , Mucosa Intestinal/inmunología , MicroARNs/genética , Células Th17/inmunología , Animales , Retroalimentación Fisiológica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Proto-Oncogénicas c-maf/metabolismo , Receptores de Interleucina/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
CLP1 is a RNA kinase involved in tRNA splicing. Recently, CLP1 kinase-dead mice were shown to display a neuromuscular disorder with loss of motor neurons and muscle paralysis. Human genome analyses now identified a CLP1 homozygous missense mutation (p.R140H) in five unrelated families, leading to a loss of CLP1 interaction with the tRNA splicing endonuclease (TSEN) complex, largely reduced pre-tRNA cleavage activity, and accumulation of linear tRNA introns. The affected individuals develop severe motor-sensory defects, cortical dysgenesis, and microcephaly. Mice carrying kinase-dead CLP1 also displayed microcephaly and reduced cortical brain volume due to the enhanced cell death of neuronal progenitors that is associated with reduced numbers of cortical neurons. Our data elucidate a neurological syndrome defined by CLP1 mutations that impair tRNA splicing. Reduction of a founder mutation to homozygosity illustrates the importance of rare variations in disease and supports the clan genomics hypothesis.
Asunto(s)
Enfermedades del Sistema Nervioso Central/genética , Mutación Missense , Proteínas Nucleares/metabolismo , Enfermedades del Sistema Nervioso Periférico/genética , Fosfotransferasas/metabolismo , ARN de Transferencia/metabolismo , Factores de Transcripción/metabolismo , Anomalías Múltiples/genética , Anomalías Múltiples/patología , Animales , Enfermedades del Sistema Nervioso Central/patología , Cerebro/patología , Preescolar , Endorribonucleasas/metabolismo , Femenino , Fibroblastos/metabolismo , Humanos , Lactante , Masculino , Ratones , Ratones Endogámicos CBA , Microcefalia/genética , Enfermedades del Sistema Nervioso Periférico/patología , ARN de Transferencia/genética , Proteínas de Unión al ARNRESUMEN
Mitochondria contain a specific translation machinery for the synthesis of mitochondria-encoded respiratory chain components. Mitochondrial tRNAs (mt-tRNAs) are also generated from the mitochondrial DNA and, similar to their cytoplasmic counterparts, are post-transcriptionally modified. Here, we find that the RNA methyltransferase METTL8 is a mitochondrial protein that facilitates 3-methyl-cytidine (m3C) methylation at position C32 of the mt-tRNASer(UCN) and mt-tRNAThr. METTL8 knockout cells show a reduction in respiratory chain activity, whereas overexpression increases activity. In pancreatic cancer, METTL8 levels are high, which correlates with lower patient survival and an enhanced respiratory chain activity. Mitochondrial ribosome profiling uncovered mitoribosome stalling on mt-tRNASer(UCN)- and mt-tRNAThr-dependent codons. Further analysis of the respiratory chain complexes using mass spectrometry revealed reduced incorporation of the mitochondrially encoded proteins ND6 and ND1 into complex I. The well-balanced translation of mt-tRNASer(UCN)- and mt-tRNAThr-dependent codons through METTL8-mediated m3C32 methylation might, therefore, facilitate the optimal composition and function of the mitochondrial respiratory chain.
Asunto(s)
Metiltransferasas/metabolismo , ARN Mitocondrial/química , ARN de Transferencia/química , Animales , Anticodón , Proliferación Celular , Codón , Citoplasma , ADN Mitocondrial/metabolismo , Transporte de Electrón , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Ratones , Mitocondrias/metabolismo , Membranas Mitocondriales , Proteínas Mitocondriales/química , Consumo de Oxígeno , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/mortalidad , Ribosomas/metabolismo , Regulación hacia ArribaRESUMEN
p53 is an intensely studied tumor-suppressive transcription factor. Recent studies suggest that the RNA-binding protein (RBP) ZMAT3 is important in mediating the tumor-suppressive effects of p53. Here, we globally identify ZMAT3-regulated RNAs and their binding sites at nucleotide resolution in intact colorectal cancer (CRC) cells. ZMAT3 binds to thousands of mRNA precursors, mainly at intronic uridine-rich sequences and affects their splicing. The strongest alternatively spliced ZMAT3 target was CD44, a cell adhesion gene and stem cell marker that controls tumorigenesis. Silencing ZMAT3 increased inclusion of CD44 variant exons, resulting in significant up-regulation of oncogenic CD44 isoforms (CD44v) and increased CRC cell growth that was rescued by concurrent knockdown of CD44v Silencing p53 phenocopied the loss of ZMAT3 with respect to CD44 alternative splicing, suggesting that ZMAT3-mediated regulation of CD44 splicing is vital for p53 function. Collectively, our findings uncover a p53-ZMAT3-CD44 axis in growth suppression in CRC cells.
Asunto(s)
Empalme Alternativo/genética , Receptores de Hialuranos/genética , Empalme del ARN/genética , Proteínas de Unión al ARN/metabolismo , Carcinogénesis/genética , Neoplasias Colorrectales/genética , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Células HCT116 , Células HEK293 , Humanos , Receptores de Hialuranos/metabolismo , Unión Proteica/genética , Precursores del ARN/metabolismo , Proteínas de Unión al ARN/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Somatic mutations in the genes encoding components of the spliceosome occur frequently in human neoplasms, including myeloid dysplasias and leukemias, and less often in solid tumors. One of the affected factors, U2AF1, is involved in splice site selection, and the most common change, S34F, alters a conserved nucleic acid-binding domain, recognition of the 3' splice site, and alternative splicing of many mRNAs. However, the role that this mutation plays in oncogenesis is still unknown. Here, we uncovered a noncanonical function of U2AF1, showing that it directly binds mature mRNA in the cytoplasm and negatively regulates mRNA translation. This splicing-independent role of U2AF1 is altered by the S34F mutation, and polysome profiling indicates that the mutation affects translation of hundreds of mRNA. One functional consequence is increased synthesis of the secreted chemokine interleukin 8, which contributes to metastasis, inflammation, and cancer progression in mice and humans.
Asunto(s)
Regulación Neoplásica de la Expresión Génica/genética , Neoplasias/fisiopatología , Factor de Empalme U2AF/metabolismo , Línea Celular Tumoral , Citoplasma/patología , Progresión de la Enfermedad , Células HEK293 , Humanos , Interleucina-8/genética , Interleucina-8/metabolismo , Células MCF-7 , Mutación/genética , Neoplasias/genética , Unión Proteica , ARN Mensajero/metabolismo , Factor de Empalme U2AF/genéticaRESUMEN
Fetal hematopoietic stem and progenitor cells (HSPCs) hold promise to cure a wide array of hematological diseases, and we previously found a role for the RNA-binding protein (RBP) Lin28b in respecifying adult HSPCs to resemble their fetal counterparts. Here we show by single-cell RNA sequencing that Lin28b alone was insufficient for complete reprogramming of gene expression from the adult toward the fetal pattern. Using proteomics and in situ analyses, we found that Lin28b (and its closely related paralog, Lin28a) directly interacted with Igf2bp3, another RBP, and their enforced co-expression in adult HSPCs reactivated fetal-like B-cell development in vivo more efficiently than either factor alone. In B-cell progenitors, Lin28b and Igf2bp3 jointly stabilized thousands of mRNAs by binding at the same sites, including those of the B-cell regulators Pax5 and Arid3a as well as Igf2bp3 mRNA itself, forming an autoregulatory loop. Our results suggest that Lin28b and Igf2bp3 are at the center of a gene regulatory network that mediates the fetal-adult hematopoietic switch. A method to efficiently generate induced fetal-like hematopoietic stem cells (ifHSCs) will facilitate basic studies of their biology and possibly pave a path toward their clinical application.
Asunto(s)
Reprogramación Celular/genética , Proteínas de Unión al ADN/metabolismo , Redes Reguladoras de Genes , Células Madre Hematopoyéticas/fisiología , Proteínas de Unión al ARN/metabolismo , Animales , Sitios de Unión , Células Cultivadas , Proteínas de Unión al ADN/genética , Ratones , MicroARNs/metabolismo , Modelos Animales , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genéticaRESUMEN
In mammals, gene silencing by the RNA-induced silencing complex (RISC) is a well-understood cytoplasmic posttranscriptional gene regulatory mechanism. Here, we show that embryonic stem cells (ESCs) contain high levels of nuclear AGO proteins and that in ESCs nuclear AGO protein activity allows for the onset of differentiation. In the nucleus, AGO proteins interact with core RISC components, including the TNRC6 proteins and the CCR4-NOT deadenylase complex. In contrast to cytoplasmic miRNA-mediated gene silencing that mainly operates on cis-acting elements in mRNA 3' untranslated (UTR) sequences, in the nucleus AGO binding in the coding sequence and potentially introns also contributed to post-transcriptional gene silencing. Thus, nuclear localization of AGO proteins in specific cell types leads to a previously unappreciated expansion of the miRNA-regulated transcriptome.
Asunto(s)
Proteínas Argonautas/fisiología , Silenciador del Gen/fisiología , MicroARNs/fisiología , Animales , Proteínas Argonautas/genética , Diferenciación Celular/genética , Línea Celular , Núcleo Celular , Citoplasma , Células Madre Embrionarias/metabolismo , Humanos , Mamíferos , Ratones , MicroARNs/genética , Interferencia de ARN , Estabilidad del ARN , ARN Mensajero , ARN Interferente Pequeño , Proteínas de Unión al ARN , Complejo Silenciador Inducido por ARN/genética , Complejo Silenciador Inducido por ARN/metabolismo , Factores de TranscripciónRESUMEN
The enhancer regions of the myogenic master regulator MyoD give rise to at least two enhancer RNAs. Core enhancer eRNA (CEeRNA) regulates transcription of the adjacent MyoD gene, whereas DRReRNA affects expression of Myogenin in trans. We found that DRReRNA is recruited at the Myogenin locus, where it colocalizes with Myogenin nascent transcripts. DRReRNA associates with the cohesin complex, and this association correlates with its transactivating properties. Despite being expressed in undifferentiated cells, cohesin is not loaded on Myogenin until the cells start expressing DRReRNA, which is then required for cohesin chromatin recruitment and maintenance. Functionally, depletion of either cohesin or DRReRNA reduces chromatin accessibility, prevents Myogenin activation, and hinders muscle cell differentiation. Thus, DRReRNA ensures spatially appropriate cohesin loading in trans to regulate gene expression.
Asunto(s)
Proteínas de Ciclo Celular/biosíntesis , Proteínas Cromosómicas no Histona/biosíntesis , Elementos de Facilitación Genéticos , Músculo Esquelético/metabolismo , Miogenina/biosíntesis , ARN no Traducido/metabolismo , Transcripción Genética , Animales , Proteínas de Ciclo Celular/genética , Diferenciación Celular , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/genética , Células HEK293 , Humanos , Ratones , Músculo Esquelético/citología , Proteína MioD/biosíntesis , Proteína MioD/genética , Miogenina/genética , ARN no Traducido/genética , CohesinasRESUMEN
RNA transcripts are subject to posttranscriptional gene regulation involving hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) expressed in a cell-type dependent fashion. We developed a cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs. The crosslinked sites are revealed by thymidine to cytidine transitions in the cDNAs prepared from immunopurified RNPs of 4-thiouridine-treated cells. We determined the binding sites and regulatory consequences for several intensely studied RBPs and miRNPs, including PUM2, QKI, IGF2BP1-3, AGO/EIF2C1-4 and TNRC6A-C. Our study revealed that these factors bind thousands of sites containing defined sequence motifs and have distinct preferences for exonic versus intronic or coding versus untranslated transcript regions. The precise mapping of binding sites across the transcriptome will be critical to the interpretation of the rapidly emerging data on genetic variation between individuals and how these variations contribute to complex genetic diseases.
Asunto(s)
Técnicas Genéticas , MicroARNs/metabolismo , ARN no Traducido/genética , Proteínas de Unión al ARN/metabolismo , Secuencias Reguladoras de Ácido Ribonucleico , Secuencia de Bases , Reactivos de Enlaces Cruzados/metabolismo , Humanos , Datos de Secuencia Molecular , Nucleósidos/metabolismo , Mutación Puntual , Alineación de SecuenciaRESUMEN
The RNA exosome is an essential 3' to 5' exoribonuclease complex that mediates degradation, processing and quality control of virtually all eukaryotic RNAs. The nucleolar RNA exosome, consisting of a nine-subunit core and a distributive 3' to 5' exonuclease EXOSC10, plays a critical role in processing and degrading nucleolar RNAs, including pre-rRNA. However, how the RNA exosome is regulated in the nucleolus is poorly understood. Here, we report that the nucleolar ubiquitin-specific protease USP36 is a novel regulator of the nucleolar RNA exosome. USP36 binds to the RNA exosome through direct interaction with EXOSC10 in the nucleolus. Interestingly, USP36 does not significantly regulate the levels of EXOSC10 and other tested exosome subunits. Instead, it mediates EXOSC10 SUMOylation at lysine (K) 583. Mutating K583 impaired the binding of EXOSC10 to pre-rRNAs, and the K583R mutant failed to rescue the defects in rRNA processing and cell growth inhibition caused by knockdown of endogenous EXOSC10. Furthermore, EXOSC10 SUMOylation is markedly reduced in cells in response to perturbation of ribosomal biogenesis. Together, these results suggest that USP36 acts as a SUMO ligase to promote EXOSC10 SUMOylation critical for the RNA exosome function in ribosome biogenesis.
Asunto(s)
Exorribonucleasas , Complejo Multienzimático de Ribonucleasas del Exosoma , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Exorribonucleasas/genética , Exorribonucleasas/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma/genética , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , ARN/metabolismo , Precursores del ARN/genética , Precursores del ARN/metabolismo , Procesamiento Postranscripcional del ARN , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , Humanos , Línea CelularRESUMEN
Hyperlactatemia often occurs in critically ill patients during severe sepsis/septic shock and is a powerful predictor of mortality. Lactate is the end product of glycolysis. While hypoxia due to inadequate oxygen delivery may result in anaerobic glycolysis, sepsis also enhances glycolysis under hyperdynamic circulation with adequate oxygen delivery. However, the molecular mechanisms involved are not fully understood. Mitogen-activated protein kinase (MAPK) families regulate many aspects of the immune response during microbial infections. MAPK phosphatase (MKP)-1 serves as a feedback control mechanism for p38 and JNK MAPK activities via dephosphorylation. Here, we found that mice deficient in Mkp-1 exhibited substantially enhanced expression and phosphorylation of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB) 3, a key enzyme that regulates glycolysis following systemic Escherichia coli infection. Enhanced PFKFB3 expression was observed in a variety of tissues and cell types, including hepatocytes, macrophages, and epithelial cells. In bone marrow-derived macrophages, Pfkfb3 was robustly induced by both E. coli and lipopolysaccharide, and Mkp-1 deficiency enhanced PFKFB3 expression with no effect on Pfkfb3 mRNA stability. PFKFB3 induction was correlated with lactate production in both WT and Mkp-1-/- bone marrow-derived macrophage following lipopolysaccharide stimulation. Furthermore, we determined that a PFKFB3 inhibitor markedly attenuated lactate production, highlighting the critical role of PFKFB3 in the glycolysis program. Finally, pharmacological inhibition of p38 MAPK, but not JNK, substantially attenuated PFKFB3 expression and lactate production. Taken together, our studies suggest a critical role of p38 MAPK and MKP-1 in the regulation of glycolysis during sepsis.
Asunto(s)
Fosfatasa 1 de Especificidad Dual , Glucólisis , Sepsis , Proteínas Quinasas p38 Activadas por Mitógenos , Animales , Ratones , Fosfatasa 1 de Especificidad Dual/genética , Fosfatasa 1 de Especificidad Dual/metabolismo , Escherichia coli/metabolismo , Lactatos , Lipopolisacáridos , Oxígeno , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Sepsis/genética , Fosfofructoquinasa-2/metabolismoRESUMEN
Although Argonaute (AGO) proteins have been the focus of microRNA (miRNA) studies, we observed AGO-free mature miRNAs directly interacting with RNA-binding proteins, implying the sophisticated nature of fine-tuning gene regulation by miRNAs. To investigate microRNA-binding proteins (miRBPs) globally, we analyzed PAR-CLIP data sets to identify RBP quaking (QKI) as a novel miRBP for let-7b. Potential existence of AGO-free miRNAs were further verified by measuring miRNA levels in genetically engineered AGO-depleted human and mouse cells. We have shown that QKI regulates miRNA-mediated gene silencing at multiple steps, and collectively serves as an auxiliary factor empowering AGO2/let-7b-mediated gene silencing. Depletion of QKI decreases interaction of AGO2 with let-7b and target mRNA, consequently controlling target mRNA decay. This finding indicates that QKI is a complementary factor in miRNA-mediated mRNA decay. QKI, however, also suppresses the dissociation of let-7b from AGO2, and slows the assembly of AGO2/miRNA/target mRNA complexes at the single-molecule level. We also revealed that QKI overexpression suppresses cMYC expression at post-transcriptional level, and decreases proliferation and migration of HeLa cells, demonstrating that QKI is a tumour suppressor gene by in part augmenting let-7b activity. Our data show that QKI is a new type of RBP implicated in the versatile regulation of miRNA-mediated gene silencing.
Asunto(s)
MicroARNs , Humanos , Animales , Ratones , MicroARNs/genética , MicroARNs/metabolismo , Células HeLa , Silenciador del Gen , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , ARN Mensajero/genéticaRESUMEN
Mitogen-activated protein kinase phosphatase 1 (Mkp-1) KO mice produce elevated cytokines and exhibit increased mortality and bacterial burden following systemic Escherichia coli infection. To understand how Mkp-1 affects immune defense, we analyzed the RNA-Seq datasets previously generated from control and E. coli-infected Mkp-1+/+ and Mkp-1-/- mice. We found that E. coli infection markedly induced programmed death-ligand 1 (PD-L1) expression and that Mkp-1 deficiency further amplified PD-L1 expression. Administration of a PD-L1-neutralizing monoclonal antibody (mAb) to Mkp-1-/- mice increased the mortality of the animals following E. coli infection, although bacterial burden was decreased. In addition, the PD-L1-neutralizing mAb increased serum interferon (IFN)-γ and tumor necrosis factor alpha, as well as lung- and liver-inducible nitric oxide synthase levels, suggesting an enhanced inflammatory response. Interestingly, neutralization of IFN-α/ß receptor 1 blocked PD-L1 induction in Mkp-1-/- mice following E. coli infection. PD-L1 was potently induced in macrophages by E. coli and lipopolysaccharide in vitro, and Mkp-1 deficiency exacerbated PD-L1 induction with little effect on the half-life of PD-L1 mRNA. In contrast, inhibitors of Janus kinase 1/2 and tyrosine kinase 2, as well as the IFN-α/ß receptor 1-neutralizing mAb, markedly attenuated PD-L1 induction. These results suggest that the beneficial effect of type I IFNs in E. coli-infected Mkp-1-/- mice is, at least in part, mediated by Janus kinase/signal transducer and activator of transcription-driven PD-L1 induction. Our studies also support the notion that enhanced PD-L1 expression contributes to the bactericidal defect of Mkp-1-/- mice.
Asunto(s)
Antígeno B7-H1 , Fosfatasa 1 de Especificidad Dual , Infecciones por Escherichia coli , Regulación de la Expresión Génica , Interferón Tipo I , Animales , Antígeno B7-H1/genética , Fosfatasa 1 de Especificidad Dual/metabolismo , Escherichia coli/genética , Escherichia coli/inmunología , Infecciones por Escherichia coli/inmunología , Regulación de la Expresión Génica/inmunología , Interferón Tipo I/genética , RatonesRESUMEN
Repressor element 1-silencing transcription factor (REST) plays a crucial role in the differentiation of neural progenitor cells (NPCs). C-terminal domain small phosphatases (CTDSPs) are REST effector proteins that reduce RNA polymerase II activity on genes required for neurogenesis. miR-26b regulates neurogenesis in zebrafish by targeting ctdsp2 mRNA, but the molecular events triggered by this microRNA (miR) remain unknown. Here, we show in a murine embryonic stem cell differentiation paradigm that inactivation of miR-26 family members disrupts the formation of neurons and astroglia and arrests neurogenesis at the neural progenitor level. Furthermore, we show that miR-26 directly targets Rest, thereby inducing the expression of a large set of REST complex-repressed neuronal genes, including miRs required for induction of the neuronal gene expression program. Our data identify the miR-26 family as the trigger of a self-amplifying system required for neural differentiation that acts upstream of REST-controlled miRs.
Asunto(s)
MicroARNs , Animales , Diferenciación Celular/genética , Ratones , MicroARNs/genética , Neurogénesis/genética , ARN Mensajero/genética , Proteínas Represoras , Factores de Transcripción , Pez Cebra/genéticaRESUMEN
We have previously shown that Mkp-1-deficient mice produce elevated TNF-α, IL-6, and IL-10 following systemic Escherichia coli infection, and they exhibited increased mortality, elevated bacterial burden, and profound metabolic alterations. To understand the function of Mkp-1 during bacterial infection, we performed RNA-sequencing analysis to compare the global gene expression between E. coli-infected wild-type and Mkp-1 -/- mice. A large number of IFN-stimulated genes were more robustly expressed in E. coli-infected Mkp-1 -/- mice than in wild-type mice. Multiplex analysis of the serum cytokine levels revealed profound increases in IFN-ß, IFN-γ, TNF-α, IL-1α and ß, IL-6, IL-10, IL-17A, IL-27, and GMSF levels in E. coli-infected Mkp-1 -/- mice relative to wild-type mice. Administration of a neutralizing Ab against the receptor for type I IFN to Mkp-1 -/- mice prior to E. coli infection augmented mortality and disease severity. Mkp-1 -/- bone marrow-derived macrophages (BMDM) produced higher levels of IFN-ß mRNA and protein than did wild-type BMDM upon treatment with LPS, E. coli, polyinosinic:polycytidylic acid, and herring sperm DNA. Augmented IFN-ß induction in Mkp-1 -/- BMDM was blocked by a p38 inhibitor but not by an JNK inhibitor. Enhanced Mkp-1 expression abolished IFN-ß induction by both LPS and E. coli but had little effect on the IFN-ß promoter activity in LPS-stimulated RAW264.7 cells. Mkp-1 deficiency did not have an overt effect on IRF3/7 phosphorylation or IKK activation but modestly enhanced IFN-ß mRNA stability in LPS-stimulated BMDM. Our results suggest that Mkp-1 regulates IFN-ß production primarily through a p38-mediated mechanism and that IFN-ß plays a beneficial role in E. coli-induced sepsis.
Asunto(s)
Fosfatasa 1 de Especificidad Dual/metabolismo , Infecciones por Escherichia coli/metabolismo , Interferón beta/metabolismo , Animales , Células Cultivadas , Fosfatasa 1 de Especificidad Dual/deficiencia , Fosfatasa 1 de Especificidad Dual/inmunología , Infecciones por Escherichia coli/inmunología , Interferón beta/genética , Interferón beta/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células RAW 264.7 , Proteínas Quinasas p38 Activadas por Mitógenos/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The vertebrate-conserved RNA-binding protein DND1 is required for the survival of primordial germ cells (PGCs), as well as the suppression of germ cell tumours in mice. Here we show that in mice DND1 binds a UU(A/U) trinucleotide motif predominantly in the 3' untranslated regions of mRNA, and destabilizes target mRNAs through direct recruitment of the CCR4-NOT deadenylase complex. Transcriptomic analysis reveals that the extent of suppression is dependent on the number of DND1-binding sites. This DND1-dependent mRNA destabilization is required for the survival of mouse PGCs and spermatogonial stem cells by suppressing apoptosis. The spectrum of target RNAs includes positive regulators of apoptosis and inflammation, and modulators of signalling pathways that regulate stem-cell pluripotency, including the TGFß superfamily, all of which are aberrantly elevated in DND1-deficient PGCs. We propose that the induction of the post-transcriptional suppressor DND1 synergizes with concurrent transcriptional changes to ensure precise developmental transitions during cellular differentiation and maintenance of the germ line.
Asunto(s)
Complejos Multiproteicos/metabolismo , Proteínas de Neoplasias/metabolismo , Estabilidad del ARN , ARN Mensajero/metabolismo , Ribonucleasas/metabolismo , Espermatogonias/citología , Células Madre/citología , Factores de Transcripción/metabolismo , Regiones no Traducidas 3'/genética , Animales , Apoptosis/genética , Secuencia de Bases , Sitios de Unión , Diferenciación Celular/genética , Perfilación de la Expresión Génica , Silenciador del Gen , Humanos , Inflamación/genética , Masculino , Ratones , Complejos Multiproteicos/química , Proteínas de Neoplasias/deficiencia , Motivos de Nucleótidos , Células Madre Pluripotentes/citología , Unión Proteica , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ribonucleasas/química , Transducción de Señal/genética , Espermatogonias/metabolismo , Células Madre/metabolismo , Transcripción Genética/genética , Factor de Crecimiento Transformador beta/genéticaRESUMEN
Crosslinking and immunoprecipitation (CLIP) methods are powerful techniques to interrogate direct protein-RNA interactions and dissect posttranscriptional gene regulatory networks. One widely used CLIP variant is photoactivatable ribonucleoside enhanced CLIP (PAR-CLIP) that involves in vivo labeling of nascent RNAs with the photoreactive nucleosides 4-thiouridine (4SU) or 6-thioguanosine (6SG), which can efficiently crosslink to interacting proteins using UVA and UVB light. Crosslinking of 4SU or 6SG to interacting amino acids changes their base-pairing properties and results in characteristic mutations in cDNA libraries prepared for high-throughput sequencing, which can be computationally exploited to remove abundant background from non-crosslinked sequences and help pinpoint RNA binding protein binding sites at nucleotide resolution on a transcriptome-wide scale. Here we present a streamlined protocol for fluorescence-based PAR-CLIP (fPAR-CLIP) that eliminates the need to use radioactivity. It is based on direct ligation of a fluorescently labeled adapter to the 3'end of crosslinked RNA on immobilized ribonucleoproteins, followed by isolation of the adapter-ligated RNA and efficient conversion into cDNA without the previously needed size fractionation on denaturing polyacrylamide gels. These improvements cut the experimentation by half to 2 days and increases sensitivity by 10-100-fold.
Asunto(s)
ADN Complementario/genética , Perfilación de la Expresión Génica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/metabolismo , Sitios de Unión , Línea Celular , Reactivos de Enlaces Cruzados/química , Electroforesis en Gel de Poliacrilamida , GTP Fosfohidrolasas/química , Biblioteca de Genes , Humanos , Inmunoprecipitación , Oligonucleótidos , Reacción en Cadena de la Polimerasa/métodos , Unión Proteica , ARN/química , Ribonucleoproteínas/genética , Sensibilidad y Especificidad , Programas Informáticos , Tiouridina/química , Rayos UltravioletaRESUMEN
The human genome encodes for over 1,500 RNA-binding proteins (RBPs), which coordinate regulatory events on RNA transcripts. Most studies of RBPs have concentrated on their action on host protein-encoding mRNAs, which constitute a minority of the transcriptome. A widely neglected subset of our transcriptome derives from integrated retroviral elements, termed endogenous retroviruses (ERVs), that comprise â¼8% of the human genome. Some ERVs have been shown to be transcribed under physiological and pathological conditions, suggesting that sophisticated regulatory mechanisms to coordinate and prevent their ectopic expression exist. However, it is unknown how broadly RBPs and ERV transcripts directly interact to provide a posttranscriptional layer of regulation. Here, we implemented a computational pipeline to determine the correlation of expression between individual RBPs and ERVs from single-cell or bulk RNA-sequencing data. One of our top candidates for an RBP negatively regulating ERV expression was RNA-binding motif protein 4 (RBM4). We used photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation to demonstrate that RBM4 indeed bound ERV transcripts at CGG consensus elements. Loss of RBM4 resulted in an elevated transcript level of bound ERVs of the HERV-K and -H families, as well as increased expression of HERV-K envelope protein. We pinpointed RBM4 regulation of HERV-K to a CGG-containing element that is conserved in the LTRs of HERV-K-10, -K-11, and -K-20, and validated the functionality of this site using reporter assays. In summary, we systematically identified RBPs that may regulate ERV function and demonstrate a role for RBM4 in controlling ERV expression.
Asunto(s)
Retrovirus Endógenos/genética , Retrovirus Endógenos/metabolismo , Procesamiento Postranscripcional del ARN , Proteínas de Unión al ARN/metabolismo , Sitios de Unión , Bases de Datos Genéticas , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica , Genoma Humano , Humanos , Inmunoprecipitación , ARN/genética , ARN/metabolismo , Motivos de Unión al ARN , Proteínas de Unión al ARN/genética , TranscriptomaRESUMEN
Eukaryotic gene expression is tightly regulated post-transcriptionally by RNA-binding proteins (RBPs) and microRNAs. The RBP AU-rich-binding factor 1 (AUF1) isoform p37 was found to have high affinity for the microRNA let-7b in vitro (Kd = â¼ 6 nM) in cells. Ribonucleoprotein immunoprecipitation, in vitro association, and single-molecule-binding analyses revealed that AUF1 promoted let-7b loading onto Argonaute 2 (AGO2), the catalytic component of the RNA-induced silencing complex (RISC). In turn, AGO2-let-7 triggered target mRNA decay. Our findings uncover a novel mechanism by which AUF1 binding and transfer of microRNA let-7 to AGO2 facilitates let-7-elicited gene silencing.