RESUMEN
The ADAR RNA-editing enzymes deaminate adenosine bases to inosines in cellular RNAs. Aberrant interferon expression occurs in patients in whom ADAR1 mutations cause Aicardi-Goutières syndrome (AGS) or dystonia arising from striatal neurodegeneration. Adar1 mutant mouse embryos show aberrant interferon induction and die by embryonic day E12.5. We demonstrate that Adar1 embryonic lethality is rescued to live birth in Adar1; Mavs double mutants in which the antiviral interferon induction response to cytoplasmic double-stranded RNA (dsRNA) is prevented. Aberrant immune responses in Adar1 mutant mouse embryo fibroblasts are dramatically reduced by restoring the expression of editing-active cytoplasmic ADARs. We propose that inosine in cellular RNA inhibits antiviral inflammatory and interferon responses by altering RLR interactions. Transfecting dsRNA oligonucleotides containing inosine-uracil base pairs into Adar1 mutant mouse embryo fibroblasts reduces the aberrant innate immune response. ADAR1 mutations causing AGS affect the activity of the interferon-inducible cytoplasmic isoform more severely than the nuclear isoform.
Asunto(s)
Adenosina Desaminasa/metabolismo , Inmunidad Innata , Edición de ARN , ARN Bicatenario/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Adenosina Desaminasa/genética , Animales , Enfermedades Autoinmunes del Sistema Nervioso/genética , Enfermedades Autoinmunes del Sistema Nervioso/inmunología , Cruzamientos Genéticos , Citocinas/metabolismo , Pérdida del Embrión/patología , Embrión de Mamíferos/patología , Femenino , Fibroblastos/metabolismo , Humanos , Mediadores de Inflamación/metabolismo , Inosina/metabolismo , Hígado/metabolismo , Masculino , Ratones Endogámicos C57BL , Mutación/genética , Malformaciones del Sistema Nervioso/genética , Malformaciones del Sistema Nervioso/inmunología , Fenotipo , Proteínas de Unión al ARN/genética , Receptores de Interferón/metabolismo , Análisis de Supervivencia , Transcripción Genética , Proteína p53 Supresora de Tumor/metabolismo , Uracilo/metabolismoRESUMEN
Long double-stranded RNA may undergo hyper-editing by adenosine deaminases that act on RNA (ADARs), where up to 50% of adenosine residues may be converted to inosine. However, although numerous RNAs may undergo hyper-editing, the role for inosine-containing hyper-edited double-stranded RNA in cells is poorly understood. Nevertheless, editing plays a critical role in mammalian cells, as highlighted by the analysis of ADAR-null mutants. In particular, the long form of ADAR1 (ADAR1(p150)) is essential for viability. Moreover, a number of studies have implicated ADAR1(p150) in various stress pathways. We have previously shown that ADAR1(p150) localized to cytoplasmic stress granules in HeLa cells following either oxidative or interferon-induced stress. Here, we show that the Z-DNA-binding domain (Zα(ADAR1)) exclusively found in ADAR1(p150) is required for its localization to stress granules. Moreover, we show that fusion of Zα(ADAR1) to either green fluorescent protein (GFP) or polypyrimidine binding protein 4 (PTB4) also results in their localization to stress granules. We additionally show that the Zα domain from other Z-DNA-binding proteins (ZBP1, E3L) is likewise sufficient for localization to stress granules. Finally, we show that Z-RNA or Z-DNA binding is important for stress granule localization. We have thus identified a novel role for Z-DNA-binding domains in mammalian cells.
Asunto(s)
Adenosina Desaminasa/química , Gránulos Citoplasmáticos/enzimología , ADN de Forma Z/metabolismo , Proteínas de Unión al ADN/química , Adenosina Desaminasa/análisis , Adenosina Desaminasa/metabolismo , Aminoácidos/química , Proteínas de Unión al ADN/análisis , Células HeLa , Humanos , Isoenzimas/análisis , Isoenzimas/química , Estrés Oxidativo , Poli I-C/farmacología , Unión Proteica , Estructura Terciaria de Proteína , ARN/química , ARN/metabolismo , Proteínas de Unión al ARN , Proteínas Virales/análisis , Proteínas Virales/químicaRESUMEN
Hyperediting by adenosine deaminases that acts on RNA (ADARs) may result in numerous Adenosine-to-Inosine (A-to-I) substitutions within long dsRNA. However, while countless RNAs may undergo hyperediting, the role for inosine-containing hyperedited dsRNA (IU-dsRNA) in cells is poorly understood. We have previously shown that IU-dsRNA binds specifically to various components of cytoplasmic stress granules, as well as to other proteins such as Tudor Staphylococcal Nuclease (Tudor-SN). Tudor-SN has been implicated in diverse roles in mammalian cells, including transcription, splicing, RNAi, and degradation. Moreover, we have shown that Tudor-SN interacts directly with stress granule proteins. Here we show that Tudor-SN localizes to cytoplasmic stress granules in HeLa cells undergoing arsenite-induced oxidative stress, or following transfection with long dsRNA (poly[IC]), which initiates an interferon cascade. We additionally demonstrate a novel interaction between Tudor-SN and ADAR1. Finally, we show that ADAR1 is also localized to stress granules in HeLa cells following various stresses.
Asunto(s)
Adenosina Desaminasa/metabolismo , Gránulos Citoplasmáticos/química , Gránulos Citoplasmáticos/metabolismo , Proteínas Nucleares/metabolismo , Estrés Fisiológico , Adenosina Desaminasa/genética , Arsenitos/farmacología , Línea Celular Tumoral , Endonucleasas , Células HeLa , Humanos , Proteínas Nucleares/genética , Poli I-C/farmacología , Unión Proteica/fisiología , Estructura Terciaria de Proteína , Transporte de Proteínas/efectos de los fármacos , Proteínas de Unión al ARN , Xantina Oxidasa/antagonistas & inhibidoresRESUMEN
Adenosine deaminases acting on RNA (ADARs) catalyze hyperediting of long double-stranded RNAs (dsRNAs), whereby up to 50% of adenosines are converted to inosine (I). Although hyperedited dsRNAs (IU-dsRNAs) have been implicated in various cellular functions, we now provide evidence for another role. We show that IU-dsRNA suppresses the induction of interferon-stimulated genes (ISGs) and apoptosis by poly(IC). Moreover, we show that IU-dsRNA inhibits the activation of interferon regulatory factor 3 (IRF3), which is essential for the induction of ISGs and apoptosis. Finally, we speculate that the inhibition of IRF3 results from specific binding of IU-dsRNA to MDA-5 or RIG-I, both of which are cytosolic sensors for poly(IC). Although our data are consistent with a previous study in which ADAR1 deletion resulted in increased expression of ISGs and apoptosis, we show that IU-dsRNA per se suppresses ISGs and apoptosis. We therefore propose that any IU-dsRNA generated by ADAR1 can inhibit both pathways.
Asunto(s)
Apoptosis , Emparejamiento Base , Interferones/metabolismo , ARN Bicatenario/química , ARN Bicatenario/metabolismo , Regulación de la Expresión Génica , Células HeLa , Humanos , Factores Reguladores del Interferón/genética , Factores Reguladores del Interferón/inmunología , Interferones/inmunología , Poli I-C/inmunología , Unión ProteicaRESUMEN
Inosine residues may be introduced into long dsRNA (double-stranded RNA) molecules by the action of a family of editing enzymes, ADARs (adenosine deaminases that act on RNA). Furthermore, hyperediting of dsRNA by ADARs may result in up to 50% of adenosine residues being converted into inosine. While the effect of hyperediting has traditionally been thought to be limited to the edited dsRNA, we have recently shown that hyperedited dsRNA [I-dsRNA (inosine-containing dsRNA)] is able to down-regulate the expression of both reporter and endogenous mRNAs in cells, in trans. Down-regulation by I-dsRNA occurs both by reducing mRNA levels and by inhibiting of translation. This finding has important functional consequences for hyperediting by ADARs.
Asunto(s)
Regulación hacia Abajo/efectos de los fármacos , Inosina/farmacología , ARN Bicatenario/farmacología , Secuencia de Bases , Gránulos Citoplasmáticos/efectos de los fármacos , Gránulos Citoplasmáticos/metabolismo , Células HeLa , Humanos , Modelos Genéticos , Datos de Secuencia Molecular , Biosíntesis de Proteínas/efectos de los fármacos , ARN Bicatenario/genética , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Long double-stranded RNAs (dsRNAs) may undergo extensive modification (hyperediting) by adenosine deaminases that act on RNA (ADARs), where up to 50% of adenosine (A) residues are changed to inosine (I). Traditionally, consequences of A-to-I editing were thought to be limited to modified RNA itself. We show here, however, that hyperedited dsRNA (I-dsRNA) is able to downregulate gene expression in trans. Furthermore, we show that both endogenous expression and reporter gene expression were substantially reduced in the presence of I-dsRNA. This was due to a reduction in reporter mRNA levels and also translation inhibition. Importantly, we show that I-dsRNA interferes with translation initiation. We also show that I-dsRNA specifically binds a stress-granule-like complex. Stress granules (SGs) are important for translational silencing during stress. Finally, we propose a model whereby editing by ADARs results in downregulation of gene expression via SG formation.
Asunto(s)
Regulación hacia Abajo , Modelos Genéticos , ARN Bicatenario/fisiología , Genes Reporteros , Globinas/análisis , Proteínas Fluorescentes Verdes/análisis , Células HeLa , Humanos , Inosina/química , Luciferasas de Luciérnaga/análisis , Luciferasas de Renilla/análisis , Biosíntesis de Proteínas , ARN Bicatenario/química , ARN Bicatenario/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismoRESUMEN
Long double-stranded RNAs (dsRNAs) may undergo covalent modification (hyper-editing) by adenosine deaminases that act on RNA (ADARs), whereby up to 50-60% of adenosine residues are converted to inosine. Previously, we have described a ribonuclease activity in various cell extracts that specifically targets dsRNAs hyper-edited by ADARs. Such a ribonuclease may play an important role in viral defense, or may alternatively be involved in down-regulation of other RNA duplexes. Cleavage of hyper-edited dsRNA occurs within sequences containing multiple IU pairs but not in duplexes that contain either isosteric GU pairs or Watson-Crick base pairs. Here, we describe experiments aimed at further characterizing cleavage of hyper-edited dsRNA. Using various inosine-containing dsRNAs we show that cleavage occurs preferentially at a site containing both IU and UI pairs, and that inclusion of even a single GU pair inhibits cleavage. We also show that cleavage occurs on both strands within a single dsRNA molecule and requires a 2'-OH group. Strikingly, we show that ADAR1, ADAR2 or dADAR all preferentially generate the preferred cleavage site when hyper-editing a long dsRNA.
Asunto(s)
Adenosina Desaminasa/metabolismo , Edición de ARN , ARN Bicatenario/metabolismo , Animales , Emparejamiento Base , Guanosina/química , Inosina/química , Oocitos/metabolismo , ARN Bicatenario/química , Proteínas de Unión al ARN , Especificidad por Sustrato , Uridina/química , Xenopus laevisRESUMEN
Long perfect double-stranded RNA (dsRNA) molecules play a role in various cellular pathways. dsRNA may undergo extensive covalent modification (hyper-editing) by adenosine deaminases that act on RNA (ADARs), resulting in conversion of up to 50% of adenosine residues to inosine (I). Alternatively, dsRNA may trigger RNA interference (RNAi), resulting in silencing of the cognate mRNA. These two pathways have previously been shown to be antagonistic. We show a novel interaction between components of the ADAR and RNAi pathways. Tudor staphylococcal nuclease (Tudor-SN) is a subunit of the RNA-induced silencing complex, which is central to the mechanism of RNAi. Here we show that Tudor-SN specifically interacts with and promotes cleavage of model hyper-edited dsRNA substrates containing multiple I.U and U.I pairs. This interaction suggests a novel unsuspected interplay between the two pathways that is more complex than mutual antagonism.
Asunto(s)
ARN Bicatenario/genética , Complejo Silenciador Inducido por ARN/metabolismo , Adenosina Desaminasa/genética , Adenosina Desaminasa/metabolismo , Animales , Emparejamiento Base , Secuencia de Bases , Cartilla de ADN , Nucleasa Microcócica/genética , Nucleasa Microcócica/metabolismo , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Edición de ARN/genética , Interferencia de ARN , ARN Bicatenario/metabolismo , Complejo Silenciador Inducido por ARN/genética , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/fisiologíaRESUMEN
Regulated switching of the mutually exclusive exons 2 and 3 of alpha-tropomyosin (TM) involves repression of exon 3 in smooth muscle cells. Polypyrimidine tract-binding protein (PTB) is necessary but not sufficient for regulation of TM splicing. Raver1 was identified in two-hybrid screens by its interactions with the cytoskeletal proteins actinin and vinculin, and was also found to interact with PTB. Consistent with these interactions raver1 can be localized in either the nucleus or cytoplasm. Here we show that raver1 is able to promote the smooth muscle-specific alternative splicing of TM by enhancing PTB-mediated repression of exon 3. This activity of raver1 is dependent upon characterized PTB-binding regulatory elements and upon a region of raver1 necessary for interaction with PTB. Heterologous recruitment of raver1, or just its C-terminus, induced very high levels of exon 3 skipping, bypassing the usual need for PTB binding sites downstream of exon 3. This suggests a novel mechanism for PTB-mediated splicing repression involving recruitment of raver1 as a potent splicing co-repressor.
Asunto(s)
Empalme Alternativo/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteína de Unión al Tracto de Polipirimidina/genética , Tropomiosina/genética , Animales , Secuencia de Bases , Sitios de Unión , Cartilla de ADN , Exones , Vectores Genéticos , Ligandos , Ratones , Sistemas de Lectura Abierta , Reacción en Cadena de la Polimerasa , Proteína de Unión al Tracto de Polipirimidina/metabolismo , Proteínas de Unión al ARN , Proteínas Recombinantes/metabolismo , Proteínas Represoras/metabolismo , Ribonucleoproteínas , TransfecciónRESUMEN
RNA interference (RNAi) and adenosine to inosine conversion are both mechanisms that respond to double-stranded RNA (dsRNA) and have been suggested to have antiviral roles. RNAi involves processing of dsRNA to short interfering RNAs (siRNAs), which subsequently mediate degradation of the cognate mRNAs. Deamination of adenosines changes the coding capacity of the RNA, as inosine is decoded as guanosine, and alters the structure because A-U base pairs are replaced by I*U wobble pairs. Here we show that RNAi is inhibited if the triggering dsRNA is first deaminated by ADAR2. Moreover, we show that production of siRNAs is progressively inhibited with increasing deamination and that this is sufficient to explain the inhibition of RNAi upon hyper-editing of dsRNAs.
Asunto(s)
Adenosina/metabolismo , Inosina/metabolismo , Edición de ARN/genética , ARN Bicatenario/antagonistas & inhibidores , ARN Bicatenario/metabolismo , Adenosina Desaminasa/metabolismo , Animales , Drosophila/genética , Electroforesis en Gel de Poliacrilamida , ARN Bicatenario/biosíntesis , ARN Bicatenario/genética , ARN Interferente Pequeño , ARN no Traducido/biosíntesis , ARN no Traducido/genética , ARN no Traducido/metabolismo , Proteínas de Unión al ARN , Transcripción Genética/genéticaRESUMEN
Extended double-stranded DNA (dsRNA) duplexes can be hyper-edited by adenosine deaminases that act on RNA (ADARs). Long uninterrupted dsRNA is relatively uncommon in cells, and is frequently associated with infection by DNA or RNA viruses. Moreover, extensive adenosine to inosine editing has been reported for various viruses. A number of cellular antiviral defence strategies are stimulated by dsRNA. An additional mechanism to remove dsRNA from cells may involve hyper-editing of dsRNA by ADARs, followed by targeted cleavage. We describe here a cytoplasmic endonuclease activity that specifically cleaves hyper-edited dsRNA. Cleavage occurs at specific sites consisting of alternating IU and UI base pairs. In contrast, unmodified dsRNA and even deaminated dsRNAs that contain four consecutive IU base pairs are not cleaved. Moreover, dsRNAs in which alternating IU and UI base pairs are replaced by isomorphic GU and UG base pairs are not cleaved. Thus, the cleavage of deaminated dsRNA appears to require an RNA structure that is unique to hyper-edited RNA, providing a molecular target for the disposal of hyper-edited viral RNA.
Asunto(s)
Adenosina Desaminasa/metabolismo , Edición de ARN , ARN Bicatenario/metabolismo , Animales , Emparejamiento Base , Secuencia de Bases , Citoplasma/enzimología , Guanina , Células HeLa , Humanos , Datos de Secuencia Molecular , Proteínas de Unión al ARN , Ribonucleasas/metabolismo , Especificidad por Sustrato , Uracilo , Xenopus laevis/genéticaRESUMEN
RNA transcripts in which all guanosine residues are replaced by inosine are degraded at a highly accelerated rate when incubated in extracts from HeLa cells, sheep uterus or pig brain. We report here the partial purification and characterization of a novel ribonuclease, referred to as I-RNase, that is responsible for the degradation of inosine-containing RNA (I-RNA). I-RNase is Mg2+ dependent and specifically degrades single-stranded I-RNA. Comparison of the Km of the enzyme for I-RNA with the Ki for inhibition by normal RNA suggests a approximately 300-fold preferential binding to I-RNA, which can account for the specificity of degradation. The site of cleavage by I-RNase is non-specific; I-RNase acts as a 3'-->5' exonuclease generating 5'-NMPs as products. The presence of alternative unconventional nucleotides in RNA does not result in degradation unless inosine residues are also present. We show that I-RNase is able to degrade RNAs that previously have been modified by the RED-1 double-stranded RNA adenosine deaminase (dsRAD). dsRADs destabilize dsRNA by converting adenosine to inosine, and some of these enzymes are interferon inducible. We therefore speculate that I-RNase in concert with dsRAD may form part of a novel cellular antiviral defence mechanism that acts to degrade dsRNA.