m6A Reader Protein Research Articles

Severe fever with thrombocytopenia syndrome virus induces lactylation of m6A reader protein YTHDF1 to facilitate viral replication.

Severe fever with thrombocytopenia syndrome virus (SFTSV), an emerging infectious pathogen with a high fatality rate, is an enveloped tripartite segmented single-stranded negative-sense RNA virus. SFTSV infection is characterized by suppressed host innate immunity, proinflammatory cytokine storm, failure of B-cell immunity, and robust viral replication. m6A modification has been shown to play a role in viral infections. However, interactions between m6A modification and SFTSV infection remain poorly understood. Through MeRIP-seq, we identify m6A modifications on SFTSV RNA. We show that YTHDF1 can bind to m6A modification sites on SFTSV, decreasing the stability of SFTSV RNA and reducing the translation efficiency of SFTSV proteins. The SFTSV virulence factor NSs increases lactylation of YTHDF1 and YTHDF1 degradation, thus facilitating SFTSV replication. Our findings indicate that the SFTSV protein NSs induce lactylation to inhibit YTHDF1 as a countermeasure to host's YTHDF1-mediated degradation of m6A-marked viral mRNAs.

M6A reader YTHDF2 governs the onset of atrial fibrillation by modulating Cacna1c translation.

Atrial fibrillation (AF) is the most common arrhythmia, which is tightly associated with the abnormal expression and function of ion channels in the atrial cardiomyocytes. N6-methyladenosine (m6A), a widespread chemical modification in eukaryotic mRNA, is known to play a significant regulatory role in the pathogenesis of heart disease. However, the significance of m6A regulatory proteins in the onset of AF remains unclear. Here, we demonstrate that the m6A reader protein YTHDF2 regulates atrial electrical remodeling and AF onset by modulating the Cav1.2 expression. Firstly, YTHDF2 expression was selectively upregulated in rat atrial cardiomyocytes with AF. Secondly, YTHDF2 knockout reduced AF susceptibility in mice. Thirdly, the knockout of YTHDF2 increased Cav1.2 protein levels in an m6A-in-dependent manner, ultimately prolonging the atrial myocardial refractory period, a critical electrophysiological substrate for the onset of AF. Fourthly, the N-terminal domain of YTHDF2 was identified as critical for Cacna1c mRNA translation regulation. Overall, our findings unveil that YTHDF2 can alter Cav1.2 protein expression in an m6A-independent manner, thereby facilitating the onset of AF. Our study suggests that YTHDF2 may be a potential intervention target for AF.

Data-augmented machine learning scoring functions for virtual screening of YTHDF1 m6A reader protein

Machine learning is rapidly advancing the drug discovery process, significantly enhancing speed and efficiency. Innovation in computer-aided drug design is primarily driven by structure- and ligand-based approaches. When the number of known inhibitors for a target is limited, data augmentation strategies are often preferred to enhance model performance. In this study, we developed predictive machine learning models for structure-based drug discovery leveraging multiple traditional machine learning algorithms trained with target and ligand dynamics-aware datasets.To illustrate our approach, we present a composite model that combines classification and regression to predict YTHDF1 inhibitors, utilizing PLEC features. YTHDF1, a key m6A reader protein involved in mRNA translation, is implicated in various cancers, making it a promising therapeutic target. Traditional structure-based virtual screening (SBVS) using generic scoring functions has struggled to identify potent YTHDF1 inhibitors due to the protein's unique binding characteristics. To overcome this, we developed YTHDF1-specific machine learning scoring functions (MLSFs) to enhance SBVS efficacy.We employed various data augmentation techniques to generate a comprehensive dataset, incorporating multiple conformations of ligands and the YTHDF1 protein. We have trained 64 YTHDF1-specific MLSFs using four machine learning algorithms and evaluated them on ten test sets, focusing on their predictive and ranking power. Our results demonstrate that the artificial neural network with protein-ligand extended connectivity fingerprints (ANN-PLEC) outperforms other MLSFs, consistently achieving high area under the precision-recall curve (PR-AUC) of 0.87. This method shows promise for targets with limited quantities of active molecules, providing a viable path forward for drug discovery research. The ANN-PLEC scoring function is made freely available on GitHub for other researchers to access and utilize https://github.com/JuniML/SBVS-YTHDF1/.

Advanced paternal age exacerbates neuroinflammation in offspring via m6A modification-mediated intergenerational inheritance

BackgroundThe trend of postponing childbearing age is prevalent worldwide. Advanced paternal age (APA) is associated with adverse pregnancy outcomes and offspring health. However, the underlying mechanism by which paternal aging affects the risk of offspring neuropsychiatric disorders is unclear. Our study aims to explore the behavioral phenotypes and the pathologic epigenetic alterations of APA offspring inherited from aging sperm.MethodsBehavioral tests, ELISA assay, immunofluorescence and western blotting were performed on offspring mice. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA immunoprecipitation sequencing (RIP-seq) were used to investigate the modified N6-methyladenosine (m6A) profiles of paternal sperm and offspring hippocampus. Intervention of gene expression by lentivirus and adeno-associated virus in both vivo and vitro examined the potential therapeutic targets of intergenerational inherited neuroinflammation.ResultsIn our study, APA offspring exhibit cognitive impairment and autism-like behavior. An increase in neuroinflammation in APA offspring is associated with microglial overactivation, which manifests as abnormal morphology and augmented engulfment. MeRIP-seq of F0 sperm and F1 hippocampus reveal that Nr4a2 is hypermethylated with decreased expression in APA offspring involving in synaptic plasticity and microglial function. In addition, Ythdc1, an m6A reader protein, is markedly elevated in aging sperm and remains elevated in adult hippocampus of APA group. Enhanced Ythdc1 recognizes and suppresses the hypermethylated Nr4a2, thereby contributing to the abnormal phenotype in offspring. The overexpression of Ythdc1 triggers microglial activation in vitro and its suppression in the hippocampus of APA progeny alleviates behavioral aberrations and attenuates neuroinflammation.ConclusionOur study provides additional evidence of the abnormal behavioral phenotypes of APA offspring and reveals potential epigenetic inheritance signatures and targeted genes for future research.

FTO-mediated SMAD2 m6A modification protects cartilage against Osteoarthritis

N6-methyladenosine (m6A) modification is one of the most prevalent forms of epigenetic modification and plays an important role in the development of degenerative diseases such as osteoarthritis (OA). However, the evidence concerning the role of m6A modification in OA is insufficient. Here, m6A modification was increased in human OA cartilage and degenerated chondrocytes. Among all of the m6A enzymes, the expression of the demethylase fat mass and obesity-associated protein (FTO) decreased dramatically. Conditional knockout of FTO in chondrocytes accelerates OA progression. FTO transcription is regulated by runt-related transcription factor-1 (RUNX1). Reduced FTO elevates m6A modification at the adenosine N6 position in SMAD family member 2 (SMAD2) mRNA, whose stability is subsequently modulated by the recruited m6A reader protein YTH N6-methyladenosine RNA binding protein F2 (YTHDF2). Collectively, these findings reveal the function and mechanism of the m6A family member FTO in OA progression. Therefore, reducing m6A modification to increase SMAD2 stability by activating FTO might be a potential therapeutic strategy for OA treatment.

A novel protein SPECC1-415aa encoded by N6-methyladenosine modified circSPECC1 regulates the sensitivity of glioblastoma to TMZ

BackgroundCircular RNAs (circRNAs) can influence a variety of biological functions and act as a significant role in the progression and recurrence of glioblastoma (GBM). However, few coding circRNAs have been discovered in cancer, and their role in GBM is still unknown. The aim of this study was to identify coding circRNAs and explore their potential roles in the progression and recurrence of GBM.MethodsCircSPECC1 was screened via circRNAs microarray of primary and recurrent GBM samples. To ascertain the characteristics and coding ability of circSPECC1, we conducted a number of experiments. Afterward, through in vivo and in vitro experiments, we investigated the biological functions of circSPECC1 and its encoded novel protein (SPECC1-415aa) in GBM, as well as their effects on TMZ sensitivity.ResultsBy analyzing primary and recurrent GBM samples via circRNAs microarray, circSPECC1 was found to be a downregulated circRNA with coding potential in recurrent GBM compared with primary GBM. CircSPECC1 suppressed the proliferation, migration, invasion, and colony formation abilities of GBM cells by encoding a new protein known as SPECC1-415aa. CircSPECC1 restored TMZ sensitivity in TMZ-resistant GBM cells by encoding the new protein SPECC1-415aa. The m6A reader protein IGF2BP1 can bind to circSPECC1 to promote its expression and stability. Mechanistically, SPECC1-415aa can bind to ANXA2 and competitively inhibit the binding of ANXA2 to EGFR, thus resulting in the inhibition of the phosphorylation of EGFR (Tyr845) and its downstream pathway protein AKT (Ser473). In vivo experiments showed that the overexpression of circSPECC1 could combine with TMZ to treat TMZ-resistant GBM, thereby restoring the sensitivity of TMZ-resistant GBM to TMZ.ConclusionsCircSPECC1 was downregulated in recurrent GBM compared with primary GBM. The m6A reader protein IGF2BP1 could promote the expression and stability of circSPECC1. The sequence of SPECC1-415aa, which is encoded by circSPECC1, can inhibit the binding of ANXA2 to EGFR by competitively binding to ANXA2 and inhibiting the phosphorylation of EGFR and AKT, thereby restoring the sensitivity of TMZ-resistant GBM cells to TMZ.

Knockdown of IGF2BP3 Down-Regulates PDCD4 Levels to Attenuate Hypoxic-Ischemic Brain Damage

Background: Hypoxic-ischemic brain damage (HIBD) is a prevalent brain injury with high mortality and morbidity. It results from hypoxia and ischemia of the brain due to various perinatal factors. A previous study showed that knockdown of programmed cell death factor 4 (PDCD4) could reduce infarction injury resulting from ischemia/reperfusion injury. However, exact mechanism by which PDCD4 acts in HIBD is not yet understood. Our aim in present investigation was to investigate the function and mechanism of PDCD4 in alleviating HIBD. Methods: An HIBD model was developed using neonatal rats. After 48 h of modeling, short-term neurological function was evaluated and the brain tissue removed for assessment of cerebral infarct volume and brain water content (BWC). A cell model of oxygen glucose deprivation/reoxygenation (OGD/R) was also constructed. Overexpression or knockdown of insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3) or PDCD4 was performed in pretreated cells. Results: The geotaxis reflex time, cerebral infarct volume, and BWC all increased after HIBD in this neonatal rat model. Additionally, the levels of PDCD4 and of the N6-Methyladenosine (m6A) reader protein IGF2BP3 were increased in HIBD rats and OGD/R-stimulated pheochromocytoma (PC12) cells relative to controls. Moreover, OGD/R-stimulated pheochromocytoma PC12 cells showed decreased cell viability, increased apoptosis, and elevated Interleukin 6 (IL-6), Interleukin 1 β (IL-1β), and tumor necrosis factor-α (TNF-α) contents. These features were reversed after knocking down IGF2BP3. The interaction between IGF2BP3 protein and PDCD4 mRNA was confirmed by RNA immunoprecipitation and RNA pull-down assays. Furthermore, knockdown of IGF2BP3 in OGD/R-stimulated PC12 cells reduced cell damage via down-regulation of PDCD4. Finally, the IGF2BP3/PDCD4 axis alleviated OGD/R-induced cell injury in primary cortical neurons (PCNs). Conclusions: PDCD4 and m6A reader protein IGF2BP3 were up-regulated in an HIBD neonatal rat model. Knockdown of IGF2BP3 in OGD/R-stimulated PC12 cells or PCNs alleviated cell damage through reducing PDCD4.

N6-methyladenosine RNA methyltransferase CpMTA1 mediates CpAphA mRNA stability through a YTHDF1-dependent m6A modification in the chestnut blight fungus.

In eukaryotic cells, N6-methyladenosine (m6A) is the most prevalent RNA epigenetic modification that plays crucial roles in multiple biological processes. Nevertheless, the functions and regulatory mechanisms of m6A in phytopathogenic fungi are poorly understood. Here, we showed that CpMTA1, an m6A methyltransferase in Cryphonectria parasitica, plays a crucial role in fungal phenotypic traits, virulence, and stress tolerance. Furthermore, the acid phosphatase gene CpAphA was implicated to be a target of CpMTA1 by integrated analysis of m6A-seq and RNA-seq, as in vivo RIP assay data confirmed that CpMTA1 directly interacts with CpAphA mRNA. Deletion of CpMTA1 drastically lowered the m6A level of CpAphA and reduced its mRNA expression. Moreover, we found that an m6A reader protein CpYTHDF1 recognizes CpAphA mRNA and increases its stability. Typically, the levels of CpAphA mRNA and protein exhibited a positive correlation with CpMTA1 and CpYTHDF1. Importantly, site-specific mutagenesis demonstrated that the m6A sites, A1306 and A1341, of CpAphA mRNA are important for fungal phenotypic traits and virulence in C. parasitica. Together, our findings demonstrate the essential role of the m6A methyltransferase CpMTA1 in C. parasitica, thereby advancing our understanding of fungal gene regulation through m6A modification.

MiR-340-3p-modified bone marrow mesenchymal stem cell-derived exosomes inhibit ferroptosis through METTL3-mediated m6A modification of HMOX1 to promote recovery of injured rat uterus

BackgroundFerroptosis is associated with the pathological progression of hemorrhagic injury and ischemia–reperfusion injury. According to our previous study, exosomes formed through bone marrow mesenchymal stem cells modified with miR-340-3p (MB-exos) can restore damaged endometrium. However, the involvement of ferroptosis in endometrial injury and the effect of MB-exos on ferroptosis remain elusive.MethodsThe endometrial injury rat model was developed. Exosomes were obtained from the supernatants of bone marrow mesenchymal stromal cells (BMSCs) and miR-340/BMSCs through differential centrifugation. We conducted RNA-seq analysis on endometrial tissues obtained from the PBS and MB-exos groups. Ferroptosis was induced in endometrial stromal cells (ESCs) by treating them with erastin or RSL3, followed by treatment with B-exos or MB-exos. We assessed the endometrial total m6A modification level after injury and subsequent treatment with B-exos or MB-exos by methylation quantification assay. We performed meRIP-qPCR to analyze m6A modification-regulated endogenous mRNAs.ResultsWe reveal that MB-exos facilitate the injured endometrium to recover by suppressing ferroptosis in endometrial stromal cells. The injured endometrium showed significantly upregulated N6-methyladenosine (m6A) modification levels; these levels were attenuated by MB-exos through downregulation of the methylase METTL3. Intriguingly, METTL3 downregulation appears to repress ferroptosis by stabilizing HMOX1 mRNA, thereby potentially elucidating the mechanism through which MB-exos inhibit ferroptosis in ESCs. We identified YTHDF2 as a critical m6A reader protein that contributes to HMOX1 mRNA degradation. YTHDF2 facilitates HMOX1 mRNA degradation by identifying the m6A binding site in the 3′-untranslated regions of HMOX1. In a rat model, treatment with MB-exos ameliorated endometrial injury-induced fibrosis by inhibiting ferroptosis in ESCs. Moreover, METTL3 short hairpin RNA-mediated inhibition of m6A modification enhanced the inhibitory effect of MB-exos on ferroptosis in endometrial injury.ConclusionsThus, these observations provide new insights regarding the molecular mechanisms responsible for endometrial recovery promotion by MB-exos and highlight m6A modification-dependent ferroptosis inhibition as a prospective therapeutic target to attenuate endometrial injury.

YTHDF2 Regulates Advanced Glycation End Products-Induced Melanogenesis through Inhibiting A20 Expression in Human Dermal Fibroblasts.

Fibroblast A20 suppresses advanced glycation end products (AGEs)-induced melanogenesis by inhibiting NLRP3 inflammasome activation. AGEs repress A20 expression and significantly m6A-methylate A20 mRNA in fibroblasts. YTHDF2 is the most studied m6A reader protein and can accelerate degradation of m6A-modified mRNA. Whether YTHDF2 regulates AGEs-induced A20 expression and pigmentation is unknown. In this study, we confirmed that YTHDF2 inversely regulated AGEs-BSA-inhibited A20 expression but facilitated AGEs-BSA-activated NF-κB signaling and NLRP3 inflammasome in fibroblasts via YTHDF2 knockdown and overexpression experiments. Mechanistically, YTHDF2 bound to m6A-modified A20 mRNA induced by AGEs-BSA and increased its degradation. Moreover, fibroblast YTHDF2 robustly promoted AGEs-BSA-induced IL-18 level in coculture supernatants and melanin content, tyrosinase activity, and expression of microphthalmia-associated transcription factor and tyrosinase in melanocytes, which were significantly blocked by IL-18 binding protein. Further, fibroblast YTHDF2 markedly increased AGEs-BSA-induced epidermal melanin level in cocultured ex vivo skin and MAPKs activation in melanocytes. Importantly, upregulated dermal YTHDF2 expression was negatively correlated with dermal A20 level and positively associated with both epidermal melanin and dermal AGEs content in sun-exposed skin and lesions of melasma and solar lentigo. These findings suggest that fibroblast YTHDF2 positively regulates AGEs-induced melanogenesis mainly via A20/ NF-κB /NLRP3 inflammasome/ IL-18 /MAPKs axis in an m6A-dependent manner and functions in photoaging-induced hyperpigmentation skin disorders.

RNA m6A modification regulates cell fate transition between pluripotent stem cells and 2-cell-like cells.

N6-methyladenosine (m6A) exerts essential roles in early embryos, especially in the maternal-to-zygotic transition stage. However, the landscape and roles of RNA m6A modification during the transition between pluripotent stem cells and 2-cell-like (2C-like) cells remain elusive. Here, we utilised ultralow-input RNA m6A immunoprecipitation to depict the dynamic picture of transcriptome-wide m6A modifications during 2C-like transitions. We found that RNA m6A modification was preferentially enriched in zygotic genome activation (ZGA) transcripts and MERVL with high expression levels in 2C-like cells. During the exit of the 2C-like state, m6A facilitated the silencing of ZGA genes and MERVL. Notably, inhibition of m6A methyltransferase METTL3 and m6A reader protein IGF2BP2 is capable of significantly delaying 2C-like state exit and expanding 2C-like cells population. Together, our study reveals the critical roles of RNA m6A modification in the transition between 2C-like and pluripotent states, facilitating the study of totipotency and cell fate decision in the future.

Hsa_circ_0007590/PTBP1 complex reprograms glucose metabolism by reducing the stability of m6A-modified PTEN mRNA in pancreatic ductal adenocarcinoma.

The role of circular RNAs (circRNAs) in glucose metabolism in pancreatic duct adenocarcinoma (PDAC) remains elusive. Through RNA sequencing of cells cultured under conditions of glucose deprivation, we identified hsa_circ_0007590. Sanger sequencing and RNase R and Act D treatments were performed to confirm the circular RNA features of hsa_circ_0007590. RNA in situ hybridization (RNA-ISH) and quantitative reverse transcription PCR (qRT-PCR) were used to estimate hsa_circ_0007590 expression in PDAC clinical specimens and cell lines. hsa_circ_0007590 expression was higher in PDAC patients and closely related to the clinicopathological characteristics of the disease. Cytoplasm‒nuclear fractionation and FISH assays demonstrated that hsa_circ_0007590 was located in the nucleus. Gain-of-function and loss-of-function assays were performed to assess the biological behaviors of PDAC cells. Seahorse XF assays were performed to validate the Warburg effect. hsa_circ_0007590 facilitated the proliferation, migration, and invasion of PDAC cells and promoted the Warburg effect. Mass spectrometry, RNA pulldown, RNA immunoprecipitation (RIP), RNA m6A quantification, m6A dot blot, MeRIP, and Western blotting were conducted to investigate the detailed mechanism through which hsa_circ_0007590 produces these effects. Mechanistically, hsa_circ_0007590 targeted PTBP1 and increased the expression of the m6A reader protein YTHDF2, leading to PTEN mRNA degradation and PI3K/AKT/mTOR pathway activation. Overall, hsa_circ_0007590, which targets PTBP1, reprograms glucose metabolism by attenuating the stability of m6A-modified PTEN mRNA and holds potential promise as a therapeutic target for PDAC.

The m6A reader SlYTH2 negatively regulates tomato fruit aroma by impeding the translation process

N6-methyladenosine (m6A) is a fundamentally important RNA modification for gene regulation, whose function is achieved through m6A readers. However, whether and how m6A readers play regulatory roles during fruit ripening and quality formation remains unclear. Here, we characterized SlYTH2 as a tomato m6A reader protein and profiled the binding sites of SlYTH2 at the transcriptome-wide level. SlYTH2 undergoes liquid-liquid phase separation and promotes RNA-protein condensate formation. The target mRNAs of SlYTH2, namely m6A-modified SlHPL and SlCCD1B associated with volatile synthesis, are enriched in SlYTH2-induced condensates. Through polysome profiling assays and proteomic analysis, we demonstrate that knockout of SlYTH2 expedites the translation process of SlHPL and SlCCD1B, resulting in augmented production of aroma-associated volatiles. This aroma enrichment significantly increased consumer preferences for CRISPR-edited fruit over wild type. These findings shed light on the underlying mechanisms of m6A in plant RNA metabolism and provided a promising strategy to generate fruits that are more attractive to consumers.

DUSP5 regulated by YTHDF1-mediated m6A modification promotes epithelial-mesenchymal transition and EGFR-TKI resistance via the TGF-β/Smad signaling pathway in lung adenocarcinoma

BackgroundLung adenocarcinoma (LUAD) patients have a dismal survival rate because of cancer metastasis and drug resistance. The study aims to identify the genes that concurrently modulate EMT, metastasis and EGFR-TKI resistance, and to investigate the underlying regulatory mechanisms.MethodsCox regression and Kaplan–Meier analyses were applied to identify prognostic oncogenes in LUAD. Gene set enrichment analysis (GSEA) was used to indicate the biological functions of the gene. Wound-healing and Transwell assays were used to detect migratory and invasive ability. EGFR-TKI sensitivity was evaluated by assessing the proliferation, clonogenic survival and metastatic capability of cancer cells with treatment with gefitinib. Methylated RNA immunoprecipitation (MeRIP) and RNA immunoprecipitation (RIP) analyses established the level of m6A modification present on the target gene and the protein’s capability to interact with RNA, respectively. Single-sample gene set enrichment (ssGSEA) algorithm used to investigate levels of immune cell infiltration.ResultsOur study identified dual-specificity phosphatase 5 (DUSP5) as a novel and powerful predictor of adverse outcomes for LUAD by using public datasets. Functional enrichment analysis found that DUSP5 was positively enriched in EMT and transforming growth factor-beta (TGF-β) signaling pathway, a prevailing pathway involved in the induction of EMT. As expected, DUSP5 knockdown suppressed EMT via inhibiting the canonical TGF-β/Smad signaling pathway in in vitro experiments. Consistently, knockdown of DUSP5 was first found to inhibit migratory ability and invasiveness of LUAD cells in in vitro and prevent lung metastasis in in vivo. DUSP5 knockdown re-sensitized gefitinib-resistant LUAD cells to gefitinib, accompanying reversion of EMT progress. In LUAD tissue samples, we found 14 cytosine-phosphate-guanine (CpG) sites of DUSP5 that were negatively associated with DUSP5 gene expression. Importantly, 5′Azacytidine (AZA), an FDA-approved DNA methyltransferase inhibitor, restored DUSP5 expression. Moreover, RIP experiments confirmed that YTH N6-methyladenosine RNA binding protein 1 (YTHDF1), a m6A reader protein, could bind DUSP5 mRNA. YTHDF1 promoted DUSP5 expression and the malignant phenotype of LUAD cells. In addition, the DUSP5-derived genomic model revealed the two clusters with distinguishable immune features and tumor mutational burden (TMB).ConclusionsBriefly, our study discovered DUSP5 which was regulated by epigenetic modification, might be a potential therapeutic target, especially in LUAD patients with acquired EGFR-TKI resistance.Graphical

Histone acetyltransferase Kat2a regulates ferroptosis via enhancing Tfrc and Hmox1 expression in diabetic cardiomyopathy

Diabetic cardiomyopathy (DCM) is a prevalent myocardial microvascular complication of the myocardium with a complex pathogenesis. Investigating the pathogenesis of DCM can significantly contribute to enhancing its prevention and treatment strategies. Our study revealed an upregulation of lysine acetyltransferase 2 A (Kat2a) expression in DCM, accompanied by a decrease in N6-methyladenosine (m6A) modified Kat2a mRNA levels. Our study revealed an upregulation of lysine acetyltransferase 2 A (Kat2a) expression in DCM, accompanied by a decrease in N6-methyladenosine (m6A) modified Kat2a mRNA levels. Functionally, inhibition of Kat2a effectively ameliorated high glucose-induced cardiomyocyte injury both in vitro and in vivo by suppressing ferroptosis. Mechanistically, Demethylase alkB homolog 5 (Alkbh5) was found to reduce m6A methylation levels on Kat2a mRNA, leading to its upregulation. YTH domain family 2 (Ythdf2) played a crucial role as an m6A reader protein mediating the degradation of Kat2a mRNA. Furthermore, Kat2a promoted ferroptosis by increasing Tfrc and Hmox1 expression via enhancing the enrichment of H3K27ac and H3K9ac on their promoter regions. In conclusion, our findings unveil a novel role for the Kat2a-ferroptosis axis in DCM pathogenesis, providing valuable insights for potential clinical interventions.

The m6A reader ECT8 is an abiotic stress sensor that accelerates mRNA decay in Arabidopsis.

N 6-methyladenosine (m6A) is the most abundant mRNA modification and plays diverse roles in eukaryotes, including plants. It regulates various processes, including plant growth, development, and responses to external or internal stress responses. However, the mechanisms underlying how m6A is related to environmental stresses in both mammals and plants remain elusive. Here, we identified EVOLUTIONARILY CONSERVED C-TERMINAL REGION 8 (ECT8) as an m6A reader protein and showed that its m6A-binding capability is required for salt stress responses in Arabidopsis (Arabidopsis thaliana). ECT8 accelerates the degradation of its target transcripts through direct interaction with the decapping protein DECAPPING 5 within processing bodies. We observed a significant increase in the ECT8 expression level under various environmental stresses. Using salt stress as a representative stressor, we found that the transcript and protein levels of ECT8 rise in response to salt stress. The increased abundance of ECT8 protein results in the enhanced binding capability to m6A-modified mRNAs, thereby accelerating their degradation, especially those of negative regulators of salt stress responses. Our results demonstrated that ECT8 acts as an abiotic stress sensor, facilitating mRNA decay, which is vital for maintaining transcriptome homeostasis and enhancing stress tolerance in plants. Our findings not only advance the understanding of epitranscriptomic gene regulation but also offer potential applications for breeding more resilient crops in the face of rapidly changing environmental conditions.

N6-Methyladenosine Promotes TNF mRNA Degradation In CD4+ T Lymphocytes.

N6-methyladenosine (m6A) is a RNA modification that can regulate post-transcriptional processes including RNA stability, translation, splicing and nuclear export. In CD4+ lymphocytes, m6A modifications have been demonstrated to play a role in early differentiation processes. The role of m6A in CD4+ T cell activation and effector function remains incompletely understood. To assess the role of m6A in CD4+ T lymphocyte activation and function, we assessed the transcriptome-wide m6A landscape of human primary CD4+ T cells by methylated RNA immunoprecipitation (meRIP) sequencing. Stimulation of the T cells impacted the m6A pattern of hundreds of transcripts including tumor necrosis factor (TNF). m6A methylation was increased on TNF mRNA after activation, predominantly in the 3' untranslated region (UTR) of the transcript. Manipulation of m6A levels in primary human T cells, the directly affected the expression of TNF. Furthermore, we identified that the m6A reader protein YT521-B homology domain family-2 (YTHDF2) binds m6A-methylated TNF mRNA, and promotes its degradation. Taken together, this study demonstrates that TNF expression in CD4+ T lymphocytes is regulated via m6A and YTHDF2, thereby providing novel insight into the regulation of T cell effector functions.

Inhibition of METTL3 ameliorates doxorubicin-induced cardiotoxicity through suppression of TFRC-mediated ferroptosis

BackgroundDoxorubicin (DOX) is a chemotherapeutic drug, while its clinical use is greatly limited by the life-threatening cardiotoxicity. N6-methyladenosine (m6A) RNA modification participates in varieties of cellular processes. Nonetheless, it remains elusive whether m6A modification and its methyltransferase METTL3 are involved in the progression of DOX-induced cardiotoxicity (DIC). MethodsMice were administrated with DOX (accumulative dosage of 20 mg/kg) repeatedly to establish a chronic DIC model. Cardiomyocyte-specific conditional METTL3 knockout mice were employed to evaluate the effects of altered m6A RNA modification on DIC. The effects of METTL3 on cardiomyocyte ferroptosis were also examined in response to DOX stimulation. ResultsDOX led to increased levels in m6A modification and METTL3 expression in cardiomyocytes in a c-Jun-dependent manner. METTL3-knockout mice exhibited improved cardiac function, remodeling and injury following DOX insult. Besides, inhibition of METTL3 alleviated DOX-induced iron accumulation and ferroptosis in cardiomyocytes, whereas METTL3 overexpression exerted the opposite effects. Mechanistically, METTL3 promoted m6A modification of TFRC mRNA, a critical gene governing iron uptake, and enhanced its stability through recognition of the m6A reader protein, IGF2BP2. Moreover, pharmacological administration of a highly selective METTL3 inhibitor STM2457 effectively ameliorated DIC in mice. ConclusionMETTL3 plays a cardinal role in the etiology of DIC by regulating cardiac iron metabolism and ferroptosis through TFRC m6A modification. Inhibition of METTL3 might be a potential therapeutic avenue for DIC.

Lopinavir enhances anoikis by remodeling autophagy in a circRNA-dependent manner

ABSTRACT Macroautophagy/autophagy-mediated anoikis resistance is crucial for tumor metastasis. As a key autophagy-related protein, ATG4B has been demonstrated to be a prospective anti-tumor target. However, the existing ATG4B inhibitors are still far from clinical application, especially for tumor metastasis. In this study, we identified a novel circRNA, circSPECC1, that interacted with ATG4B. CircSPECC1 facilitated liquid-liquid phase separation of ATG4B, which boosted the ubiquitination and degradation of ATG4B in gastric cancer (GC) cells. Thus, pharmacological addition of circSPECC1 may serve as an innovative approach to suppress autophagy by targeting ATG4B. Specifically, the circSPECC1 underwent significant m6A modification in GC cells and was subsequently recognized and suppressed by the m6A reader protein ELAVL1/HuR. The activation of the ELAVL1-circSPECC1-ATG4B pathway was demonstrated to mediate anoikis resistance in GC cells. Moreover, we also verified that the above pathway was closely related to metastasis in tissues from GC patients. Furthermore, we determined that the FDA-approved compound lopinavir efficiently enhanced anoikis and prevented metastasis by eliminating repression of ELAVL1 on circSPECC1. In summary, this study provides novel insights into ATG4B-mediated autophagy and introduces a viable clinical inhibitor of autophagy, which may be beneficial for the treatment of GC with metastasis.

Depletion of the N6-Methyladenosine (m6A) reader protein IGF2BP3 induces ferroptosis in glioma by modulating the expression of GPX4

Emerging evidence highlights the multifaceted contributions of m6A modifications to glioma. IGF2BP3, a m6A modification reader protein, plays a crucial role in post-transcriptional gene regulation. Though several studies have identified IGF2BP3 as a poor prognostic marker in glioma, the underlying mechanism remains unclear. In this study, we demonstrated that IGF2BP3 knockdown is detrimental to cell growth and survival in glioma cells. Notably, we discovered that IGF2BP3 regulated ferroptosis by modulating the protein expression level of GPX4 through direct binding to a specific motif on GPX4 mRNA. Strikingly, the m6A modification at this motif was found to be critical for GPX4 mRNA stability and translation. Furthermore, IGF2BP3 knockdown glioma cells were incapable of forming tumors in a mouse xenograft model and were more susceptible to phagocytosis by microglia. Our findings shed light on an unrecognized regulatory function of IGF2BP3 in ferroptosis. The identification of a critical m6A site within the GPX4 transcript elucidates the significance of post-transcriptional control in ferroptosis.