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1.
Theriogenology ; 177: 140-150, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34700071

RESUMO

It has been reported that N6-methyladenosine (m6A) methyltransferase-like 3 (METTL3) plays an important role in zygote genome activation during embryonic development, but the effects of METTL3 under oxidative stress in the early development of goat embryos remain largely unknown. In this study, zygotes were monitored at 72 and 168 h after fertilization, and they developed to the 8-cell stage and blastocyst stage under hypoxic conditions and normoxic conditions. Single-cell transcriptome sequencing was performed at the 8-cell stage and the blastocyst stage in the goat embryos, the differentially expressed METTL3 was screened from the sequencing results. We found that microinjection of small interfering RNA (siRNA) against METTL3 caused developmental arrest, both 8-cell rates (37.45 ± 2.21% vs. 47.09 ± 1.38%; P < 0.01) and blastocyst rates of Si-METTL3 (12.17% ± 2.84 vs. 20.83 ± 3.61%; P < 0.01) in Si-METTL3 group were significantly decreased compared with that of control under hypoxic conditions, significant changes were found in the m6A-related genes and the expression levels of critical transcription factors, such as, NANOG, GATA3, CDX2 and SOX17, were decreased. This study revealed the key role of METTL3 in the regulation of embryonic development under oxidative stress, and laid the foundation for further study of the crucial mechanism of oxidative stress during the early embryonic development of goats.


Assuntos
Cabras , Metiltransferases , Adenosina , Animais , Desenvolvimento Embrionário , Metiltransferases/genética , RNA Mensageiro
3.
Medicina (B Aires) ; 81(5): 837-839, 2021.
Artigo em Espanhol | MEDLINE | ID: mdl-34633958

RESUMO

Ticagrelor is anantiplatelet agent which acts through reversible binding to the P2Y12 adenosine-diphosphate receptors. In acute coronary syndromes it has been shown to reduce the risk of major cardiovascular events such as myocardial infarction, stroke and death. Although some hemorrhagic, kidney, liver and respiratory complications have been described in detail with the use of ticagrelor, other less frequent adverse effects are not so well clarified. We report the case of a patient with a systemic inflammatory response syndrome secondary to the use of ticagrelor.


Assuntos
Síndrome Coronariana Aguda , Infarto do Miocárdio , Síndrome Coronariana Aguda/induzido quimicamente , Síndrome Coronariana Aguda/tratamento farmacológico , Adenosina/efeitos adversos , Humanos , Síndrome de Resposta Inflamatória Sistêmica/induzido quimicamente , Ticagrelor/efeitos adversos
4.
Zhonghua Gan Zang Bing Za Zhi ; 29(9): 904-907, 2021 Sep 20.
Artigo em Chinês | MEDLINE | ID: mdl-34638217

RESUMO

Adenosine, as an endogenous purine nucleoside, is widely distributed in various tissues and organs of the body. It binds to adenosine receptors to regulate a variety of important biological processes. Adenosine 2A receptors have a close relationship with the occurrence and development of various clinical diseases. This article reviews the research progress of adenosine 2A receptors in non-alcoholic fatty liver disease, acute immune hepatitis, liver ischemia-reperfusion injury, liver fibrosis, etc., in order to provide new research strategies for the prevention and treatment of these diseases.


Assuntos
Receptores Purinérgicos P1 , Traumatismo por Reperfusão , Adenosina , Humanos , Cirrose Hepática
5.
Sci Rep ; 11(1): 19998, 2021 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-34620963

RESUMO

Understanding the effects of metabolism on the rational design of novel and more effective drugs is still a considerable challenge. To the best of our knowledge, there are no entirely computational strategies that make it possible to predict these effects. From this perspective, the development of such methodologies could contribute to significantly reduce the side effects of medicines, leading to the emergence of more effective and safer drugs. Thereby, in this study, our strategy is based on simulating the electron ionization mass spectrometry (EI-MS) fragmentation of the drug molecules and combined with molecular docking and ADMET models in two different situations. In the first model, the drug is docked without considering the possible metabolic effects. In the second model, each of the intermediates from the EI-MS results is docked, and metabolism occurs before the drug accesses the biological target. As a proof of concept, in this work, we investigate the main antiviral drugs used in clinical research to treat COVID-19. As a result, our strategy made it possible to assess the biological activity and toxicity of all potential by-products. We believed that our findings provide new chemical insights that can benefit the rational development of novel drugs in the future.


Assuntos
Antivirais/metabolismo , COVID-19/tratamento farmacológico , Descoberta de Drogas , SARS-CoV-2/efeitos dos fármacos , Adenina/efeitos adversos , Adenina/análogos & derivados , Adenina/metabolismo , Adenina/farmacologia , Adenosina/efeitos adversos , Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina/farmacologia , Monofosfato de Adenosina/efeitos adversos , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/metabolismo , Monofosfato de Adenosina/farmacologia , Alanina/efeitos adversos , Alanina/análogos & derivados , Alanina/metabolismo , Alanina/farmacologia , Amidas/efeitos adversos , Amidas/metabolismo , Amidas/farmacologia , Antivirais/efeitos adversos , Antivirais/farmacologia , COVID-19/metabolismo , Cloroquina/efeitos adversos , Cloroquina/análogos & derivados , Cloroquina/metabolismo , Cloroquina/farmacologia , Desenho de Fármacos , Humanos , Redes e Vias Metabólicas , Simulação de Acoplamento Molecular , Nitrocompostos/efeitos adversos , Nitrocompostos/metabolismo , Nitrocompostos/farmacologia , Pirazinas/efeitos adversos , Pirazinas/metabolismo , Pirazinas/farmacologia , Pirrolidinas/efeitos adversos , Pirrolidinas/metabolismo , Pirrolidinas/farmacologia , Ribavirina/efeitos adversos , Ribavirina/metabolismo , Ribavirina/farmacologia , SARS-CoV-2/metabolismo , Tiazóis/efeitos adversos , Tiazóis/metabolismo , Tiazóis/farmacologia
6.
Anal Chim Acta ; 1182: 338946, 2021 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-34602192

RESUMO

This work reports the first electrochemical bioplatform developed for the multidetection of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in DNA, DNA N6-methyladenine (6mA) and RNA N6-methyladenosine (m6A) methylations at global level. Direct competitive immunoassays were implemented on the surface of magnetic beads (MBs) and optimized for the single amperometric determination of different targets varying in length, sequence and number of methylations on screen-printed carbon electrodes. After evaluating the sensitivity and selectivity of such determinations and the confirmation of no cross-reactivity, a multiplexed disposable platform allowing the simultaneous determination of the mentioned four methylation events in only 45 min has been prepared. The multiplexed bioplatform was successfully applied to the determination of m6A in cellular total RNA and of 5-mC, 5-hmC and 6mA in genomic DNA extracted from tissues. The developed bioplatform showed its usefulness to discriminate the aggressiveness of cancerous cells and between healthy and tumor tissues of colorectal cancer patients.


Assuntos
Ácidos Nucleicos , Adenosina , Humanos , Fenômenos Magnéticos , Metilação , RNA
7.
Nat Commun ; 12(1): 5911, 2021 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-34625545

RESUMO

Immune cells at sites of inflammation are continuously activated by local antigens and cytokines, and regulatory mechanisms must be enacted to control inflammation. The stepwise hydrolysis of extracellular ATP by ectonucleotidases CD39 and CD73 generates adenosine, a potent immune suppressor. Here we report that human effector CD8 T cells contribute to adenosine production by releasing CD73-containing extracellular vesicles upon activation. These extracellular vesicles have AMPase activity, and the resulting adenosine mediates immune suppression independently of regulatory T cells. In addition, we show that extracellular vesicles isolated from the synovial fluid of patients with juvenile idiopathic arthritis contribute to T cell suppression in a CD73-dependent manner. Our results suggest that the generation of adenosine upon T cell activation is an intrinsic mechanism of human effector T cells that complements regulatory T cell-mediated suppression in the inflamed tissue. Finally, our data underscore the role of immune cell-derived extracellular vesicles in the control of immune responses.


Assuntos
5'-Nucleotidase/metabolismo , Adenosina/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Vesículas Extracelulares/metabolismo , Proteínas Ligadas por GPI/metabolismo , Imunossupressão , 5'-Nucleotidase/genética , Trifosfato de Adenosina , Animais , Autoimunidade , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Proliferação de Células , Vesículas Extracelulares/imunologia , Humanos , Inflamação , Ativação Linfocitária , Camundongos , Linfócitos T , Linfócitos T Reguladores/imunologia
8.
Int J Mol Sci ; 22(19)2021 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-34638933

RESUMO

Lung cancer is the leading cause of cancer-related mortality worldwide, and its tumorigenesis involves the accumulation of genetic and epigenetic events in the respiratory epithelium. Epigenetic modifications, such as DNA methylation, RNA modification, and histone modifications, have been widely reported to play an important role in lung cancer development and in other pulmonary diseases. Whereas the functionality of DNA and chromatin modifications referred to as epigenetics is widely characterized, various modifications of RNA nucleotides have recently come into prominence as functionally important. N6-methyladosine (m6A) is the most prevalent internal modification in mRNAs, and its machinery of writers, erasers, and readers is well-characterized. However, several other nucleotide modifications of mRNAs and various noncoding RNAs have also been shown to play an important role in the regulation of biological processes and pathology. Such epitranscriptomic modifications play an important role in regulating various aspects of RNA metabolism, including transcription, translation, splicing, and stability. The dysregulation of epitranscriptomic machinery has been implicated in the pathological processes associated with carcinogenesis including uncontrolled cell proliferation, migration, invasion, and epithelial-mesenchymal transition. In recent years, with the advancement of RNA sequencing technology, high-resolution maps of different modifications in various tissues, organs, or disease models are being constantly reported at a dramatic speed. This facilitates further understanding of the relationship between disease development and epitranscriptomics, shedding light on new therapeutic possibilities. In this review, we summarize the basic information on RNA modifications, including m6A, m1A, m5C, m7G, pseudouridine, and A-to-I editing. We then demonstrate their relation to different kinds of lung diseases, especially lung cancer. By comparing the different roles RNA modifications play in the development processes of different diseases, this review may provide some new insights and offer a better understanding of RNA epigenetics and its involvement in pulmonary diseases.


Assuntos
Epigênese Genética , Pneumopatias/genética , Neoplasias Pulmonares/genética , Processamento Pós-Transcricional do RNA , RNA/genética , Adenosina/análogos & derivados , Adenosina/metabolismo , Animais , Humanos , Pneumopatias/metabolismo , Neoplasias Pulmonares/metabolismo , RNA/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
9.
J Biomed Sci ; 28(1): 70, 2021 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-34635103

RESUMO

In modern societies, with an increase in the older population, age-related neurodegenerative diseases have progressively become greater socioeconomic burdens. To date, despite the tremendous effort devoted to understanding neurodegenerative diseases in recent decades, treatment to delay disease progression is largely ineffective and is in urgent demand. The development of new strategies targeting these pathological features is a timely topic. It is important to note that most degenerative diseases are associated with the accumulation of specific misfolded proteins, which is facilitated by several common features of neurodegenerative diseases (including poor energy homeostasis and mitochondrial dysfunction). Adenosine is a purine nucleoside and neuromodulator in the brain. It is also an essential component of energy production pathways, cellular metabolism, and gene regulation in brain cells. The levels of intracellular and extracellular adenosine are thus tightly controlled by a handful of proteins (including adenosine metabolic enzymes and transporters) to maintain proper adenosine homeostasis. Notably, disruption of adenosine homeostasis in the brain under various pathophysiological conditions has been documented. In the past two decades, adenosine receptors (particularly A1 and A2A adenosine receptors) have been actively investigated as important drug targets in major degenerative diseases. Unfortunately, except for an A2A antagonist (istradefylline) administered as an adjuvant treatment with levodopa for Parkinson's disease, no effective drug based on adenosine receptors has been developed for neurodegenerative diseases. In this review, we summarize the emerging findings on proteins involved in the control of adenosine homeostasis in the brain and discuss the challenges and future prospects for the development of new therapeutic treatments for neurodegenerative diseases and their associated disorders based on the understanding of adenosine homeostasis.


Assuntos
Adenosina/fisiologia , Encéfalo/fisiopatologia , Homeostase , Doenças Neurodegenerativas/fisiopatologia , Proteínas/metabolismo , Humanos
10.
Nat Plants ; 7(10): 1397-1408, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34650267

RESUMO

Cryptochromes (CRYs) are photoreceptors that mediate light regulation of the circadian clock in plants and animals. Here we show that CRYs mediate blue-light regulation of N6-methyladenosine (m6A) modification of more than 10% of messenger RNAs in the Arabidopsis transcriptome, especially those regulated by the circadian clock. CRY2 interacts with three subunits of the METTL3/14-type N6-methyladenosine RNA methyltransferase (m6A writer): MTA, MTB and FIP37. Photo-excited CRY2 undergoes liquid-liquid phase separation (LLPS) to co-condense m6A writer proteins in vivo, without obviously altering the affinity between CRY2 and the writer proteins. mta and cry1cry2 mutants share common defects of a lengthened circadian period, reduced m6A RNA methylation and accelerated degradation of mRNA encoding the core component of the molecular oscillator circadian clock associated 1 (CCA1). These results argue for a photoregulatory mechanism by which light-induced phase separation of CRYs modulates m6A writer activity, mRNA methylation and abundance, and the circadian rhythms in plants.


Assuntos
Adenosina/análogos & derivados , Arabidopsis/genética , Relógios Circadianos/genética , Criptocromos/metabolismo , Fotorreceptores de Plantas/metabolismo , Adenosina/metabolismo , Arabidopsis/metabolismo , Arabidopsis/efeitos da radiação
11.
Int J Mol Sci ; 22(19)2021 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-34638947

RESUMO

Perilipin5 (Plin5) is a scaffold protein that plays an important role in lipid droplets (LD) formation, but the regulatory effect of leptin on it is unclear. Our study aimed to explore the underlying mechanisms by which leptin reduces the N6-methyladenosine (m6A) methylation of Plin5 through fat mass and obesity associated genes (FTO) and regulates the lipolysis. To this end, 24 Landrace male piglets (7.73 ± 0.38 kg) were randomly sorted into two groups, either a control group (Control, n = 12) or a 1 mg/kg leptin recombinant protein treatment group (Leptin, n = 12). After 4 weeks of treatment, the results showed that leptin treatment group had lower body weight, body fat percentage and blood lipid levels, but the levels of Plin5 mRNA and protein increased significantly in adipose tissue (p < 0.05). Leptin promotes the up-regulation of FTO expression level in vitro, which in turn leads to the decrease of Plin5 M6A methylation (p < 0.05). In in vitro porcine adipocytes, overexpression of FTO aggravated the decrease of M6A methylation and increased the expression of Plin5 protein, while the interference fragment of FTO reversed the decrease of m6A methylation (p < 0.05). Finally, the overexpression in vitro of Plin5 significantly reduces the size of LD, promotes the metabolism of triglycerides and the operation of the mitochondrial respiratory chain, and increases thermogenesis. This study clarified that leptin can regulate Plin5 M6A methylation by promoting FTO to affect the lipid metabolism and energy consumption, providing a theoretical basis for treating diseases related to obesity.


Assuntos
Adenosina/análogos & derivados , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , Leptina/administração & dosagem , Lipólise/efeitos dos fármacos , Perilipina-5/metabolismo , Adenosina/metabolismo , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Animais , Peso Corporal/efeitos dos fármacos , Células Cultivadas , Expressão Gênica/efeitos dos fármacos , Lipólise/genética , Masculino , Metilação/efeitos dos fármacos , Perilipina-5/genética , Interferência de RNA , RNA Mensageiro/genética , Proteínas Recombinantes/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Suínos , Transfecção , Triglicerídeos/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética
12.
ACS Chem Neurosci ; 12(18): 3410-3417, 2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34469110

RESUMO

Adenosine receptor (AR) radiotracers for positron emission tomography (PET) have provided knowledge on the in vivo biodistribution of ARs in the central nervous system (CNS), which is of therapeutic interest for various neuropsychiatric disorders. Additionally, radioligands that can image changes in endogenous adenosine levels in different physiological and pathological conditions are still lacking. The binding of known antagonist adenosine A1 receptor (A1R) radiotracer, [11C]MDPX, failed to be inhibited by elevated endogenous adenosine in a rodent PET study. Since most of the known AR PET radiotracers were antagonists, we propose that an A1R agonist radioligand may possess higher sensitivity to measure changes in endogenous adenosine concentration. Herein, we report our latest findings toward the development of a full agonist adenosine A1 radioligand for PET. Based on a 3,5-dicyanopyridine template, 16 new derivatives were designed and synthesized to optimize both binding affinity and functional activity, resulting in two full agonists (compounds 27 and 29) with single-digit nanomolar affinities and good subtype selectivity (A1/A2A selectivity of ∼1000-fold for compound 27 and 29-fold for compound 29). Rapid O-[11C]methylation provided [11C]27 and [11C]29 in high radiochemical yields and radiochemical purity. However, subsequent brain PET imaging in rodents showed poor brain permeability for both radioligands. An in vivo PET study using knockout mice for MDR 1a/a, BCRP, and MRP1 indicated that these compounds might be substrates for brain efflux pumps. In addition, in silico evaluation using multiparameter optimization identified high molecular weight and high polar surface area as the main molecular descriptors responsible for low brain penetration. These results will provide further insight toward development of full agonist adenosine A1 radioligands and also highly potent CNS A1AR drugs.


Assuntos
Proteínas de Neoplasias , Agonistas do Receptor Purinérgico P1 , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Adenosina , Animais , Camundongos , Tomografia por Emissão de Pósitrons , Distribuição Tecidual
13.
Methods Enzymol ; 658: 161-190, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34517946

RESUMO

The RNA methyltransferase (MTase) complex METTL3-METTL14 transfers methyl groups from S-adenosyl-l-methionine (AdoMet) to the N6-position of adenosines within its consensus sequence, the DRACH motif (D=A, G, U; R=A, G; H=A, C, U). Interestingly, this MTase complex shows remarkable promiscuity regarding the cosubstrate. This can be exploited to install nonnatural modifications, like clickable or photocaging groups. Clickable groups are widely used for subsequent functionalization and open a broad range of possibilities for downstream applications. Here, we elaborate on click chemistry for coupling of RNA to biotin to enrich MTase targets via streptavidin-coated magnetic beads. Importantly, after clicking and coupling to beads the modification becomes sterically demanding and stalls reverse transcriptases, leading to termination adjacent to the MTase target site. Using radioactively labeled primers in the reverse transcription, the modified position can be precisely identified on a sequencing gel via phosphor imaging.


Assuntos
Metiltransferases , RNA , Adenosina , Metionina , Metiltransferases/genética , S-Adenosilmetionina
14.
Biosens Bioelectron ; 194: 113625, 2021 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-34534950

RESUMO

N6-methyladenosine (m6A) is the most abundant post-transcriptional modification in RNA and has important implications in physiological processes and tumor development. However, sensitive and specific quantification of locus-specific m6A modification levels remains a challenging task. In the present work, a novel m6A-sensitive DNAzyme was utilized to directly detect m6A by coupling with a three-way junction-mediated isothermal exponential CRISPR amplification reaction for the first time. This method was built on the fact that the binding arm of the DNAzyme bound to the specific site and its core structure catalyzed the selective cleavage of unmodified adenine instead of methylated adenines. Subsequently, the intact RNA was identified by the proximity effect of the three-way junction. Enormous amounts of single-stranded DNA products were generated through a combination of SDA and EXPAR for signal amplification. The specific real-time curve of products was recorded through detecting the fluorescence intensity triggered by CRISPR Cas12a. As a result, methylation target of abundance down to 1% was successfully identified. In addition, this strategy could be used for the analysis of cell RNA extracts. Combined with an electrochemical sensor for quantitative detection of RNA methylation, we demonstrated the generality of as-proposed strategy. We envision the present method would provide a new platform for the analysis of m6A in RNA and promote its application in clinical diseases.


Assuntos
Técnicas Biossensoriais , DNA Catalítico , Adenosina/análogos & derivados , Metilação , RNA/metabolismo
16.
ACS Chem Neurosci ; 12(20): 3818-3828, 2021 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-34491720

RESUMO

The pathogenesis of Alzheimer's disease (AD), the most prevalent form of dementia, remains unclear. Over the past few years, evidence has accumulated indicating that perturbed cerebral bioenergetics and neuroinflammation may compromise cognitive functions and precedes the onset of AD and that impaired function of glial cells can likely contribute to the development of the disease. Recently, N6-methyladenosine (m6A) modification of RNA has been implicated in the regulation of different processes in the brain and to play a potential role in neurodegeneration. In the present study, we investigated the potential role of the m6A machinery enzymes in a streptozotocin (STZ) model of AD in human astrocytoma CCF-STTG1 cells. We observed that STZ-treated astrocytes expressed significantly higher levels of m6A demethylase fat mass and obesity-associated protein (FTO) and m6A reader YTHDF1 (YTH domain-containing family protein 1). Our experiments revealed that MO-I-500, a novel pharmacological inhibitor of FTO, can strongly reduce the adverse effects of STZ. Inhibition of FTO enhanced the survival of cells exposed to STZ and suppressed oxidative stress, apoptosis, elevated expression of glial fibrillary acidic protein, mitochondrial dysfunction, and bioenergetic disturbances induced by this compound. Overall, the results of this study indicate that perturbed m6A signaling may be contributing to AD pathogenesis, likely by compromising astrocyte bioenergetics.


Assuntos
Dioxigenase FTO Dependente de alfa-Cetoglutarato , Astrócitos , Adenosina , Humanos , Mitocôndrias , Estreptozocina/toxicidade
17.
Analyst ; 146(20): 6306-6314, 2021 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-34550117

RESUMO

Reliable and cost-effective quantification of RNA modifications at a specific gene locus is essential to elucidate the pathogenic mechanism encoded by RNA epigenetics. Current methods to quantify N6-methyladenosine (m6A) at specific sites can hardly satisfy the requirement of clinical application because epigenetic information is easily lost through polymerase chain reaction (PCR) assay or other isothermal amplification methods unless tedious pretreatment is applied. Herein, we propose a simple xeno nucleic acid (XNA) as a blocker probe to mediate the methylation specific reverse transcription quantitative polymerase chain reaction (MsRT-qPCR) assay to directly magnify the minor differences between epigenetic bases and unmodified bases in RNA. Strand displacement reactions selectively initiated between the reverse transcription primer (RT-primer) and the XNA probe at the m6A template given the affinity differences between the blocker probes and the m6A-modified RNA (m6A-RNA) and unmodified RNA (A-RNA). Thus, preferential amplification of m6A-RNA was allowed. Integration of a well-established oligo-modified Fe3O4@UiO-66-NH4 allowed purification of mRNA and lncRNA from cellular total RNA samples and greatly reduced the non-specific interference of m6A detection in real samples. Multiple specific sites of m6A in mRNA and lncRNA samples are also successfully quantified. The XNA probe-based m6A assay required only common and available lab equipment and materials, which can be applied in m6A-related fundamental studies and clinical diagnosis.


Assuntos
Adenosina , RNA Longo não Codificante , Adenosina/análogos & derivados , Metilação , RNA Mensageiro , Reação em Cadeia da Polimerase em Tempo Real
18.
Nat Commun ; 12(1): 5373, 2021 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-34508078

RESUMO

Ankylosing spondylitis (AS) is a type of rheumatic disease characterized by chronic inflammation and pathological osteogenesis in the entheses. Previously, we demonstrated that enhanced osteogenic differentiation of MSC from AS patients (AS-MSC) resulted in pathological osteogenesis, and that during the enhanced osteogenic differentiation course, AS-MSC induced TNF-α-mediated local inflammation. However, whether TNF-α in turn affects AS-MSC remains unknown. Herein, we further demonstrate that a high-concentration TNF-α treatment triggers enhanced directional migration of AS-MSC in vitro and in vivo, which enforces AS pathogenesis. Mechanistically, TNF-α leads to increased expression of ELMO1 in AS-MSC, which is mediated by a METTL14 dependent m6A modification in ELMO1 3'UTR. Higher ELMO1 expression of AS-MSC is found in vivo in AS patients, and inhibiting ELMO1 in SKG mice produces therapeutic effects in this spondyloarthritis model. This study may provide insight into not only the pathogenesis but also clinical therapy for AS.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Células-Tronco Mesenquimais/patologia , Osteogênese/genética , Espondilite Anquilosante/patologia , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Animais , Biópsia , Medula Óssea/patologia , Estudos de Casos e Controles , Diferenciação Celular/genética , Movimento Celular/genética , Metilação de DNA , Modelos Animais de Doenças , Epigênese Genética , Feminino , Células HEK293 , Voluntários Saudáveis , Humanos , Masculino , Camundongos , Cultura Primária de Células , Espondilite Anquilosante/induzido quimicamente , Espondilite Anquilosante/diagnóstico , Espondilite Anquilosante/genética , Microtomografia por Raio-X , beta-Glucanas/administração & dosagem , beta-Glucanas/efeitos adversos
19.
Nat Commun ; 12(1): 5522, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34535671

RESUMO

Natural killer (NK) cells exert critical roles in anti-tumor immunity but how their functions are regulated by epitranscriptional modification (e.g., N6-methyladenosine (m6A) methylation) is unclear. Here we report decreased expression of the m6A "writer" METTL3 in tumor-infiltrating NK cells, and a positive correlation between protein expression levels of METTL3 and effector molecules in NK cells. Deletion of Mettl3 in NK cells alters the homeostasis of NK cells and inhibits NK cell infiltration and function in the tumor microenvironment, leading to accelerated tumor development and shortened survival in mice. The gene encoding SHP-2 is m6A modified, and its protein expression is decreased in METTL3-deficient NK cells. Reduced SHP-2 activity renders NK cells hyporesponsive to IL-15, which is associated with suppressed activation of the AKT and MAPK signaling pathway in METTL3-deficient NK cells. These findings show that m6A methylation safeguards the homeostasis and tumor immunosurveillance function of NK cells.


Assuntos
Adenosina/análogos & derivados , Células Matadoras Naturais/imunologia , Metiltransferases/metabolismo , Neoplasias/imunologia , Neoplasias/metabolismo , RNA/metabolismo , Adenosina/metabolismo , Animais , Carcinogênese/patologia , Linhagem Celular Tumoral , Deleção de Genes , Homeostase , Interleucina-15/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Metilação , Metiltransferases/deficiência , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Microambiente Tumoral
20.
Nat Commun ; 12(1): 5543, 2021 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-34545078

RESUMO

N6,2'-O-dimethyladenosine (m6Am) is an abundant RNA modification located adjacent to the 5'-end of the mRNA 7-methylguanosine (m7G) cap structure. m6A methylation on 2'-O-methylated A at the 5'-ends of mRNAs is catalyzed by the methyltransferase Phosphorylated CTD Interacting Factor 1 (PCIF1). The role of m6Am and the function of PCIF1 in regulating host-pathogens interactions are unknown. Here, we investigate the dynamics and reprogramming of the host m6Am RNA methylome during HIV infection. We show that HIV infection induces a dramatic decrease in m6Am of cellular mRNAs. By using PCIF1 depleted T cells, we identify 2237 m6Am genes and 854 are affected by HIV infection. Strikingly, we find that PCIF1 methyltransferase function restricts HIV replication. Further mechanism studies show that HIV viral protein R (Vpr) interacts with PCIF1 and induces PCIF1 ubiquitination and degradation. Among the m6Am genes, we find that PCIF1 inhibits HIV infection by enhancing a transcription factor ETS1 (ETS Proto-Oncogene 1, transcription factor) stability that binds HIV promoter to regulate viral transcription. Altogether, our study discovers the role of PCIF1 in HIV-host interactions, identifies m6Am modified genes in T cells which are affected by viral infection, and reveals how HIV regulates host RNA epitranscriptomics through PCIF1 degradation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenosina/análogos & derivados , HIV-1/metabolismo , Proteínas Nucleares/metabolismo , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/metabolismo , Regiões 5' não Traduzidas/genética , Adenosina/metabolismo , Genoma Viral , Infecções por HIV/virologia , HIV-1/genética , Humanos , Metilação , Estabilidade Proteica , Proteólise , Proteína Proto-Oncogênica c-ets-1/metabolismo , RNA/metabolismo , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , Transcrição Genética , Replicação Viral
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