Dynamic modulation of N6-methyladenosine by ionizing radiation in human cells

BackgroundThe role of epigenetic modulation in immunity is receiving increased recognition—particularly in the context of RNA N6-methyladenosine (m6A) modifications. Nevertheless, it is still uncertain whether m6A methylation plays a role in the onset and progression of intracranial aneurysms (IAs). This study aimed to establish the function of m6A RNA methylation in IA, as well as its correlation with the immunological microenvironment.MethodsOur study included a total of 97 samples (64 IA, 33 normal) in the training set and 60 samples (44 IA, 16 normal) in the validation set to systematically assess the pattern of RNA modifications mediated by 22 m6A regulators. The effects of m6A modifications on immune microenvironment features, i.e., immune response gene sets, human leukocyte antigen (HLA) genes, and infiltrating immune cells were explored. We employed Lasso, machine learning, and logistic regression for the purpose of identifying an m6A regulator gene signature of IA with external data validation. For the unsupervised clustering analysis of m6A modification patterns in IA, consensus clustering methods were employed. Enrichment analysis was used to assess immune response activity along with other functional pathways. The identification of m6A methylation markers was identified based on a protein–protein interaction network and weighted gene co-expression network analysis.ResultsWe identified an m6A regulator signature of IGFBP2, IGFBP1, IGF2BP2, YTHDF3, ALKBH5, RBM15B, LRPPRC, and ELAVL1, which could easily distinguish individuals with IA from healthy individuals. Unsupervised clustering revealed three m6A modification patterns. Gene enrichment analysis illustrated that the tight junction, p53 pathway, and NOTCH signaling pathway varied significantly in m6A modifier patterns. In addition, the three m6A modification patterns showed significant differences in m6A regulator expression, immune microenvironment, and bio-functional pathways. Furthermore, macrophages, activated T cells, and other immune cells were strongly correlated with m6A regulators. Eight m6A indicators were discovered—each with a statistically significant correlation with IA—suggesting their potential as prognostic biological markers.ConclusionOur study demonstrates that m6A RNA methylation and the immunological microenvironment are both intricately correlated with the onset and progression of IA. The novel insight into patterns of m6A modification offers a foundation for the development of innovative treatment approaches for IA.

N6-methyladenosine (m6A) RNA methylation has recently emerged as a significant co-transcriptional modification involved in regulating various RNA functions. It plays a vital function in numerous biological processes. Enzymes referred to as m6A methyltransferases, such as the methyltransferaselike (METTL) 3-METTL14-Wilms tumor 1 (WT1)-associated protein (WTAP) complex, are responsible for adding m6A modifications, while m6A demethylases, including fat mass and obesity-associated protein (FTO) and alkB homolog 5 (ALKBH5), can remove m6A methylation. The functions of m6A-methylated RNA are regulated through the recognition and interaction of m6A reader proteins. Recent research has shown that m6A methylation takes place at multiple sites within hepatitis B virus (HBV) RNAs, and the location of these modifications can differentially impact the HBV infection. The addition of m6A modifications to HBV RNA can influence its stability and translation, thereby affecting viral replication and pathogenesis. Furthermore, HBV infection can also alter the m6A modification pattern of host RNA, indicating the virus's ability to manipulate host cellular processes, including m6A modification. This manipulation aids in establishing chronic infection, promoting liver disease, and contributing to pathogenesis. A comprehensive understanding of the functional roles of m6A modification during HBV infection is crucial for developing innovative approaches to combat HBV-mediated liver disease. In this review, we explore the functions of m6A modification in HBV replication and its impact on the development of liver disease.

Random Forest model reveals the interaction between N6-methyladenosine modifications and RNA-binding proteins

BackgroundDNA 5-methylcytosine (5mC) and RNA N6-methyladenosine (m6A) methylation are prevalent modifications in eukaryotes, both playing crucial roles in gene regulation. Recent studies have explored their crosstalk and impact on transcription. However, the intricate relationships among 5mC, m6A, and gene expression remain incompletely elucidated.ResultsWe collect data on 5mC, m6A, and gene expression from samples from three tissues from each of four pregnant cattle and sheep. We construct a comprehensive genome-wide self-interaction (same gene) and across-interaction (across genes) network of 5mC and m6A within gene-bodies or promoters and gene expression in both species. Qualitative analysis identifies uniquely expressed genes with specific m6A methylation in each tissue from both species. A quantitative comparison of gene expression ratio between methylated and unmethylated genes for m6A within gene body and promoter, and 5mC within gene body and promoter confirms the positive effect of RNA methylation on gene expression. Importantly, the influence of RNA methylation on gene expression is stronger than that of DNA methylation. The predominant self- and across-interactions are between RNA methylation within gene bodies and gene expression, as well as between RNA methylation within promoters and gene expression in both species.ConclusionsRNA methylation has a stronger effect on gene expression than does DNA methylation within gene bodies and promoters. DNA and RNA methylation in gene-bodies has a greater impact on gene expression than those in promoters. These findings deepen comprehension of the dynamics and complex relationships among the epigenome, epitranscriptome, and transcriptome, offering fresh insights for advancing epigenetics research.

The role of m6A methylation in prenatal maternal psychological distress and birth outcome

Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer deaths. Afatinib is the first-line anti-cancer agent for treatment of NSCLC. However, unexpected resistance has been a major obstacle for its clinical efficacy. In this study, we dissected afatinib resistance from the perspective of N6-Methyladenosine (m6A) modification. First, we depicted the m6A modification profiles for the afatinib resistant and sensitive NSCLC cell lines (H1299 and A549). We found that the sum enrichment scores of the resistant cell line (H1299) was much higher than that of the sensitive cell line (A549). Next, we identified the functionally m6A-modified genes, which were the intersection of the differentially m6A methylated genes and the differentially expressed genes between H1299 and A549, as well as negative correlation between m6A modification levels and gene expression levels. In addition, functional enrichment analysis of the functionally m6A-modified genes indicated that m6A methylation might modify cell cycle to affect afatinib response. Furthermore, the functionally m6A-modified genes were over-represented in the putative drug resistance-associated genes and the FDA-approved drug targets, and had significantly higher average degree and clustering coefficient than other genes in protein-protein interaction (PPI) network. We also identified five network modules, which were all related to drug resistance functions. Finally, survival analysis demonstrated that m6A modification could affect prognosis of NSCLC patients. In conclusion, we conducted a first attempt to dissect m6A methylation affection on afatinib resistance in NSCLC, and brought inspiration for the study of epigenetic roles in drug resistance.

N6-methyladenosine (m6A) modification is the most prevalent chemical modification in eukaryotic mRNA and is associated with the development of various immune diseases. However, the role of m6A methylation in rheumatoid arthritis (RA) development is unclear. We preliminarily explored the role of m6A methylation-related mRNAs in RA for its clinical application. The discovery of m6A methylation-modifying genes in this study may provide a fresh perspective on the development of drugs for RA treatment. High-throughput sequencing combined with methylated RNA immunoprecipitation (MeRIP-seq) and RNA sequencing were used to assess whole-transcriptome m6A modifications in the synovium of patients with RA. The relationship between m6A-modified target genes and RA inflammation and macrophages was determined. The expression of the m6A-modified significant transcript-enriched inflammatory signaling pathway was assessed through animal experiments. Differentially expressed m6A genes were correlated with macrophage activation involved in immune response, vascular endothelium, MAPK signaling pathway, PI3K − Akt signaling pathway, and other inflammatory processes. Furthermore, combined analysis with m6A-seq and RNA-seq revealed 120 genes with significant changes in both m6A modification and mRNA expression. We selected the top 3 candidate mRNAs that were upregulated and downregulated simultaneously. The expression of phosphatase and tensin homolog deleted on chromosome ten (PTEN) mRNA and protein in RA patients was lower than that in healthy control (HC). SHC-binding protein 1 (SHCBP1) and neurexophilin-3 (NXPH3) mRNA expressions were increased in RA patients. The expression of M1 macrophages was increased in RA patients. RA markers are such as rheumatoid factor (RF) and peptide containing citrulline (CCP). Further animal experiments showed that the expression of synovial MAPK, PI3K, and Akt1 proteins in the RA model was increased, and the PTEN, p-PTEN protein expression was decreased. PI3K, Akt1, PTEN, and p-PTEN were correlated to RA joint inflammation. This study revealed a unique pattern of differential m6A methylation modifications in RA and concluded that m6A modification is related to the occurrence of RA synovial inflammation.

Methylating RNA post-transcriptionally is emerging as a significant mechanism of gene regulation in eukaryotes. The crosstalk between RNA methylation and histone modification is critical for chromatin state and gene expression in mammals. However, it is not well understood mechanistically in plants. Here, the authors report a genome-wide correlation between RNA 5-cytosine methylation (m5 C) and histone 3 lysine27 trimethylation (H3K27me3) in Arabidopsis. The plant-specific Polycomb group (PcG) protein EMBRYONIC FLOWER1 (EMF1) plays dual roles as activators or repressors. Transcriptome-wide RNA m5 C profilingrevealed that m5 C peaks are mostly enriched in chromatin regions that lacked H3K27me3 in both wild type and emf1 mutants. EMF1 repressed the expression of m5 C methyltransferase tRNA specific methyltransferase 4B (TRM4B) through H3K4me3, independent of PcG-mediated H3K27me3 mechanism. The 5-Cytosine methylation on targets is increased in emf1 mutants, thereby decreased the mRNA transcripts of photosynthesis and chloroplast genes. In addition, impairing EMF1 activity reduced H3K27me3 levels of PcG targets, such as starch genes, which are de-repressed in emf1 mutants. Both EMF1-mediated promotion and repression of gene activities via m5 C and H3K27me3 are required for normal vegetative growth. Collectively, t study reveals a previously undescribed epigenetic mechanism of RNA m5 C modifications and histone modifications to regulate gene expression in eukaryotes.

N6-methyladenosine (m6A) RNA methylation is profoundly involved in epigenetic regulation, especially for carcinogenesis and tumor progression. Mounting evidence suggests that methyltransferase METTL3 regulates malignant behaviors of gastric cancer (GC). However, the clinical significance and biological implication of SPHK2 and its related m6A modification in GC remain unclear. In this study, quantitative real-time PCR (qRT-PCR), western blot and immunohistochemistry were utilized to detect the expression profiles and prognostic significance of SPHK2 in GC. Here, we showed that increased SPHK2 was signified a poor prognosis of GC patients. Phosphorylation and ubiquitination assays were used to investigate the possible mechanisms of SPHK2-mediated KLF2 expression. SPHK2 can promote the phosphorylation of KLF2, which triggers the ubiquitination and degradation of KLF2 protein in GC. Methylated RNA immunoprecipitation (MeRIP) was performed to uncover the m6A modification of SPHK2 mRNA. METTL3 promotes translation of SPHK2 mRNA via an m6A-YTHDF1-dependent manner. Functionally, SPHK2 facilitates GC cell proliferation, migration and invasion by inhibiting KLF2 expression. SPHK2/KLF2 regulates the cell proliferation, migration, and invasion induced by METTL3 in GC. Overall, our findings reveal that METTL3-mediated m6A modification of SPHK2 contributes to GC progression, which extends the understanding of the importance m6A methylation in GC and represents a potential target for GC therapy.

DNA double strand breaks are induced by ionizing radiation (IR), leading to the phosphorylation of the core histone protein H2AX (termed γH2AX). The understanding of the γH2AX responses in irradiated human buccal cells is still very limited. We used visual scoring and laser scanning cytometry (LSC) methods to investigate γH2AX signaling following exposure of human buccal cells (from six individuals) to ionizing radiation at 0-4 Gy. The frequency of nuclei containing 15-30 γH2AX foci was significantly elevated 30 min post-IR exposure (by visual scoring). Concomitantly, there was a significant decrease in the frequency of cells without foci following exposure to IR. IR-induced γH2AX signal as determined by laser scanning cytometry (which included γH2AX integral and MaxPixel value) increased significantly in all individual's 2N nuclei 30 min post-IR and was similar for all three nuclear shapes identified. Individuals with the lowest baseline γH2AX integral (i.e., in nonirradiated cells) showed the greatest fold stimulation of γH2AX and significant dose-responses to IR doses of 1, 2, and 4 Gy. In 5 out of 6 individuals, the frequency of visually scored γH2AX in nuclei showed a strong correlation (up to r = 0.999) with LSC scored γH2AX integrals. The γH2AX response and subsequent decline varied between individuals but remained elevated above baseline levels 24 h post IR exposure. γH2AX response in irradiated human buccal cells has potential to be used as an index of baseline DNA damage in population studies. The variable response to IR exposure between individuals should be taken into consideration when using the γH2AX assay for radiation biodosimetry.

Background: The N6-methyladenosine (m6A) modification plays a critical role in cancer development. Little is known about the m6A modification in triple-negative breast cancer (TNBC), the most aggressive subtype of breast cancer. Thus, the prognostic value of m6A RNA methylation in TNBC deserves exploration.Methods: The expression levels of the 13 m6A methylation regulators were compared between the 98 TNBC tumor samples and normal tissue samples based on the transcriptome profiles from The Cancer Genome Atlas (TCGA). The association between the m6A regulators and patients' overall survival was assessed by Kaplan-Meier survival analysis and Cox regression analysis. Lasso regression analysis was conducted to construct a prognostic model based on the m6A methylation system. The prognostic performance of the identified model was validated in GSE88847 and GSE135565 datasets. A nomogram combining the TNM stage and the m6A prognostic model was further constructed for the survival prediction of TNBC patients.Results: The m6A regulator genes were remarkably dysregulated in TNBC tumor tissues, with ALKBH5, YTHDF2, HNRNPC, KIAA1429, and RBM15 significantly up-regulated and FTO, YTHDC1, YTHDC2, METTL3, METTL14, and ZC3H13 significantly down-regulated (P < 0.01). The expression level of ALKBH5 was an independent unfavorable prognostic factor (HR = 3.327, P = 0.006), while METTL14 (HR = 0.425, P = 0.009) was an independent favorable prognostic factor for TNBC patients. A prognostic model consisting of ALKBH5 and METTL14 was therefore proposed displaying higher accuracy of risk prediction when combined with TNM stage with an AUC of 0.791. The prognostic value of the identified signature remained consistent within the two external validation datasets.Conclusion: The m6A methylation regulators were significantly dysregulated in TNBC tissues and could constitute a novel prognostic signature for the survival prediction of TNBC patients.

Middle ear cholesteatoma is a chronic middle ear disease characterized by severe hearing loss and adjacent bone erosion, resulting in numerous complications. This study sought to identify pathways involved in N6-methyladenosine (m6A) modification of circRNA in middle ear cholesteatoma. A m6A circRNA epitranscriptomic microarray analysis was performed in middle ear cholesteatoma tissues (n = 5) and normal post-auricular skin samples (n = 5). Bioinformatics analyses subsequently explored the biological functions (Gene Ontology, GO) and signaling pathways (Kyoto Encyclopedia of Genes and Genomes, KEGG) underlying middle ear cholesteatoma pathogenesis. Methylated RNA immunoprecipitation qPCR (MeRIP-qPCR) was performed to verify the presence of circRNAs with m6A modifications in middle ear cholesteatoma and normal skin samples. Microarray analysis identified 3,755 circRNAs as significantly differentially modified by m6A methylation in middle ear cholesteatoma compared with the normal post-auricular skin. Among these, 3,742 were hypermethylated (FC ≥ 2, FDR < 0.05) and 13 were hypomethylated (FC ≤ 1/2, FDR < 0.05). GO analysis terms with the highest enrichment score were localization, cytoplasm, and ATP-dependent activity for biological processes, cellular components, and molecular functions respectively. Of the eight hypermethylated circRNA pathways, RNA degradation pathway has the highest enrichment score. Peroxisome Proliferator-Activated Receptor (PPAR) signaling pathway was hypomethylated. To validate the microarray analysis, we conducted MeRIP-qPCR to assess the methylation levels of five specific m6A-modified circRNAs: hsa_circRNA_061554, hsa_circRNA_001454, hsa_circRNA_031526, hsa_circRNA_100833, and hsa_circRNA_022382. The validation was highly consistent with the findings from the microarray analysis. Our study firstly presents m6A modification patterns of circRNAs in middle ear cholesteatoma. This finding suggests a direction for circRNA m6A modification research in the etiology of cholesteatoma and provides potential therapeutic targets for the treatment of middle ear cholesteatoma.

RBM15 drives the progression of lung adenocarcinoma by regulating N6-methyladenosine-mediated LDHA mRNA stability

Rationale: N6-methyladenosine (m6A), a prevalent post-transcriptional methylation of RNA in eukaryotes, plays a crucial role in the modulation of antiviral immunity. However, the mechanisms regulating m6A modification during virus infection remain largely unclear. Gut microbiota that is indispensable in priming host's defense to influenza virus infection was recently reported to be involved in driving m6A modification in extrapulmonary organs. We hypothesized that gut microbiota-mediated m6A modification in the lung was linked to its anti-influenza effect. Methods: Specific pathogen-free (SPF) mice were intranasally challenged with an influenza A virus (H1N1) strain (A/PR8) or PBS. Depletion of gut microbiota was performed by a cocktail of antibiotics (ampicillin, metronidazole, neomycin sulfate, and vancomycin) in drinking water for 4 weeks. Level of m6A methylation in lung tissue was quantified by mRNA dot blot assay and immunohistochemistry. Molecules involved in m6A modification were measured by WB and qPCR. Bronchoalveolar lavage fluid was collected for the quantification of inflammatory cells and virus-associated interferons (IFN-α and IFN-β). Protein and mRNA level of IFN-α and IFN-β in lung tissue were also measured. Viral load was quantified by detection of viral nucleoprotein in lung tissue. Results: Level of m6A methylation in the lung was significantly elevated in SPF mice after infection with H1N1. Consistently, a key m6A writer protein (METTL3) that drives methylation was highly expressed after viral infection. Of note, two m6A erasers (ALKBH5 and FTO) that mediate demethylation were also elevated, which potentially represents protective feedback that limits the overmethylation of lung mRNAs. Microbiota depletion amplified m6A modification in mice lungs as evidenced by higher level of m6A methylation in antibiotic-treated mice compared to naïve ones. Strikingly, depletion of microbiota not only enhanced the expression of METTL3, but suppressed the expression of ALKBH5 and FTO. Concomitantly, mice devoid of microbiota showed impaired antiviral immunity characterized by decreased secretion of IFN-α and IFN-β and increased viral load. Microbiota depletion was also associated with worse infection-related consequences including heightened airway inflammation, more weight-loss, and increased mortality. Conclusion: Gut microbiota inhibits influenza virus-induced m6A methylation in the lung which is linked to its antiviral immunity. Precise mechanisms underlying this microbiota-mediated epigenetic modification in viral infection deserve further investigation.

BackgroundStudies on biological functions of N6‐methyladenosine (m6A) modification in mRNA have sprung up in recent years. Previous studies have reported m6A can determine mRNA fate and play a pivotal role in tumour development and progression. The zinc finger protein 677 (ZNF677) belongs to the zinc finger protein family and possesses transcription factor activity by binding sequence‐specific DNA.MethodsThe expression of ZNF677 and its clinicopathological impact were evaluated in renal cell carcinoma (RCC) patients. The m6A level of ZNF677 was determined by m6A methylated RNA immunoprecipitation‐sequencing (MeRIP‐seq) and MeRIP‐qPCR in RCC tissues and adjacent normal tissues. RNA immunoprecipitation‐qPCR (RIP‐qPCR) and luciferase assays were performed to identify the targeted effect of IGF2BP2 and YTHDF1 on ZNF677. RCC cells and subcutaneous models uncovered the role of ZNF677 methylated by CRISPR/dCas13b‐METTL3 in tumour growth. ZNF677‐binding sites in the CDKN3 promoter were investigated by chromatin immunoprecipitation (ChIP) and luciferase assays.ResultsZNF677 is frequently downregulated in RCC tissues and its low expression is associated with unfavourable prognosis and decreased m6A modification level. Further, we find the m6A‐modified coding sequence (CDS) of ZNF677 positively regulates its translation and mRNA stability via binding with YTHDF1 and IGF2BP2, respectively. Targeted specific methylation of ZNF677 m6A by CRISPR/dCas13b‐METLL3 system can significantly increase the m6A and expression level of ZNF677, and dramatically inhibit cell proliferation and induce cell apoptosis of RCC cells. In addition, ZNF677 exerted its tumour suppressor functions in RCC cells through transcriptional repression of CDKN3 via binding to its promoter. In vitro and clinical data confirm the negative roles of ZNF677/CDKN3 in tumour growth and progression of RCC.ConclusionZNF677 functions as a tumour suppressor and is frequently silenced via m6A modification in RCC, which may highlight m6A methylation‐based approach for RCC diagnosis and therapy.