RESUMO
Nonsense-mediated decay (NMD) is a eukaryotic mRNA surveillance system that selectively degrades transcripts with premature termination codons (PTC). Many RNA-binding proteins (RBP) regulate their expression levels by a negative feedback loop, in which RBP binds its own pre-mRNA and causes alternative splicing to introduce a PTC. We present a bioinformatic analysis integrating three data sources, eCLIP assays for a large RBP panel, shRNA inactivation of NMD pathway, and shRNA-depletion of RBPs followed by RNA-seq, to identify novel such autoregulatory feedback loops. We show that RBPs frequently bind their own pre-mRNAs, their exons respond prominently to NMD pathway disruption, and that the responding exons are enriched with nearby eCLIP peaks. We confirm previously proposed models of autoregulation in SRSF7 and U2AF1 genes and present two novel models, in which (i) SFPQ binds its mRNA and promotes switching to an alternative distal 3'-UTR that is targeted by NMD, and (ii) RPS3 binding activates a poison 5'-splice site in its pre-mRNA that leads to a frame shift and degradation by NMD. We also suggest specific splicing events that could be implicated in autoregulatory feedback loops in RBM39, HNRNPM, and U2AF2 genes. The results are available through a UCSC Genome Browser track hub.
Assuntos
Códon sem Sentido , Degradação do RNAm Mediada por Códon sem Sentido , Splicing de RNA , RNA Interferente Pequeno/metabolismo , Transcriptoma , Regiões 3' não Traduzidas , Processamento Alternativo , Biologia Computacional , Éxons , Mutação da Fase de Leitura , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/metabolismo , Humanos , Proteínas Nucleares/metabolismo , Precursores de RNA/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Processamento de Serina-Arginina/metabolismo , Spliceossomos , Fator de Processamento U2AF/metabolismoRESUMO
The Armed Forces has seen an increase in the number of operational deployments overseas and a greater demand for Accident and Emergency (A&E) trained nurses. This article describes a modified Delphi study used to contribute to the development of a strategy for emergency nursing in the Defence Nursing Services. Twenty-eight A&E specialists took part and the key issues raised were recruitment and retention, staff development, new roles, research priorities, increased internal recruitment of A&E nurses to meet operational demands, and the need for a structured career pathway to help retention. The most pressing areas requiring research were evaluation of the nurse practitioner role, clinical competencies and managing heat injuries in the operational setting. The modified Delphi study provided a valuable and detailed insight into the challenges and aspirations of the military A&E nursing cadre and has assisted in developing a strategy for emergency nursing.
Assuntos
Atitude do Pessoal de Saúde , Competência Clínica/normas , Enfermagem em Emergência/organização & administração , Enfermagem Militar/organização & administração , Papel do Profissional de Enfermagem , Mobilidade Ocupacional , Consenso , Currículo , Técnica Delphi , Educação de Pós-Graduação em Enfermagem/organização & administração , Enfermagem em Emergência/educação , Planejamento em Saúde/organização & administração , Necessidades e Demandas de Serviços de Saúde , Transtornos de Estresse por Calor/enfermagem , Humanos , Enfermagem Militar/educação , Enfermeiros Administradores/educação , Enfermeiros Administradores/organização & administração , Enfermeiros Administradores/psicologia , Enfermeiros Clínicos/educação , Enfermeiros Clínicos/organização & administração , Profissionais de Enfermagem/educação , Profissionais de Enfermagem/organização & administração , Auditoria de Enfermagem/organização & administração , Pesquisa em Enfermagem/organização & administração , Seleção de Pessoal , Técnicas de Planejamento , Medicina Estatal/organização & administração , Reino UnidoRESUMO
Wide variation in glucocorticoid (Gc) sensitivity exists between individuals which may influence susceptibility to, and treatment response of, inflammatory diseases. To determine a genetic fingerprint of Gc sensitivity 100 healthy human volunteers were polarized into the 10% most Gc-sensitive and 10% most Gc-resistant following a low dose dexamethasone (0.25 mg) suppression test. Gene expression profiling of primary lymphocytes identified the 98 most significantly Gc regulated genes. These genes were used to build a subnetwork of Gc signaling, with 54 genes mapping as nodes, and 6 non-Gc regulated genes inferred as signaling nodes. Twenty four of the 98 genes showed a difference in Gc response in vitro dependent on the Gc sensitivity of their donor individuals in vivo. A predictive model was built using both partial least squares discriminate analysis and support vector machines that predicted donor glucocorticoid sensitivity with 87% accuracy. Discriminating genes included bone morphogenetic protein receptor, type II (BMPRII). Transfection studies showed that BMPRII modulated Gc action. These studies reveal a broad base of gene expression that predicts Gc sensitivity and determine a Gc signaling network in human primary T lymphocytes. Furthermore, this combined gene profiling, and functional analysis approach has identified BMPRII as a modulator of Gc signaling.
Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Glucocorticoides/farmacologia , Adulto , Análise por Conglomerados , Dexametasona/farmacologia , Feminino , Humanos , Análise dos Mínimos Quadrados , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismoRESUMO
Unlike other nuclear receptors, transactivation by the glucocorticoid receptor (GR) is increased by the inhibition of the ubiquitin/proteasome pathway. Here, we demonstrate that the ubiquitin-conjugating enzyme (E2), UbcH7, physically interacts with the GR and, when overexpressed, reduces the ability of the receptor to upregulate gene expression. Chemical inhibition of the 26S proteasome abolished the downregulation effect of overexpressed UbcH7, suggesting a role for the 26S proteasome, and GR protein stability in mediating the UbcH7 effect. Furthermore, a UbcH7 dominant negative mutant (C89S), unable to transfer ubiquitin, failed to repress GR transactivation. Indeed, overexpression of the mutant UbcH7 was sufficient to augment GR transactivation to levels achieved using the proteasome inhibitor MG132, but there was no further induction when MG132 and the UbcH7 mutant were used together. Expression of the dominant negative UbcH7 abolished ligand-dependent downregulation of GR protein, suggesting that the UbcH7 effect was mediated by regulation of GR protein concentration. Taken together, these data show that UbcH7 is a key regulator of GR turnover and glucocorticoid sensitivity.
Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Receptores de Glucocorticoides/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Animais , Células COS , Chlorocebus aethiops , Expressão Gênica , Regulação da Expressão Gênica , Células HeLa , Homeostase , Humanos , Immunoblotting , Imunoprecipitação , Leupeptinas/farmacologia , Mutação , Receptores de Glucocorticoides/genética , Transfecção/métodos , Técnicas do Sistema de Duplo-Híbrido , Enzimas de Conjugação de Ubiquitina/genéticaRESUMO
Glucocorticoids inhibit inflammation by acting through the glucocorticoid receptor (GR) and powerfully repressing NF-kappaB function. Ligand binding to the C-terminal of GR promotes the nuclear translocation of the receptor and binding to NF-kappaB through the GR DNA binding domain. We sought how ligand recognition influences the interaction between NF-kappaB and GR. Both dexamethasone (agonist) and RU486 (antagonist) promote efficient nuclear translocation, and we show occupancy of the same intranuclear compartment as NF-kappaB with both ligands. However, unlike dexamethasone, RU486 had negligible activity to inhibit NF-kappaB transactivation. This failure may stem from altered co-factor recruitment or altered interaction with NF-kappaB. Using both glutathione S-transferase pull-down and bioluminescence resonance energy transfer approaches, we identified a major glucocorticoid ligand effect on interaction between the GR and the p65 component of NF-kappaB, with RU486 inhibiting recruitment compared with dexamethasone. Using the bioluminescence resonance energy transfer assay, we found that RU486 efficiently recruited NCoR to the GR, unlike dexamethasone, which recruited SRC1. Therefore, RU486 promotes differential protein recruitment to both the C-terminal and DNA binding domain of the receptor. Importantly, using chromatin immunoprecipitation, we show that impaired interaction between GR and p65 with RU486 leads to reduced recruitment of the GR to the NF-kappaB-responsive region of the interleukin-8 promoter, again in contrast to dexamethasone that significantly increased GR binding. We demonstrate that ligand-induced conformation of the GR C-terminal has profound effects on the functional surface generated by the DNA binding domain of the GR. This has implications for understanding ligand-dependent interdomain communication.