RESUMEN
Modified bases act as marks on cellular RNAs so that they can be distinguished from foreign RNAs, reducing innate immune responses to endogenous RNA. In humans, mutations giving reduced levels of one base modification, adenosine-to-inosine deamination, cause a viral infection mimic syndrome, a congenital encephalitis with aberrant interferon induction. These Aicardi-Goutières syndrome 6 mutations affect adenosine deaminase acting on RNA 1 (ADAR1), which generates inosines in endogenous double-stranded (ds)RNA. The inosine base alters dsRNA structure to prevent aberrant activation of antiviral cytosolic helicase RIG-I-like receptors. We review how effects of inosines, ADARs, and other modified bases have been shown to be important in innate immunity and cancer.
Asunto(s)
Inmunidad Innata , Edición de ARN , Proteínas de Unión al ARN , Adenosina Desaminasa/genética , Adenosina Desaminasa/metabolismo , Humanos , ARN Bicatenario , Proteínas de Unión al ARN/metabolismo , TranscriptomaRESUMEN
The adenosine deaminase acting on RNA (ADAR) enzymes are essential for neuronal function and innate immune control. ADAR1 RNA editing prevents aberrant activation of antiviral dsRNA sensors through editing of long, double-stranded RNAs (dsRNAs). In this review, we focus on the ADAR2 proteins involved in the efficient, highly site-specific RNA editing to recode open reading frames first discovered in the GRIA2 transcript encoding the key GLUA2 subunit of AMPA receptors; ADAR1 proteins also edit many of these sites. We summarize the history of ADAR2 protein research and give an up-to-date review of ADAR2 structural studies, human ADARBI (ADAR2) mutants causing severe infant seizures, and mouse disease models. Structural studies on ADARs and their RNA substrates facilitate current efforts to develop ADAR RNA editing gene therapy to edit disease-causing single nucleotide polymorphisms (SNPs). Artificial ADAR guide RNAs are being developed to retarget ADAR RNA editing to new target transcripts in order to correct SNP mutations in them at the RNA level. Site-specific RNA editing has been expanded to recode hundreds of sites in CNS transcripts in Drosophila and cephalopods. In Drosophila and C. elegans, ADAR RNA editing also suppresses responses to self dsRNA.
Asunto(s)
Adenosina Desaminasa , Adenosina Desaminasa/metabolismo , Animales , Antivirales , Caenorhabditis elegans/genética , Drosophila/genética , Terapia Genética , Humanos , Ratones , ARN Bicatenario/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Receptores AMPA/genética , Receptores AMPA/metabolismoRESUMEN
ConspectusThe adenosine deaminase acting on RNA (ADAR) enzymes that catalyze the conversion of adenosine to inosine in double-stranded (ds)RNA are evolutionarily conserved and are essential for many biological functions including nervous system function, hematopoiesis, and innate immunity. Initially it was assumed that the wide-ranging biological roles of ADARs are due to inosine in mRNA being read as guanosine by the translational machinery, allowing incomplete RNA editing in a target codon to generate two different proteins from the same primary transcript. In humans, there are approximately seventy-six positions that undergo site-specific editing in tissues at greater than 20% efficiency that result in recoding. Many of these transcripts are expressed in the central nervous system (CNS) and edited by ADAR2. Exploiting mouse genetic models revealed that transgenic mice lacking the gene encoding Adar2 die within 3 weeks of birth. Therefore, the role of ADAR2 in generating protein diversity in the nervous system is clear, but why is ADAR RNA editing activity essential in other biological processes, particularly editing mainly involving ADAR1? ADAR1 edits human transcripts having embedded Alu element inverted repeats (AluIRs), but the link from this activity to innate immunity activation was elusive. Mice lacking the gene encoding Adar1 are embryonically lethal, and a major breakthrough was the discovery that the role of Adar1 in innate immunity is due to its ability to edit such repetitive element inverted repeats which have the ability to form dsRNA in transcripts. The presence of inosine prevents activation of the dsRNA sensor melanoma differentiation-associated protein 5 (Mda5). Thus, inosine helps the cell discriminate self from non-self RNA, acting like a barcode on mRNA. As innate immunity is key to many different biological processes, the basis for this widespread biological role of the ADAR1 enzyme became evident.Our group has been studying ADARs from the outset of research on these enzymes. In this Account, we give a historical perspective, moving from the initial purification of ADAR1 and ADAR2 and cloning of their encoding genes up to the current research focus in the field and what questions still remain to be addressed. We discuss the characterizations of the proteins, their localizations, posttranslational modifications, and dimerization, and how all of these affect their biological activities. Another aspect we explore is the use of mouse and Drosophila genetic models to study ADAR functions and how these were crucial in determining the biological functions of the ADAR proteins. Finally, we describe the severe consequences of rare mutations found in the human genes encoding ADAR1 and ADAR2.
Asunto(s)
Adenosina Desaminasa , ARN Bicatenario , Animales , Ratones , Humanos , Adenosina Desaminasa/genética , Adenosina Desaminasa/metabolismo , ARN Bicatenario/genética , Inmunidad Innata , ARN Mensajero/genética , Inosina/genética , Inosina/metabolismoRESUMEN
ADAR1 edits adenosines to inosines in cellular double-stranded (ds)RNA, thereby preventing aberrant activation of antiviral dsRNA sensors, as well as interferon (IFN) induction in Aicardi-Goutières syndrome (AGS) encephalopathy. Recently, Nakahama et al., Tang et al., Maurano et al., and de Reuver et al. demonstrated that Adar1 Zα domain-mutant mice show aberrant MDA5 and PKR activation, developing encephalopathies; short Z-RNA patches within cellular dsRNA are unexpectedly crucial in causing aberrant antiviral responses.
Asunto(s)
Adenosina Desaminasa , Enfermedades Autoinmunes del Sistema Nervioso , Adenosina Desaminasa/genética , Adenosina Desaminasa/metabolismo , Animales , Antivirales , Enfermedades Autoinmunes del Sistema Nervioso/genética , Humanos , Ratones , Edición de ARN , ARN BicatenarioRESUMEN
The RNA editing enzyme ADAR2 is essential for the recoding of brain transcripts. Impaired ADAR2 editing leads to early-onset epilepsy and premature death in a mouse model. Here, we report bi-allelic variants in ADARB1, the gene encoding ADAR2, in four unrelated individuals with microcephaly, intellectual disability, and epilepsy. In one individual, a homozygous variant in one of the double-stranded RNA-binding domains (dsRBDs) was identified. In the others, variants were situated in or around the deaminase domain. To evaluate the effects of these variants on ADAR2 enzymatic activity, we performed in vitro assays with recombinant proteins in HEK293T cells and ex vivo assays with fibroblasts derived from one of the individuals. We demonstrate that these ADAR2 variants lead to reduced editing activity on a known ADAR2 substrate. We also demonstrate that one variant leads to changes in splicing of ADARB1 transcript isoforms. These findings reinforce the importance of RNA editing in brain development and introduce ADARB1 as a genetic etiology in individuals with intellectual disability, microcephaly, and epilepsy.
Asunto(s)
Adenosina Desaminasa/genética , Predisposición Genética a la Enfermedad/genética , Variación Genética/genética , Discapacidad Intelectual/genética , Microcefalia/genética , Proteínas de Unión al ARN/genética , Convulsiones/genética , Alelos , Empalme Alternativo/genética , Niño , Preescolar , Células HEK293 , Humanos , Masculino , Empalme del ARN/genéticaRESUMEN
Adenosine-to-inosine (A-to-I) RNA editing is a conserved post-transcriptional mechanism mediated by ADAR enzymes that diversifies the transcriptome by altering selected nucleotides in RNA molecules. Although many editing sites have recently been discovered, the extent to which most sites are edited and how the editing is regulated in different biological contexts are not fully understood. Here we report dynamic spatiotemporal patterns and new regulators of RNA editing, discovered through an extensive profiling of A-to-I RNA editing in 8,551 human samples (representing 53 body sites from 552 individuals) from the Genotype-Tissue Expression (GTEx) project and in hundreds of other primate and mouse samples. We show that editing levels in non-repetitive coding regions vary more between tissues than editing levels in repetitive regions. Globally, ADAR1 is the primary editor of repetitive sites and ADAR2 is the primary editor of non-repetitive coding sites, whereas the catalytically inactive ADAR3 predominantly acts as an inhibitor of editing. Cross-species analysis of RNA editing in several tissues revealed that species, rather than tissue type, is the primary determinant of editing levels, suggesting stronger cis-directed regulation of RNA editing for most sites, although the small set of conserved coding sites is under stronger trans-regulation. In addition, we curated an extensive set of ADAR1 and ADAR2 targets and showed that many editing sites display distinct tissue-specific regulation by the ADAR enzymes in vivo. Further analysis of the GTEx data revealed several potential regulators of editing, such as AIMP2, which reduces editing in muscles by enhancing the degradation of the ADAR proteins. Collectively, our work provides insights into the complex cis- and trans-regulation of A-to-I editing.
Asunto(s)
Adenosina Desaminasa , Primates/genética , Edición de ARN/genética , Proteínas de Unión al ARN , Adenosina Desaminasa/genética , Adenosina Desaminasa/metabolismo , Animales , Femenino , Genotipo , Células HEK293 , Humanos , Masculino , Ratones , Músculos/metabolismo , Proteínas Nucleares/metabolismo , Especificidad de Órganos/genética , Proteolisis , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Análisis Espacio-Temporal , Especificidad de la Especie , Transcriptoma/genéticaRESUMEN
BACKGROUND: Adenosine-to-inosine RNA editing is a co-transcriptional/post-transcriptional modification of double-stranded RNA, catalysed by one of two active adenosine deaminases acting on RNA (ADARs), ADAR1 and ADAR2. ADARB1 encodes the enzyme ADAR2 that is highly expressed in the brain and essential to modulate the function of glutamate and serotonin receptors. Impaired ADAR2 editing causes early onset progressive epilepsy and premature death in mice. In humans, ADAR2 dysfunction has been very recently linked to a neurodevelopmental disorder with microcephaly and epilepsy in four unrelated subjects. METHODS: We studied three children from two consanguineous families with severe developmental and epileptic encephalopathy (DEE) through detailed physical and instrumental examinations. Exome sequencing (ES) was used to identify ADARB1 mutations as the underlying genetic cause and in vitro assays with transiently transfected cells were performed to ascertain the impact on ADAR2 enzymatic activity and splicing. RESULTS: All patients showed global developmental delay, intractable early infantile-onset seizures, microcephaly, severe-to-profound intellectual disability, axial hypotonia and progressive appendicular spasticity. ES revealed the novel missense c.1889G>A, p.(Arg630Gln) and deletion c.1245_1247+1 del, p.(Leu415PhefsTer14) variants in ADARB1 (NM_015833.4). The p.(Leu415PhefsTer14) variant leads to incorrect splicing resulting in frameshift with a premature stop codon and loss of enzyme function. In vitro RNA editing assays showed that the p.(Arg630Gln) variant resulted in a severe impairment of ADAR2 enzymatic activity. CONCLUSION: In conclusion, these data support the pathogenic role of biallelic ADARB1 variants as the cause of a distinctive form of DEE, reinforcing the importance of RNA editing in brain function and development.
Asunto(s)
Adenosina Desaminasa/genética , Adenosina Desaminasa/metabolismo , Encefalopatías/genética , Epilepsia/genética , Trastornos del Neurodesarrollo/genética , ARN Bicatenario/metabolismo , Proteínas de Unión al ARN/genética , Alelos , Encefalopatías/enzimología , Encefalopatías/metabolismo , Niño , Preescolar , Consanguinidad , Epilepsia/enzimología , Femenino , Células HEK293 , Humanos , Mutación , Trastornos del Neurodesarrollo/enzimología , Linaje , Edición de ARN , Proteínas de Unión al ARN/metabolismoRESUMEN
Viral and cellular double-stranded RNA (dsRNA) is recognized by cytosolic innate immune sensors, including RIG-I-like receptors. Some cytoplasmic dsRNA is commonly present in cells, and one source is mitochondrial dsRNA, which results from bidirectional transcription of mitochondrial DNA (mtDNA). Here we demonstrate that Trp53 mutant mouse embryonic fibroblasts contain immune-stimulating endogenous dsRNA of mitochondrial origin. We show that the immune response induced by this dsRNA is mediated via RIG-I-like receptors and leads to the expression of type I interferon and proinflammatory cytokine genes. The mitochondrial dsRNA is cleaved by RNase L, which cleaves all cellular RNA including mitochondrial mRNAs, increasing activation of RIG-I-like receptors. When mitochondrial transcription is interrupted there is a subsequent decrease in this immune-stimulatory dsRNA. Our results reveal that the role of p53 in innate immunity is even more versatile and complex than previously anticipated. Our study, therefore, sheds new light on the role of endogenous RNA in diseases featuring aberrant immune responses.
Asunto(s)
Adenosina Desaminasa/genética , Proteína 58 DEAD Box/genética , Inmunidad Innata/genética , ARN Bicatenario/genética , ARN Mitocondrial/genética , Proteína p53 Supresora de Tumor/genética , Proteínas Adaptadoras Transductoras de Señales , Adenosina Desaminasa/deficiencia , Adenosina Desaminasa/inmunología , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Proteína 58 DEAD Box/inmunología , Embrión de Mamíferos , Endorribonucleasas/genética , Endorribonucleasas/inmunología , Fibroblastos/citología , Fibroblastos/inmunología , Factor 7 Regulador del Interferón/genética , Factor 7 Regulador del Interferón/inmunología , Helicasa Inducida por Interferón IFIH1/genética , Helicasa Inducida por Interferón IFIH1/inmunología , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas/genética , Proteínas/inmunología , ARN Bicatenario/inmunología , ARN Mitocondrial/inmunología , Proteínas de Unión al ARN , Transcripción Genética , Transfección , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/inmunologíaRESUMEN
Eukaryotic mRNAs are modified by several chemical marks which have significant impacts on mRNA biology, gene expression, and cellular metabolism as well as on the survival and development of the whole organism. The most abundant and well-studied mRNA base modifications are m6A and ADAR RNA editing. Recent studies have also identified additional mRNA marks such as m6Am, m5C, m1A and Ψ and studied their roles. Each type of modification is deposited by a specific writer, many types of modification are recognized and interpreted by several different readers and some types of modifications can be removed by eraser enzymes. Several works have addressed the functional relationships between some of the modifications. In this review we provide an overview on the current status of research on the different types of mRNA modifications and about the crosstalk between different marks and its functional consequences.
Asunto(s)
Epigénesis Genética , Epigenómica/métodos , Procesamiento Postranscripcional del ARN , ARN Mensajero/metabolismo , Transcriptoma , Animales , Humanos , ARN Mensajero/genéticaRESUMEN
The universal nine-amino-acid transactivation domains (9aaTADs) have been identified in numerous transcription activators. Here, we identified the conserved 9aaTAD motif in all nine members of the specificity protein (SP) family. Previously, the Sp1 transcription factor has been defined as a glutamine-rich activator. We showed by amino acid substitutions that the glutamine residues are completely dispensable for 9aaTAD function and are not conserved in the SP family. We described the origin and evolutionary history of 9aaTADs. The 9aaTADs of the ancestral Sp2 gene became inactivated in early chordates. We next discovered that an accumulation of valines in 9aaTADs inactivated their transactivation function and enabled their strict conservation during evolution. Subsequently, in chordates, Sp2 has duplicated and created new paralogs, Sp1, Sp3, and Sp4 (the SP1-4 clade). During chordate evolution, the dormancy of the Sp2 activation domain lasted over 100 million years. The dormant but still intact ancestral Sp2 activation domains allowed diversification of the SP1-4 clade into activators and repressors. By valine substitution in the 9aaTADs, Sp1 and Sp3 regained their original activator function found in ancestral lower metazoan sea sponges. Therefore, the vertebrate SP1-4 clade could include both repressors and activators. Furthermore, we identified secondary 9aaTADs in Sp2 introns present from fish to primates, including humans. In the gibbon genome, introns containing 9aaTADs were used as exons, which turned the Sp2 gene into an activator. Similarly, we identified introns containing 9aaTADs used conditionally as exons in the (SP family-unrelated) transcription factor SREBP1, suggesting that the intron-9aaTAD reservoir is a general phenomenon.
Asunto(s)
Evolución Molecular , Regulación de la Expresión Génica , Intrones/genética , Factor de Transcripción Sp2/antagonistas & inhibidores , Factor de Transcripción Sp2/genética , Valina/metabolismo , Secuencia de Aminoácidos , Animales , Duplicación de Gen , Humanos , Filogenia , Homología de Secuencia , Factor de Transcripción Sp2/metabolismo , Activación Transcripcional , Valina/genéticaRESUMEN
BACKGROUND: In fly brains, the Drosophila Adar (adenosine deaminase acting on RNA) enzyme edits hundreds of transcripts to generate edited isoforms of encoded proteins. Nearly all editing events are absent or less efficient in larvae but increase at metamorphosis; the larger number and higher levels of editing suggest editing is most required when the brain is most complex. This idea is consistent with the fact that Adar mutations affect the adult brain most dramatically. However, it is unknown whether Drosophila Adar RNA editing events mediate some coherent physiological effect. To address this question, we performed a genetic screen for suppressors of Adar mutant defects. Adar5G1 null mutant flies are partially viable, severely locomotion defective, aberrantly accumulate axonal neurotransmitter pre-synaptic vesicles and associated proteins, and develop an age-dependent vacuolar brain neurodegeneration. RESULTS: A genetic screen revealed suppression of all Adar5G1 mutant phenotypes tested by reduced dosage of the Tor gene, which encodes a pro-growth kinase that increases translation and reduces autophagy in well-fed conditions. Suppression of Adar5G1 phenotypes by reduced Tor is due to increased autophagy; overexpression of Atg5, which increases canonical autophagy initiation, reduces aberrant accumulation of synaptic vesicle proteins and suppresses all Adar mutant phenotypes tested. Endosomal microautophagy (eMI) is another Tor-inhibited autophagy pathway involved in synaptic homeostasis in Drosophila. Increased expression of the key eMI protein Hsc70-4 also reduces aberrant accumulation of synaptic vesicle proteins and suppresses all Adar5G1 mutant phenotypes tested. CONCLUSIONS: These findings link Drosophila Adar mutant synaptic and neurotransmission defects to more general cellular defects in autophagy; presumably, edited isoforms of CNS proteins are required for optimum synaptic response capabilities in the brain during the behaviorally complex adult life stage.
Asunto(s)
Adenosina Desaminasa/genética , Autofagia , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiología , Transmisión Sináptica/genética , Adenosina Desaminasa/metabolismo , Animales , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Larva/genética , Larva/crecimiento & desarrollo , Larva/fisiología , Masculino , MutaciónRESUMEN
Our knowledge of the variety and abundances of RNA base modifications is rapidly increasing. Modified bases have critical roles in tRNAs, rRNAs, translation, splicing, RNA interference, and other RNA processes, and are now increasingly detected in all types of transcripts. Can new biological principles associated with this diversity of RNA modifications, particularly in mRNAs and long non-coding RNAs, be identified? This review will explore this question by focusing primarily on adenosine to inosine (A-to-I) RNA editing by the adenine deaminase acting on RNA (ADAR) enzymes that have been intensively studied for the past 20 years and have a wide range of effects. Over 100 million adenosine to inosine editing sites have been identified in the human transcriptome, mostly in embedded Alu sequences that form potentially innate immune-stimulating dsRNA hairpins in transcripts. Recent research has demonstrated that inosine in the epitranscriptome and ADAR1 protein establish innate immune tolerance for host dsRNA formed by endogenous sequences. Innate immune sensors that detect viral nucleic acids are among the readers of epitranscriptome RNA modifications, though this does preclude a wide range of other modification effects.
Asunto(s)
Adenosina Desaminasa/genética , Aminohidrolasas/genética , Inmunidad Innata/genética , Proteínas de Unión al ARN/genética , Transcriptoma/genética , Adenosina/genética , Humanos , Inosina/genética , Edición de ARN/genética , Procesamiento Postranscripcional del ARN/genética , ARN Mensajero/genéticaRESUMEN
We review the structures and functions of ADARs and their involvements in human diseases. ADAR1 is widely expressed, particularly in the myeloid component of the blood system, and plays a prominent role in promiscuous editing of long dsRNA. Missense mutations that change ADAR1 residues and reduce RNA editing activity cause Aicardi-Goutières Syndrome, a childhood encephalitis and interferonopathy that mimics viral infection and resembles an extreme form of Systemic Lupus Erythmatosus (SLE). In Adar1 mouse mutant models aberrant interferon expression is prevented by eliminating interferon activation signaling from cytoplasmic dsRNA sensors, indicating that unedited cytoplasmic dsRNA drives the immune induction. On the other hand, upregulation of ADAR1 with widespread promiscuous RNA editing is a prominent feature of many cancers and particular site-specific RNA editing events are also affected. ADAR2 is most highly expressed in brain and is primarily required for site-specific editing of CNS transcripts; recent findings indicate that ADAR2 editing is regulated by neuronal excitation for synaptic scaling of glutamate receptors. ADAR2 is also linked to the circadian clock and to sleep. Mutations in ADAR2 could contribute to excitability syndromes such as epilepsy, to seizures, to diseases involving neuronal plasticity defects, such as autism and Fragile-X Syndrome, to neurodegenerations such as ALS, or to astrocytomas or glioblastomas in which reduced ADAR2 activity is required for oncogenic cell behavior. The range of human disease associated with ADAR1 mutations may extend further to include other inflammatory conditions while ADAR2 mutations may affect psychiatric conditions.
Asunto(s)
Adenosina Desaminasa , Trastornos Mentales , Mutación , Enfermedades del Sistema Nervioso , Edición de ARN/genética , ARN Bicatenario , Proteínas de Unión al ARN , Adenosina Desaminasa/genética , Adenosina Desaminasa/metabolismo , Animales , Humanos , Trastornos Mentales/genética , Trastornos Mentales/metabolismo , Ratones , Ratones Mutantes , Enfermedades del Sistema Nervioso/genética , Enfermedades del Sistema Nervioso/metabolismo , ARN Bicatenario/genética , ARN Bicatenario/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
We show that RNA editing sites can be called with high confidence using RNA sequencing data from multiple samples across either individuals or species, without the need for matched genomic DNA sequence. We identified many previously unidentified editing sites in both humans and Drosophila; our results nearly double the known number of human protein recoding events. We also found that human genes harboring conserved editing sites within Alu repeats are enriched for neuronal functions.
Asunto(s)
Edición de ARN , Análisis de Secuencia de ARN/métodos , Adenosina/genética , Elementos Alu , Animales , Biología Computacional/métodos , Drosophila melanogaster/genética , Humanos , Inosina/genéticaRESUMEN
RNA editing by deamination of specific adenosine bases to inosines during pre-mRNA processing generates edited isoforms of proteins. Recoding RNA editing is more widespread in Drosophila than in vertebrates. Editing levels rise strongly at metamorphosis, and Adar(5G1) null mutant flies lack editing events in hundreds of CNS transcripts; mutant flies have reduced viability, severely defective locomotion and age-dependent neurodegeneration. On the other hand, overexpressing an adult dADAR isoform with high enzymatic activity ubiquitously during larval and pupal stages is lethal. Advantage was taken of this to screen for genetic modifiers; Adar overexpression lethality is rescued by reduced dosage of the Rdl (Resistant to dieldrin), gene encoding a subunit of inhibitory GABA receptors. Reduced dosage of the Gad1 gene encoding the GABA synthetase also rescues Adar overexpression lethality. Drosophila Adar(5G1) mutant phenotypes are ameliorated by feeding GABA modulators. We demonstrate that neuronal excitability is linked to dADAR expression levels in individual neurons; Adar-overexpressing larval motor neurons show reduced excitability whereas Adar(5G1) null mutant or targeted Adar knockdown motor neurons exhibit increased excitability. GABA inhibitory signalling is impaired in human epileptic and autistic conditions, and vertebrate ADARs may have a relevant evolutionarily conserved control over neuronal excitability.
Asunto(s)
Adenosina Desaminasa/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimología , Neuronas Motoras/enzimología , Potenciales de Acción , Adenosina Desaminasa/genética , Animales , Cromosomas de Insectos , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/fisiología , Genes Letales , Genotipo , Larva/enzimología , Neuronas Motoras/fisiología , Fenotipo , Edición de ARN , Receptores de GABA-A/genética , Transducción de Señal , Ácido gamma-Aminobutírico/metabolismoRESUMEN
ADAR2 catalyses the deamination of adenosine to inosine at the GluR2 Q/R site in the pre-mRNA encoding the critical subunit of AMPA receptors. Among ADAR2 substrates this is the vital one as editing at this position is indispensable for normal brain function. However, the regulation of ADAR2 post-translationally remains to be elucidated. We demonstrate that the phosphorylation-dependent prolyl-isomerase Pin1 interacts with ADAR2 and is a positive regulator required for the nuclear localization and stability of ADAR2. Pin1(-/-) mouse embryonic fibroblasts show mislocalization of ADAR2 in the cytoplasm and reduced editing at the GluR2 Q/R and R/G sites. The E3 ubiquitin ligase WWP2 plays a negative role by binding to ADAR2 and catalysing its ubiquitination and subsequent degradation. Therefore, ADAR2 protein levels and catalytic activity are coordinately regulated in a positive manner by Pin1 and negatively by WWP2 and this may have downstream effects on the function of GluR2. Pin1 and WWP2 also regulate the large subunit of RNA Pol II, so these proteins may also coordinately regulate other key cellular proteins.
Asunto(s)
Adenosina Desaminasa/metabolismo , Isomerasa de Peptidilprolil/metabolismo , Edición de ARN , Receptores AMPA/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Línea Celular , Fibroblastos/metabolismo , Ratones , Peptidilprolil Isomerasa de Interacción con NIMA , ARN Polimerasa II/metabolismo , Proteínas de Unión al ARN , UbiquitinaciónRESUMEN
Nearly 150 different enzymatically modified forms of the four canonical residues in RNA have been identified. For instance, enzymes of the ADAR (adenosine deaminase acting on RNA) family convert adenosine residues into inosine in cellular dsRNAs. Recent findings show that DNA endonuclease V enzymes have undergone an evolutionary transition from cleaving 3' to deoxyinosine in DNA and ssDNA to cleaving 3' to inosine in dsRNA and ssRNA in humans. Recent work on dsRNA-binding domains of ADARs and other proteins also shows that a degree of sequence specificity is achieved by direct readout in the minor groove. However, the level of sequence specificity observed is much less than that of DNA major groove-binding helix-turn-helix proteins. We suggest that the evolution of DNA-binding proteins following the RNA to DNA genome transition represents the major advantage that DNA genomes have over RNA genomes. We propose that a hypothetical RNA modification, a RRAR (ribose reductase acting on genomic dsRNA) produced the first stretches of DNA in RNA genomes. We discuss why this is the most satisfactory explanation for the origin of DNA. The evolution of this RNA modification and later steps to DNA genomes are likely to have been driven by cellular genome co-evolution with viruses and intragenomic parasites. RNA modifications continue to be involved in host-virus conflicts; in vertebrates, edited cellular dsRNAs with inosine-uracil base pairs appear to be recognized as self RNA and to suppress activation of innate immune sensors that detect viral dsRNA.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , ARN/metabolismo , Animales , Drosophila , Evolución Molecular , Edición de ARN/fisiologíaRESUMEN
Adar null mutant mouse embryos die with aberrant double-stranded RNA (dsRNA)-driven interferon induction, and Adar Mavs double mutants, in which interferon induction is prevented, die soon after birth. Protein kinase R (Pkr) is aberrantly activated in Adar Mavs mouse pup intestines before death, intestinal crypt cells die, and intestinal villi are lost. Adar Mavs Eifak2 (Pkr) triple mutant mice rescue all defects and have long-term survival. Adenosine deaminase acting on RNA 1 (ADAR1) and PKR co-immunoprecipitate from cells, suggesting PKR inhibition by direct interaction. AlphaFold studies on an inhibitory PKR dsRNA binding domain (dsRBD)-kinase domain interaction before dsRNA binding and on an inhibitory ADAR1 dsRBD3-PKR kinase domain interaction on dsRNA provide a testable model of the inhibition. Wild-type or editing-inactive human ADAR1 expressed in A549 cells inhibits activation of endogenous PKR. ADAR1 dsRNA binding is required for, but is not sufficient for, PKR inhibition. Mutating the ADAR1 dsRBD3-PKR contact prevents co-immunoprecipitation, ADAR1 inhibition of PKR activity, and co-localization of ADAR1 and PKR in cells.
RESUMEN
Adenosine deaminases acting on RNA (ADARs) are best known for altering the coding sequences of mRNA through RNA editing, as in the GluR-B Q/R site. ADARs have also been shown to affect RNA interference (RNAi) and microRNA processing by deamination of specific adenosines to inosine. Here, we show that ADAR proteins can affect RNA processing independently of their enzymatic activity. We show that ADAR2 can modulate the processing of mir-376a2 independently of catalytic RNA editing activity. In addition, in a Drosophila assay for RNAi deaminase-inactive ADAR1 inhibits RNAi through the siRNA pathway. These results imply that ADAR1 and ADAR2 have biological functions as RNA-binding proteins that extend beyond editing per se and that even genomically encoded ADARs that are catalytically inactive may have such functions.
Asunto(s)
Adenosina Desaminasa/metabolismo , MicroARNs/genética , Edición de ARN/genética , ARN Interferente Pequeño/genética , Transducción de Señal/fisiología , Adenosina Desaminasa/genética , Animales , Northern Blotting , Línea Celular , Drosophila , Humanos , Interferencia de ARN , Proteínas de Unión al ARN , Transducción de Señal/genéticaRESUMEN
Drosophila melanogaster has a single Adar gene encoding a protein related to mammalian ADAR2 that edits transcripts encoding glutamate receptor subunits. We describe the structure of the Drosophila Adar locus and use ModENCODE information to supplement published data on Adar gene transcription, and splicing. We discuss the roles of ADAR in Drosophila in terms of the two main types of RNA molecules edited and roles of ADARs as RNA-binding proteins. Site-specific RNA editing events in transcripts encoding ion channel subunits were initially found serendipitously and subsequent directed searches for editing sites and transcriptome sequencing have now led to 972 edited sites being identified in 597 transcripts. Four percent of D. melanogaster transcripts are site-specifically edited and these encode a wide range of largely membrane-associated proteins expressed particularly in CNS. Electrophysiological studies on the effects of specific RNA editing events on ion channel subunits do not suggest that loss of RNA editing events in ion channels consistently produce a particular outcome such as making Adar mutant neurons more excitable. This possibility would have been consistent with neurodegeneration seen in Adar mutant fly brains. A further set of ADAR targets are dsRNA intermediates in siRNA generation, derived from transposons and from structured RNA loci. Transcripts with convergent overlapping 3' ends are also edited and the first discovered instance of RNA editing in Drosophila, in the Rnp4F transcript, is an example. There is no evidence yet to show that Adar antagonizes RNA interference in Drosophila. Evidence has been obtained that catalytically inactive ADAR proteins exert effects on microRNA generation and RNA interference. Whether all effects of inactive ADARs are due to RNA-binding or to even further roles of these proteins remains to be determined.