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1.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 36(11): 1097-1099, 2019 Nov 10.
Artigo em Chinês | MEDLINE | ID: mdl-31703134

RESUMO

OBJECTIVE: To explore the genetic etiology of two unrelated patients with dyschromatosis symmetrica hereditaria. METHODS: Variant analysis of the ADAR gene was carried out by Sanger sequencing. RESULTS: Patient 1 was found to harbor a c.2633_2634delCT (p.Ser878fs) in exon 8 of the ADAR gene. The same variant was not found among 100 unrelated individuals. No pathogenic variant of the ADAR gene was found in patient 2. Functional prediction of the ADAR c.2633_2634delCT (p.Ser878fs) variant indicated it to be pathogenic by losing a catalytic structural domain. CONCLUSION: The c.2633_2634delCT (p.Ser878fs) variant of the ADAR gene probably underlies the pathogenesis of DSH in one of the patients.


Assuntos
Adenosina Desaminase/genética , Transtornos da Pigmentação/congênito , Proteínas de Ligação a RNA/genética , Humanos , Mutação , Linhagem , Transtornos da Pigmentação/genética , Tomografia Computadorizada por Raios X
2.
Chem Biol Interact ; 311: 108796, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31421116

RESUMO

Lambda-cyhalothrin (LCT) is a broad-spectrum pesticide widely used in agriculture throughout the world. This pesticide is considered a potential contaminant of surface and underground water as well as food, posing a risk to ecosystems and humans. In this sense, we decided to evaluate the activity of enzymes belonging to the purinergic system, which is linked with regulation of extracellular nucleotides and nucleosides, such as adenosine triphosphate (ATP) and adenosine (Ado) molecules involved in the regulation of inflammatory response. However, there are no data concerning the effects of LCT exposure on the purinergic system, where extracellular nucleotides act as signaling molecules. The aim of this study was to evaluate nucleotide hydrolysis by E-NTPDase (ecto-nucleoside triphosphate diphosphohydrolase), Ecto-NPP (ecto-nucleotide pyrophosphatase/phosphodiesterase), ecto-5'-nucleotidase and ecto-adenosine deaminase (E-ADA) in platelets and liver of adult rats on days 7, 30, 45 and 60 after daily gavage with 6.2 and 31.1 mg/kg bw of LCT. Gene expression patterns of NTPDases1-3 and 5'-nucleotidase were also determined in those tissues. In parallel, lambda-cyhalothrin metabolites [3-(2-chloro-3,3,3- trifluoroprop-1-enyl)-2,2-dimethyl-cyclopropane carboxylic acid (CFMP), 4-hydroxyphenoxybenzoic acid (4-OH-3-PBA), and 3-phenoxybenzoic acid (3-PBA)] were measured in plasma. Results showed that exposure rats to LCT caused a significant increase in the assessed enzymes activities. Gene expression pattern of ectonucleotidases further revealed a significant increase in E-NTPDase1, E-NTPDase2, and E-NTPDase3 mRNA levels after LCT administration at all times. A dose-dependent increase in LCT metabolite levels was also observed but there no significant variations in levels from weeks to week, suggesting steady-steady equilibrium. Correlation analyses revealed that LCT metabolites in the liver and plasma were positively correlated with the adenine nucleotides hydrolyzing enzyme, E-ADA and E-NPP activities in platelets and liver of rats exposed to lambda-cyhalothin. Our results show that LCT and its metabolites may affect purinergic enzymatic cascade and cause alterations in energy metabolism.


Assuntos
Plaquetas/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Fígado/efeitos dos fármacos , Nitrilos/farmacologia , Nucleotidases/genética , Nucleosídeos de Purina/metabolismo , Piretrinas/farmacologia , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Animais , Plaquetas/enzimologia , Plaquetas/metabolismo , Cromatografia Líquida de Alta Pressão , Hidrólise , Fígado/enzimologia , Fígado/metabolismo , Masculino , Espectrometria de Massas , Nitrilos/sangue , Nitrilos/metabolismo , Nucleotidases/metabolismo , Piretrinas/sangue , Piretrinas/metabolismo , Pirofosfatases/genética , Pirofosfatases/metabolismo , Ratos , Ratos Wistar
3.
Nat Biotechnol ; 37(9): 1059-1069, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31308540

RESUMO

Current tools for targeted RNA editing rely on the delivery of exogenous proteins or chemically modified guide RNAs, which may lead to aberrant effector activity, delivery barrier or immunogenicity. Here, we present an approach, called leveraging endogenous ADAR for programmable editing of RNA (LEAPER), that employs short engineered ADAR-recruiting RNAs (arRNAs) to recruit native ADAR1 or ADAR2 enzymes to change a specific adenosine to inosine. We show that arRNA, delivered by a plasmid or viral vector or as a synthetic oligonucleotide, achieves editing efficiencies of up to 80%. LEAPER is highly specific, with rare global off-targets and limited editing of non-target adenosines in the target region. It is active in a broad spectrum of cell types, including multiple human primary cell types, and can restore α-L-iduronidase catalytic activity in Hurler syndrome patient-derived primary fibroblasts without evoking innate immune responses. As a single-molecule system, LEAPER enables precise, efficient RNA editing with broad applicability for therapy and basic research.


Assuntos
Adenosina Desaminase/classificação , Adenosina Desaminase/metabolismo , Edição de RNA , Proteínas de Ligação a RNA/metabolismo , RNA/genética , Adenosina Desaminase/genética , Animais , Linhagem Celular , Engenharia Genética , Humanos , Proteínas de Ligação a RNA/genética
4.
Nat Biotechnol ; 37(9): 1070-1079, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31332326

RESUMO

Base editors use DNA-modifying enzymes targeted with a catalytically impaired CRISPR protein to precisely install point mutations. Here, we develop phage-assisted continuous evolution of base editors (BE-PACE) to improve their editing efficiency and target sequence compatibility. We used BE-PACE to evolve cytosine base editors (CBEs) that overcome target sequence context constraints of canonical CBEs. One evolved CBE, evoAPOBEC1-BE4max, is up to 26-fold more efficient at editing cytosine in the GC context, a disfavored context for wild-type APOBEC1 deaminase, while maintaining efficient editing in all other sequence contexts tested. Another evolved deaminase, evoFERNY, is 29% smaller than APOBEC1 and edits efficiently in all tested sequence contexts. We also evolved a CBE based on CDA1 deaminase with much higher editing efficiency at difficult target sites. Finally, we used data from evolved CBEs to illuminate the relationship between deaminase activity, base editing efficiency, editing window width and byproduct formation. These findings establish a system for rapid evolution of base editors and inform their use and improvement.


Assuntos
Adenosina Desaminase/metabolismo , Evolução Molecular Direcionada , Edição de Genes , Adenosina Desaminase/genética , Animais , Sequência de Bases , Sistemas CRISPR-Cas , Linhagem Celular , Regulação Enzimológica da Expressão Gênica , Marcação de Genes , Humanos , Mutação INDEL , Camundongos
5.
DNA Res ; 26(3): 261-272, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31231762

RESUMO

Adenosine-to-inosine (A-to-I) RNA editing meditated by adenosine deaminases acting on RNA (ADARs) enzymes is a widespread post-transcriptional event in mammals. However, A-to-I editing in skeletal muscle remains poorly understood. By integrating strand-specific RNA-seq, whole genome bisulphite sequencing, and genome sequencing data, we comprehensively profiled the A-to-I editome in developing skeletal muscles across 27 prenatal and postnatal stages in pig, an important farm animal and biomedical model. We detected 198,892 A-to-I editing sites and found that they occurred more frequently at prenatal stages and showed low conservation among pig, human, and mouse. Both the editing level and frequency decreased during development and were positively correlated with ADAR enzymes expression. The hyper-edited genes were functionally related to the cell cycle and cell division. A co-editing module associated with myogenesis was identified. The developmentally differential editing sites were functionally enriched in genes associated with muscle development, their editing levels were highly correlated with expression of their host mRNAs, and they potentially influenced the gain/loss of miRNA binding sites. Finally, we developed a database to visualize the Sus scrofa RNA editome. Our study presents the first profile of the dynamic A-to-I editome in developing animal skeletal muscle and provides evidences that RNA editing is a vital regulator of myogenesis.


Assuntos
Adenosina Desaminase/metabolismo , Músculo Esquelético/crescimento & desenvolvimento , Edição de RNA , RNA Mensageiro/metabolismo , Sus scrofa/crescimento & desenvolvimento , Adenosina Desaminase/genética , Animais , Bases de Dados Genéticas , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Músculo Esquelético/enzimologia , Músculo Esquelético/metabolismo , Análise de Sequência de RNA , Sus scrofa/genética , Sus scrofa/metabolismo , Sequenciamento Completo do Genoma
6.
Nat Commun ; 10(1): 1605, 2019 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-30962428

RESUMO

Colonies of the bumblebee Bombus terrestris are characterized by wide phenotypic variability among genetically similar full-sister workers, suggesting a major role for epigenetic processes. Here, we report a high level of ADAR-mediated RNA editing in the bumblebee, despite the lack of an ADAR1-homolog. We identify 1.15 million unique genomic sites, and 164 recoding sites residing in 100 protein coding genes, including ion channels, transporters, and receptors predicted to affect brain function and behavior. Some edited sites are similarly edited in other insects, cephalopods and even mammals. The global editing level of protein coding and non-coding transcripts weakly correlates with task performance (brood care vs. foraging), but not affected by dominance rank or juvenile hormone known to influence physiology and behavior. Taken together, our findings show that brain editing levels are high in naturally behaving bees, and may be regulated by relatively short-term effects associated with brood care or foraging activities.


Assuntos
Abelhas/fisiologia , Comportamento Animal/fisiologia , Edição de RNA/fisiologia , RNA/genética , Comportamento Social , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Animais , Encéfalo/metabolismo , Epigênese Genética/fisiologia , Feminino , Variação Genética/genética , Variação Genética/fisiologia , Masculino , RNA/isolamento & purificação , RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Análise de Sequência de RNA
8.
RNA ; 25(6): 713-726, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30894411

RESUMO

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.


Assuntos
Adenosina Desaminase/genética , Proteína DEAD-box 58/genética , Imunidade Inata/genética , RNA de Cadeia Dupla/genética , RNA Mitocondrial/genética , Proteína Supressora de Tumor p53/genética , Adenosina Desaminase/deficiência , Adenosina Desaminase/imunologia , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Proteína DEAD-box 58/imunologia , Embrião de Mamíferos , Endorribonucleases/genética , Endorribonucleases/imunologia , Fibroblastos/citologia , Fibroblastos/imunologia , Fator Regulador 7 de Interferon/genética , Fator Regulador 7 de Interferon/imunologia , Helicase IFIH1 Induzida por Interferon/genética , Helicase IFIH1 Induzida por Interferon/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas/genética , Proteínas/imunologia , RNA de Cadeia Dupla/imunologia , RNA Mitocondrial/imunologia , Transcrição Genética , Transfecção , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/imunologia
10.
Methods ; 156: 40-45, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30827465

RESUMO

The deamination of adenosine to inosine by RNA editing is a widespread post-transcriptional process that expands genetic diversity. Selective substitution of inosine for adenosine in pre-mRNA transcripts can alter splicing, mRNA stability, and the amino acid sequence of the encoded protein. The functional consequences of RNA editing-dependent amino acid substitution are known for only a handful of RNA editing substrates. Many of these studies began in heterologous mammalian expression systems; however, the gold-standard for determining the functional significance of transcript-specific re-coding A-to-I editing events is the generation of a mouse model that expresses only one RNA editing-dependent isoform. The frequency of site-specific RNA editing varies spatially, temporally, and in some diseases, therefore, determining the profile of RNA editing frequency is also an important element of research. Here we review the strengths and weaknesses of existing mouse models for the study of RNA editing, as well as methods for quantifying RNA editing frequencies in vivo. Importantly, we highlight opportunities for future RNA editing studies in mice, projecting that improvements in genome editing and high-throughput sequencing technologies will allow the field to excel in coming years.


Assuntos
Adenosina/metabolismo , Inosina/metabolismo , Edição de RNA , RNA Mensageiro/genética , Análise de Sequência de RNA/métodos , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Processamento Alternativo , Animais , Pareamento de Bases , Sequência de Bases , Humanos , Camundongos , Modelos Biológicos , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
11.
Methods ; 156: 46-52, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30827466

RESUMO

Over 150 unique RNA modifications are now known including several nonstandard nucleotides present in the body of messenger RNAs. These modifications can alter a transcript's function and are collectively referred to as the epitrancriptome. Chemically modified nucleoside analogs are poised to play an important role in the study of these epitranscriptomic marks. Introduced chemical features on nucleic acid strands provide unique structures or reactivity that can be used for downstream detection or quantification. Three methods are used in the field to synthesize RNA containing chemically modified nucleoside analogs. Nucleoside analogs can be introduced by metabolic labeling, via polymerases with modified nucleotide triphosphates or via phosphoramidite-based chemical synthesis. In this review, these methods for incorporation of nucleoside analogs will be discussed with specific recently published examples pertaining to the study of the epitranscriptome.


Assuntos
Edição de RNA , RNA de Cadeia Dupla/química , Ribonucleotídeos/química , S-Adenosilmetionina/metabolismo , Coloração e Rotulagem/métodos , Transcriptoma , Adenosina/análogos & derivados , Adenosina/química , Adenosina/metabolismo , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Animais , Humanos , Inosina/metabolismo , Conformação de Ácido Nucleico , Compostos Organofosforados/química , Compostos Organofosforados/metabolismo , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonucleotídeos/metabolismo , S-Adenosilmetionina/análogos & derivados , Selênio/química , Selênio/metabolismo
12.
RNA ; 25(5): 607-619, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30737359

RESUMO

Adenosine deaminase acting on transfer RNA (ADAT) is an essential eukaryotic enzyme that catalyzes the deamination of adenosine to inosine at the first position of tRNA anticodons. Mammalian ADATs modify eight different tRNAs, having increased their substrate range from a bacterial ancestor that likely deaminated exclusively tRNAArg Here we investigate the recognition mechanisms of tRNAArg and tRNAAla by human ADAT to shed light on the process of substrate expansion that took place during the evolution of the enzyme. We show that tRNA recognition by human ADAT does not depend on conserved identity elements, but on the overall structural features of tRNA. We find that ancestral-like interactions are conserved for tRNAArg, while eukaryote-specific substrates use alternative mechanisms. These recognition studies show that human ADAT can be inhibited by tRNA fragments in vitro, including naturally occurring fragments involved in important regulatory pathways.


Assuntos
Adenosina Desaminase/metabolismo , Anticódon/química , RNA de Transferência de Alanina/química , RNA de Transferência de Arginina/química , Adenosina/metabolismo , Adenosina Desaminase/genética , Anticódon/genética , Anticódon/metabolismo , Sequência de Bases , Desaminação , Evolução Molecular , Expressão Gênica , Humanos , Inosina/metabolismo , Conformação de Ácido Nucleico , RNA de Transferência de Alanina/genética , RNA de Transferência de Alanina/metabolismo , RNA de Transferência de Arginina/genética , RNA de Transferência de Arginina/metabolismo , Alinhamento de Sequência , Especificidade por Substrato
13.
Med Sci Monit ; 25: 1469-1479, 2019 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-30798327

RESUMO

BACKGROUND Intrahepatic and distant metastases could be the major cause of treatment failure in hepatocellular carcinoma (HCC). The deep mechanism of HCC metastasis is closely related to the interaction between integrins and extracellular matrix (ECM) in tumor microenvironment. MATERIAL AND METHODS In vitro cell adhesion assay was performed to determine the capability of adhering to ECM elements of HCC cells. To modulate the expression status of ADAR1 p110 in tumor cells, lentivirus system was applied. Meanwhile, patients' HCC samples and orthotopic xenograft mouse model were used for verifying our in vitro data. RESULTS ADAR1 p110 could strongly enhance the adhesion of HCC tumor cells to ECM, which was usually regarded as the initiation of tumor invasion. Such phenotype was caused due to up-regulation of ITGA2 both in mRNA and protein level. Moreover, specimen collected from HCC patients revealed a positive correlation between ADAR1 and ITGA2. Finally, ADAR1 p110 promoted HCC metastasis was verified when we applied orthotopic xenograft mouse model. CONCLUSIONS ADAR1 could enhance HCC metastasis by promoting tumor cells adhering to ECM via increasing ITGA2 expression. This phenomenon could provide novel information to better understanding the mechanism of HCC metastasis procedure.


Assuntos
Adenosina Desaminase/metabolismo , Carcinoma Hepatocelular/patologia , Integrina alfa2/metabolismo , Neoplasias Hepáticas/patologia , Proteínas de Ligação a RNA/metabolismo , Adenosina Desaminase/genética , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Feminino , Xenoenxertos , Humanos , Integrina alfa2/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Camundongos Nus , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Ativação Transcricional , Microambiente Tumoral , Regulação para Cima
14.
Nat Commun ; 10(1): 823, 2019 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-30778076

RESUMO

Follicular helper T cells (Tfh) play critical roles instructing, and initiating T-cell dependent antibody responses. The underlying mechanisms that enhance their function is therefore critical for vaccine development. Here we apply gene array analysis identifying adenosine deaminase (ADA) as a key molecule that delineates a human Tfh helper program in proliferating circulating Tfh (cTfh) cells and Germinal Centers Tfh (GC-Tfh). ADA-1 expression and enzymatic activity are increased in efficient cTfh2-17/GC-Tfh cells. Exogenous ADA-1 enhances less efficient cTfh1 and pro-follicular Tfh PD-1+ CXCR5+ cells to provide B cell help, while pharmacological inhibition of ADA-1 activity impedes cTfh2-17/GC-Tfh function and diminished antibody response. Mechanistically, ADA-1 controls the Tfh program by influencing IL6/IL-2 production, controlling CD26 extracellular expression and could balance signals through adenosine receptors. Interestingly, dysfunctional Tfh from HIV infected-individual fail to regulate the ADA pathway. Thus, ADA-1 regulates human Tfh and represents a potential target for development of vaccine strategy.


Assuntos
Adenosina Desaminase/metabolismo , Infecções por HIV/patologia , Linfócitos T Auxiliares-Indutores/fisiologia , Adenosina Desaminase/genética , Adenilil Ciclases/metabolismo , Linfócitos B/citologia , Técnicas de Cocultura , Dipeptidil Peptidase 4/metabolismo , Centro Germinativo/metabolismo , Infecções por HIV/metabolismo , Humanos , Interleucina-2/metabolismo , Interleucina-6/metabolismo , Linfócitos T Auxiliares-Indutores/virologia
16.
Int J Legal Med ; 133(3): 863-869, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30617847

RESUMO

BACKGROUND: There is evidence that inflammation plays a role in the etiology of sudden infant death syndrome (SIDS). Immune system dysregulation seems to be the background of higher infection susceptibility in SIDS infants. This phenotype is possibly determined by genetic factors. METHODS: Twenty-three single nucleotide polymorphisms (SNPs) in the following 13 candidate genes governing the immune system were successfully genotyped in 251 Caucasian SIDS cases and 336 controls from Germany: ADAR1, CSF2RB, DDX58, IFNA1, IFNA21, IFNA8, IFNAR2, IFNG, IL6, MX2, OAS1, OAS3, and TNFA. Associations between genotypes and SIDS were then statistically evaluated using logistic regression analyses. RESULTS: Overall analysis revealed statistically significant results for two variants in interferon gamma (IFNG) (rs2069705: OR 1.40 (1.07; 1.83), p = 0.01; and rs2069727: OR 0.75 (0.59; 0.96), p = 0.02) and for one variant in interferon alpha 8 (IFNA8) (rs1330321: OR 1.85 (1.06; 3.21), p = 0.03). Haplotype analyses identified a three-marker risk IFNG haplotype rs2069727-rs2069718-rs2069705 associated with SIDS (OR = 1.62, 95% CI 1.23-2.13; p = 0.0003). Subgroup associations were found for variants in adenosine deaminase acting on RNA1 (ADAR1), 2',5'-oligoadenylate synthetase-1 (OAS1) and colony stimulating factor 2 receptor beta common subunit (CSF2RB). CONCLUSION: In summary, this large study of 251 SIDS cases for common variants in 13 candidate genes governing the immune system has provided first evidence for a role of IFNG in the etiology of SIDS and should stimulate further research into the clinicopathological relevance of immunomodulatory genes for this fatal syndrome.


Assuntos
Predisposição Genética para Doença , Polimorfismo de Nucleotídeo Único , Morte Súbita do Lactente/genética , 2',5'-Oligoadenilato Sintetase/genética , Adenosina Desaminase/genética , Estudos de Casos e Controles , Subunidade beta Comum dos Receptores de Citocinas/genética , Feminino , Genótipo , Haplótipos , Humanos , Lactente , Recém-Nascido , Interferon-alfa/genética , Interferon gama/genética , Modelos Logísticos , Masculino , Proteínas de Ligação a RNA/genética
17.
Nat Biotechnol ; 37(2): 133-138, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30692694

RESUMO

Site-directed RNA editing might provide a safer or more effective alternative to genome editing in certain clinical scenarios. Until now, RNA editing has relied on overexpression of exogenous RNA editing enzymes or of endogenous human ADAR (adenosine deaminase acting on RNA) enzymes. Here we describe the engineering of chemically optimized antisense oligonucleotides that recruit endogenous human ADARs to edit endogenous transcripts in a simple and programmable way, an approach we call RESTORE (recruiting endogenous ADAR to specific transcripts for oligonucleotide-mediated RNA editing). We observed almost no off-target editing, and natural editing homeostasis was not perturbed. We successfully applied RESTORE to a panel of standard human cell lines and human primary cells and demonstrated repair of the clinically relevant PiZZ mutation, which causes α1-antitrypsin deficiency, and editing of phosphotyrosine 701 in STAT1, the activity switch of the signaling factor. RESTORE requires only the administration of an oligonucleotide, circumvents ectopic expression of proteins, and represents an attractive approach for drug development.


Assuntos
Adenosina Desaminase/genética , Oligonucleotídeos Antissenso/genética , Edição de RNA , Proteínas de Ligação a RNA/genética , Regiões 3' não Traduzidas , Motivos de Aminoácidos , Células Cultivadas , Desenho de Drogas , Células HeLa , Células Hep G2 , Humanos , Interferon-alfa/farmacologia , Mutação , Fases de Leitura Aberta , Fosfotirosina/química , RNA Mensageiro/metabolismo , Fator de Transcrição STAT1/genética , Transdução de Sinais , alfa 1-Antitripsina/genética , Deficiência de alfa 1-Antitripsina/genética
18.
J Dermatol Sci ; 93(2): 75-81, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30692041

RESUMO

Dyschromatosis symmetrica hereditaria (DSH) and reticulate acropigmentation of Kitamura (RAK) are rare, inherited pigmentary diseases. DSH shows a mixture of pigmented and depigmented macules on the extremities. RAK shows reticulated, slightly depressed pigmented macules on the extremities. The causative gene of DSH was clarified as ADAR1 by positional cloning including linkage analysis and haplotype analysis in 2003. Ten years later, the causative gene of RAK was identified as ADAM10 by whole-exome sequencing, in 2013. ADAR1 is an RNA-editing enzyme which catalyzes the deamination of adenosine to inosine (A-to-I) in double-stranded RNA substrates during post-transcription processing. Inosine acts as guanine during translation, resulting in codon alterations or alternative splice sites that lead to functional changes in proteins when they occur in coding regions. In 2012, it was clarified that ADAR1 mutations cause Aicardi-Goutières syndrome 6, which is a severe genetic inflammatory disease that affects the brain and the skin. A zinc metalloprotease, a disintegrin and metalloprotease domain-containing protein 10 (ADAM10), is involved in the ectodomain shedding of various membrane proteins and shows various functions in vivo. ADAM10 is known to be involved in the ectodomain shedding of Notch proteins as substrates in the skin. We speculate that the pathogenesis of RAK and Dowling-Degos disease (DDD, a pigmentary disease similar to RAK) is associated with the Notch signaling pathway. In addition, ADAM10 mutations proved to be associated with late-onset Alzheimer disease. This review comprehensively discusses the updated pathophysiology of those genetic pigmentary disorders.


Assuntos
Proteína ADAM10/genética , Adenosina Desaminase/genética , Secretases da Proteína Precursora do Amiloide/genética , Hiperpigmentação/genética , Proteínas de Membrana/genética , Transtornos da Pigmentação/congênito , Proteínas de Ligação a RNA/genética , Receptores Notch/metabolismo , Dermatopatias Genéticas/genética , Dermatopatias Papuloescamosas/genética , Humanos , Hiperpigmentação/patologia , Mutação , Transtornos da Pigmentação/genética , Transtornos da Pigmentação/patologia , Doenças Raras/genética , Pele/patologia , Dermatopatias Genéticas/patologia , Dermatopatias Papuloescamosas/patologia , Pigmentação da Pele/genética
19.
J Virol ; 93(7)2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30651363

RESUMO

Orf virus (ORFV) infects sheep and goats and is also an important zoonotic pathogen. The viral protein OV20.0 has been shown to suppress innate immunity by targeting the double-stranded RNA (dsRNA)-activated protein kinase (PKR) by multiple mechanisms. These mechanisms include a direct interaction with PKR and binding with two PKR activators, dsRNA and the cellular PKR activator (PACT), which ultimately leads to the inhibition of PKR activation. In the present study, we identified a novel association between OV20.0 and adenosine deaminase acting on RNA 1 (ADAR1). OV20.0 bound directly to the dsRNA binding domains (RBDs) of ADAR1 in the absence of dsRNA. Additionally, OV20.0 preferentially interacted with RBD1 of ADAR1, which was essential for its dsRNA binding ability and for the homodimerization that is critical for intact adenosine-to-inosine (A-to-I)-editing activity. Finally, the association with OV20.0 suppressed the A-to-I-editing ability of ADAR1, while ADAR1 played a proviral role during ORFV infection by inhibiting PKR phosphorylation. These observations revealed a new strategy used by OV20.0 to evade antiviral responses via PKR.IMPORTANCE Viruses evolve specific strategies to counteract host innate immunity. ORFV, an important zoonotic pathogen, encodes OV20.0 to suppress PKR activation via multiple mechanisms, including interactions with PKR and two PKR activators. In this study, we demonstrated that OV20.0 interacts with ADAR1, a cellular enzyme responsible for converting adenosine (A) to inosine (I) in RNA. The RNA binding domains, but not the catalytic domain, of ADAR1 are required for this interaction. The OV20.0-ADAR1 association affects the functions of both proteins; OV20.0 suppressed the A-to-I editing of ADAR1, while ADAR1 elevated OV20.0 expression. The proviral role of ADAR1 is likely due to the inhibition of PKR phosphorylation. As RNA editing by ADAR1 contributes to the stability of the genetic code and the structure of RNA, these observations suggest that in addition to serving as a PKR inhibitor, OV20.0 might modulate ADAR1-dependent gene expression to combat antiviral responses or achieve efficient viral infection.


Assuntos
Adenosina Desaminase/genética , Vírus do Orf/genética , RNA Viral/genética , Proteínas de Ligação a RNA/genética , Proteínas Virais/genética , Replicação Viral/genética , Células A549 , Adenosina/genética , Animais , Linhagem Celular , Linhagem Celular Tumoral , Ectima Contagioso/genética , Proteínas Ativadoras de GTPase/genética , Células HEK293 , Humanos , Imunidade Inata/genética , Inosina/genética , Fosforilação/genética , Edição de RNA/genética , RNA de Cadeia Dupla/genética , Ovinos
20.
Immunogenetics ; 71(4): 299-305, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30610243

RESUMO

Deficiency of adenosine deaminase 2 (DADA2) is an autoinflammatory disease caused by autosomal recessive mutations in Cat Eye Syndrome Chromosome Region 1 (CECR1) gene. In this report, we aimed to describe the clinical manifestations, immunological features, genotype, and treatments of one Chinese patient with novel CECR1 gene mutations. This patient initially presented with recurrent fever and rashes from the age of 3 months, but no pathogen was found. She then developed dry gangrene of the fingers at 5 months of age. Laboratory examinations revealed elevated levels of C-reactive protein and thrombocytes. The expression of interleukin-6 (IL-6) and IL-8 were both elevated. Sequencing results revealed that she had compound heterozygous mutations in CECR1 gene (c.1211T>C, p.Phe404Ser and c.1114 G>A, p.Val372Met). Subsequently, treatment with anti-IL-6 (tocilizumab) was started. However, she developed blurred vision in the right eye with occlusion of the central retinal artery, accompanied by unsteady gait. Magnetic resonance imaging (MRI) showed infarction of the right thalamus. Finally, she underwent hematopoietic stem cell transplantation (HSCT) and is currently in remission. Our findings suggest that HSCT could cure this disease.


Assuntos
Adenosina Desaminase/deficiência , Agamaglobulinemia/terapia , Transplante de Células-Tronco Hematopoéticas/métodos , Doenças Hereditárias Autoinflamatórias/terapia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Mutação , Imunodeficiência Combinada Severa/terapia , Adenosina Desaminase/genética , Agamaglobulinemia/diagnóstico por imagem , Agamaglobulinemia/genética , Grupo com Ancestrais do Continente Asiático , Sequência de Bases , China , Feminino , Doenças Hereditárias Autoinflamatórias/diagnóstico por imagem , Doenças Hereditárias Autoinflamatórias/genética , Humanos , Lactente , Imagem por Ressonância Magnética , Indução de Remissão , Análise de Sequência de DNA , Imunodeficiência Combinada Severa/diagnóstico por imagem , Imunodeficiência Combinada Severa/genética
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