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As the most common and aggressive primary malignant brain tumor, glioblastoma is still lacking a satisfactory curative approach. The standard management consisting of gross total resection followed by radiotherapy and chemotherapy with temozolomide only prolongs patients' life moderately. In recent years, many therapeutics have failed to give a breakthrough in GBM treatment. In the search for new treatment solutions, we became interested in the repurposing of existing medicines, which have established safety profiles. We focused on the possible implementation of well-known drugs, metformin, and arginine. Metformin is widely used in diabetes treatment, but arginine is mainly a cardiovascular protective drug. We evaluated the effects of metformin and arginine on total DNA methylation, as well as the oxidative stress evoked by treatment with those agents. In glioblastoma cell lines, a decrease in 5-methylcytosine contents was observed with increasing drug concentration. When combined with temozolomide, both guanidines parallelly increased DNA methylation and decreased 8-oxo-deoxyguanosine contents. These effects can be explained by specific interactions of the guanidine group with m5CpG dinucleotide. We showed that metformin and arginine act on the epigenetic level, influencing the foreground and potent DNA regulatory mechanisms. Therefore, they can be used separately or in combination with temozolomide, in various stages of disease, depending on desired treatment effects.
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Arginina , Metilación de ADN , Reposicionamiento de Medicamentos , Glioblastoma , Metformina , Temozolomida , Metformina/farmacología , Metformina/uso terapéutico , Glioblastoma/tratamiento farmacológico , Glioblastoma/metabolismo , Humanos , Arginina/metabolismo , Reposicionamiento de Medicamentos/métodos , Metilación de ADN/efectos de los fármacos , Línea Celular Tumoral , Temozolomida/uso terapéutico , Temozolomida/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/metabolismo , Estrés Oxidativo/efectos de los fármacos , Epigénesis Genética/efectos de los fármacos , 5-Metilcitosina/metabolismo , 5-Metilcitosina/análogos & derivadosRESUMEN
BACKGROUND: Ribosomal RNA Processing 8 (RRP8) is a gene associated with RNA modification and has been implicated in the development of several types of tumors in recent research. Nevertheless, the biological importance of RRP8 in pan-cancer has not yet been thoroughly and comprehensively investigated. METHODS: In this study, we conducted an analysis of various public databases to investigate the biological functions of RRP8. Our analysis included examining its correlation with pan-cancer prognosis, heterogeneity, stemness, immune checkpoint genes, and immune cell infiltration. Furthermore, we utilized the GDSC and CTRP databases to assess the sensitivity of RRP8 to small molecule drugs. RESULTS: Our findings indicate that RRP8 exhibits differential expression between tumor and normal samples, particularly impacting the prognosis of various cancers such as Adrenocortical carcinoma (ACC) and Kidney Chromophobe (KICH). The expression of RRP8 is intricately linked to tumor heterogeneity and stemness markers. Additionally, RRP8 shows a positive correlation with the presence of tumor-infiltrating cells, with TP53 being the predominant mutated gene in these malignancies. CONCLUSION: Our findings suggest that RRP8 may serve as a potential prognostic marker and therapeutic target in a variety of cancer types.
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BACKGROUND: N6-methyladenosine (m6A) and 5-methylcytosine (m5C) play a role in modifying long non-coding RNAs (lncRNAs) implicated in tumorigenesis and progression. This study was performed to evaluate prognostic value of m6A- and m5C-related lncRNAs and develop an efficient model for prognosis prediction in cervical cancer (CC). METHODS: Using gene expression data of TCGA set, we identified m6A- and m5C-related lncRNAs. Consensus Clustering Analysis was performed for samples subtyping based on survival-related lncRNAs, followed by analyzing tumor infiltrating immune cells (TIICs). Optimal signature lncRNAs were obtained using lasso Cox regression analysis for constructing a prognostic model and a nomogram to predict prognosis. RESULTS: We built a co-expression network of 23 m6A-related genes, 15 m5C-related genes, and 62 lncRNAs. Based on 9 m6A- and m5C-related lncRNAs significantly associated with overall survival (OS) time, two molecular subtypes were obtained, which had significantly different OS time and fractions of TIICs. A prognostic model based on six m6A- and m5C-related signature lncRNAs was constructed, which could dichotomize patients into two risk subgroups with significantly different OS time. Prognostic power of the model was successfully validated in an independent dataset. We subsequently constructed a nomogram which could accurately predict survival probabilities. Drug sensitivity analysis found preferred chemotherapeutic agents for high and low-risk patients, respectively. CONCLUSION: Our study reveals that m6A- and m5C-related lncRNAs are associated with prognosis and immune microenvironment of CC. The m6A- and m5C-related six-lncRNA signature may be a useful tool for survival stratification in CC and open new avenues for individualized therapies.
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5-Metilcitosina , Adenosina , ARN Largo no Codificante , Neoplasias del Cuello Uterino , ARN Largo no Codificante/genética , Humanos , Adenosina/análogos & derivados , Adenosina/genética , Adenosina/metabolismo , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/mortalidad , Neoplasias del Cuello Uterino/diagnóstico , Femenino , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Pronóstico , Regulación Neoplásica de la Expresión Génica , Nomogramas , Biomarcadores de Tumor/genética , Perfilación de la Expresión GénicaRESUMEN
Epigenetic modifications, particularly RNA methylation and histone alterations, play a crucial role in heredity, development, and disease. Among these, RNA 5-methylcytosine (m5C) is the most prevalent RNA modification in mammalian cells, essential for processes such as ribosome synthesis, translational fidelity, mRNA nuclear export, turnover, and translation. The increasing volume of nucleotide sequences has led to the development of machine learning-based predictors for m5C site prediction. However, these predictors often face challenges related to training data limitations and overfitting due to insufficient external validation. This study introduces m5C-Seq, an ensemble learning approach for RNA modification profiling, designed to address these issues. m5C-Seq employs a meta-classifier that integrates 15 probabilities generated from a novel, large dataset using systematic encoding methods to make final predictions. Demonstrating superior performance compared to existing predictors, m5C-Seq represents a significant advancement in accurate RNA modification profiling. The code and the newly established datasets are made available through GitHub at https://github.com/Z-Abbas/m5C-Seq.
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BACKGROUND: Breast tumorigenesis is a complex and multistep process accompanied by both genetic and epigenetic dysregulation. In contrast to the extensive studies on DNA epigenetic modifications 5-hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC) in malignant breast tumors, their roles in the early phases of breast tumorigenesis remain ambiguous. RESULTS: DNA 5hmC and 5mC exhibited a consistent and significant decrease from usual ductal hyperplasia to atypical ductal hyperplasia and subsequently to ductal carcinoma in situ (DCIS). However, 5hmC showed a modest increase in invasive ductal breast cancer compared to DCIS. Genomic analyses showed that the changes in 5hmC and 5mC levels occurred around the transcription start sites (TSSs), and the modification levels were strongly correlated with gene expression levels. Meanwhile, it was found that differentially hydroxymethylated regions (DhMRs) and differentially methylated regions (DMRs) were overlapped in the early phases and accompanied by the enrichment of active histone marks. In addition, TET2-related DNA demethylation was found to be involved in breast tumorigenesis, and four transcription factor binding sites (TFs: ESR1, FOXA1, GATA3, FOS) were enriched in TET2-related DhMRs/DMRs. Intriguingly, we also identified a certain number of common DhMRs between tumor samples and cell-free DNA (cfDNA). CONCLUSIONS: Our study reveals that dynamic changes in DNA 5hmC and 5mC play a vital role in propelling breast tumorigenesis. Both TFs and active histone marks are involved in TET2-related DNA demethylation. Concurrent changes in 5hmC signals in primary breast tumors and cfDNA may play a promising role in breast cancer screening.
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5-Metilcitosina , Neoplasias de la Mama , Proteínas de Unión al ADN , Dioxigenasas , Proteínas Proto-Oncogénicas , Humanos , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Femenino , Neoplasias de la Mama/genética , Proteínas de Unión al ADN/genética , Dioxigenasas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Carcinogénesis/genética , Metilación de ADN/genética , Epigénesis Genética/genética , Regulación Neoplásica de la Expresión Génica , Desmetilación del ADNRESUMEN
Brain derived neurotrophic factor (BDNF) is the most studied trophic factor in the central nervous system (CNS), and its role in the maturation of neurons, including synapse development and maintenance has been investigated intensely for over three decades. The primary receptor for BDNF is the tropomyosin receptor kinase B (TrkB), which is broadly expressed as two primary isoforms in the brain; the full length TrkB (TrkB.FL) receptor, expressed mainly in neurons and the truncated TrkB (TrkB.T1) receptor. We recently demonstrated that TrkB.T1 is predominately expressed in astrocytes, and appears critical for astrocyte morphological maturation. Given the critical role of BDNF/TrkB pathway in healthy brain development and mature CNS function, we aimed to identify molecular underpinnings of cell-type specific expression of each TrkB isoform. Using Nanopore sequencing which enables direct, long read sequencing of native DNA, we profiled DNA methylation patterns of the entire TrkB gene, Ntrk2, in both neurons and astrocytes. Here, we identified robust differences in cell-type specific isoform expression associated with significantly different methylation patterns of the Ntrk2 gene in each cell type. Notably, astrocytes demonstrated lower 5mC methylation, and higher 5hmC across the entire gene when compared to neurons, including differentially methylated sites (DMSs) found in regions flanking the unique TrkB.T1 protein coding sequence (CDS). These data suggest DNA methylation patterns may provide instruction for isoform specific TrkB expression across unique CNS cell types.
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Hepatocellular carcinoma (HCC) is a highly aggressive cancer with a poor prognosis. The molecular mechanisms underlying its development remain unclear. Recent studies have highlighted the crucial role of RNA modifications in HCC progression, which indicates their potential as therapeutic targets and biomarkers for managing HCC. In this review, we discuss the functional role and molecular mechanisms of RNA modifications in HCC through a review and summary of relevant literature, to explore the potential therapeutic agents and biomarkers for diagnostic and prognostic of HCC. This review indicates that specific RNA modification pathways, such as N6-methyladenosine, 5-methylcytosine, N7-methylguanosine, and N1-methyladenosine, are erroneously regulated and are involved in the proliferation, autophagy, innate immunity, invasion, metastasis, immune cell infiltration, and drug resistance of HCC. These findings provide a new perspective for understanding the molecular mechanisms of HCC, as well as potential targets for the diagnosis and treatment of HCC by targeting specific RNA-modifying enzymes or recognition proteins. More than ten RNA-modifying regulators showed the potential for use for the diagnosis, prognosis and treatment decision utility biomarkers of HCC. Their application value for HCC biomarkers necessitates extensive multi-center sample validation in the future. A growing number of RNA modifier inhibitors are being developed, but the lack of preclinical experiments and clinical studies targeting RNA modification in HCC poses a significant obstacle, and further research is needed to evaluate their application value in HCC treatment. In conclusion, this review provides an in-depth understanding of the complex interplay between RNA modifications and HCC while emphasizing the promising potential of RNA modifications as therapeutic targets and biomarkers for managing HCC.
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Biomarcadores de Tumor , Carcinoma Hepatocelular , Neoplasias Hepáticas , Procesamiento Postranscripcional del ARN , Humanos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/terapia , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/terapia , Neoplasias Hepáticas/diagnóstico , Biomarcadores de Tumor/metabolismo , Animales , Regulación Neoplásica de la Expresión Génica , Pronóstico , ARN/genética , ARN/metabolismoRESUMEN
Selective C-H activation on complex biological macromolecules is a key goal in the field of organic chemistry. It requires thermodynamically challenging chemical transformations to be delivered in mild, aqueous conditions. 5-Methylcytosine (5mC) is a fundamentally important epigenetic modification in DNA that has major implications for biology and has emerged as a vital biomarker. Selective functionalisation of 5mC would enable new chemical approaches to tag, detect and map DNA methylation to enhance the study and exploitation of this epigenetic feature. We demonstrate the first example of direct and selective chemical oxidation of 5mC to 5-formylcytosine (5fC) in DNA, employing a photocatalytic system. This transformation was used to selectively tag 5mC. We also provide proof-of-concept for deploying this chemistry for single-base resolution sequencing of 5mC and genetic bases adenine (A), cytosine (C), guanine (G), thymine (T) in DNA on a next-generation sequencing system. This work exemplifies how photocatalysis has the potential to transform the analysis of DNA.
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Mitochondrial double-stranded RNA (dsRNA) can form spontaneously in mitochondria, blocking mitochondrial gene expression and triggering an immune response. A recent study by Kim, Tan, et al. identified a safeguard mechanism in which NOP2/Sun RNA methyltransferase 4 (NSUN4)-mediated RNA methylation (m5C) recruits the RNA degradation machinery to prevent dsRNA formation.
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As advances in RNA modification research progress, the significance of 5-methylcytosine (m5C) modification is being increasingly acknowledged. m5C undergoes modification by the methyltransferase NOP2/Sun domain (NSUN) family/DNA methyltransferase (DNMT) family (writer) and is removed by demethylases (eraser), including the ten-eleven translocation (TET) family and Alkb homolog 1 (ALKBH1). Moreover, m5C interacts with RNA-binding proteins (reader), such as Y-box-binding protein 1 (YBX1) and Aly/REF export factor (ALYREF). Expanding on this structural framework, m5C modification possesses the capacity to regulate various physiological and pathological processes. Recent studies indicate that m5C plays a pivotal regulatory role in the central nervous system, and its dysregulation may correlate with the onset and progression of various central nervous system diseases. In this review, we summarize recent research on m5C components and delve into the potential mechanisms of m5C involvement in central nervous system disorders, such as Alzheimer's disease, brain tumors, epilepsy, and stroke.
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Enfermedades del Sistema Nervioso Central , Humanos , Enfermedades del Sistema Nervioso Central/metabolismo , Enfermedades del Sistema Nervioso Central/genética , 5-Metilcitosina/metabolismo , Animales , ARN/metabolismo , ARN/genéticaRESUMEN
Enzymatic modification of DNA nucleobases can coordinate gene expression, nuclease protection, or mutagenesis. We recently discovered a clade of phage-specific cytosine methyltransferase (MT) and 5-methylpyrimidine dioxygenase (5mYOX) enzymes that produce 5-hydroxymethylcytosine (5hmC) as a precursor for enzymatic hypermodifications on viral genomes. Here, we identify phage MT- and 5mYOX-associated glycosyltransferases (GTs) that catalyze linkage of diverse sugars to 5hmC nucleobase substrates. Metavirome mining revealed thousands of biosynthetic gene clusters containing enzymes with predicted roles in cytosine sugar hypermodification. We developed a platform for high-throughput screening of GT-containing pathways, relying on the Escherichia coli metabolome as a substrate pool. We successfully reconstituted several pathways and isolated diverse sugar modifications appended to cytosine, including mono-, di-, or tri-saccharides comprised of hexoses, N-acetylhexosamines, or heptose. These findings expand our knowledge of hypermodifications on nucleic acids and the origins of corresponding sugar-installing enzymes.
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Glicosiltransferasas , Polisacáridos , Polisacáridos/metabolismo , Glicosiltransferasas/metabolismo , Glicosiltransferasas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , 5-Metilcitosina/metabolismo , 5-Metilcitosina/análogos & derivados , ADN/metabolismoRESUMEN
Tamoxifen, a selective estrogen receptor modulator (SERM), exhibits dual agonist or antagonist effects contingent upon its binding to either G-protein-coupled estrogen receptor (GPER) or estrogen nuclear receptor (ESR). Estrogen signaling plays a pivotal role in initiating epigenetic alterations and regulating estrogen-responsive genes in breast cancer. Employing three distinct breast cancer cell lines-MCF-7 (ESR+; GPER+), MDA-MB-231 (ESR-; GPER-), and SkBr3 (ESR-; GPER+)-this study subjected them to treatment with two tamoxifen derivatives: 4-hydroxytamoxifen (4-HT) and endoxifen (Endox). Through 2D high-performance liquid chromatography with tandem mass spectrometry detection (HPLC-MS/MS), varying levels of 5-methylcytosine (5-mC) were found, with MCF-7 displaying the highest levels. Furthermore, TET3 mRNA expression levels varied among the cell lines, with MCF-7 exhibiting the lowest expression. Notably, treatment with 4-HT induced significant changes in TET3 expression across all cell lines, with the most pronounced increase seen in MCF-7 and the least in MDA-MB-231. These findings underscore the influence of tamoxifen derivatives on DNA methylation patterns, particularly through modulating TET3 expression, which appears to be contingent on the presence of estrogen receptors. This study highlights the potential of targeting epigenetic modifications for personalized anti-cancer therapy, offering a novel avenue to improve treatment outcomes.
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Neoplasias de la Mama , Dioxigenasas , Regulación Neoplásica de la Expresión Génica , Moduladores Selectivos de los Receptores de Estrógeno , Tamoxifeno , Humanos , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Tamoxifeno/farmacología , Tamoxifeno/análogos & derivados , Femenino , Dioxigenasas/genética , Dioxigenasas/metabolismo , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Células MCF-7 , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Línea Celular Tumoral , Receptores de Estrógenos/metabolismo , Receptores de Estrógenos/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/genética , Metilación de ADN/efectos de los fármacos , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Espectrometría de Masas en TándemRESUMEN
DNA methylation plays an important role in the development and tissue differentiation of eukaryotes. In this study, bisulfite sequencing (BS-seq) technology was used to analyze the DNA methylation profiles of liver tissues taken from Rongchang pigs at three postnatal feeding stages, including newborn, suckling, and adult. The DNA methylation pattern across the genomes or genic region showed little difference between the three stages. We observed 419 differentially methylated regions (DMRs) in promoters, corresponding to 323 genes between newborn and suckling stages, in addition to 288 DMRs, corresponding to 134 genes, between suckling and adult stages and 351 DMRs, corresponding to 293 genes, between newborn and adult stages. These genes with DMRs were mainly enriched in metabolic, immune-related functional processes. Correlation analysis showed that the methylation level of gene promoters was significantly negatively correlated with gene expression. Further, we found that genes related to nutritional metabolism, e.g., carbohydrate metabolism (FAHD1 and GUSB) or fatty acid metabolism (LPIN1 and ACOX2), lost DNA methylation in their promoter, with mRNA expression increased in newborn pigs compared with those in the suckling stage. A few fatty acid metabolism-related genes (SLC27A5, ACOX2) were hypomethylated and highly expressed in the newborn stage, which might satisfy the nutritional requirements of Rongchang pigs with high neonatal birth rates. In the adult stage, HMGCS2-which is related to fatty acid ß-oxidation-was hypomethylated and highly expressed, which explains that the characteristics of high energy utilization in adult Rongchang pigs and their immune-related genes (CD68, STAT2) may be related to the establishment of liver immunity. This study provides a comprehensive analysis of genome-wide DNA methylation patterns in pig liver postnatal development and growth. Our findings will serve as a valuable resource in hepatic metabolic studies and the agricultural food industry.
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Metilación de ADN , Hígado , Regiones Promotoras Genéticas , Animales , Hígado/metabolismo , Hígado/crecimiento & desarrollo , Porcinos/crecimiento & desarrollo , Porcinos/genética , Animales Recién Nacidos/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Epigénesis GenéticaRESUMEN
Hepatocellular carcinoma (HCC) is a malignancy that occurs worldwide and is generally associated with poor prognosis. The development of resistance to targeted therapies such as sorafenib is a major challenge in clinical cancer treatment. In the present study, Ten-eleven translocation protein 1 (TET1) was found to be highly expressed in sorafenib-resistant HCC cells and knockdown of TET1 can substantially improve the therapeutic effect of sorafenib on HCC, indicating the potential important roles of TET1 in sorafenib resistance in HCC. Mechanistic studies determined that TET1 and Yes-associated protein 1 (YAP1) synergistically regulate the promoter methylation and gene expression of DNA repair-related genes in sorafenib-resistant HCC cells. RNA sequencing indicated the activation of DNA damage repair signaling was extensively suppressed by the TET1 inhibitor Bobcat339. We also identified TET1 as a direct transcriptional target of YAP1 by promoter analysis and chromatin-immunoprecipitation assays in sorafenib-resistant HCC cells. Furthermore, we showed that Bobcat339 can overcome sorafenib resistance and synergized with sorafenib to induce tumor eradication in HCC cells and mouse models. Finally, immunostaining showed a positive correlation between TET1 and YAP1 in clinical samples. Our findings have identified a previously unrecognized molecular pathway underlying HCC sorafenib resistance, thus revealing a promising strategy for cancer therapy.
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Carcinoma Hepatocelular , Reparación del ADN , Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas , Transducción de Señal , Sorafenib , Animales , Humanos , Ratones , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Metilación de ADN/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Reparación del ADN/genética , Resistencia a Antineoplásicos/genética , Epigénesis Genética/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Vía de Señalización Hippo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Ratones Endogámicos BALB C , Ratones Desnudos , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/genética , Transducción de Señal/efectos de los fármacos , Sorafenib/farmacología , Sorafenib/uso terapéutico , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas Señalizadoras YAP/metabolismoRESUMEN
Cardiomyopathy (CM) represents a heterogeneous group of diseases primarily affecting cardiac structure and function, with genetic and epigenetic dysregulation playing a pivotal role in its pathogenesis. Emerging evidence from the burgeoning field of epitranscriptomics has brought to light the significant impact of various RNA modifications, notably N6-methyladenosine (m6A), 5-methylcytosine (m5C), N7-methylguanosine (m7G), N1-methyladenosine (m1A), 2'-O-methylation (Nm), and 6,2'-O-dimethyladenosine (m6Am), on cardiomyocyte function and the broader processes of cardiac and vascular remodelling. These modifications have been shown to influence key pathological mechanisms including mitochondrial dysfunction, oxidative stress, cardiomyocyte apoptosis, inflammation, immune response, and myocardial fibrosis. Importantly, aberrations in the RNA methylation machinery have been observed in human CM cases and animal models, highlighting the critical role of RNA methylating enzymes and their potential as therapeutic targets or biomarkers for CM. This review underscores the necessity for a deeper understanding of RNA methylation processes in the context of CM, to illuminate novel therapeutic avenues and diagnostic tools, thereby addressing a significant gap in the current management strategies for this complex disease.
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Cardiomiopatías , Epigénesis Genética , ARN , Humanos , Animales , Cardiomiopatías/genética , Cardiomiopatías/metabolismo , Cardiomiopatías/terapia , ARN/genética , ARN/metabolismo , Metilación , Metilación de ARNRESUMEN
Mitochondria are essential regulators of innate immunity. They generate long mitochondrial double-stranded RNAs (mt-dsRNAs) and release them into the cytosol to trigger an immune response under pathological stress conditions. Yet the regulation of these self-immunogenic RNAs remains largely unknown. Here, we employ CRISPR screening on mitochondrial RNA (mtRNA)-binding proteins and identify NOP2/Sun RNA methyltransferase 4 (NSUN4) as a key regulator of mt-dsRNA expression in human cells. We find that NSUN4 induces 5-methylcytosine (m5C) modification on mtRNAs, especially on the termini of light-strand long noncoding RNAs. These m5C-modified RNAs are recognized by complement C1q-binding protein (C1QBP), which recruits polyribonucleotide nucleotidyltransferase to facilitate RNA turnover. Suppression of NSUN4 or C1QBP results in increased mt-dsRNA expression, while C1QBP deficiency also leads to increased cytosolic mt-dsRNAs and subsequent immune activation. Collectively, our study unveils the mechanism underlying the selective degradation of light-strand mtRNAs and establishes a molecular mark for mtRNA decay and cytosolic release.
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5-Metilcitosina , Citosol , Mitocondrias , Estabilidad del ARN , ARN Bicatenario , ARN Mitocondrial , Humanos , Citosol/metabolismo , 5-Metilcitosina/metabolismo , Mitocondrias/metabolismo , Mitocondrias/genética , ARN Bicatenario/metabolismo , ARN Bicatenario/genética , ARN Mitocondrial/genética , ARN Mitocondrial/metabolismo , Células HEK293 , Células HeLa , Metiltransferasas/metabolismo , Metiltransferasas/genética , Inmunidad Innata , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Animales , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Sistemas CRISPR-CasRESUMEN
5-methylcytosine (m5C) is a prevalent RNA modification crucial for gene expression regulation. However, accurate and sensitive m5C sites identification remains challenging due to severe RNA degradation and reduced sequence complexity during bisulfite sequencing (BS-seq). Here, we report m5C-TAC-seq, a bisulfite-free approach combining TET-assisted m5C-to-f5C oxidation with selective chemical labeling, therefore enabling direct base-resolution m5C detection through pre-enrichment and C-to-T transitions at m5C sites. With m5C-TAC-seq, we comprehensively profiled the m5C methylomes in human and mouse cells, identifying a substantially larger number of confident m5C sites. Through perturbing potential m5C methyltransferases, we deciphered the responsible enzymes for most m5C sites, including the characterization of NSUN5's involvement in mRNA m5C deposition. Additionally, we characterized m5C dynamics during mESC differentiation. Notably, the mild reaction conditions and preservation of nucleotide composition in m5C-TAC-seq allow m5C detection in chromatin-associated RNAs. The accurate and robust m5C-TAC-seq will advance research into m5C methylation functional investigation.
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5-Metilcitosina , Sulfitos , Transcriptoma , 5-Metilcitosina/metabolismo , 5-Metilcitosina/química , Animales , Humanos , Ratones , Sulfitos/química , Metiltransferasas/metabolismo , Metiltransferasas/genética , Perfilación de la Expresión Génica/métodos , Diferenciación CelularRESUMEN
Allergic diseases, characterized by a broad spectrum of clinical manifestations and symptoms, encompass a significant category of IgE-mediated atopic disorders, including asthma, allergic rhinitis, atopic dermatitis, and food allergies. These complex conditions arise from the intricate interplay between genetic and environmental factors and are known to contribute to socioeconomic burdens globally. Recent advancements in the study of allergic diseases have illuminated the crucial role of DNA methylation (DNAm) in their pathogenesis. This review explores the factors influencing DNAm in allergic diseases and delves into their mechanisms, offering valuable perspectives for clinicians. Understanding these epigenetic modifications aims to lay the groundwork for improved early prevention strategies. Moreover, our analysis of DNAm mechanisms in these conditions seeks to enhance diagnostic and therapeutic approaches, paving the way for more effective management of allergic diseases in the future.
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Epigenetic cytosine methylation covers most of genomic CpG dinucleotides in human cells. In addition to common deamination-mediated mutagenesis at CpG sites, an alternative deamination-independent pathway associated with DNA polymerase activity was previously described. This mutagenesis is characterized by the TCGâTTG mutational signature and is believed to arise from dAMP misincorporation opposite 5-methylcytosine (mC) or its oxidized derivative 5-hydroxymethylcytosine (hmC) by B-family replicative DNA polymerases with disrupted proofreading 3â5'-exonuclease activity. In addition to being less stable and pro-mutagenic themselves, cytosine modifications also increase the risk of adjacent nucleotides damage, including the formation of 8-oxo-2'-deoxyguanosine (8-oxoG), a well-known mutagenic lesion. The effect of cytosine methylation on error-prone DNA polymerases lacking proofreading activity and involved in repair and DNA translesion synthesis remains unexplored. Here we analyze the efficiency and fidelity of translesion Y-family polymerases (Pol κ, Pol η, Pol ι and REV1) and primase-polymerase PrimPol opposite mC and hmC as well as opposite 8-oxoG adjacent to mC in the TCG context. We demonstrate that epigenetic cytosine modifications suppress Pol ι and REV1 activities and lead to increasing dAMP misincorporation by PrimPol, Pol κ and Pol ι in vitro. Cytosine methylation also increases misincorporation of dAMP opposite the adjacent 8-oxoG by PrimPol, decreases the TLS activity of Pol η opposite the lesion but increases dCMP incorporation opposite 8-oxoG by REV1. Altogether, these data suggest that methylation and hydroxymethylation of cytosine alter activity and fidelity of translesion DNA polymerases.
Asunto(s)
5-Metilcitosina , Citosina , Metilación de ADN , ADN Polimerasa Dirigida por ADN , Humanos , ADN Polimerasa Dirigida por ADN/metabolismo , Citosina/metabolismo , Citosina/análogos & derivados , 5-Metilcitosina/metabolismo , 5-Metilcitosina/análogos & derivados , Reparación del ADN , Daño del ADN , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/genética , ADN Polimerasa iota , ADN/metabolismo , Enzimas Multifuncionales/metabolismo , Replicación del ADN , 8-Hidroxi-2'-Desoxicoguanosina/metabolismoRESUMEN
Obesity is marked by excessive fat accumulation in the adipose tissue, which disrupts metabolic processes and causes chronic systemic inflammation. Commonly, body mass index (BMI) is used to assess obesity-related risks, predicting potential metabolic disorders. However, for a better clustering of obese patients, we must consider molecular and epigenetic changes which may be responsible for inflammation and metabolic changes. Our study involved two groups of patients, obese and healthy donors, on which routine analysis were performed, focused on BMI, leukocytes count, and C-reactive protein (CRP) and completed with global DNA methylation and gene expression analysis for genes involved in inflammation and adipogenesis. Our results indicate that obese patients exhibited elevated leukocytes levels, along with increased BMI and CRP. The obese group revealed a global hypomethylation and upregulation of proinflammatory genes, with adipogenesis genes following the same trend of being overexpressed. The study confirms that obesity is linked to systematic inflammation and metabolic dysfunction through epigenetic and molecular alterations. The CRP was correlated with the hypomethylation status in obese patients, and this fact may contribute to a better understanding of the roles of specific genes in adipogenesis and inflammation, leading to a better personalized therapy.