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Global mRNA abundance depends on the balance of synthesis and decay of a population of mRNAs. To account for this balance during activation of T cells, we used metabolic labeling to quantify the contributions of RNA transcription and decay over a 4 h time course during activation of leukemia-derived Jurkat T cells. While prior studies suggested more than half of the changes in mRNA abundance were due to RNA stability, we found a smaller but more interesting population of mRNAs changed stability. These mRNAs clustered into functionally related subpopulations that included replicative histones, ribosomal biogenesis and cell motility functions. We then applied a novel analysis based on integrating global protein-RNA binding with concurrent changes in RNA stability at specific time points following activation. This analysis demonstrated robust stabilization of mRNAs by the HuR RNA-binding protein 4 h after activation. Our unexpected findings demonstrate that the temporal regulation of mRNA stability coordinates vital cellular pathways and is in part controlled by the HuR RNA binding protein in Jurkat T cells following activation.
Assuntos
Proteína Semelhante a ELAV 1/metabolismo , Ativação Linfocitária/genética , Estabilidade de RNA , RNA Mensageiro/genética , Linfócitos T/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Células Jurkat , Ativação Linfocitária/imunologia , RNA Mensageiro/metabolismo , Linfócitos T/imunologia , Transcrição GênicaRESUMO
The cellular growth cycle is initiated and maintained by punctual, yet agile, regulatory events involving modifications of cell cycle proteins as well as coordinated gene expression to support cyclic checkpoint decisions. Recent evidence indicates that post-transcriptional partitioning of messenger RNA subsets by RNA-binding proteins help physically localize, temporally coordinate, and efficiently translate cell cycle proteins. This dynamic organization of mRNAs encoding cell cycle components contributes to the overall economy of the cell cycle consistent with the post-transcriptional RNA regulon model of gene expression. This review examines several recent studies demonstrating the coordination of mRNA subsets encoding cell cycle proteins during nuclear export and subsequent coupling to protein synthesis, and discusses evidence for mRNA coordination of p53 targets and the DNA damage response pathway. We consider how these observations may connect to upstream and downstream post-transcriptional coordination and coupling of splicing, export, localization, and translation. Published examples from yeast, nematode, insect, and mammalian systems are discussed, and we consider genetic evidence supporting the conclusion that dysregulation of RNA regulons may promote pathogenic states of growth such as carcinogenesis.
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Ciclo Celular , Proliferação de Células , RNA Mensageiro/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Dano ao DNA , Expressão Gênica , Humanos , Interferência de RNA , Transporte de RNA , RNA Mensageiro/genética , RegulonRESUMO
UNLABELLED: The ability of CD8+ T cells to effectively limit HIV-1 replication and block HIV-1 acquisition is determined by the capacity to rapidly respond to HIV-1 antigens. Understanding both the functional properties and regulation of an effective CD8+ response would enable better evaluation of T cell-directed vaccine strategies and may inform the design of new therapies. We assessed the antigen specificity, cytokine signature, and mechanisms that regulate antiviral gene expression in CD8+ T cells from a cohort of HIV-1-infected virus controllers (VCs) (<5,000 HIV-1 RNA copies/ml and CD4+ lymphocyte counts of >400 cells/µl) capable of soluble inhibition of HIV-1. Gag p24 and Nef CD8+ T cell-specific soluble virus inhibition was common among the VCs and correlated with substantial increases in the abundance of mRNAs encoding the antiviral cytokines macrophage inflammatory proteins MIP-1α, MIP-1αP (CCL3L1), and MIP-1ß; granulocyte-macrophage colony-stimulating factor (GM-CSF); lymphotactin (XCL1); tumor necrosis factor receptor superfamily member 9 (TNFRSF9); and gamma interferon (IFN-γ). The induction of several of these mRNAs was driven through a coordinated response of both increased transcription and stabilization of mRNA, which together accounted for the observed increase in mRNA abundance. This coordinated response allows rapid and robust induction of mRNA messages that can enhance the CD8+ T cells' ability to inhibit virus upon antigen encounter. IMPORTANCE: We show that mRNA stability, in addition to transcription, is key in regulating the direct anti-HIV-1 function of antigen-specific memory CD8+ T cells. Regulation at the level of RNA helps enable rapid recall of memory CD8+ T cell effector functions for HIV-1 inhibition. By uncovering and understanding the mechanisms employed by CD8+ T cell subsets with antigen-specific anti-HIV-1 activity, we can identify new strategies for comprehensive identification of other important antiviral genes. This will, in turn, enhance our ability to inhibit virus replication by informing both cure strategies and HIV-1 vaccine designs that aim to reduce transmission and can aid in blocking HIV-1 acquisition.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Citocinas/biossíntese , Regulação da Expressão Gênica , Antígenos HIV/imunologia , HIV-1/imunologia , Biossíntese de Proteínas , Transcrição Gênica , Estudos de Coortes , Citocinas/genética , Perfilação da Expressão Gênica , Sobreviventes de Longo Prazo ao HIV , Humanos , RNA Mensageiro/análise , RNA Mensageiro/genéticaRESUMO
Mutations in DJ-1, PINK1 (PTEN-induced putative kinase 1) and parkin all cause recessive parkinsonism in humans, but the relationships between these genes are not clearly defined. One event associated with loss of any of these genes is altered mitochondrial function. Recent evidence suggests that turnover of damaged mitochondria by autophagy might be central to the process of recessive parkinsonism. Here, we show that loss of DJ-1 leads to loss of mitochondrial polarization, fragmentation of mitochondria and accumulation of markers of autophagy (LC3 punctae and lipidation) around mitochondria in human dopaminergic cells. These effects are due to endogenous oxidative stress, as antioxidants will reverse all of them. Similar to PINK1 and parkin, DJ-1 also limits mitochondrial fragmentation in response to the mitochondrial toxin rotenone. Furthermore, overexpressed parkin will protect against loss of DJ-1 and, although DJ-1 does not alter PINK1 mitochondrial phenotypes, DJ-1 is still active against rotenone-induced damage in the absence of PINK1. None of the three proteins complex together using size exclusion chromatography. These data suggest that DJ-1 works in parallel to the PINK1/parkin pathway to maintain mitochondrial function in the presence of an oxidative environment.
Assuntos
Autofagia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Oncogênicas/metabolismo , Proteínas Quinases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Linhagem Celular Tumoral , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Mitocôndrias/genética , Mitocôndrias/fisiologia , Mutação , Proteínas Oncogênicas/genética , Estresse Oxidativo/genética , Doença de Parkinson/genética , Proteína Desglicase DJ-1 , Proteínas Quinases/genética , Rotenona/farmacologia , Ubiquitina-Proteína Ligases/genéticaRESUMO
Parkinson's disease (PD) is a major neurodegenerative condition with several rare Mendelian forms. Oxidative stress and mitochondrial function have been implicated in the pathogenesis of PD but the molecular mechanisms involved in the degeneration of neurons remain unclear. DJ-1 mutations are one cause of recessive parkinsonism, but this gene is also reported to be involved in cancer by promoting Ras signaling and suppressing PTEN-induced apoptosis. The specific function of DJ-1 is unknown, although it is responsive to oxidative stress and may play a role in the maintenance of mitochondria. Here, we show, using four independent methods, that DJ-1 associates with RNA targets in cells and the brain, including mitochondrial genes, genes involved in glutathione metabolism, and members of the PTEN/PI3K cascade. Pathogenic recessive mutants are deficient in this activity. We show that DJ-1 is sufficient for RNA binding at nanomolar concentrations. Further, we show that DJ-1 binds RNA but dissociates after oxidative stress. These data implicate a single mechanism for the pleiotropic effects of DJ-1 in different model systems, namely that the protein binds multiple RNA targets in an oxidation-dependent manner.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Oncogênicas/metabolismo , Transtornos Parkinsonianos/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Sequência de Bases , Encéfalo/metabolismo , Linhagem Celular , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/genética , Genes Mitocondriais , Genes Recessivos , Glutationa/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Proteínas Oncogênicas/antagonistas & inibidores , Proteínas Oncogênicas/deficiência , Proteínas Oncogênicas/genética , Estresse Oxidativo , PTEN Fosfo-Hidrolase/metabolismo , Transtornos Parkinsonianos/genética , Peroxirredoxinas , Fosfatidilinositol 3-Quinases/metabolismo , Proteína Desglicase DJ-1 , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
Mutations in DJ-1 lead to a monogenic form of early onset recessive parkinsonism. DJ-1 can respond to oxidative stress, which has been proposed to be involved in the pathogenesis of sporadic Parkinson disease (PD). We have recently reported that DJ-1 interacts with mRNA in an oxidation-dependent manner. Here, we confirm interaction of DJ-1 and RNA in human brain using immunoprecipitation followed by quantitative real time PCR. We confirmed previous reports that DJ-1 is more oxidized in cortex from cases of sporadic PD compared to controls. In the same samples, protein and RNA expression was measured for four DJ-1 target genes GPx4, MAPK8IP1, ND2 and ND5. While no alterations in mRNA expression were observed, an increase in protein expression was observed in PD cases for GPx4 and MAPK8IP1. In the same patients, we saw decreased mRNA and protein levels of two mitochondrial targets, ND2 and ND5. These results suggest that these proteins undergo regulation at the post-transcriptional level that may involve translational regulation by DJ-1.
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Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Oncogênicas/genética , Doença de Parkinson/genética , Processamento Pós-Transcricional do RNA/fisiologia , RNA Mensageiro/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Encéfalo/metabolismo , Eletroforese em Gel Bidimensional/métodos , Glutationa Peroxidase/genética , Humanos , Imunoprecipitação/métodos , NADH Desidrogenase/genética , Doença de Parkinson/patologia , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Proteína Desglicase DJ-1 , Proteína Fosfatase 2/genética , Proteínas Proto-Oncogênicas/genética , Selenoproteína W/genética , Proteína de Morte Celular Associada a bcl/genéticaRESUMO
Mutations in PINK1 (PTEN-induced putative kinase 1) are causal for early onset recessive parkinsonism in humans, characterized by damage to the nigrostriatal system. In situ hybridization studies in rodent brains have suggested a predominantly neuronal expression of PINK1 mRNA but immunocytochemistry of human brain tissue has shown PINK1-like immunoreactivity in both neurons and glia. In this study, we assessed the comparative distribution of PINK1 mRNA in human, rat and mouse brain. We observe that in humans PINK1 message is expressed in neurons with very little to no signal in glia and confirms similar findings in rodent tissue. Highest levels of expression were observed in hippocampus, substantia nigra and cerebellar Purkinje cells. We also show that PINK1 mRNA expression is similar in nigral neurons from neurologically normal controls and sporadic Parkinson's disease cases.
Assuntos
Encéfalo/metabolismo , Expressão Gênica/fisiologia , Doença de Parkinson/metabolismo , Proteínas Quinases/metabolismo , RNA Mensageiro/metabolismo , Animais , Encéfalo/patologia , Humanos , Hibridização In Situ , Camundongos , Neurônios/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/patologia , Proteínas Quinases/genética , RatosRESUMO
Mutations in the DJ-1 gene are associated with recessive, early onset Parkinson's disease (PD). We reported previously that one of the point mutations, L166P, destabilizes the protein and thus produces an effective knockout of the gene. Here, we have expanded this analysis to include a series of mutations and polymorphisms identified throughout the gene. The M26I point mutation was also unstable, although the effect was not as dramatic as with L166P. Protein levels were rescued in part, but not completely, by proteasome inhibition. Other variants, such as R98Q, were generally stable. We noted that M26I and L166P are both in helical regions near the dimer interface. However, M26I retains the ability to dimerize. We also examined the subcellular localization of DJ-1 and found that most mutations were similar to the wild-type (wt) protein in that a few cells showed mitochondrial staining. However, in all cases, the proportion of cells with mitochondrial DJ-1 staining was increased in oxidative conditions, suggesting that oxidation promotes the mitochondrial localization of DJ-1.
Assuntos
Acetilcisteína/análogos & derivados , Proteínas Oncogênicas/genética , Mutação Puntual , Polimorfismo Genético , Acetilcisteína/farmacologia , Cloreto de Amônio/farmacologia , Análise de Variância , Animais , Western Blotting/métodos , Linhagem Celular Tumoral , Cicloeximida/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Dimerização , Imunofluorescência/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imunoprecipitação/métodos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Modelos Moleculares , Neuroblastoma , Proteínas Oncogênicas/química , Proteína Desglicase DJ-1 , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Frações Subcelulares/metabolismo , Fatores de Tempo , Transfecção/métodosRESUMO
Mutations in DJ-1 can cause early onset parkinsonism. Various antibodies have been generated to detect this protein, one of which is a commonly used monoclonal antibody (clone 3E8). Since results of in situ examinations of DJ-1 expression with this antibody have differed from analyses with species-specific antibodies (e.g. rat), it would be useful to know the epitope for this antibody. Using GFP-tagged deletion constructs of human DJ-1, we have localized the epitope region for this antibody to within residues 56-78 of human DJ-1. Mapping this region to the published three-dimensional structure of DJ-1 indicates that this is a solvent-accessible surface epitope. Immunonegativity of E64D mutant DJ-1 with the monoclonal antibody suggests that glutamate 64 of human DJ-1 contributes to the epitope recognized by this antibody. Moreover, the loss of immunoreactivity due to such a small substitution demonstrates the remarkable sensitivity of the monoclonal antibody 3E8 to DJ-1.
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Anticorpos Monoclonais/química , Especificidade de Anticorpos , Epitopos , Proteínas Oncogênicas/imunologia , Sequência de Aminoácidos , Animais , Reações Antígeno-Anticorpo , Western Blotting/métodos , Linhagem Celular , Epitopos/imunologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Mutagênese Sítio-Dirigida/imunologia , Proteína Desglicase DJ-1 , Estrutura Secundária de Proteína , Ratos , Transfecção/métodosRESUMO
Mutations in the PARK7 gene, DJ-1, have been reported to cause early-onset and familial Parkinson's disease (PD). The function of DJ-1 and how it contributes to the development of the disease is not clear today, but several studies report that DJ-1 is responsive to oxidative stress and important for the maintenance of mitochondria. We have screened three coding regions of DJ-1 (exon 2, 5 and 7) in a Swedish Parkinson cohort. The Swedish PD material consisted of 67 patients with a self reported positive family history of PD and 77 patients with early-onset of disease (≤50 years old). We detected two patients with the previously reported synonymous mutation, Ala167Ala (c.501A>G, rs71653621), in exon 7. No Ala167Ala carriers were identified among 213 neurologically healthy Swedish controls. Mechanisms by which the synonymous Ala167Ala mutation can have consequences are unknown. It may affect the mRNA stability, secondary structure of mRNA, synthesis, turnover, protein folding and function. We could show a 1.3% decrease in DJ-1 mRNA folding energy in the A
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The formation of cysteine-sulfinic acid has recently become appreciated as a modification that links protein function to cellular oxidative status. Human DJ-1, a protein associated with inherited parkinsonism, readily forms cysteine-sulfinic acid at a conserved cysteine residue (Cys106 in human DJ-1). Mutation of Cys106 causes the protein to lose its normal protective function in cell culture and model organisms. However, it is unknown whether the loss of DJ-1 protective function in these mutants is due to the absence of Cys106 oxidation or the absence of the cysteine residue itself. To address this question, we designed a series of substitutions at a proximal glutamic acid residue (Glu18) in human DJ-1 that alter the oxidative propensity of Cys106 through changes in hydrogen bonding. We show that two mutations, E18N and E18Q, allow Cys106 to be oxidized to Cys106-sulfinic acid under mild conditions. In contrast, the E18D mutation stabilizes a cysteine-sulfenic acid that is readily reduced to the thiol in solution and in vivo. We show that E18N and E18Q can both partially substitute for wild-type DJ-1 using mitochondrial fission and cell viability assays. In contrast, the oxidatively impaired E18D mutant behaves as an inactive C106A mutant and fails to protect cells. We therefore conclude that formation of Cys106-sulfinic acid is a key modification that regulates the protective function of DJ-1.
Assuntos
Cisteína/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Oncogênicas/metabolismo , Ácidos Sulfínicos/metabolismo , Substituição de Aminoácidos , Animais , Linhagem Celular Tumoral , Cisteína/genética , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/metabolismo , Ácido Glutâmico/genética , Ácido Glutâmico/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Proteínas Mitocondriais/genética , Mutação , Proteínas Oncogênicas/genética , Oxirredução , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Proteína Desglicase DJ-1RESUMO
Mutations in the LRRK2 gene, coding for dardarin, cause dominantly inherited Parkinson's disease (PD). Dardarin is a large protein, and mutations are found throughout the gene including the kinase domain. However, it is not clear if kinase activity is important for the damaging effects of pathogenic mutations. In this study, we noted two cellular phenotypes associated with mutant dardarin. First, pathogenic mutations increase the tendency of dardarin to form inclusion bodies. Secondly, neurons and neuronal cell lines undergo cell death after expression of mutant protein. Manipulating activity by replacing the kinase domain with a 'kinase-dead' version blocks inclusion body formation and strongly delays cell death. This predicts that kinase inhibitors will be useful therapeutic agents in patients with LRRK2 mutations and, perhaps, in sporadic PD. We also show that dardarin protein is expressed within human midbrain neurons and that C-terminal epitopes are also found in some Lewy bodies.