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1.
Gene ; 805: 145909, 2021 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-34419568

RESUMO

BACKGROUND: Adenosine deaminase acting on RNA 3 (ADAR3) was known as a prognosis factor in gliomas, while its function on neuropathic pain (NP) is barely investigated. Therefore, our present study concentrated on the potential role of ADAR3 in NP. METHODS: The chronic constriction injury (CCI) mouse model was established to induce NP in vivo. Behavioral experiments were carried out to analyze mechanical allodynia and thermal hyperalgesia. RT-qPCR and western blotting assays were used to detect the mRNA and protein expressions. The ADAR3-overexpressed adenovirus was injected into the CCI mice through an intrathecal catheter. ELISA was used to detect the contents of IL (interleukin)-6, IL-10, TNF (tumor necrosis factor)-α, IL-1ß and IL-18. NLR Family Pyrin Domain Containing 3 (NLRP3) was predicted to be the target gene of ADAR3 using Starbase. The interaction between ADAR3 and NLRP3 was verified via RNA pull-down, RNA immunoprecipitation and Pearson's correlation coefficient assays. Immunohistochemical staining assay visualized the expressions of NLRP3 and caspase1. RESULTS: Allodynia and hyperalgesia were exacerbated in the CCI mice, which implied a successful establishment of the NP model, while ADAR3 expression level was suppressed. After injecting ADAR3-overexpressed adenovirus into the CCI mice, allodynia, hyperalgesia and inflammation were all restrained. Moreover, NLRP3 was verified to negatively correlated with ADAR3. Additionally, the pyroptosis-related protein NLRP3, ASC, caspase1, IL-1ß, IL-18 and GSDMD expressions were all decreased by ADAR3. CONCLUSION: In conclusion, ADAR3 alleviated inflammation and pyroptosis of NP through targeting NLRP3, which suggested a therapeutical target for NP.


Assuntos
Adenosina Desaminase/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Neuralgia/genética , Adenosina Desaminase/metabolismo , Animais , Constrição Patológica/fisiopatologia , Hiperalgesia/genética , Inflamação/genética , Inflamação/metabolismo , Interleucina-10/metabolismo , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Neuralgia/metabolismo , Piroptose/genética , Piroptose/fisiologia , Fator de Necrose Tumoral alfa/metabolismo
2.
Science ; 373(6558): 984-991, 2021 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-34446600

RESUMO

Protein kinase activity must be precisely regulated, but how a cell governs hyperactive kinases remains unclear. In this study, we generated a constitutively active mitogen-activated protein kinase DYF-5 (DYF-5CA) in Caenorhabditis elegans that disrupted sensory cilia. Genetic suppressor screens identified that mutations of ADR-2, an RNA adenosine deaminase, rescued ciliary phenotypes of dyf-5CA We found that dyf-5CA animals abnormally transcribed antisense RNAs that pair with dyf-5CA messenger RNA (mRNA) to form double-stranded RNA, recruiting ADR-2 to edit the region ectopically. RNA editing impaired dyf-5CA mRNA splicing, and the resultant intron retentions blocked DYF-5CA protein translation and activated nonsense-mediated dyf-5CA mRNA decay. The kinase RNA editing requires kinase hyperactivity. The similar RNA editing-dependent feedback regulation restricted the other ciliary kinases NEKL-4/NEK10 and DYF-18/CCRK, which suggests a widespread mechanism that underlies kinase regulation.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimologia , Cílios/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Edição de RNA , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Animais , Caenorhabditis elegans/genética , Núcleo Celular/metabolismo , Cílios/enzimologia , Ativação Enzimática , Fenótipo , Biossíntese de Proteínas , Proteínas Serina-Treonina Quinases/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA , Estabilidade de RNA , RNA Antissenso/genética , RNA Antissenso/metabolismo , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , RNA de Helmintos/genética , RNA de Helmintos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Transcrição Genética
3.
J Virol ; 95(19): e0065221, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34346762

RESUMO

The filovirus family includes deadly pathogens such as Ebola virus (EBOV) and Marburg virus (MARV). A substantial portion of filovirus genomes encode 5' and 3' untranslated regions (UTRs) of viral mRNAs. Select viral genomic RNA sequences corresponding to 3' UTRs are prone to editing by adenosine deaminase acting on RNA 1 (ADAR1). A reporter mRNA approach, in which different 5' or 3' UTRs were inserted into luciferase-encoding mRNAs, demonstrates that MARV 3' UTRs yield different levels of reporter gene expression, suggesting modulation of translation. The modulation occurs in cells unable to produce microRNAs (miRNAs) and can be recapitulated in a MARV minigenome assay. Deletion mutants identified negative regulatory regions at the ends of the MARV nucleoprotein (NP) and large protein (L) 3' UTRs. Apparent ADAR1 editing mutants were previously identified within the MARV NP 3' UTR. Introduction of these changes into the MARV nucleoprotein (NP) 3' UTR or deletion of the region targeted for editing enhances translation, as indicated by reporter assays and polysome analysis. In addition, the parental NP 3' UTR, but not the edited or deletion mutant NP 3' UTRs, induces a type I interferon (IFN) response upon transfection into cells. Because some EBOV isolates from the West Africa outbreak exhibited ADAR1 editing of the viral protein of 40 kDa (VP40) 3' UTR, VP40 3' UTRs with parental and edited sequences were similarly assayed. The EBOV VP40 3' UTR edits also enhanced translation, but neither the wild-type nor the edited 3' UTRs induced IFN. These findings implicate filoviral mRNA 3' UTRs as negative regulators of translation that can be inactivated by innate immune responses that induce ADAR1. IMPORTANCE UTRs comprise a large percentage of filovirus genomes and are apparent targets of editing by ADAR1, an enzyme with pro- and antiviral activities. However, the functional significance of the UTRs and ADAR1 editing has been uncertain. This study demonstrates that MARV and EBOV 3' UTRs can modulate translation, in some cases negatively. ADAR1 editing or deletion of select regions within the translation suppressing 3' UTRs relieves the negative effects of the UTRs. These data indicate that filovirus 3' UTRs contain translation regulatory elements that are modulated by activation of ADAR1, suggesting a complex interplay between filovirus gene expression and innate immunity.


Assuntos
Regiões 3' não Traduzidas , Adenosina Desaminase/metabolismo , Ebolavirus/genética , Marburgvirus/genética , Biossíntese de Proteínas , Proteínas de Ligação a RNA/metabolismo , Animais , Linhagem Celular , Ebolavirus/metabolismo , Genes Reporter , Humanos , Interferon Tipo I/biossíntese , Marburgvirus/metabolismo , MicroRNAs/genética , Mutação , Proteínas do Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/metabolismo , Polirribossomos/metabolismo , Edição de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo
4.
Enzyme Microb Technol ; 149: 109851, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34311888

RESUMO

The popularity and promise of gene therapy for common genetic diseases are currently increasing. Although effective treatments for genetic disorders are rare, editing of the mutated gene is a possible therapeutic approach for conditions caused by stop codon mutations, including either amber (TAG), opal (TGA) or ochre (TAA) stop codons. Restoration of point-mutated RNAs using artificial RNA editing can be used to modify gene-encoded information and generate functionally distinct proteins from a single gene. By linking the catalytic domain of the RNA editing enzyme, adenosine deaminase acting on RNA (ADAR), to an antisense guide RNA, specific adenosines (A) can be converted to inosine (I), which is recognized as guanosine (G) during translation. In this study, we engineered the deaminase domain of ADAR1 and the MS2 system to target a specific adenosine and restore the G to A mutations. To this end, the ADAR1 deaminase domain was fused with the RNA binding protein, MS2, which binds to MS2 RNA. Guide RNAs of 19 bp were designed to be complementary to target mRNAs, with either 6X stem-loops downstream of the guide RNA and a CMV promoter, or a 1X MS2 stem-loop on either side of the guide RNA and a U6 promoter. The engineered ADAR1 deaminase domain could convert adenosine to inosine at the desired editing site in EGFP, which was edited to contain an amber (TAG), opal (TGA) or ochre (TAA) stop codon. The system could convert the stop codons to a read-through tryptophan codon (TGG) in a cellular system, leading to fluorescence emission, observed using JuLi microscopy. PCR-RFLP and Sanger sequencing of the target transcript were also conducted, revealing an editing efficiency of 20.97 % for the opal stop codon, and 26 % and 17 % for the 5' and 3' A residues, respectively, in the ochre stop codon, using the double MS2. This was a higher editing rate than that achieved using the MS2-6X guide RNA. Observation of restoration of the read-through codon from the three different stop codons over time demonstrated a relatively low percentage of edited codons after 24 h, which increased after 48 h, but decreased again after 72 h. Successful establishment of this system has the potential to represent a new era in the field of gene therapy.


Assuntos
Código Genético , Edição de RNA , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Códon de Terminação/genética , Humanos , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
5.
J Chem Phys ; 154(18): 185101, 2021 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-34241023

RESUMO

The effect of ligand binding on the conformational transitions of the add A-riboswitch in cellular environments is investigated theoretically within the framework of the generalized Langevin equation combined with steered molecular dynamics simulations. Results for the transition path time distribution provide an estimate of the transit times, which are difficult to determine experimentally. The time for the conformational transitions of the riboswitch aptamer is longer for the ligand bound state as compared to that of the unbound one. The transition path time of the riboswitch follows a counterintuitive trend as it decreases with an increase in the barrier height. The mean transition path time of either transitions of the riboswitch in the ligand bound/unbound state increases with an increase in the complexity of the surrounding environment due to the caging effect. The results of the probability density function, transition path time distribution, and mean transition path time obtained from the theory qualitatively agree with those obtained from the simulations and with earlier experimental and theoretical studies.


Assuntos
Adenosina Desaminase/química , Teoria da Densidade Funcional , Simulação de Dinâmica Molecular , Adenosina Desaminase/metabolismo , Ligantes
6.
RNA ; 27(10): 1220-1240, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34244459

RESUMO

Metabotropic glutamate receptor 4 (mGlu4) is one of eight mGlu receptors within the Class C G protein-coupled receptor superfamily. mGlu4 is primarily localized to the presynaptic membrane of neurons where it functions as an auto and heteroreceptor controlling synaptic release of neurotransmitter. mGlu4 is implicated in numerous disorders and is a promising drug target; however, more remains to be understood about its regulation and pharmacology. Using high-throughput sequencing, we have validated and quantified an adenosine-to-inosine (A-to-I) RNA editing event that converts glutamine 124 to arginine in mGlu4; additionally, we have identified a rare but novel K129R site. Using an in vitro editing assay, we then validated the pre-mRNA duplex that allows for editing by ADAR enzymes and predicted its conservation across the mammalian species. Structural modeling of the mGlu4 protein predicts the Q124R substitution to occur in the B helix of the receptor that is critical for receptor dimerization and activation. Interestingly, editing of a receptor homodimer does not disrupt G protein activation in response to the endogenous agonist, glutamate. Using an assay designed to specifically measure heterodimer populations at the surface, however, we found that Q124R substitution decreased the propensity of mGlu4 to heterodimerize with mGlu2 and mGlu7 Our study is the first to extensively describe the extent and regulatory factors of RNA editing of mGlu4 mRNA transcripts. In addition, we have proposed a novel functional consequence of this editing event that provides insights regarding its effects in vivo and expands the regulatory capacity for mGlu receptors.


Assuntos
Edição de RNA , RNA Mensageiro/genética , Receptores de Glutamato Metabotrópico/genética , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Sequência de Aminoácidos , Animais , Pareamento de Bases , Sequência de Bases , Aves , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Corpo Estriado/citologia , Corpo Estriado/metabolismo , Células HEK293 , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Modelos Moleculares , Neurônios/citologia , Neurônios/metabolismo , Conformação de Ácido Nucleico , Mutação Puntual , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Glutamato Metabotrópico/química , Receptores de Glutamato Metabotrópico/metabolismo , Répteis , Homologia de Sequência de Aminoácidos
7.
PLoS Biol ; 19(7): e3001342, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34329302

RESUMO

A new study in PLOS Biology finds that interferon (IFN)-induced adenosine deaminase acting on RNA 1 (ADAR1) mRNA is N6-methyladenosine (m6A) modified to promote its translation, enabling ADAR1 to modify self-double-stranded RNAs (dsRNAs) generated during the IFN response and preventing activation of the melanoma differentiation-associated protein 5 (MDA5)-mediated host antiviral response.


Assuntos
Adenosina Desaminase , RNA de Cadeia Dupla , Adenosina , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Interferons/metabolismo , RNA de Cadeia Dupla/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
8.
BMC Infect Dis ; 21(1): 575, 2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-34130662

RESUMO

BACKGROUND: A pleural fluid adenosine deaminase (ADA) has been used globally to assist in the diagnosis of a tuberculous pleural effusion (TPE) with a notable negative predictive value. CASE PRESENTATION: We report a case of a patient with a negative pleural fluid ADA who was found to have culture-positive and biopsy-proven Mycobacterium tuberculosis. CONCLUSIONS: This case shows the importance of pursuing gold standard diagnostic studies when clinical suspicion remains high despite negative preliminary testing. We further describe gaps in research to improve pleural fluid biomarkers for TPE.


Assuntos
Adenosina Desaminase/análise , Mycobacterium tuberculosis/isolamento & purificação , Tuberculose Pleural/diagnóstico , Adenosina Desaminase/metabolismo , Adulto , Antituberculosos/uso terapêutico , Biomarcadores/análise , Biópsia/métodos , Exsudatos e Transudatos , Humanos , Masculino , Derrame Pleural/diagnóstico , Derrame Pleural/microbiologia , Valor Preditivo dos Testes , Resultado do Tratamento , Tuberculose Pleural/tratamento farmacológico , Tuberculose Pleural/enzimologia
9.
Chemistry ; 27(48): 12300-12304, 2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34169589

RESUMO

Various RNA-targeting approaches have been engineered to modify specific sites on endogenous transcripts, breaking new ground for a variety of basic research tools and promising clinical applications in the future. Here, we combine site-directed adenosine-to-inosine RNA editing with chemically induced dimerization. Specifically, we achieve tight and dose-dependent control of the editing reaction with gibberellic acid, and obtain editing yields up to 20 % and 44 % in the endogenous STAT1 and GAPDH transcript in cell culture. Furthermore, the disease-relevant MECP2 R106Q mutation was repaired with editing yields up to 42 %. The introduced principle will enable new applications where temporal or spatiotemporal control of an RNA-targeting mechanism is desired.


Assuntos
Adenosina Desaminase , Edição de RNA , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Dimerização , RNA/genética , Proteínas de Ligação a RNA/metabolismo
10.
Nat Genet ; 53(6): 881-894, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33972779

RESUMO

Esophageal squamous cell carcinomas (ESCCs) harbor recurrent chromosome 3q amplifications that target the transcription factor SOX2. Beyond its role as an oncogene in ESCC, SOX2 acts in development of the squamous esophagus and maintenance of adult esophageal precursor cells. To compare Sox2 activity in normal and malignant tissue, we developed engineered murine esophageal organoids spanning normal esophagus to Sox2-induced squamous cell carcinoma and mapped Sox2 binding and the epigenetic and transcriptional landscape with evolution from normal to cancer. While oncogenic Sox2 largely maintains actions observed in normal tissue, Sox2 overexpression with p53 and p16 inactivation promotes chromatin remodeling and evolution of the Sox2 cistrome. With Klf5, oncogenic Sox2 acquires new binding sites and enhances activity of oncogenes such as Stat3. Moreover, oncogenic Sox2 activates endogenous retroviruses, inducing expression of double-stranded RNA and dependence on the RNA editing enzyme ADAR1. These data reveal SOX2 functions in ESCC, defining targetable vulnerabilities.


Assuntos
Adenosina Desaminase/metabolismo , Epigenoma , Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas do Esôfago/genética , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Regiões 3' não Traduzidas/genética , Animais , Sequência de Bases , Carcinogênese/genética , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Retrovirus Endógenos/genética , Elementos Facilitadores Genéticos/genética , Regulação Neoplásica da Expressão Gênica , Genoma Humano , Humanos , Interferons/metabolismo , Íntrons/genética , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Organoides/patologia , Ligação Proteica , RNA de Cadeia Dupla/metabolismo , Fatores de Transcrição SOXB1/genética , Proteína Supressora de Tumor p53/metabolismo
11.
Nucleic Acids Res ; 49(11): 6529-6548, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34057470

RESUMO

Post-transcriptional modification of tRNA wobble adenosine into inosine is crucial for decoding multiple mRNA codons by a single tRNA. The eukaryotic wobble adenosine-to-inosine modification is catalysed by the ADAT (ADAT2/ADAT3) complex that modifies up to eight tRNAs, requiring a full tRNA for activity. Yet, ADAT catalytic mechanism and its implication in neurodevelopmental disorders remain poorly understood. Here, we have characterized mouse ADAT and provide the molecular basis for tRNAs deamination by ADAT2 as well as ADAT3 inactivation by loss of catalytic and tRNA-binding determinants. We show that tRNA binding and deamination can vary depending on the cognate tRNA but absolutely rely on the eukaryote-specific ADAT3 N-terminal domain. This domain can rotate with respect to the ADAT catalytic domain to present and position the tRNA anticodon-stem-loop correctly in ADAT2 active site. A founder mutation in the ADAT3 N-terminal domain, which causes intellectual disability, does not affect tRNA binding despite the structural changes it induces but most likely hinders optimal presentation of the tRNA anticodon-stem-loop to ADAT2.


Assuntos
Adenosina Desaminase/química , Adenosina/metabolismo , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Animais , Domínio Catalítico , Linhagem Celular Tumoral , Movimento Celular , Cristalografia por Raios X , Ferredoxinas/química , Inosina/metabolismo , Camundongos , Modelos Moleculares , Mutação , Neurônios/fisiologia , Domínios Proteicos , RNA de Transferência/química , RNA de Transferência/metabolismo
12.
J Cell Sci ; 134(10)2021 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-34037233

RESUMO

Zα domains recognize the left-handed helical Z conformation of double-stranded nucleic acids. They are found in proteins involved in the nucleic acid sensory pathway of the vertebrate innate immune system and host evasion by viral pathogens. Previously, it has been demonstrated that ADAR1 (encoded by ADAR in humans) and DAI (also known as ZBP1) localize to cytoplasmic stress granules (SGs), and this localization is mediated by their Zα domains. To investigate the mechanism, we determined the interactions and localization pattern for the N-terminal region of human DAI (ZαßDAI), which harbours two Zα domains, and for a ZαßDAI mutant deficient in nucleic acid binding. Electrophoretic mobility shift assays demonstrated the ability of ZαßDAI to bind to hyperedited nucleic acids, which are enriched in SGs. Furthermore, using immunofluorescence and immunoprecipitation coupled with mass spectrometry, we identified several interacting partners of the ZαßDAI-RNA complex in vivo under conditions of arsenite-induced stress. These interactions are lost upon loss of nucleic acid-binding ability or upon RNase treatment. Thus, we posit that the mechanism for the translocation of Zα domain-containing proteins to SGs is mainly mediated by the nucleic acid-binding ability of their Zα domains. This article has an associated First Person interview with Bharath Srinivasan, joint first author of the paper.


Assuntos
DNA Forma Z , Ácidos Nucleicos , Adenosina Desaminase/metabolismo , Grânulos Citoplasmáticos/metabolismo , Humanos , Conformação de Ácido Nucleico , RNA , Proteínas de Ligação a RNA
13.
PLoS Genet ; 17(5): e1009516, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33983932

RESUMO

Adenosine deaminase acting on RNA 1 (ADAR1), an enzyme responsible for adenosine-to-inosine RNA editing, is composed of two isoforms: nuclear p110 and cytoplasmic p150. Deletion of Adar1 or Adar1 p150 genes in mice results in embryonic lethality with overexpression of interferon-stimulating genes (ISGs), caused by the aberrant recognition of unedited endogenous transcripts by melanoma differentiation-associated protein 5 (MDA5). However, among numerous RNA editing sites, how many RNA sites require editing, especially by ADAR1 p150, to avoid MDA5 activation and whether ADAR1 p110 contributes to this function remains elusive. In particular, ADAR1 p110 is abundant in the mouse brain where a subtle amount of ADAR1 p150 is expressed, whereas ADAR1 mutations cause Aicardi-Goutières syndrome, in which the brain is one of the most affected organs accompanied by the elevated expression of ISGs. Therefore, understanding RNA editing-mediated prevention of MDA5 activation in the brain is especially important. Here, we established Adar1 p110-specific knockout mice, in which the upregulated expression of ISGs was not observed. This result suggests that ADAR1 p150-mediated RNA editing is enough to suppress MDA5 activation. Therefore, we further created Adar1 p110/Adar2 double knockout mice to identify ADAR1 p150-mediated editing sites. This analysis demonstrated that although the elevated expression of ISGs was not observed, only less than 2% of editing sites were preserved in the brains of Adar1 p110/Adar2 double knockout mice. Of note, we found that some sites were highly edited, which was comparable to those found in wild-type mice, indicating the presence of ADAR1 p150-specific sites. These data suggest that RNA editing at a very limited sites, which is mediated by a subtle amount of ADAR1 p150, is sufficient to prevents MDA5 activation, at least in the mouse brain.


Assuntos
Adenosina Desaminase/metabolismo , Encéfalo/metabolismo , Helicase IFIH1 Induzida por Interferon/metabolismo , Edição de RNA , Regiões 3' não Traduzidas/genética , Adenosina Desaminase/deficiência , Adenosina Desaminase/genética , Animais , Animais Recém-Nascidos , Feminino , Íntrons/genética , Isoenzimas/metabolismo , Camundongos , Camundongos Knockout , Mutação , Especificidade de Órgãos , Proteínas de Ligação a RNA/genética , Taxa de Sobrevida
14.
PLoS Genet ; 17(5): e1009513, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33983939

RESUMO

Double-stranded RNA (dsRNA) is produced both by virus and host. Its recognition by the melanoma differentiation-associated gene 5 (MDA5) initiates type I interferon responses. How can a host distinguish self-transcripts from nonself to ensure that responses are targeted correctly? Here, I discuss a role for MDA5 helicase in inducing Z-RNA formation by Alu inverted repeat (AIR) elements. These retroelements have highly conserved sequences that favor Z-formation, creating a site for the dsRNA-specific deaminase enzyme ADAR1 to dock. The subsequent editing destabilizes the dsRNA, ending further interaction with MDA5 and terminating innate immune responses directed against self. By enabling self-recognition, Alu retrotransposons, once invaders, now are genetic elements that keep immune responses in check. I also discuss the possible but less characterized roles of the other helicases in modulating innate immune responses, focusing on DExH-box helicase 9 (DHX9) and Mov10 RISC complex RNA helicase (MOV10). DHX9 and MOV10 function differently from MDA5, but still use nucleic acid structure, rather than nucleotide sequence, to define self. Those genetic elements encoding the alternative conformations involved, referred to as flipons, enable helicases to dynamically shape a cell's repertoire of responses. In the case of MDA5, Alu flipons switch off the dsRNA-dependent responses against self. I suggest a number of genetic systems in which to study interactions between flipons and helicases further.


Assuntos
Helicase IFIH1 Induzida por Interferon/metabolismo , Conformação de Ácido Nucleico , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/metabolismo , Adenosina Desaminase/química , Adenosina Desaminase/metabolismo , Animais , RNA Helicases DEAD-box/metabolismo , Doença , Edição de Genes , Humanos , Proteínas de Neoplasias/metabolismo , RNA Helicases/metabolismo , RNA de Cadeia Dupla/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Especificidade por Substrato , Vírus
15.
Int J Mol Sci ; 22(7)2021 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-33801676

RESUMO

Atrial fibrillation (AF) is the most common form of cardiac arrhythmia seen in clinical practice. While some clinical parameters may predict the transition from paroxysmal to persistent AF, the molecular mechanisms behind the AF perpetuation are poorly understood. Thus, oxidative stress, calcium overload and inflammation, among others, are believed to be involved in AF-induced atrial remodelling. Interestingly, adenosine and its receptors have also been related to AF development and perpetuation. Here, we investigated the expression of adenosine A2A receptor (A2AR) both in right atrium biopsies and peripheral blood mononuclear cells (PBMCs) from non-dilated sinus rhythm (ndSR), dilated sinus rhythm (dSR) and AF patients. In addition, plasma adenosine content and adenosine deaminase (ADA) activity in these subjects were also determined. Our results revealed increased A2AR expression in the right atrium from AF patients, as previously described. Interestingly, increased levels of adenosine content and reduced ADA activity in plasma from AF patients were detected. An increase was observed when A2AR expression was assessed in PBMCs from AF subjects. Importantly, a positive correlation (P=0.001) between A2AR expression in the right atrium and PBMCs was observed. Overall, these results highlight the importance of the A2AR in AF and suggest that the evaluation of this receptor in PBMCs may be potentially be useful in monitoring disease severity and the efficacy of pharmacological treatments in AF patients.


Assuntos
Fibrilação Atrial/sangue , Leucócitos Mononucleares/citologia , Receptor A2A de Adenosina/sangue , Regulação para Cima , Adenosina/metabolismo , Adenosina Desaminase/metabolismo , Idoso , Idoso de 80 Anos ou mais , Remodelamento Atrial , Feminino , Células HEK293 , Átrios do Coração , Humanos , Masculino , Microscopia Confocal , Pessoa de Meia-Idade
16.
Clin Chem Lab Med ; 59(9): 1592-1599, 2021 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-33908223

RESUMO

OBJECTIVES: The aim of the present study was to validate a commercially available automated assay for the measurement of total adenosine deaminase (tADA) and its isoenzymes (ADA1 and ADA2) in saliva in a fast and accurate way, and evaluate the possible changes of these analytes in individuals with SARS-CoV-2 infection. METHODS: The validation, in addition to the evaluation of precision and accuracy, included the analysis of the effects of the main procedures that are currently being used for SARS-CoV-2 inactivation in saliva and a pilot study to evaluate the possible changes in salivary tADA and isoenzymes in individuals infected with SARS-CoV-2. RESULTS: The automated assay proved to be accurate and precise, with intra- and inter-assay coefficients of variation below 8.2%, linearity under dilution linear regression with R2 close to 1, and recovery percentage between 80 and 120% in all cases. This assay was affected when the sample is treated with heat or SDS for virus inactivation but tolerated Triton X-100 and NP-40. Individuals with SARS-CoV-2 infection (n=71) and who recovered from infection (n=11) had higher mean values of activity of tADA and its isoenzymes than healthy individuals (n=35). CONCLUSIONS: tADA and its isoenzymes ADA1 and ADA2 can be measured accurately and precisely in saliva samples in a rapid, economical, and reproducible way and can be analyzed after chemical inactivation with Triton X-100 and NP-40. Besides, the changes observed in tADA and isoenzymes in individuals with COVID-19 open the possibility of their potential use as non-invasive biomarkers in this disease.


Assuntos
Adenosina Desaminase/metabolismo , Bioensaio/métodos , Biomarcadores/metabolismo , COVID-19/diagnóstico , SARS-CoV-2/enzimologia , Saliva/enzimologia , Adulto , COVID-19/virologia , Estudos de Casos e Controles , Feminino , Humanos , Isoenzimas , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Adulto Jovem
17.
Nat Commun ; 12(1): 2165, 2021 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-33846332

RESUMO

Adenosine-to-inosine (A-to-I) RNA editing catalyzed by ADAR enzymes occurs in double-stranded RNAs. Despite a compelling need towards predictive understanding of natural and engineered editing events, how the RNA sequence and structure determine the editing efficiency and specificity (i.e., cis-regulation) is poorly understood. We apply a CRISPR/Cas9-mediated saturation mutagenesis approach to generate libraries of mutations near three natural editing substrates at their endogenous genomic loci. We use machine learning to integrate diverse RNA sequence and structure features to model editing levels measured by deep sequencing. We confirm known features and identify new features important for RNA editing. Training and testing XGBoost algorithm within the same substrate yield models that explain 68 to 86 percent of substrate-specific variation in editing levels. However, the models do not generalize across substrates, suggesting complex and context-dependent regulation patterns. Our integrative approach can be applied to larger scale experiments towards deciphering the RNA editing code.


Assuntos
Adenosina Desaminase/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Mutagênese/genética , Edição de RNA/genética , Sequências Reguladoras de Ácido Nucleico/genética , Algoritmos , Sequência de Bases , Proteína 9 Associada à CRISPR/metabolismo , Células HEK293 , Humanos , Aprendizado de Máquina , Modelos Genéticos , Mutação/genética , Conformação de Ácido Nucleico , RNA/química , RNA/genética , Especificidade por Substrato
18.
Mol Cell ; 81(11): 2374-2387.e3, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-33905683

RESUMO

Adenosine-to-inosine editing is catalyzed by ADAR1 at thousands of sites transcriptome-wide. Despite intense interest in ADAR1 from physiological, bioengineering, and therapeutic perspectives, the rules of ADAR1 substrate selection are poorly understood. Here, we used large-scale systematic probing of ∼2,000 synthetic constructs to explore the structure and sequence context determining editability. We uncover two structural layers determining the formation and propagation of A-to-I editing, independent of sequence. First, editing is robustly induced at fixed intervals of 35 bp upstream and 30 bp downstream of structural disruptions. Second, editing is symmetrically introduced on opposite sites on a double-stranded structure. Our findings suggest a recursive model for RNA editing, whereby the structural alteration induced by the editing at one site iteratively gives rise to the formation of an additional editing site at a fixed periodicity, serving as a basis for the propagation of editing along and across both strands of double-stranded RNA structures.


Assuntos
Adenosina Desaminase/genética , Adenosina/metabolismo , Inosina/metabolismo , Edição de RNA , RNA de Cadeia Dupla/genética , Proteínas de Ligação a RNA/genética , Células A549 , Adenosina/genética , Adenosina Desaminase/metabolismo , Animais , Pareamento de Bases , Células HEK293 , Humanos , Inosina/genética , Células MCF-7 , Camundongos , Células NIH 3T3 , Conformação de Ácido Nucleico , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/metabolismo , Proteínas de Ligação a RNA/metabolismo
19.
Nat Commun ; 12(1): 2287, 2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33863894

RESUMO

Both adenine base editors (ABEs) and cytosine base editors (CBEs) have been recently revealed to induce transcriptome-wide RNA off-target editing in a guide RNA-independent manner. Here we construct a reporter system containing E.coli Hokb gene with a tRNA-like motif for robust detection of RNA editing activities as the optimized ABE, ABEmax, induces highly efficient A-to-I (inosine) editing within an E.coli tRNA-like structure. Then, we design mutations to disrupt the potential interaction between TadA and tRNAs in structure-guided principles and find that Arginine 153 (R153) within TadA is essential for deaminating RNAs with core tRNA-like structures. Two ABEmax or mini ABEmax variants (TadA* fused with Cas9n) with deletion of R153 within TadA and/or TadA* (named as del153/del153* and mini del153) are successfully engineered, showing minimized RNA off-targeting, but comparable DNA on-targeting activities. Moreover, R153 deletion in recently reported ABE8e or ABE8s can also largely reduce their RNA off-targeting activities. Taken together, we develop a strategy to generate engineered ABEs (eABEs) with minimized RNA off-targeting activities.


Assuntos
Adenosina Desaminase/genética , Proteína 9 Associada à CRISPR/genética , DNA/genética , Proteínas de Escherichia coli/genética , Edição de Genes/métodos , Adenina/metabolismo , Adenosina Desaminase/metabolismo , Toxinas Bacterianas/genética , Proteína 9 Associada à CRISPR/metabolismo , Linhagem Celular Tumoral , Citosina/metabolismo , DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Genes Reporter , Células HEK293 , Humanos , Inosina/genética , Inosina/metabolismo , Engenharia de Proteínas , Edição de RNA/genética , RNA de Transferência/genética , RNA de Transferência/metabolismo , RNA-Seq , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
20.
Int J Mol Sci ; 22(7)2021 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-33805331

RESUMO

Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion spectroscopy is commonly used for quantifying conformational changes of protein in µs-to-ms timescale transitions. To elucidate the dynamics and mechanism of protein binding, parameters implementing CPMG relaxation dispersion results must be appropriately determined. Building an analytical model for multi-state transitions is particularly complex. In this study, we developed a new global search algorithm that incorporates a random search approach combined with a field-dependent global parameterization method. The robust inter-dependence of the parameters carrying out the global search for individual residues (GSIR) or the global search for total residues (GSTR) provides information on the global minimum of the conformational transition process of the Zα domain of human ADAR1 (hZαADAR1)-DNA complex. The global search results indicated that a α-helical segment of hZαADAR1 provided the main contribution to the three-state conformational changes of a hZαADAR1-DNA complex with a slow B-Z exchange process. The two global exchange rate constants, kex and kZB, were found to be 844 and 9.8 s-1, respectively, in agreement with two regimes of residue-dependent chemical shift differences-the "dominant oscillatory regime" and "semi-oscillatory regime". We anticipate that our global search approach will lead to the development of quantification methods for conformational changes not only in Z-DNA binding protein (ZBP) binding interactions but also in various protein binding processes.


Assuntos
Adenosina Desaminase/química , DNA de Forma B/química , DNA Forma Z/química , Modelos Moleculares , Proteínas de Ligação a RNA/química , Adenosina Desaminase/metabolismo , Algoritmos , DNA de Forma B/metabolismo , DNA Forma Z/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Conformação Proteica , Proteínas de Ligação a RNA/metabolismo , Termodinâmica
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