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1.
PLoS Pathog ; 16(9): e1008842, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32898178

RESUMO

Signaling through retinoic acid inducible gene I (RIG-I) like receptors (RLRs) is tightly regulated, with activation occurring upon sensing of viral nucleic acids, and suppression mediated by negative regulators. Under homeostatic conditions aberrant activation of melanoma differentiation-associated protein-5 (MDA5) is prevented through editing of endogenous dsRNA by RNA editing enzyme Adenosine Deaminase Acting on RNA (ADAR1). In addition, ADAR1 is postulated to play pro-viral and antiviral roles during viral infections that are dependent or independent of RNA editing activity. Here, we investigated the importance of ADAR1 isoforms in modulating influenza A virus (IAV) replication and revealed the opposing roles for ADAR1 isoforms, with the nuclear p110 isoform restricting versus the cytoplasmic p150 isoform promoting IAV replication. Importantly, we demonstrate that p150 is critical for preventing sustained RIG-I signaling, as p150 deficient cells showed increased IFN-ß expression and apoptosis during IAV infection, independent of RNA editing activity. Taken together, the p150 isoform of ADAR1 is important for preventing sustained RIG-I induced IFN-ß expression and apoptosis during viral infection.


Assuntos
Adenosina Desaminase/metabolismo , Apoptose , Proteína DEAD-box 58/metabolismo , Vírus da Influenza A/fisiologia , Influenza Humana/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Replicação Viral , Células A549 , Adenosina Desaminase/genética , Proteína DEAD-box 58/genética , Células HEK293 , Humanos , Influenza Humana/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas de Ligação a RNA/genética
2.
DNA Cell Biol ; 39(10): 1862-1871, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32845709

RESUMO

Sepsis is a life-threatening disorder and leads to organ dysfunction and death. Therefore, searching for more alternative biomarkers is of great significance for sepsis assessment and surveillance. In our study, the gene expression profiles of 163 samples from healthy controls and septic patients were analyzed and 8 gene co-expression modules were identified by constructing weighted gene co-expression network. The blue and yellow modules showed close correlations with the phenotypic trait "days postsepsis." Besides, differentially expressed genes (DEGs) over time in septic patients were screened using Short Time-series Expression Miner (STEM) program. The intersection of genes in the blue and yellow modules and DEGs, which were significantly enriched in "HTLV-1 infection" pathway, was analyzed with protein-protein interaction network. The logistic regression model based on these eight mRNAs was constructed to determine the type of the sample reliably. Eight vital genes CECR1, ANXA2, ELANE, CTSG, AZU1, PRTN3, LYZ, and DEFA4 presented high scores and may be associated with sepsis, which provided candidate biomarkers for sepsis.


Assuntos
Sepse/genética , Transcriptoma , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Anexina A2/genética , Anexina A2/metabolismo , Biomarcadores/metabolismo , Catepsina G/genética , Catepsina G/metabolismo , Redes Reguladoras de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Elastase de Leucócito/genética , Elastase de Leucócito/metabolismo , Mieloblastina/genética , Mieloblastina/metabolismo , Sepse/metabolismo , Sepse/patologia , Análise de Sobrevida , alfa-Defensinas/genética , alfa-Defensinas/metabolismo
3.
Genet Sel Evol ; 52(1): 35, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32611306

RESUMO

Base editing has the potential to improve important economic traits in agriculture and can precisely convert single nucleotides in DNA or RNA sequences into minimal double-strand DNA breaks (DSB). Adenine base editors (ABE) have recently emerged as a base editing tool for the conversion of targeted A:T to G:C, but have not yet been used in sheep. ABEmax is one of the latest versions of ABE, which consists of a catalytically-impaired nuclease and a laboratory-evolved DNA-adenosine deaminase. The Booroola fecundity (FecBB) mutation (g.A746G, p.Q249R) in the bone morphogenetic protein receptor 1B (BMPR1B) gene influences fecundity in many sheep breeds. In this study, by using ABEmax we successfully obtained lambs with defined point mutations that result in an amino acid substitution (p.Gln249Arg). The efficiency of the defined point mutations was 75% in newborn lambs, since six lambs were heterozygous at the FecBB mutation site (g.A746G, p.Q249R), and two lambs were wild-type. We did not detect off-target mutations in the eight edited lambs. Here, we report the validation of the first gene-edited sheep generated by ABE and highlight its potential to improve economically important traits in livestock.


Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo I/genética , Fertilidade/genética , Edição de Genes/métodos , Adenina/metabolismo , Adenosina Desaminase/metabolismo , Adenosina Desaminase/fisiologia , Animais , Cruzamento , Feminino , Engenharia Genética/métodos , Genótipo , Heterozigoto , Tamanho da Ninhada de Vivíparos/genética , Masculino , Mutação , Fenótipo , Polimorfismo de Nucleotídeo Único , Gravidez , Ovinos/genética
4.
Parasitol Res ; 119(9): 2897-2905, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32677001

RESUMO

The central nervous system of the intermediate host plays a central role in lifelong persistence of Toxoplasma gondii as well as the pathogenesis of congenital toxoplasmosis and reactivated infection in immunocompromised individuals. The purinergic system has been implicated in a wide range of immunological pathways for controlling intracellular responses to pathogens, including T. gondii. In the present study, we investigated the effect of resveratrol (RSV) on ectonucleotidases, adenosine deaminase (ADA), and purinergic receptors during chronic infection by T. gondii. For this study, Swiss mice were divided into control (CTL), resveratrol (RSV), infected (INF), and INF+RSV groups. The animals were orally infected with the VEG strain and treated with RSV (100 mg/kg, orally). Ectonucleotidase activities, P2X7, P2Y1, A1, and A2A purinergic receptor density, ROS, and thiobarbituric acid reactive substances levels were measured in the cerebral cortex of mice. T. gondii infection increased NTPDase and reduced ADA activities. Treatment with RSV also affected enzymes hydrolysing extracellular nucleotides and nucleosides. Finally, RSV affected P1 and P2 purinergic receptor expression during T. gondii infection. Overall, RSV-mediated beneficial changes in purinergic signalling and oxidative stress, possibly improving cerebral cortex homeostasis in T. gondii infection.


Assuntos
Córtex Cerebral/parasitologia , Inibidores Enzimáticos/farmacologia , Fármacos Neuroprotetores/farmacologia , Resveratrol/farmacologia , Toxoplasmose Animal/tratamento farmacológico , Adenosina Desaminase/metabolismo , Animais , Camundongos , Receptores Purinérgicos/metabolismo , Transdução de Sinais , Toxoplasma/imunologia
5.
Sci Adv ; 6(25): eabb5813, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32596474

RESUMO

The COVID-19 outbreak has become a global health risk, and understanding the response of the host to the SARS-CoV-2 virus will help to combat the disease. RNA editing by host deaminases is an innate restriction process to counter virus infection, but it is not yet known whether this process operates against coronaviruses. Here, we analyze RNA sequences from bronchoalveolar lavage fluids obtained from coronavirus-infected patients. We identify nucleotide changes that may be signatures of RNA editing: adenosine-to-inosine changes from ADAR deaminases and cytosine-to-uracil changes from APOBEC deaminases. Mutational analysis of genomes from different strains of Coronaviridae from human hosts reveals mutational patterns consistent with those observed in the transcriptomic data. However, the reduced ADAR signature in these data raises the possibility that ADARs might be more effective than APOBECs in restricting viral propagation. Our results thus suggest that both APOBECs and ADARs are involved in coronavirus genome editing, a process that may shape the fate of both virus and patient.


Assuntos
Betacoronavirus/genética , Betacoronavirus/metabolismo , Infecções por Coronavirus/genética , Interações Hospedeiro-Patógeno/genética , Pneumonia Viral/genética , Edição de RNA/genética , Transcriptoma , Desaminases APOBEC/genética , Desaminases APOBEC/metabolismo , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Sequência de Bases/genética , Líquido da Lavagem Broncoalveolar/virologia , Infecções por Coronavirus/virologia , Genoma Viral/genética , Humanos , Taxa de Mutação , Nucleotídeos/genética , Nucleotídeos/metabolismo , Pandemias , Pneumonia Viral/virologia , RNA Viral/genética , Replicação Viral/genética
6.
Nucleic Acids Res ; 48(14): 7958-7972, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32597966

RESUMO

Adenosine deaminases acting on RNA (ADARs) are enzymes that convert adenosine to inosine in duplex RNA, a modification that exhibits a multitude of effects on RNA structure and function. Recent studies have identified ADAR1 as a potential cancer therapeutic target. ADARs are also important in the development of directed RNA editing therapeutics. A comprehensive understanding of the molecular mechanism of the ADAR reaction will advance efforts to develop ADAR inhibitors and new tools for directed RNA editing. Here we report the X-ray crystal structure of a fragment of human ADAR2 comprising its deaminase domain and double stranded RNA binding domain 2 (dsRBD2) bound to an RNA duplex as an asymmetric homodimer. We identified a highly conserved ADAR dimerization interface and validated the importance of these sequence elements on dimer formation via gel mobility shift assays and size exclusion chromatography. We also show that mutation in the dimerization interface inhibits editing in an RNA substrate-dependent manner for both ADAR1 and ADAR2.


Assuntos
Adenosina Desaminase/química , Adenosina Desaminase/metabolismo , Edição de RNA , RNA de Cadeia Dupla/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Adenosina Desaminase/genética , Cristalografia por Raios X , Humanos , Modelos Moleculares , Mutação , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , RNA de Cadeia Dupla/química , Proteínas de Ligação a RNA/genética
7.
PLoS Genet ; 16(6): e1008836, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32479508

RESUMO

Codon usage bias is a universal feature of all genomes and plays an important role in regulating protein expression levels. Modification of adenosine to inosine at the tRNA anticodon wobble position (I34) by adenosine deaminases (ADATs) is observed in all eukaryotes and has been proposed to explain the correlation between codon usage and tRNA pool. However, how the tRNA pool is affected by I34 modification to influence codon usage-dependent gene expression is unclear. Using Neurospora crassa as a model system, by combining molecular, biochemical and bioinformatics analyses, we show that silencing of adat2 expression severely impaired the I34 modification levels for the ADAT-related tRNAs, resulting in major ADAT-related tRNA profile changes and reprogramming of translation elongation kinetics on ADAT-related codons. adat2 silencing also caused genome-wide codon usage-biased ribosome pausing on mRNAs and proteome landscape changes, leading to selective translational repression or induction of different mRNAs. The induced expression of CPC-1, the Neurospora ortholog of yeast GCN4p, mediates the transcriptional response after adat2 silencing and amino acid starvation. Together, our results demonstrate that the tRNA I34 modification by ADAT plays a major role in driving codon usage-biased translation to shape proteome landscape.


Assuntos
Anticódon/genética , Uso do Códon , Elongação Traducional da Cadeia Peptídica/genética , Proteoma/genética , RNA de Transferência de Arginina/genética , Adenosina/metabolismo , Adenosina Desaminase/metabolismo , Anticódon/metabolismo , Biologia Computacional , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Inosina/metabolismo , Neurospora crassa/genética , RNA de Transferência de Arginina/metabolismo , Ribossomos/metabolismo
8.
Proc Natl Acad Sci U S A ; 117(19): 10265-10270, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32350138

RESUMO

Coformycin and pentostatin are structurally related N-nucleoside inhibitors of adenosine deaminase characterized by an unusual 1,3-diazepine nucleobase. Herein, the cof gene cluster responsible for coformycin biosynthesis is identified. Reconstitution of the coformycin biosynthetic pathway in vitro demonstrates that it overlaps significantly with the early stages of l-histidine biosynthesis. Committed entry into the coformycin pathway takes place via conversion of a shared branch point intermediate to 8-ketocoformycin-[Formula: see text]-monophosphate catalyzed by CofB, which is a homolog of succinylaminoimidazolecarboxamide ribotide (SAICAR) synthetase. This reaction appears to proceed via a Dieckmann cyclization and a retro-aldol elimination, releasing ammonia and D-erythronate-4-phosphate as coproducts. Completion of coformycin biosynthesis involves reduction and dephosphorylation of the CofB product, with the former reaction being catalyzed by the NADPH-dependent dehydrogenase CofA. CofB also shows activation by adenosine triphosphate (ATP) despite the reaction requiring neither a phosphorylated nor an adenylated intermediate. This may serve to help regulate metabolic partitioning between the l-histidine and coformycin pathways.


Assuntos
Adenosina Desaminase/química , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/genética , Coformicina/biossíntese , Família Multigênica , Streptomyces/genética , Adenosina Desaminase/metabolismo , Monofosfato de Adenosina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Vias Biossintéticas , Fosforilação , Streptomyces/metabolismo
9.
Nat Neurosci ; 23(6): 718-729, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32367065

RESUMO

DNA forms conformational states beyond the right-handed double helix; however, the functional relevance of these noncanonical structures in the brain remains unknown. Here we show that, in the prefrontal cortex of mice, the formation of one such structure, Z-DNA, is involved in the regulation of extinction memory. Z-DNA is formed during fear learning and reduced during extinction learning, which is mediated, in part, by a direct interaction between Z-DNA and the RNA-editing enzyme Adar1. Adar1 binds to Z-DNA during fear extinction learning, which leads to a reduction in Z-DNA at sites where Adar1 is recruited. Knockdown of Adar1 leads to an inability to modify a previously acquired fear memory and blocks activity-dependent changes in DNA structure and RNA state-effects that are fully rescued by the introduction of full-length Adar1. These findings suggest a new mechanism of learning-induced gene regulation that is dependent on proteins that recognize alternate DNA structure states, which are required for memory flexibility.


Assuntos
Adenosina Desaminase/metabolismo , Adenosina Desaminase/fisiologia , DNA Forma Z/fisiologia , Extinção Psicológica/fisiologia , Edição de RNA/fisiologia , Animais , DNA Forma Z/metabolismo , Medo , Aprendizagem/fisiologia , Camundongos , Córtex Pré-Frontal/metabolismo , RNA Interferente Pequeno/farmacologia
10.
Anticancer Res ; 40(3): 1307-1314, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32132027

RESUMO

BACKGROUND/AIM: Malignant pleural mesothelioma (MPM) is an intractable cancer, and causes of its malignant transformation are not well known. Adenosine deaminase acting on RNA (ADAR) is an RNA-editing enzyme that converts adenosine into inosine in double-stranded RNAs potentially involved in malignant development. MATERIALS AND METHODS: To examine the role of ADAR1 and ADAR2 in MPM, small interfering RNAs (siRNAs) against ADAR1 or ADAR2 were used. RESULTS: Transfection of siRNA against ADAR2 suppressed proliferation, motility, and invasiveness of MPM cells expressing both ADAR1 and ADAR2; however, siRNA against ADAR1 did not affect these cellular activities. Overexpression of ADAR2, that was incapable of binding to RNA, suppressed growth, motility, and invasion of MPM cells. However, overexpression of ADAR2 that had no enzyme activity did not alter the malignant properties of MPM cells. CONCLUSION: Enhancement of the malignant characteristics of cultured MPM cells via ADAR2 was independent of RNA-editing activity.


Assuntos
Adenosina Desaminase/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Mesotelioma/genética , Mesotelioma/metabolismo , Edição de RNA , Proteínas de Ligação a RNA/metabolismo , Adenosina Desaminase/biossíntese , Adenosina Desaminase/genética , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologia , Mesotelioma/enzimologia , Mesotelioma/patologia , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/biossíntese , Proteínas de Ligação a RNA/genética , Transfecção
11.
Proc Natl Acad Sci U S A ; 117(11): 5987-5996, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32123111

RESUMO

Endogenous retroviruses and long terminal repeat (LTR) retrotransposons are mobile genetic elements that are closely related to retroviruses. Desilenced endogenous retroviruses are associated with human autoimmune disorders and neurodegenerative diseases. Caenorhabditis elegans and related Caenorhabditis spp. contain LTR retrotransposons and, as described here, numerous integrated viral genes including viral envelope genes that are part of LTR retrotransposons. We found that both LTR retrotransposons and endogenous viral elements are silenced by ADARs [adenosine deaminases acting on double-stranded RNA (dsRNA)] together with the endogenous RNA interference (RNAi) factor ERI-6/7, a homolog of MOV10 helicase, a retrotransposon and retrovirus restriction factor in human. siRNAs corresponding to integrated viral genes and LTR retrotransposons, but not to DNA transposons, are dependent on the ADARs and ERI-6/7. siRNAs corresponding to palindromic repeats are independent of the ADARs and ERI-6/7, and are in fact increased in adar- and eri-6/7-defective mutants because of an antiviral RNAi response to dsRNA. Silencing of LTR retrotransposons is dependent on downstream RNAi factors and P granule components but is independent of the viral sensor DRH-1/RIG-I and the nuclear Argonaute NRDE-3. The activation of retrotransposons in the ADAR- and ERI-6/7/MOV10-defective mutant is associated with the induction of the unfolded protein response (UPR), a common response to viral infection. The overlap between genes induced upon viral infection and infection with intracellular pathogens and genes coexpressed with retrotransposons suggests that there is a common response to different types of foreign elements that includes a response to proteotoxicity presumably caused by the burden of replicating pathogens and expressed retrotransposons.


Assuntos
Caenorhabditis elegans/genética , Retrovirus Endógenos/genética , Interações entre Hospedeiro e Microrganismos/genética , Interferência de RNA , Retroelementos/genética , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Animais , Caenorhabditis elegans/virologia , Proteínas de Caenorhabditis elegans/metabolismo , DNA Helicases/metabolismo , DNA Viral/metabolismo , Estresse do Retículo Endoplasmático/genética , Regulação Viral da Expressão Gênica , Genes Virais/genética , Humanos , RNA de Cadeia Dupla/metabolismo , RNA Viral/metabolismo , Homologia de Sequência de Aminoácidos , Sequências Repetidas Terminais/genética , Resposta a Proteínas não Dobradas/genética
12.
Nucleic Acids Res ; 48(8): 3999-4012, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32201888

RESUMO

In eukaryotic cells, with the exception of the specialized genomes of mitochondria and plastids, all genetic information is sequestered within the nucleus. This arrangement imposes constraints on how the information can be tailored for different cellular regions, particularly in cells with complex morphologies like neurons. Although messenger RNAs (mRNAs), and the proteins that they encode, can be differentially sorted between cellular regions, the information itself does not change. RNA editing by adenosine deamination can alter the genome's blueprint by recoding mRNAs; however, this process too is thought to be restricted to the nucleus. In this work, we show that ADAR2 (adenosine deaminase that acts on RNA), an RNA editing enzyme, is expressed outside of the nucleus in squid neurons. Furthermore, purified axoplasm exhibits adenosine-to-inosine activity and can specifically edit adenosines in a known substrate. Finally, a transcriptome-wide analysis of RNA editing reveals that tens of thousands of editing sites (>70% of all sites) are edited more extensively in the squid giant axon than in its cell bodies. These results indicate that within a neuron RNA editing can recode genetic information in a region-specific manner.


Assuntos
Adenosina Desaminase/metabolismo , Neurônios/enzimologia , Edição de RNA , Adenosina/metabolismo , Animais , Axônios/enzimologia , Citoplasma/enzimologia , Decapodiformes/enzimologia , Células HEK293 , Humanos , Inosina/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Sinapses/enzimologia
13.
Nat Commun ; 11(1): 799, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32034135

RESUMO

RNA editing and splicing are the two major processes that dynamically regulate human transcriptome diversity. Despite growing evidence of crosstalk between RNA editing enzymes (mainly ADAR1) and splicing machineries, detailed mechanistic explanations and their biological importance in diseases, such as cancer are still lacking. Herein, we identify approximately a hundred high-confidence splicing events altered by ADAR1 and/or ADAR2, and ADAR1 or ADAR2 protein can regulate cassette exons in both directions. We unravel a binding tendency of ADARs to dsRNAs that involves GA-rich sequences for editing and splicing regulation. ADAR1 edits an intronic splicing silencer, leading to recruitment of SRSF7 and repression of exon inclusion. We also present a mechanism through which ADAR2 binds to dsRNA formed between GA-rich sequences and polypyrimidine (Py)-tract and precludes access of U2AF65 to 3' splice site. Furthermore, we find these ADARs-regulated splicing changes per se influence tumorigenesis, not merely byproducts of ADARs editing and binding.


Assuntos
Adenosina Desaminase/metabolismo , Neoplasias/genética , Precursores de RNA/genética , Proteínas de Ligação a RNA/metabolismo , Adenosina Desaminase/genética , Processamento Alternativo , Animais , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Éxons , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas de Membrana/genética , Camundongos Endogâmicos NOD , Degradação do RNAm Mediada por Códon sem Sentido , Edição de RNA , Sítios de Splice de RNA , Processamento de RNA , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Fatores de Processamento de Serina-Arginina/genética , Fator de Processamento U2AF/genética
14.
Nucleic Acids Res ; 48(7): e41, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32083657

RESUMO

RNAs are post-transcriptionally modified by dedicated writer or eraser enzymes that add or remove specific modifications, respectively. Mass spectrometry (MS) of RNA is a useful tool to study the modification state of an oligonucleotide (ON) in a sensitive manner. Here, we developed an ion-pairing reagent free chromatography for positive ion detection of ONs by low- and high-resolution MS, which does not interfere with other types of small compound analyses done on the same instrument. We apply ON-MS to determine the ONs from an RNase T1 digest of in vitro transcribed tRNA, which are purified after ribozyme-fusion transcription by automated size exclusion chromatography. The thus produced tRNAValAAC is substrate of the human tRNA ADAT2/3 enzyme and we confirm the deamination of adenosine to inosine and the formation of tRNAValIACin vitro by ON-MS. Furthermore, low resolution ON-MS is used to monitor the demethylation of ONs containing 1-methyladenosine by bacterial AlkB in vitro. The power of high-resolution ON-MS is demonstrated by the detection and mapping of modified ONs from native total tRNA digested with RNase T1. Overall, we present an oligonucleotide MS method which is broadly applicable to monitor in vitro RNA (de-)modification processes and native RNA.


Assuntos
Espectrometria de Massas , Oligonucleotídeos/análise , Processamento Pós-Transcricional do RNA , RNA de Transferência/química , RNA de Transferência/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina Desaminase/metabolismo , Cromatografia em Gel , Células HEK293 , Células HeLa , Humanos , Oxigenases de Função Mista/metabolismo , Oligonucleotídeos/isolamento & purificação , RNA de Transferência/biossíntese , RNA de Transferência/isolamento & purificação , RNA de Transferência de Valina/química , RNA de Transferência de Valina/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonuclease T1/metabolismo
15.
BMC Biol ; 18(1): 15, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-32059717

RESUMO

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.


Assuntos
Adenosina Desaminase/genética , Autofagia , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiologia , Transmissão Sináptica/genética , Adenosina Desaminase/metabolismo , Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Larva/genética , Larva/crescimento & desenvolvimento , Larva/fisiologia , Masculino , Mutação
16.
Nat Commun ; 11(1): 198, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31924792

RESUMO

The neural crest gives rise to numerous cell types, dysfunction of which contributes to many disorders. Here, we report that adenosine deaminase acting on RNA (ADAR1), responsible for adenosine-to-inosine editing of RNA, is required for regulating the development of two neural crest derivatives: melanocytes and Schwann cells. Neural crest specific conditional deletion of Adar1 in mice leads to global depigmentation and absence of myelin from peripheral nerves, resulting from alterations in melanocyte survival and differentiation of Schwann cells, respectively. Upregulation of interferon stimulated genes precedes these defects, which are associated with the triggering of a signature resembling response to injury in peripheral nerves. Simultaneous extinction of MDA5, a key sensor of unedited RNA, rescues both melanocytes and myelin defects in vitro, suggesting that ADAR1 safeguards neural crest derivatives from aberrant MDA5-mediated interferon production. We thus extend the landscape of ADAR1 function to the fields of neural crest development and disease.


Assuntos
Adenosina Desaminase/metabolismo , Melanócitos/metabolismo , Crista Neural/metabolismo , Células de Schwann/metabolismo , Adenosina Desaminase/genética , Animais , Diferenciação Celular , Modelos Animais de Doenças , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Coração , Interferons/metabolismo , Camundongos , Camundongos Knockout , Neurogênese , Edição de RNA , Nervo Isquiático/citologia , Pele/patologia , Transcriptoma , Regulação para Cima
17.
DNA Cell Biol ; 39(3): 343-348, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31999481

RESUMO

RNA editing is a process by which nascent RNA transcripts are covalently modified, thus enhancing the complexity of the transcriptome. The most common modifications are deaminations of adenosine to inosine at sites of complex RNA secondary structure, a process that is carried out by the adenosine deaminase acting on double-strand RNA (ADAR) family of RNA editases. Although much has been learned about the ADAR family members since their discovery, very little information on their post-transcriptional regulation has been reported. Similar to most proteins, the ADAR family members are post-translationally modified at multiple sites. We recently reported that members of the AKT kinase family directly phosphorylate ADAR1p110 and ADAR2 on a conserved threonine within the catalytic domain of the protein. Phosphorylation was observed to differentially inhibit the enzymatic activity of the ADAR proteins toward known RNA substrates. The direct downstream involvement of the AKT kinases in multiple major signaling pathways associated with cell survival, growth, glucose metabolism (insulin signaling), and differentiation is well established; thus, the AKT kinases represent a link between ADAR-dependent A-to-I editing and major signal transduction pathways that are necessary for cell maintenance and development.


Assuntos
Adenosina Desaminase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Edição de RNA , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Animais , Humanos , Fosforilação
18.
Cell ; 180(2): 278-295.e23, 2020 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-31978345

RESUMO

Mutations in FAMIN cause arthritis and inflammatory bowel disease in early childhood, and a common genetic variant increases the risk for Crohn's disease and leprosy. We developed an unbiased liquid chromatography-mass spectrometry screen for enzymatic activity of this orphan protein. We report that FAMIN phosphorolytically cleaves adenosine into adenine and ribose-1-phosphate. Such activity was considered absent from eukaryotic metabolism. FAMIN and its prokaryotic orthologs additionally have adenosine deaminase, purine nucleoside phosphorylase, and S-methyl-5'-thioadenosine phosphorylase activity, hence, combine activities of the namesake enzymes of central purine metabolism. FAMIN enables in macrophages a purine nucleotide cycle (PNC) between adenosine and inosine monophosphate and adenylosuccinate, which consumes aspartate and releases fumarate in a manner involving fatty acid oxidation and ATP-citrate lyase activity. This macrophage PNC synchronizes mitochondrial activity with glycolysis by balancing electron transfer to mitochondria, thereby supporting glycolytic activity and promoting oxidative phosphorylation and mitochondrial H+ and phosphate recycling.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Adenina/metabolismo , Adenosina/metabolismo , Adenosina Desaminase/metabolismo , Cromatografia Líquida/métodos , Células HEK293 , Células Hep G2 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Espectrometria de Massas/métodos , Enzimas Multifuncionais/genética , Fosforilação , Proteínas/genética , Nucleotídeos de Purina/metabolismo , Purinas/metabolismo
19.
Res Vet Sci ; 129: 90-95, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31954319

RESUMO

This study aims to evaluate the effect of the presence of food and the material used in a panel of biomarkers in saliva of horses. For the food effect study, clean saliva was incubated with a known amount of food consisting of oats, hay or grass. Significant changes were observed when saliva was incubated with oats for total protein (P = .050) and phosphorus (P = .008), with grass for total protein (P = .037), salivary alpha-amylase (sAA, P = .018), total esterase (TEA, P = .018), butyrilcholinesterase (BChE, P = .037), adenosine deaminase (ADA, P = .037), and total bilirubin (P = .018), and with hay for sAA (P = .018), phosphorus (P = .037), γ-glutamyl transferase (gGT, P = .004), and creatine kinase (CK, P = .016). For the material-based collection study, saliva using a sponge and a cotton role at the same time were collected and compared. Lower values were obtained in clean saliva collected with cotton role compared to sponge for sAA (P = .030), TEA (P = .034), BChE (P = .003), gGT (P = .002) and cortisol (P < .001) In conclusion, the presence of food and the material used for its collection, can influence the results obtained when analytes are measured in saliva of horses.


Assuntos
Ração Animal/análise , Contaminação de Alimentos , Cavalos , Saliva/química , Adenosina Desaminase/química , Adenosina Desaminase/metabolismo , Animais , Bilirrubina/química , Bilirrubina/metabolismo , Biomarcadores/química , Biomarcadores/metabolismo , Carboxilesterase/química , Carboxilesterase/metabolismo , Colinesterases/química , Colinesterases/metabolismo , Dieta/veterinária , Proteínas na Dieta/química , Proteínas na Dieta/metabolismo , Feminino , Humanos , Hidrocortisona , Masculino , Fósforo/química , Fósforo/metabolismo , alfa-Amilases/química , alfa-Amilases/metabolismo
20.
RNA ; 26(4): 454-469, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31941663

RESUMO

Adenosine-to-inosine RNA editing is an essential post-transcriptional modification catalyzed by adenosine deaminase acting on RNA (ADAR)1 and ADAR2 in mammals. For numerous sites in coding sequences (CDS) and microRNAs, editing is highly conserved and has significant biological consequences, for example, by altering amino acid residues and target recognition. However, no comprehensive and quantitative studies have been undertaken to determine how specific ADARs contribute to conserved sites in vivo. Here, we amplified each RNA region with editing site(s) separately and combined these for deep sequencing. Then, we compared the editing ratios of all sites that were conserved in CDS and microRNAs in the cerebral cortex and spleen of wild-type mice, Adar1E861A/E861AIfih-/- mice expressing inactive ADAR1 (Adar1 KI) and Adar2-/-Gria2R/R (Adar2 KO) mice. We found that most of the sites showed a preference for one ADAR. In contrast, some sites, such as miR-3099-3p, showed no ADAR preference. In addition, we found that the editing ratio for several sites, such as DACT3 R/G, was up-regulated in either Adar mutant mouse strain, whereas a coordinated interplay between ADAR1 and ADAR2 was required for the efficient editing of specific sites, such as the 5-HT2CR B site. We further created double mutant Adar1 KI Adar2 KO mice and observed viable and fertile animals with the complete absence of editing, demonstrating that ADAR1 and ADAR2 are the sole enzymes responsible for all editing sites in vivo. Collectively, these findings indicate that editing is regulated in a site-specific manner by the different interplay between ADAR1 and ADAR2.


Assuntos
Adenosina Desaminase/metabolismo , MicroRNAs/metabolismo , Edição de RNA , Proteínas de Ligação a RNA/metabolismo , Adenosina Desaminase/genética , Animais , Feminino , Masculino , Camundongos , MicroRNAs/genética , Mutação , Motivos de Nucleotídeos , Proteínas de Ligação a RNA/genética
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