RESUMEN
Therapeutic fluoropyrimidines 5-fluorouracil (5-FU) and 5-fluorocytosine (5-FC) are in long use for treatment of human cancers and severe invasive fungal infections, respectively. 5-Fluorouridine triphosphate represents a bioactive metabolite of both drugs and is incorporated into target cells' RNA. Here we use the model fungus Saccharomyces cerevisiae to define fluorinated tRNA as a key mediator of 5-FU and 5-FC cytotoxicity when specific tRNA methylations are absent. tRNA methylation deficiency caused by loss of Trm4 and Trm8 was previously shown to trigger an RNA quality control mechanism resulting in partial destabilization of hypomodified tRNAValAAC. We demonstrate that, following incorporation into tRNA, fluoropyrimidines strongly enhance degradation of yeast tRNAValAAC lacking Trm4 and Trm8 dependent methylations. At elevated temperature, such effect occurs already in absence of Trm8 alone. Genetic approaches and quantification of tRNA modification levels reveal that enhanced fluoropyrimidine cytotoxicity results from additional, drug induced uridine modification loss and activation of tRNAValAAC decay involving the exonuclease Xrn1. These results suggest that inhibition of tRNA methylation may be exploited to boost therapeutic efficiency of 5-FU and 5-FC.
Asunto(s)
Flucitosina , Fluorouracilo , ARN de Transferencia , Saccharomyces cerevisiae , Exorribonucleasas/metabolismo , Exorribonucleasas/genética , Flucitosina/farmacología , Fluorouracilo/farmacología , Metilación , Estabilidad del ARN/efectos de los fármacos , ARN de Transferencia/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , ARNt Metiltransferasas/metabolismo , ARNt Metiltransferasas/genética , Uridina/metabolismoRESUMEN
Many virus lysis/transport buffers used in molecular diagnostics, including the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA, contain guanidine-based chaotropic salts, primarily guanidine hydrochloride (GuHCl) or guanidine isothiocyanate (GITC). Although the virucidal effects of GuHCl and GITC alone against some enveloped viruses have been established, standardized data on their optimum virucidal concentrations against SARS-CoV-2 and effects on viral RNA stability are scarce. Thus, we aimed to determine the optimum virucidal concentrations of GuHCl and GITC against SARS-CoV-2 compared to influenza A virus (IAV), another enveloped respiratory virus. We also evaluated the effectiveness of viral RNA stabilization at the determined optimum virucidal concentrations under high-temperature conditions (35°C) using virus-specific real-time reverse transcription polymerase chain reaction. Both viruses were potently inactivated by 1.0 M GITC and 2.5 M GuHCl, but the GuHCl concentration for efficient SARS-CoV-2 inactivation was slightly higher than that for IAV inactivation. GITC showed better viral RNA stability than GuHCl at the optimum virucidal concentrations. An increased concentration of GuHCl or GITC increased viral RNA degradation at 35°C. Our findings highlight the need to standardize GuHCl and GITC concentrations in virus lysis/transport buffers and the potential application of these guanidine-based salts alone as virus inactivation solutions in SARS-CoV-2 and IAV molecular diagnostics.
Asunto(s)
Guanidina , Virus de la Influenza A , ARN Viral , SARS-CoV-2 , Manejo de Especímenes , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/genética , Virus de la Influenza A/efectos de los fármacos , Virus de la Influenza A/genética , Guanidina/farmacología , Guanidina/química , ARN Viral/genética , Humanos , Manejo de Especímenes/métodos , Genoma Viral , COVID-19/virología , COVID-19/diagnóstico , Chlorocebus aethiops , Células Vero , Inactivación de Virus/efectos de los fármacos , Animales , Estabilidad del ARN/efectos de los fármacos , Contención de Riesgos Biológicos , Guanidinas/farmacología , Guanidinas/química , Sales (Química)/farmacología , Sales (Química)/químicaRESUMEN
Atherosclerosis (AS) represents a prevalent initiating factor for cardiovascular events. Insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3) is an oncofetal RNA-binding protein that participates in cardiovascular diseases. This work aimed to elaborate the effects of IGF2BP3 on AS and the probable mechanism by using an oxidized low-density lipoprotein (ox-LDL)-induced human umbilical vein endothelial cells (HUVECs) model. Results indicated that IGF2BP3 expression was declined in the blood of AS patients and ox-LDL-induced HUVECs. IGF2BP3 elevation alleviated ox-LDL-provoked viability loss, apoptosis, oxidative DNA damage and endothelial dysfunction in HUVECs. Moreover, IGF2BP3 bound SESN1 and stabilized SESN1 mRNA. Furthermore, SESN1 interference reversed the impacts of IGF2BP3 overexpression on the apoptosis, oxidative DNA damage and endothelial dysfunction of ox-LDL-challenged HUVECs. Additionally, the activation of Nrf2 signaling mediated by IGF2BP3 up-regulation in ox-LDL-treated HUVECs was blocked by SESN1 absence. Collectively, SESN1 stabilized by IGF2BP3 might protect against AS by activating Nrf2 signaling.
Asunto(s)
Células Endoteliales de la Vena Umbilical Humana , Lipoproteínas LDL , Factor 2 Relacionado con NF-E2 , Estrés Oxidativo , ARN Mensajero , Proteínas de Unión al ARN , Transducción de Señal , Humanos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Lipoproteínas LDL/farmacología , Lipoproteínas LDL/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Factor 2 Relacionado con NF-E2/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Estrés Oxidativo/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Apoptosis/efectos de los fármacos , Aterosclerosis/metabolismo , Aterosclerosis/genética , Aterosclerosis/patología , Estabilidad del ARN/efectos de los fármacos , Daño del ADN , SestrinasRESUMEN
We report the use of trimethylsilyl azide and Selectfluor to implement a standard protocol targeted at the prenylated nucleic acid known as i6A-RNA. After optimizing the conditions, we applied this method to regulate a wide range of i6A-RNA species using synthetic imidazole-based probes (I-IV). We observed that prenylated nucleic acid plays a crucial role in the cell hemostasis in A549 cell lines.
Asunto(s)
Azidas , Química Clic , Halogenación , Imidazoles , Humanos , Imidazoles/química , Imidazoles/síntesis química , Azidas/química , Células A549 , ARN/química , ARN/metabolismo , Estructura Molecular , Estabilidad del ARN/efectos de los fármacosRESUMEN
Hippocampal neurons maintain the ability of proliferation throughout life to support neurogenesis. Deoxynivalenol (DON) is a mycotoxin that exhibits brain toxicity, yet whether and how DON affects hippocampal neurogenesis remains unknown. Here, we use mouse hippocampal neuron cells (HT-22) as a model to illustrate the effects of DON on neuron proliferation and to explore underlying mechanisms. DON exposure significantly inhibits the proliferation of HT-22 cells, which is associated with an up-regulation of cell cycle inhibitor p21 at both mRNA and protein levels. Global and site-specific m6A methylation levels on the 3'UTR of p21 mRNA are significantly increased in response to DON treatment, whereas inhibition of m6A hypermethylation significantly alleviates DON-induced cell cycle arrest. Further mechanistic studies indicate that the m6A readers YTHDF1 and IGF2BP1 are responsible for m6A-mediated increase in p21 mRNA stability. Meanwhile, 3'UTR of E3 ubiquitin ligase TRIM21 mRNA is also m6A hypermethylated, and another m6A reader YTHDF2 binds to the m6A sites, leading to decreased TRIM21 mRNA stability. Consequently, TRIM21 suppression impairs ubiquitin-mediated p21 protein degradation. Taken together, m6A-mediated upregulation of p21, at both post-transcriptional and post-translational levels, contributes to DON-induced inhibition of hippocampal neuron proliferation. These results may provide new insights for epigenetic therapy of neurodegenerative diseases.
Asunto(s)
Proliferación Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Hipocampo , Neuronas , Tricotecenos , Regulación hacia Arriba , Animales , Tricotecenos/toxicidad , Tricotecenos/farmacología , Hipocampo/metabolismo , Hipocampo/efectos de los fármacos , Hipocampo/citología , Ratones , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Regulación hacia Arriba/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Línea Celular , Regiones no Traducidas 3'/genética , Neurogénesis/efectos de los fármacos , ARN Mensajero/metabolismo , ARN Mensajero/genética , Estabilidad del ARN/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Metilación/efectos de los fármacosRESUMEN
BACKGROUND: Prolonged exposure to interleukin-17A (IL-17A) can induce autoimmune myocarditis, and MLN4924, an inhibitor of NEDD8 activating enzyme (NAE), has been reported to effectively suppress various inflammatory reactions. However, the effects of MLN4924 in IL-17A-mediated inflammation associated with autoimmune myocarditis remain uncertain. METHODS: An experimental autoimmune myocarditis (EAM) model was established and treated with MLN4924. The inflammation degree of heart tissues was assessed histopathologically. The expression levels of inflammatory cytokines and chemokines were measured using ELISA and RT-qPCR, respectively. Additionally, the interaction of biomacromolecules was detected through co-immunoprecipitation (Co-IP) and RNA immunoprecipitation (RIP). RESULTS: MLN4924 could attenuate IL-17A-induced inflammation. In the in vivo studies, MLN4924 treatment improved inflammatory responses, diminished immune cell infiltration and tissue fibrosis, and reduced the secretion of various inflammatory cytokines in serum, including IL-1ß, IL-6, TNF-α, and MCP-1. In vitro experiments further corroborated these findings, showing that MLN4924 treatment reduced the secretion and transcription of pro-inflammatory factors, particularly MCP-1. Mechanistically, we confirmed that MLN4924 promoted Act1 ubiquitination degradation and disrupted Act1's interaction with IL-17R, thereby impeding the formation of the IL-17R/Act1/TRAF6 complex and subsequent activation of TAK1, c-Jun, and p65. Moreover, MLN4924 interfered with Act1's binding to mRNA, resulting in mRNA instability. CONCLUSIONS: In conclusion, MLN4924 effectively alleviated inflammatory symptoms in EAM by disrupting the interaction between IL and 17R and Act1, thereby reducing Act1-mediated mRNA stability and resulting in decreased expression of pro-inflammatory factors.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Enfermedades Autoinmunes , Ciclopentanos , Citocinas , Miocarditis , Pirimidinas , Estabilidad del ARN , Animales , Miocarditis/tratamiento farmacológico , Miocarditis/inmunología , Miocarditis/metabolismo , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Enfermedades Autoinmunes/tratamiento farmacológico , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Ciclopentanos/farmacología , Ciclopentanos/uso terapéutico , Ratones , Estabilidad del ARN/efectos de los fármacos , Masculino , Citocinas/metabolismo , Interleucina-17/metabolismo , Modelos Animales de Enfermedad , Humanos , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , ARN Mensajero/metabolismo , Ratones Endogámicos BALB CRESUMEN
Cervical cancer (CC) is a prevalent malignancy among women worldwide. This study was designed to investigate the role of METTL14 in sorafenib-induced ferroptosis in CC. METTL14 expression and m6A methylation were determined in CC tissues, followed by analyzes correlating these factors with clinical features. Subsequently, METTL14 was knocked down in CC cell lines, and the effects on cell proliferation, mitochondrial morphology and ferroptosis were assessed using CCK-8, microscopy, and markers associated with ferroptosis, respectively. The regulatory relationship between METTL14 and FTH1 was verified using qRT-PCR and luciferase reporter assays. The functional significance of this interaction was further investigated both in vitro and in vivo by co-transfecting cells with overexpression vectors or shRNAs targeting METTL14 and FTH1 after sorafenib treatment. METTL14 expression and m6A methylation were significantly reduced in CC tissues, and lower METTL14 expression levels were associated with a poorer CC patients' prognosis. Notably, METTL14 expression increased during sorafenib-induced ferroptosis, and METTL14 knockdown attenuated the ferroptotic response induced by sorafenib in CC cells. FTH1 was identified as a direct target of METTL14, with METTL14 overexpression leading to increased m6A methylation of FTH1 mRNA, resulting in reduced stability and expression of FTH1 in CC. Furthermore, FTH1 overexpression or treatment with LY294002 partially counteracted the promotion of sorafenib-induced ferroptosis by METTL14. In vivo xenograft experiments demonstrated that inhibiting METTL14 reduced the anticancer effects of sorafenib, whereas suppression of FTH1 significantly enhanced sorafenib-induced ferroptosis and increased its anticancer efficacy. METTL14 reduces FTH1 mRNA stability through m6A methylation, thereby enhancing sorafenib-induced ferroptosis, which contributes to suppressing CC progression via the PI3K/Akt signaling pathway.