RESUMEN
Changes to our environment have led to the emergence of human pathogens such as chikungunya virus. Chikungunya virus infection is today a major public health concern. It is a debilitating chronic disease impeding patients' mobility, affecting millions of people. Disease development relies on skeletal muscle infection. The importance of skeletal muscle in chikungunya virus infection led to the hypothesis that it could serve as a viral reservoir and could participate to virus persistence. Here we questioned the interconnection between skeletal muscle cells metabolism, their differentiation stage and the infectivity of the chikungunya virus. We infected human skeletal muscle stem cells at different stages of differentiation with chikungunya virus to study the impact of their metabolism on virus production and inversely the impact of virus on cell metabolism. We observed that chikungunya virus infectivity is cell differentiation and metabolism-dependent. Chikungunya virus interferes with the cellular metabolism in quiescent undifferentiated and proliferative muscle cells. Moreover, activation of chikungunya infected quiescent muscle stem cells, induces their proliferation, increases glycolysis and amplifies virus production. Therefore, our results showed that Chikungunya virus infectivity and the antiviral response of skeletal muscle cells relies on their energetic metabolism and their differentiation stage. Then, muscle stem cells could serve as viral reservoir producing virus after their activation.
Asunto(s)
Fiebre Chikungunya , Virus Chikungunya , Humanos , Músculo Esquelético/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Diferenciación Celular , Replicación Viral/fisiologíaRESUMEN
Obesity is associated with chronic low-grade inflammation and oxidative stress that blunt insulin response in its target tissues, leading to insulin resistance (IR). IR is a characteristic feature of type 2 diabetes. Skeletal muscle is responsible for 75% of total insulin-dependent glucose uptake; consequently, skeletal muscle IR is considered to be the primary defect of systemic IR development. Interestingly, some obese people stay insulin-sensitive and metabolically healthy. With the aim of understanding this difference and identifying the mechanisms responsible for insulin sensitivity maintenance/IR development during obesity, we explored the role of the latent endoribonuclease (RNase L) in skeletal muscle cells. RNase L is a regulator of innate immunity, of double-stranded RNA sensors and of toll-like receptor (TLR) 4 signaling. It is regulated during inflammation by interferons and its activity is dependent on its binding to 2-5A, an oligoadenylate synthesized by oligoadenylate synthetases (OAS). Increased expression of RNase L or downregulation of its inhibitor (RLI) improved insulin response in mouse myogenic C2C12 cells and in primary human myotubes from normal-weight subjects treated with palmitate, a saturated free fatty acid (FFA) known to induce inflammation and oxidative stress via TLR4 activation. While RNase L and RLI levels remained unchanged, OAS level was decreased in primary myotubes from insulin-resistant obese subjects (OB-IR) compared with myotubes from insulin-sensitive obese subjects (OB-IS). TLR3 and mitochondrial manganese superoxide dismutase (MnSOD) were also underexpressed in OB-IR myotubes. Activation of RNase L by 2-5A transfection allowed to restore insulin response, OAS, MnSOD and TLR3 expression in OB-IR myotubes. Due to low expression of OAS, OB-IR myotubes present a defect in RNase L activation and TLR3 regulation. Consequently, MnSOD level is low and insulin sensitivity is reduced. These results support that RNase L activity limits FFA/obesity-induced impairment of insulin response in muscle cells via TLR3 and MnSOD expression.
Asunto(s)
Endorribonucleasas/metabolismo , Resistencia a la Insulina , Insulina/metabolismo , Mioblastos Esqueléticos/enzimología , Obesidad/enzimología , Músculo Cuádriceps/enzimología , Superóxido Dismutasa/metabolismo , Receptor Toll-Like 3/metabolismo , 2',5'-Oligoadenilato Sintetasa/genética , 2',5'-Oligoadenilato Sintetasa/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Estudios de Casos y Controles , Regulación hacia Abajo , Endorribonucleasas/genética , Activación Enzimática , Femenino , Células HeLa , Humanos , Masculino , Ratones , Persona de Mediana Edad , Obesidad/genética , Ácido Palmítico/metabolismo , Interferencia de ARN , Transducción de Señal , Superóxido Dismutasa/genética , Receptor Toll-Like 3/genética , TransfecciónRESUMEN
Adipose tissue structure is altered during obesity, leading to deregulation of whole-body metabolism. Its function depends on its structure, in particular adipocytes number and differentiation stage. To better understand the mechanisms regulating adipogenesis, we have investigated the role of an endoribonuclease, endoribonuclease L (RNase L), using wild-type and RNase L-knockout mouse embryonic fibroblasts (RNase L(-/-)-MEFs). Here, we identify C/EBP homologous protein 10 (CHOP10), a dominant negative member of the CCAAT/enhancer-binding protein family, as a specific RNase L target. We show that RNase L is associated with CHOP10 mRNA and regulates its stability. CHOP10 expression is conserved in RNase L(-/-)-MEFs, maintaining preadipocyte state while impairing their terminal differentiation. RNase L(-/-)-MEFs have decreased lipids storage capacity, insulin sensitivity and glucose uptake. Expression of ectopic RNase L in RNase L(-/-)-MEFs triggers CHOP10 mRNA instability, allowing increased lipids storage, insulin response and glucose uptake. Similarly, downregulation of CHOP10 mRNA with CHOP10 siRNA in RNase L(-/-)-MEFs improves their differentiation in adipocyte. In vivo, aged RNase L(-)/(-) mice present an expanded adipose tissue, which, however, is unable to correctly store lipids, illustrated by ectopic lipids storage in the liver and in the kidney. These findings highlight RNase L as an essential regulator of adipogenesis via the regulation of CHOP10 mRNA.
Asunto(s)
Adipocitos/metabolismo , Diferenciación Celular/fisiología , Endorribonucleasas/metabolismo , Resistencia a la Insulina/fisiología , Metabolismo de los Lípidos/fisiología , Estabilidad del ARN/fisiología , ARN Mensajero/metabolismo , Factor de Transcripción CHOP/biosíntesis , Adipocitos/citología , Adipogénesis/fisiología , Animales , Regulación hacia Abajo/fisiología , Endorribonucleasas/genética , Glucosa/genética , Glucosa/metabolismo , Ratones , Ratones Noqueados , ARN Mensajero/genética , Factor de Transcripción CHOP/genéticaRESUMEN
We have coupled a spectrophotometer with a scanning near-field optical microscope to obtain, with a single scan, simultaneously scanning near-field optical microscope fluorescence images at different wavelengths as well as topography and transmission images. Extraction of the fluorescence spectra enabled us to decompose the different wavelengths of the fluorescence signals which normally overlap. We thus obtained images of the different fluorescence emissions of acridine orange bound to single or double stranded nucleic acids in human metaphase chromosomes before and after DNAse I or RNAse A treatment. The analysis of these images allowed us to visualize some specific chromatin areas where RNA is associated with DNA showing that such a technique could be used to identify multiple components within a cell.
Asunto(s)
Cromosomas Humanos/ultraestructura , Microscopía/métodos , Espectrometría de Fluorescencia/métodos , Cromatina/ultraestructura , Colorantes Fluorescentes/farmacología , Humanos , Procesamiento de Imagen Asistido por Computador/métodos , Coloración y Etiquetado/métodosRESUMEN
BACKGROUND: With the current increasing incidence of allergies worldwide, new treatments showing efficacy and long term safety are needed for chronic conditions such as persistent allergic rhinitis (PER). New generation H1-antihistamines have demonstrated anti-allergic properties, which could possibly enhance their effectiveness in long-term periods of treatment. OBJECTIVE: To investigate the efficacy of rupatadine, in controlling symptoms of PER over a 12-week period. METHODS: A randomized, double blind, parallel-group, placebo-controlled study was carried out in patients aged older than 12 years with PER. Main inclusion criteria were: instantaneous total symptom score (i6TSS) >or=45, nasal obstruction score
Asunto(s)
Antialérgicos/uso terapéutico , Cetirizina/uso terapéutico , Ciproheptadina/análogos & derivados , Rinitis Alérgica Perenne/tratamiento farmacológico , Adolescente , Adulto , Antialérgicos/administración & dosificación , Antialérgicos/efectos adversos , Cetirizina/administración & dosificación , Cetirizina/efectos adversos , Ciproheptadina/administración & dosificación , Ciproheptadina/efectos adversos , Ciproheptadina/uso terapéutico , Método Doble Ciego , Femenino , Humanos , Masculino , Índice de Severidad de la Enfermedad , Factores de Tiempo , Resultado del TratamientoRESUMEN
Interferons (IFNs) inhibit the growth of many different cell types by altering the expression of specific genes. IFNs activities are partly mediated by the 2'-5' oligoadenylates-RNase L RNA decay pathway. RNase L is an endoribonuclease requiring activation by 2'-5' oligoadenylates to cleave single-stranded RNA. Here, we present evidence that degradation of mitochondrial mRNA by RNase L leads to cytochrome c release and caspase 3 activation during IFNalpha-induced apoptosis. We identify and characterize the mitochondrial translation initiation factor (IF2mt) as a new partner of RNase L. Moreover, we show that specific inhibition of mitochondrial translation with chloramphenicol inhibits the IFNalpha-induced degradation of mitochondrial mRNA by RNase L. Finally, we demonstrate that overexpression of IF2mt in human H9 cells stabilizes mitochondrial mRNA, inhibits apoptosis induced by IFNalpha and partially reverses IFNalpha-cell growth inhibition. On the basis of our results, we propose a model describing how RNase L regulates mitochondrial mRNA stability through its interaction with IF2mt.
Asunto(s)
Apoptosis/fisiología , Endorribonucleasas/metabolismo , Estabilidad del ARN/fisiología , ARN Mensajero/metabolismo , ARN/metabolismo , Apoptosis/efectos de los fármacos , Secuencia de Bases , Caspasa 3/metabolismo , Línea Celular , Citocromos c/metabolismo , Cartilla de ADN/genética , Factores Eucarióticos de Iniciación/genética , Factores Eucarióticos de Iniciación/metabolismo , Células HeLa , Humanos , Técnicas In Vitro , Interferón Tipo I/farmacología , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Modelos Biológicos , Biosíntesis de Proteínas , ARN/genética , Estabilidad del ARN/genética , ARN Mensajero/genética , ARN Mitocondrial , Proteínas Recombinantes , Técnicas del Sistema de Dos HíbridosRESUMEN
Interferon alpha (IFNalpha) belongs to a cytokine family that exhibits antiviral properties, immuno-modulating effects, and antiproliferative activity on normal and neoplasic cells in vitro and in vivo. IFNalpha exerts antitumor action by inducing direct cytotoxicity against tumor cells. This toxicity is at least partly due to induction of apoptosis. Although the molecular basis of the inhibition of cell growth by IFNalpha is only partially understood, there is a direct correlation between the sensitivity of cells to the antiproliferative action of IFNalpha and the down-regulation of their mitochondrial mRNAs. Here, we studied the role of the 2-5A/RNase L system and its inhibitor RLI in this regulation of the mitochondrial mRNAs by IFNalpha. We found that a fraction of cellular RNase L and RLI is localized in the mitochondria. Thus, we down-regulated RNase L activity in human H9 cells by stably transfecting (i) RNase L antisense cDNA or (ii) RLI sense cDNA constructions. In contrast to control cells, no post-transcriptional down-regulation of mitochondrial mRNAs and no cell growth inhibition were observed after IFNalpha treatment in these transfectants. These results demonstrate that IFNalpha exerts its antiproliferative effect on H9 cells at least in part via the degradation of mitochondrial mRNAs by RNase L.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Nucleótidos de Adenina/metabolismo , Chaperoninas , Endorribonucleasas/metabolismo , Interferón-alfa/farmacología , Mitocondrias/genética , Oligorribonucleótidos/metabolismo , Proteínas/metabolismo , ARN Mensajero/genética , Secuencia de Bases , División Celular/efectos de los fármacos , Cartilla de ADN , Endorribonucleasas/antagonistas & inhibidores , Humanos , ARN Mensajero/metabolismo , Células Tumorales CultivadasRESUMEN
H19 is a paternally imprinted gene whose expression produces a 2.4 kb RNA in most tissues during development and in mammalian myoblastic cell lines upon differentiation. Deletion of the active maternal allele of H19 and its flanking regions in the mouse leads to biallelic methylation and loss of imprinting of the neighbouring Igf2 gene. The function of H19 RNA remains unknown and, although polysome-associated, the absence of a conserved open reading frame suggests that it does not encode a protein product. We describe a novel post-transcriptional regulation of H19 gene expression which, in spite of this lack of coding capacity, is dependent on translational activity. We show that stabilization of the RNA is solely responsible for its accumulation during in vitro muscle cell differentiation. This conclusion is based on the finding that inhibition of protein synthesis results in a dramatic destabilization of H19 RNA in proliferating mouse C2C12 myoblastic cells but not in differentiated cells, and on run-on experiments which showed that the rate of transcription of H19 RNA remains constant during muscle cell differentiation. This mechanism could also be involved in H19 gene expression during mouse development in addition to its transcriptional activation which we have shown to occur.
Asunto(s)
Músculo Esquelético/fisiología , Procesamiento Postranscripcional del ARN/fisiología , ARN no Traducido/biosíntesis , ARN/metabolismo , Animales , Diferenciación Celular/genética , División Celular/genética , Células Cultivadas , Regulación del Desarrollo de la Expresión Génica/fisiología , Impresión Genómica , Ratones , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , ARN/genética , ARN Largo no Codificante , ARN no Traducido/genética , Regulación hacia ArribaRESUMEN
The 2',5'-oligoadenylate (2-5A)/RNase L pathway is one of several enzymatic pathways induced by interferons (IFN). RNase L is a latent endoribonuclease that is activated on its binding by 2-5A and inhibited by the ribonuclease L inhibitor (RLI). We have shown previously by coimmunoprecipitation that RNase L may be associated with a 90-kDa RNA binding protein (RNABP), identified with a monoclonal antibody (mAb) raised against an RNase L complex purified under native conditions on 2-5A-sepharose. Here we confirm, by gel-filtration and pull-down analysis, the association of RNase L and RNABP, and we demonstrate that this association is significantly increased in the presence of 2-5A. Moreover, we found that RNABP protein levels decrease during terminal differentiation in various cell lines but do not vary during vesicular stomatitis virus (VSV) or encephalomyocarditis virus (EMCV) infection or following IFN-alpha/beta treatment. In this latter case, although total cellular RNABP levels do not vary, the amount of RNABP found in the cytoplasm increases in comparison to that found in the nucleus, indicating a cytoplasmic localization of RNABP after IFN-alpha/beta treatment. Finally, we demonstrate the interaction between RNase L and RNABP in intact cells. Microinjection of an mAb against RNABP into HeLa cells inhibits RNase L antiviral activity and partially inhibits the IFN-alpha/beta-induced antiviral activity.
Asunto(s)
Endorribonucleasas/metabolismo , Proteínas de Unión al ARN/metabolismo , Nucleótidos de Adenina/metabolismo , Animales , Anticuerpos Monoclonales , Diferenciación Celular , Transformación Celular Viral , Cromatografía de Afinidad , Cromatografía en Gel , Virus de la Encefalomiocarditis/fisiología , Endorribonucleasas/química , Endorribonucleasas/aislamiento & purificación , Células HeLa , Humanos , Interferón Tipo I/farmacología , Leucemia Eritroblástica Aguda , Ratones , Peso Molecular , Oligorribonucleótidos/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/aislamiento & purificación , Células Tumorales Cultivadas , Células U937 , Virus de la Estomatitis Vesicular Indiana/fisiologíaRESUMEN
The 2'-5' oligoadenylate (2-5A)/RNase L pathway is one of the enzymatic pathways induced by interferon. RNase L is a latent endoribonuclease which is activated by 2-5A and inhibited by a specific protein known as RLI (RNase L inhibitor). This system has an important role in regulating viral infection. Additionally, variations in RNase L activity have been observed during cell growth and differentiation but the significance of the 2-5A/RNase L/RLI pathway in these latter processes is not known. To determine the roles of RNase L and RLI in muscle differentiation, C2 mouse myoblasts were transfected with sense and antisense RLI cDNA constructs. Importantly, the overexpression of RLI in C2 cells was associated with diminished RNase L activity, an increased level of MyoD mRNA, and accelerated kinetics of muscle differentiation. Inversely, transfection of the RLI antisense construct was associated with increased RNase L activity, a diminished level of MyoD mRNA, and delayed differentiation. In agreement with these data, MyoD mRNA levels were also decreased in C2 cells transfected with an inducible RNase L construct. The effect of RNase L activity on MyoD mRNA levels was relatively specific because expression of several other mRNAs was not altered in C2 transfectants. Therefore, RNase L is directly involved in myoblast differentiation, probably through its role in regulating MyoD stability. This is the first identification of a potential mRNA target for RNase L.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Chaperoninas , Endorribonucleasas/metabolismo , Músculo Esquelético/metabolismo , Proteína MioD/genética , Proteínas/metabolismo , ARN Mensajero/metabolismo , Animales , Diferenciación Celular/genética , Células Cultivadas , ADN sin Sentido , Endorribonucleasas/genética , Inhibidores Enzimáticos/metabolismo , Regulación de la Expresión Génica , Semivida , Isopropil Tiogalactósido/farmacología , Ratones , Músculo Esquelético/citología , Proteína MioD/efectos de los fármacos , Proteína MioD/metabolismo , Miogenina/genética , Miogenina/metabolismo , Proteínas/genética , Estabilidad del ARN , ARN Mensajero/química , TransfecciónRESUMEN
PURPOSE: Recent studies have revealed abnormalities in the ribonuclease L pathway in peripheral blood mononuclear cells of patients with the chronic fatigue syndrome. We conducted a blinded study to detect possible differences in the distribution of 2-5A binding proteins in the cells of patients with chronic fatigue syndrome and controls. PATIENTS AND METHODS: We studied 57 patients with chronic fatigue syndrome and 53 control subjects (28 healthy subjects and 25 patients with depression or fibromyalgia). A radioactive probe was used to label 2-5A binding proteins in unfractionated peripheral blood mononuclear cell extracts and to compare their distribution in the three groups. RESULTS: A 37 kDa 2-5A binding polypeptide was found in 50 (88%) of the 57 patients with chronic fatigue syndrome compared with 15 (28%) of the 53 controls (P < 0.01). When present, the amount of 37 kDa protein was very low in the control groups. When expressed as the ratio of the 37 kDa protein to the 80 kDa protein, 41 (72%) of the 57 patients with chronic fatigue syndrome had a ratio > 0.05, compared with 3 (11%) of the 28 healthy subjects and none of the patients with fibromyalgia or depression. CONCLUSION: The presence of a 37 kDa 2-5A binding protein in extracts of peripheral blood mononuclear cells may distinguish patients with chronic fatigue syndrome from healthy subjects and those suffering from other diseases.
Asunto(s)
Nucleótidos de Adenina/sangre , Síndrome de Fatiga Crónica/sangre , Oligorribonucleótidos/sangre , Adulto , Biomarcadores/sangre , Estudios de Casos y Controles , Depresión/sangre , Diagnóstico Diferencial , Síndrome de Fatiga Crónica/diagnóstico , Femenino , Fibromialgia/sangre , Humanos , Masculino , Persona de Mediana Edad , Monocitos/metabolismoRESUMEN
The interferon-regulated 2-5A/RNase L pathway plays a major role in the antiviral and antiproliferative activities of these cytokines. Several viruses, however, have evolved strategies to escape the antiviral activity of the 2-5A/RNase L pathway. In this context, we have cloned a cDNA coding for the RNase L inhibitor (RLI), a protein that specifically inhibits RNase L and whose regulated expression in picornavirus-infected cells down regulates the activity of the 2-5A/RNase L pathway. We show here that RLI increases during the course of human immunodeficiency virus type 1 (HIV-1) infection, which may be related to the downregulation of RNase L activity that has been described to occur in HIV-infected cells. In order to establish a possible causal relationship between these observations, we have stably transfected H9 cells with RLI sense or antisense cDNA-expressing vectors. The overexpression of RLI causes a decrease in RNase L activity and a twofold enhancement of HIV production. This increase in HIV replication correlates with an increase in HIV RNA and proteins. In contrast, reduction of RLI levels in RLI antisense cDNA-expressing clones reverses the inhibition of RNase L activity associated with HIV multiplication and leads to a threefold decrease in the viral load. This anti-HIV activity correlated with a decrease in HIV RNA and proteins. These findings demonstrate that the level of RLI, via its modulation of RNase L activity, can severely impair HIV replication and suggest the involvement of RLI in the inhibition of the 2-5A/RNase L system observed during HIV infection.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/metabolismo , Nucleótidos de Adenina/antagonistas & inhibidores , Endorribonucleasas/antagonistas & inhibidores , Inhibidores Enzimáticos/metabolismo , VIH-1/fisiología , Oligorribonucleótidos/antagonistas & inhibidores , Línea Celular , Regulación hacia Abajo , Endorribonucleasas/metabolismo , Humanos , Transfección , Replicación ViralRESUMEN
The interferon-(IFN)-inducible 2',5'-oligoadenylate (2-5A)/endoribonuclease L (RNase L) pathway plays a major role in the antiviral and antiproliferative effects of IFN. The 2-5A/RNase L pathway appears to be regulated by the cell-growth status or viral infection. Viruses, and picornaviruses in particular, have evolved strategies to escape the 2-5A/RNase L-pathway-associated antiviral activity. We have recently cloned a cDNA coding for RLI, a RNase-L-specific protein inhibitor. Its regulated expression by viral infection could provide a new strategy to modulate the 2-5A/RNase L pathway. Since RNase L had been shown to be down regulated upon encephalomyocarditis (EMCV) infection, we stably transfected HeLa cells with a RLI antisense cDNA expressing vector. Four independent clones named VAS1, VAS2, VAS3 and VAS4 and one clone transfected with the empty vector (VV) as control, were analyzed. The level of RLI was decreased by 20% for VAS1, 25% for VAS2, 75% for VAS3 and 50% for VAS4. The inactivation of RNase L observed during EMCV infection was decreased in these clones as compared to control HeLa cells. Here again the results vary between the four clones. The maximum inhibition of RNase L (90%) was observed in control cells and in VAS1 while 48% inhibition was observed in VAS4 and 25% in VAS3. The reversal in RNase L inhibition thus reflects closely the resulting RLI level, in keeping with a major role of RLI in EMCV-induced down regulation of 2-5A-binding activity of RNase L. Moreover, cells expressing a low level of RLI (VAS3 and VAS 4) are partially resistant to EMCV infection.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Infecciones por Cardiovirus/enzimología , Chaperoninas , Virus de la Encefalomiocarditis , Endorribonucleasas/antagonistas & inhibidores , Inhibidores Enzimáticos/metabolismo , Proteínas/antagonistas & inhibidores , Anticuerpos , Infecciones por Cardiovirus/genética , Infecciones por Cardiovirus/virología , ADN sin Sentido/genética , ADN sin Sentido/farmacología , Regulación hacia Abajo/efectos de los fármacos , Virus de la Encefalomiocarditis/aislamiento & purificación , Endorribonucleasas/metabolismo , Inhibidores Enzimáticos/farmacología , Células HeLa , Humanos , Proteínas/genética , Proteínas/metabolismo , TransfecciónRESUMEN
The 2-5A/RNase L system is one of the pathways induced by interferon (IFN). It plays a major role in the antiviral and antiproliferative activities of IFNs. Recently, we have shown that the activity of the RNase L could be inhibited by a proteic inhibitor, the RNase L Inhibitor (RLI). Human RLI (Hu-RLI) was cloned and characterized. We describe here the isolation and characterization of the cDNA encoding the murine RLI (Mu-RLI). Hu-RLI and Mu-RLI protein have 98% amino acid identity. Mu-RLI is functionally homologous to Hu-RLI, and all the structural features and amino acid sequence motifs of Hu-RLI are conserved in Mu-RLI. Moreover, reticulocyte lysate translated Mu-RLI protein is also able to inhibit 2-5A binding on 2-5A-dependent RNAse-L. Northern blot analysis revealed that Mu-RLI cDNA hybridizes with one mRNA of 3.5 kb except for the testis where two mRNA of 3.5 and 2.1 kb, respectively, are detected, suggesting a tissue-specific regulation.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Chaperoninas , Inhibidores Enzimáticos/química , Biosíntesis de Proteínas , Proteínas/química , Nucleótidos de Adenina/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Unión Competitiva , Clonación Molecular , Secuencia Conservada , ADN Complementario , Inhibidores Enzimáticos/farmacología , Humanos , Masculino , Methanococcus/genética , Ratones , Datos de Secuencia Molecular , Oligorribonucleótidos/metabolismo , Especificidad de Órganos , Proteínas/farmacología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología , Saccharomyces cerevisiae/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transcripción GenéticaRESUMEN
The (2-5A)-RNase L pathway is an important component of interferon (IFN) action. Its central role in the antiviral effect of IFN against Picornaviridae has been clearly demonstrated. We have characterized and cloned a new component of this pathway, the RNase L inhibitor (RLI). RLI is a cellular protein whose mRNA is not regulated by IFN but is induced by viruses, such as encephalomyocarditis virus (EMCV). RLI inhibits RNase L during the time course of EMCV infection, and overexpression of RLI in HeLa cells partially reverses the antiviral action of IFN against EMCV. The replicative complexes of several viruses consist of double-stranded RNA structures. These dsRNAs could activate gene transcription as demonstrated for IFNs and could be responsible for RLI induction. We describe the increased expression of RLI mRNA and RLI protein induced by synthetic dsRNAs, such as poly(I):poly(C). This induction gives rise to an inhibition of the 2-5A-binding activity of RNase L. The inhibition of RNase L activity is transcient, probably due to the rapid turnover of RLI protein.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Chaperoninas , Inhibidores Enzimáticos/metabolismo , Biosíntesis de Proteínas , ARN Bicatenario/biosíntesis , Línea Celular , Clonación Molecular , Virus de la Encefalomiocarditis/fisiología , Inhibidores Enzimáticos/aislamiento & purificación , Inhibidores Enzimáticos/farmacocinética , Semivida , Células HeLa , Humanos , Proteínas/aislamiento & purificación , Proteínas/farmacocinética , Replicación ViralAsunto(s)
Transportadoras de Casetes de Unión a ATP , Chaperoninas , Mapeo Cromosómico/métodos , Cromosomas Humanos Par 4 , Endorribonucleasas/antagonistas & inhibidores , Inhibidores Enzimáticos , Proteínas/genética , Cromosomas Humanos Par 1 , Cromosomas Humanos Par 7 , Sondas de ADN , Humanos , Hibridación Fluorescente in SituRESUMEN
The 2-5A/RNase L system is considered as a central pathway of interferon (IFN) action and could possibly play a more general physiological role as for instance in the regulation of RNA stability in mammalian cells. We describe here the expression cloning and initial characterization of RLI (for RNase L inhibitor), a new type of endoribonuclease inhibitor. RLI cDNA codes for a 68-kDa polypeptide whose expression is not regulated by IFN. Its expression in reticulocyte extracts antagonizes the 2-5A binding ability and the nuclease activity of endogenous RNase L or the cloned 2DR polypeptide. The inhibition requires the association of RLI with the nuclease and is dependent on the ratio between the two proteins. Likewise RLI is coimmunoprecipitated with the RNase L complex by a nuclease-specific antibody. RLI does not lead to 2-5A degradation or to irreversible modification of RNase L. The overexpression of RLI in stably transfected HeLa cells inhibits the antiviral activity of IFN on encephalomyocarditis virus but not on vesicular stomatitis virus. RLI therefore appears as the first described and potentially important mediator of the 2-5A/RNase L pathway.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Nucleótidos de Adenina/metabolismo , Chaperoninas , Endorribonucleasas/antagonistas & inhibidores , Oligorribonucleótidos/metabolismo , Proteínas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Línea Celular , Clonación Molecular , ADN Complementario , Endorribonucleasas/metabolismo , Regulación de la Expresión Génica , Humanos , Interferones/farmacología , Datos de Secuencia Molecular , Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
The subunit composition of RNase L, a key enzyme in the interferon system, has been characterized. RNase L was purified from human Daudi cells on a column of 2-5A-Sepharose and used to immunize Balb/c mice. A specific monoclonal antibody which recognizes a protein of 80 kDa has been isolated. This protein has been characterized and shown to be an RNA-binding protein with nuclease activity which is associated with, but distinct from, the 80-kDa 2-5A-binding protein known previously as RNase L. It is therefore proposed that the 2-5A-dependent RNase L is a complex of two distinct subunits: an 80-kDa RNA-binding protein (i.e. the catalytic subunit) and an 80-kDa 2-5A-binding protein (i.e. the regulatory subunit).