m6A RNA Modification Research Articles

METTL14 Promotes Lipopolysaccharide-Induced Myocardial Damage via m6A-Dependent Stabilization of TRPM7 mRNA.

Sepsis-induced myocardial injury (SIMI) is a vital pathological component of severe sepsis and septic shock. As a prevalent internal mRNA modification in eukaryotic cells, N6-methyladenosine (m6A) modification is implicated in sepsis and immune disorders. Methyltransferase-like 14 (METTL14), a core subunit of the methyltransferase complex that catalyzes messenger RNA m6A modification, is involved in the regulation of human cardiomyocyte cell line (AC16) injury. This study aimed to explore the role and mechanism of METTL14 in lipopolysaccharide (LPS) -induced myocardial injury.Cell viability and apoptosis were analyzed via 3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT), flow cytometry, and TdT-mediated dUTP nick-end labeling (TUNEL) assay. The Tumor necrosis factor alpha (TNF-α) and Interleukin-1beta (IL-1β) levels were analyzed via Enzyme linked immunosorbent assay (ELISA). Caspase-3 activity, reactive oxygen species activity, malondialdehyde level, and glutathione level were assessed using special assay kits. The levels of transient receptor potential melastatin 7 (TRPM7) and METTL14 mRNA were determined via Real-time quantitative polymerase chain reaction (RT-qPCR). Meanwhile, the protein levels of TRPM7, METTL14, phospho-p65 (p-p65), total p65 (p65), p-IκBα, and total IκBα (IκBα) were examined via western blot assay.LPS treatment repressed AC16 cell viability and induced cell apoptosis, inflammatory response, oxidative stress, and ferroptosis in vitro. METTL14 and TRPM7 were upregulated in LPS-treated AC16 cells. At the molecular level, METTL14 could increase the stability of TRPM7 mRNA via m6A methylation. Moreover, METTL14 deficiency could abolish LPS-triggered AC16 cell injury and ferroptosis via TRPM7 regulation.METTL14 knockdown reversed LPS-caused myocardial cell damage mainly by regulating the stability of TRPM7 mRNA, providing a novel therapeutic target for septic cardiomyopathy treatment.

Molecular regulators of chemotaxis in human hematopoietic stem cells.

Hematopoietic stem cells (HSCs), essential for lifelong blood cell regeneration, are clinically utilized to treat various hematological disorders. These cells originate in the aorta-gonad-mesonephros region, expand in the fetal liver, and mature in the bone marrow. Chemotaxis, involving gradient sensing, polarization, and migration, directs HSCs and is crucial for their homing and mobilization. The molecular regulation of HSC chemotaxis involves chemokines, chemokine receptors, signaling pathways, and cytoskeletal proteins. Recent advances in understanding these regulatory mechanisms have deepened insights into HSC development and hematopoiesis, offering new avenues for therapeutic innovations. Strategies including glucocorticoid receptor activation, modulation of histone acetylation, stimulation of nitric oxide signaling, and interference with m6A RNA modification have shown potential in enhancing CXCR4 expression, thereby improving the chemotactic response and homing capabilities of human HSCs. This review synthesizes current knowledge on the molecular regulation of human HSC chemotaxis and its implications for health and disease.

Visualization and Quantification of Single-Base m6A Methylation.

N 6-methyladenosine (m6A) has emerged as the most prevalent form of RNA modification found across various RNA classes. The detection and quantification of m6A RNA modifications under various physiological conditions are crucial for elucidating disease mechanisms and identifying potential therapeutic targets. However, visualizing intracellular m6A modifications at single-nucleotide resolution remains a significant challenge. Existing methods based on high-throughput sequencing or in vitro assays are not suitable for in situ m6A RNA imaging. In this work, we introduce the TadA8.20-assisted N6-methyladenosine RNA imaging at single-nucleotide resolution (TARS) method for precise visualization and quantification of both A and m6A forms at specific RNA sites within single cells. Validation studies using TARS on MALAT1 lncRNA in HeLa cells and CCND1 mRNA in breast cancer cell lines demonstrated its high specificity and efficiency in mapping and quantifying m6A modifications at single-nucleotide resolution. TARS represents a novel tool that advances m6A RNA modification research by offering accurate and detailed insights into m6A modifications at the single-nucleotide level.

N6-methyladenosine RNA modification promotes Severe Fever with Thrombocytopenia Syndrome Virus infection.

Severe Fever with Thrombocytopenia Syndrome Virus (SFTSV), a novel bunyavirus primarily transmitted by Haemaphysalis longicornis, induces severe disease with a high mortality rate. N6-methyladenosine (m6A) is a prevalent internal chemical modification in eukaryotic mRNA that has been reported to regulate viral infection. However, the role of m6A modification during SFTSV infection remains elusive. We here reported that SFTSV RNAs bear m6A modification during infection. Manipulating the expressions or activities of host m6A regulators significantly impacted SFTSV infection. Mechanistically, SFTSV recruited m6A regulators through the nucleoprotein to modulate the m6A modification of viral RNA, eventually resulting in enhanced infection by promoting viral mRNA translation efficiency and/or genome RNA stability. m6A mutations in the S genome diminished virus particle production, while m6A mutations in the G transcript impaired the replication of recombinant vesicular stomatitis virus (rVSV) expressing G protein in vitro and in vivo. Interestingly, m6A modification was evolutionarily conserved and facilitated SFTSV infection in primary tick cells. These findings may open an avenue for the development of m6A-targeted anti-SFTSV vaccines, drugs, and innovative strategies for the prevention and control of tick-borne disease.

YTHDF3 rs7464 A > G polymorphism increases Chinese neuroblastoma risk: A multiple-center case-control study.

Neuroblastoma (NB), a rare childhood cancer originating in nerve tissue. YTHDF3, a member of the YTH domain protein family, is involved in RNA m6A modification and cancer progression. Polymorphisms in YTHDF3 may influence its expression and biological function. Herein, this study estimated the association between YTHDF3 polymorphisms (rs2241753, rs2241754, and rs7464) and NB susceptibility in a multicenter study comprising 898 cases and 1734 controls. We genotyped YTHDF3 candidate polymorphisms by the TaqMan assay. Logistic regression analysis was applied to indicate the possible relationships between these polymorphisms and NB susceptibility, using odds ratios (ORs) and 95% confidence intervals (CIs). Logistic regression analysis revealed that the rs2241753 GA versus GG decreased NB risk (Adjusted OR = 0.84, 95% CI = 0.71-0.997, p = .047), while the rs7464 GG versus AA enhanced NB risk (Adjusted OR = 1.62, 95% CI = 1.20-2.18, p = .002). Additionally, rs7464 GG versus AA/AG showed a higher risk (Adjusted OR = 1.66, 95% CI = 1.24-2.22, p = .0006). Combination analysis showed that having 1-3 risk genotypes versus 0 was associated with increased NB risk (Adjusted OR = 1.28, 95%CI = 1.09-1.51, p = .003). The significance of rs7464 and the risk genotypes combination persisted across multiple subgroups, whereas rs2241754 was significant only in mediastinal NB. False-positive report probability (FPRP) confirmed the reliability of results. Notably, the interaction between rs7464 and rs2241754 may increase NB risk dramatically. Our study demonstrated that YTHDF3 rs7464 A > G significantly affected NB susceptibility, warranting validation in larger sample sizes.

Enhancing Radiofrequency Ablation for Hepatocellular Carcinoma: Nano-Epidrug Effects on Immune Modulation and Antigenicity Restoration.

Radiofrequency ablation (RFA), a critical therapy for hepatocellular carcinoma (HCC), carries a significant risk of recurrence and metastasis, particularly owing to mechanisms involving immune evasion and antigen downregulation via epigenetic modifications. This study introduces a "nano-epidrug" named MFMP. MFMP, which is composed of hollow mesoporous manganese dioxide (MnO2) nanoparticles, FIDAS-5 as an MAT2A inhibitor, macrophage membrane, and anti-PD-L1 (aPD-L1), targets HCC cells. By selectively binding to these cells, MFMP initially reverses immune suppression via PD-L1 inhibition. After endocytosis, MFMP disassembles in the tumor microenvironment, releasing FIDAS-5 and Mn2+. FIDAS-5 prevents cGAS methylation, whereas Mn2+ aids STING pathway restoration. In addition, FIDAS-5 reduces m6A RNA modification, suppressing EGFR expression. These changes enhance HCC antigenicity to promote cytotoxic T cell recognition and cytotoxic killing. Furthermore, MFMP mediates immunogenic cell death in HCC by synergizing with RFA through cGAS DNA demethylation, EGFR mRNA demethylation, and TBK1 protein phosphorylation, thereby inhibiting recurrence and metastasis and enhancing immune memory. Thus, MFMP is a potential adjunctive therapy requiring clinical validation.

Current insights on m6A RNA modification in acute leukemia: therapeutic targets and future prospects.

RNA modification is the critical mechanism for regulating post-transcriptional processes. There are more than 150 RNA modifications reported so far, among which N6-Methyladenosine is the most prevalent one. M6A RNA modification complex consists of 'writers', 'readers' and 'erasers' which together in a group catalyze, recognize and regulate the methylation process of RNA and thereby regulate the stability and translation of mRNA. The discovery of erasers also known as demethylases, revolutionized the research on RNA modifications as it revealed that this modification is reversible. Since then, various studies have focused on discovering the role of m6A modification in various diseases especially cancers. Aberrant expression of these 'readers', 'writers', and 'erasers' is found to be altered in various cancers resulting in disturbance of cellular homeostasis. Acute leukemias are the most common cancer found in pediatric patients and account for 20% of adult cases. Dysregulation of the RNA modifying complex have been reported in development and progression of hematopoietic malignancies. Further, targeting m6A modification is the new approach for cancer immunotherapy and is being explored extensively. This review provides detailed information about current information on the role of m6A RNA modification in acute leukemia and their therapeutic potential.

DDDR-08. PIM1 INHIBITION: A NOVEL THERAPEUTIC APPROACH FOR IDH-MUTANT GLIOMAS

Abstract BACKGROUND PIM1, a constitutively active serine/threonine kinase has emerged as a relevant target in cancer treatment. Previously, we discovered that IDH-mutant glioma exhibited high kinase activity of PIM1, suggesting an increased sensitivity to PIM1 inhibition. Here we sought to investigate PIM1 inhibition as a therapeutic approach for IDH-mutant gliomas. METHODS Patient-derived IDH-mutant and wildtype glioma cell lines were used to evaluate the impact of PIM1inhibition on cell viability, proliferation, and colony formation. mRNA and protein expression of PIM1 were determined by qRT-PCR, Western-blot, and immunohistochemistry. m6A dot-blot assay was conducted to detect m6A RNA levels in glioma cells. m6A modification status and its location in PIM1 transcript were evaluated by m6A RNA immunoprecipitation array. RNA-binding protein immunoprecipitation was performed using anti-YTHDF1 antibody to identify PIM1 RNA-YTHDF1 protein interactions. RESULTS An upregulation of PIM1 protein is demonstrated in IDH-mutant patient tumor tissues and cells. A pan-PIM inhibitor, AZD1208 significantly suppressed cell viability, cell proliferation, and colony formation in IDH-mutant cells but had a lesser effect in IDH-wildtype cells. Interestingly, a lower mRNA expression of PIM1 is observed, while the protein level is significantly elevated in IDH-mutant cells compared to wildtype cells, suggesting post-transcriptional and translational regulation of PIM1 associated with IDH mutation. Mechanistically, 2-hydroxyglutarate, unique to IDH-mutant gliomas, competitively inhibits α-KG-dependent RNA demethylase FTO. We demonstrated suppressed FTO activity and an increased m6A RNA level in IDH-mutant glioma cells. An enrichment of m6A modification at motif “GG (m6A) CU” located in the 3’UTR is found in the PIM1 transcript. Furthermore, we demonstrated that the m6A modification site is recognized by the m6A reader YTHDF1 to promote protein translation, leading to an elevated PIM1 protein expression. CONCLUSION m6A RNA modification contributes to an enhanced PIM1 activity and thereby a selective sensitivity to PIM1 inhibition in IDH-mutant gliomas.

YTHDC1 is a therapeutic target for B-cell acute lymphoblastic leukemia by attenuating DNA damage response through the KMT2C-H3K4me1/me3 epigenetic axis.

B-cell acute lymphoblastic leukemia (B-ALL) is an aggressive malignancy characterized by the aberrant accumulation of immature and dysfunctional B cells in bone marrow (BM). Although chemotherapy and other therapies have been widely applied, some patients such as relapsed ordrug-refractory (R/R) B-ALL patients exhibit limited response. YT521-B homologous domain-containing protein 1 (YTHDC1) is a nuclear reader of N6-methyladenosine (m6A) RNA modification, which has been implicated in different malignancies including leukemia. In the current study, we show that YTHDC1 is highly expressed in B-ALL cells. YTHDC1 knockdown attenuated B-ALL cell proliferation and cell cycle progression in vitro, and prolonged survival of mice in the human B-ALL xenograft model in vivo attributable to compromised leukemogenesis. Mechanistically, YTHDC1 knockdown significantly increased the accumulation of endogenous and chemotherapeutic agents-induced DNA damage in B-ALL cells. Furthermore, we identified that YTHDC1 binds to and stabilizes m6A-modified KMT2C mRNA. KMT2C is a key enzyme catalyzing histone H3K4 methylation required for the expression of DNA damage response (DDR)-related genes, implying that YTHDC1 inhibitors might improve chemotherapy by attenuating DDR via reducing KMT2C. Indeed, with molecular docking and biochemical experiments, we identified EPZ-5676 as a YTHDC1 inhibitor, and combination of EPZ-5676 with Cytarabine (Ara-c) significantly improved the efficacy of chemotherapy in B-ALL mouse models using YTHDC1high primary and lined B-ALL cells. Collectively, YTHDC1 is required for DDR in B-ALL cells by upregulating DDR-related gene expression via stabilizing m6A-modified KMT2C mRNA, thereby leading to increased histone H3K4 methylation, and targeted inhibition of YTHDC1 is a potentially new therapeutic strategy against B-ALL, especially YTHDC1high B-ALL.

LAMP-MS for Locus-Specific Visual Quantification of DNA 5mC and RNA m6A Using Ultra-Low Input.

Enhancing the effectiveness of utilizing circulating cell-free DNA (cfDNA) for disease screening remains a challenge, necessitating improved sensitivity, specificity, cost-efficiency, and patient adherence. We present here LAMP-MS, an innovative technology that integrates linear amplification with single-base quantitative nucleic acid mass spectrometry on silicon chips. This approach overcomes several limitations in utilizing cfDNA 5-methylcytosine (5mC) status for colorectal cancer (CRC) screening. LAMP-MS enables unbiased amplification of as little as 1 ng of cfDNA, site-specifically quantify methylation levels of tens to hundreds of 5mC sites, thereby facilitating cost-effective, high-resolution quantitative detection of cfDNA methylation markers. We have validated the accuracy of DNA methylation determination using DNA probes and cfDNA from patient plasma samples, confirmed by mass spectrometric peak areas. Additionally, we have further shown this Mass Array technology could be expanded to also quantify RNA m6A modification sites. Combining the ability to work with ultra-low input materials and a visually interpretable output, LAMP-MS stands out as a promising method for real-world applications in clinics and laboratories for nucleic acid methylation detection and quantification.

LAMP‐MS for Locus‐Specific Visual Quantification of DNA 5mC and RNA m6A Using Ultra‐Low Input

Enhancing the effectiveness of utilizing circulating cell‐free DNA (cfDNA) for disease screening remains a challenge, necessitating improved sensitivity, specificity, cost‐efficiency, and patient adherence. We present here LAMP‐MS, an innovative technology that integrates linear amplification with single‐base quantitative nucleic acid mass spectrometry on silicon chips. This approach overcomes several limitations in utilizing cfDNA 5‐methylcytosine (5mC) status for colorectal cancer (CRC) screening. LAMP‐MS enables unbiased amplification of as little as 1 ng of cfDNA, site‐specifically quantify methylation levels of tens to hundreds of 5mC sites, thereby facilitating cost‐effective, high‐resolution quantitative detection of cfDNA methylation markers. We have validated the accuracy of DNA methylation determination using DNA probes and cfDNA from patient plasma samples, confirmed by mass spectrometric peak areas. Additionally, we have further shown this Mass Array technology could be expanded to also quantify RNA m6A modification sites. Combining the ability to work with ultra‐low input materials and a visually interpretable output, LAMP‐MS stands out as a promising method for real‐world applications in clinics and laboratories for nucleic acid methylation detection and quantification.

MST-m6A: A Novel Multi-Scale Transformer-based Framework for Accurate Prediction of m6A Modification Sites Across Diverse Cellular Contexts

N6-methyladenosine (m6A) modification, a prevalent epigenetic mark in eukaryotic cells, is crucial in regulating gene expression and RNA metabolism. Accurately identifying m6A modification sites is essential for understanding their functions within biological processes and the intricate mechanisms that regulate them. Recent advances in high-throughput sequencing technologies have enabled the generation of extensive datasets characterizing m6A modification sites at single-nucleotide resolution, leading to the development of computational methods for identifying m6A RNA modification sites. However, most current methods focus on specific cell lines, limiting their generalizability and practical application across diverse biological contexts. To address the limitation, we propose MST-m6A, a novel approach for identifying m6A modification sites with higher accuracy across various cell lines and tissues. MST-m6A utilizes a multi-scale transformer-based architecture, employing dual k-mer tokenization to capture rich feature representations and global contextual information from RNA sequences at multiple levels of granularity. These representations are then effectively combined using a channel fusion mechanism and further processed by a convolutional neural network to enhance prediction accuracy. Rigorous validation demonstrates that MST-m6A significantly outperforms conventional machine learning models, deep learning models, and state-of-the-art predictors. We anticipate that the high precision and cross-cell-type adaptability of MST-m6A will provide valuable insights into m6A biology and facilitate advancements in related fields. The proposed approach is available at https://github.com/cbbl-skku-org/MST-m6A/ for prediction and reproducibility purposes.

Investigating N6-methyladenosin RNA modification as a mediator of microRNA mechanical regulation in the heart

Abstract Background The heart is exposed to mechanical forces and continuously adapts to its environment in a process known as cardiac remodeling. Excessive mechanical load is a primary driver of pathological cardiac remodeling, leading to heart failure (HF), a prominent cause of morbidity and mortality worldwide. A set of microRNAs (miRs), termed mechano-miRs, has been identified to respond to mechanical forces and contribute to heart pathophysiology and HF. N6-methyladenosine (m6A) RNA modification is emerging as a novel regulatory layer impacting gene expression and occurring both in coding and non-coding RNAs. In this context, the N6-adenosine-methyltransferase METTL-3 regulates the processing of miR primary transcripts (pri-miRs) to functional miRs, promoting angiogenesis and cardiac repair after myocardial infarction. On the other side, dysregulation of m6A due to a defect in FTO, a m6A demethylase, has been implicated in HF. However, the role of m6A RNA modifications in heart mechanical responses and mechano-miR regulation remains unexplored. Purpose This project aims to: 1) investigate the impact of mechanical overload on the expression of m6A regulatory machinery components; 2) unveil the role of m6A-mechano-miRs axis in mechanically induced responses driving pathological cardiac remodeling leading to heart failure. Methods and Results Using living myocardial slices (LMS) technology, we modulate mechanical load in a multicellular environment while maintaining physiological behavior. Human LMS, obtained from the left ventricle of human donor non-failing hearts (NHS Blood and Transplant INOAR program, IRAS project ID: 189069) and rat LMS are subjected to two degrees of mechanical preload: physiological sarcomere length (SL) (SL = 2.2 μm) and overload (SL = 2.4 μm). This enables us to mimic the effects of mechanical stress on the heart during HF (volume overload model). LMS are maintained for 48 hours under electrical stimulation in circulating oxygenated media at 37°C. Mechanically overloaded LMS exhibit decreased contractility, accompanied by an increase in global m6A levels compared to physiological controls. Consistent with this finding, the analysis of the expression of m6A machinery reveals a downregulation of m6A demethylases (FTO and ALKBH5) in overloaded LMS. We identified putative cardiac mechano-miRs by small-RNA sequencing in both human and rat LMS and next predicted potential m6A sites on their primary transcripts via SRAMP (sequence-based RNA adenosine methylation site predictor). Conclusions Mechanical overload affects global m6A levels and the expression of m6A demethylases in the heart. Ongoing analysis of the m6A profile on the identified mechano-miR primary transcripts and mRNA targets holds promise for unveiling novel mechanically regulated m6A events, providing insights for transformative therapeutic strategies.

ALKBH5 Protects Against Hepatic Ischemia-Reperfusion Injury by Regulating YTHDF1-Mediated YAP Expression.

Ischemia/reperfusion (I/R) injury with severe cell death is a major complication involved in liver transplantation and resection. The identification of key regulators improving hepatocyte activity may provide potential strategies to clinically resolve I/R-induced injury. N6-methyladenosine (m6A) RNA modification is essential for tissue homeostasis and pathogenesis. However, the potential involvement of m6A in the regulation of hepatocyte activity and liver injury has not been fully explored. In the present study, we found that hepatocyte AlkB homolog H5 (ALKBH5) levels were decreased both in vivo and in vitro I/R models. Hepatocyte-specific ALKBH5 overexpression effectively attenuated I/R-induced liver necrosis and improved cell proliferation in mice. Mechanistically, ALKBH5-mediated m6A demethylation improved the mRNA stability of YTH N6-methyladenosine RNA-binding protein 1 (YTHDF1), thereby increasing its expression, which consequently promoted the translation of Yes-associated protein (YAP). In conclusion, ALKBH5 is a regulator of hepatic I/R injury that improves hepatocyte repair and proliferation by maintaining YTHDF1 stability and YAP content. The ALKBH5-m6A-YTHDF1-YAP axis represents promising therapeutic targets for hepatic I/R injury to improve the prognosis of liver surgery.

Coxsackievirus B3-Induced m6A Modification of RNA Enhances Viral Replication via Suppression of YTHDF-Mediated Stress Granule Formation.

N6-methyladenosine (m6A) is the most prevalent internal RNA modification. Here, we demonstrate that coxsackievirus B3 (CVB3), a common causative agent of viral myocarditis, induces m6A modification primarily at the stop codon and 3' untranslated regions of its genome. As a positive-sense single-stranded RNA virus, CVB3 replicates exclusively in the cytoplasm through a cap-independent translation initiation mechanism. Our study shows that CVB3 modulates the expression and nucleo-cytoplasmic transport of the m6A machinery components-METTL3, ALKBH5 and YTHDFs-resulting in increased m6A modifications that enhance viral replication. Mechanistically, this enhancement is mediated through YTHDF-driven stress granule (SG) formation. We observed that YTHDF proteins co-localize with human antigen R (HuR), a protein facilitating cap-independent translation, in SGs during early infection. Later in infection, YTHDFs are cleaved, suppressing SG formation. Notably, for the first time, we identified that during early infection CVB3's RNA-dependent RNA polymerase (3D) and double-stranded RNA (dsRNA) are stored in SGs, co-localizing with HuR. This early-stage sequestration likely protects viral components for use in late-phase replication, when SGs are disrupted due to YTHDF cleavage. In summary, our findings reveal that CVB3-induced m6A modifications enhance viral replication by regulating YTHDF-mediated SG dynamics. This study provides a potential therapeutic strategy for CVB3-induced myocarditis.

Lactylation-Driven IGF2BP3-Mediated Serine Metabolism Reprogramming and RNA m6A-Modification Promotes Lenvatinib Resistance in HCC.

Acquired resistance remains a bottleneck for molecular-targeted therapy in advanced hepatocellular carcinoma (HCC). Metabolic adaptation and epigenetic remodeling are recognized as hallmarks of cancer that may contribute to acquired resistance. In various lenvatinib-resistant models, increased glycolysis leads to lactate accumulation and lysine lactylation of IGF2BP3. This lactylation is crucial for capturing PCK2 and NRF2 mRNAs, thereby enhancing their expression. This process reprograms serine metabolism and strengthens the antioxidant defense system. Additionally, altered serine metabolism increases the availability of methylated substrates, such as S-adenosylmethionine (SAM), for N6-methyladenosine (m6A) methylation of PCK2 and NRF2 mRNAs. The lactylated IGF2BP3-PCK2-SAM-m6A loop maintains elevated PCK2 and NRF2 levels, enhancing the antioxidant system and promoting lenvatinib resistance in HCC. Treatment with liposomes carrying siRNAs targeting IGF2BP3 or the glycolysis inhibitor 2-DG restored lenvatinib sensitivity in vivo. These findings highlight the connection between metabolic reprogramming and epigenetic regulation and suggest that targeting metabolic pathways may offer new strategies to overcome lenvatinib resistance in HCC.

Detecting m6A RNA modification from nanopore sequencing using a semisupervised learning framework.

Direct nanopore-based RNA sequencing can be used to detect posttranscriptional base modifications, such as N6-methyladenosine (m6A) methylation, based on the electric current signals produced by the distinct chemical structures of modified bases. A key challenge is the scarcity of adequate training data with known methylation modifications. We present Xron, a hybrid encoder-decoder framework that delivers a direct methylation-distinguishing basecaller by training on synthetic RNA data and immunoprecipitation (IP)-based experimental data in two steps. First, we generate data with more diverse modification combinations through in silico cross-linking. Second, we use this data set to train an end-to-end neural network basecaller followed by fine-tuning on IP-based experimental data with label smoothing. The trained neural network basecaller outperforms existing methylation detection methods on both read-level and site-level prediction scores. Xron is a standalone, end-to-end m6A-distinguishing basecaller capable of detecting methylated bases directly from raw sequencing signals, enabling de novo methylome assembly.

M6A modification of mutant huntingtin RNA promotes the biogenesis of pathogenic huntingtin transcripts

In Huntington’s disease (HD), aberrant processing of huntingtin (HTT) mRNA produces HTT1a transcripts that encode the pathogenic HTT exon 1 protein. The mechanisms behind HTT1a production are not fully understood. Considering the role of m6A in RNA processing and splicing, we investigated its involvement in HTT1a generation. Here, we show that m6A methylation is increased before the cryptic poly(A) sites (IpA1 and IpA2) within the huntingtin RNA in the striatum of Hdh+/Q111 mice and human HD samples. We further assessed m6A’s role in mutant Htt mRNA processing by pharmacological inhibition and knockdown of METTL3, as well as targeted demethylation of Htt intron 1 using a dCas13-ALKBH5 system in HD mouse cells. Our data reveal that Htt1a transcript levels are regulated by both METTL3 and the methylation status of Htt intron 1. They also show that m6A methylation in intron 1 depends on expanded CAG repeats. Our findings highlight a potential role for m6A in aberrant splicing of Htt mRNA.

ALKBH5-Mediated m6A Modification of XBP1 Facilitates NSCLC Progression Through the IL-6-JAK-STAT3 Pathway.

The X-box-binding protein 1 (XBP1) is an important transcription factor during endoplasmic reticulum stress response, which was reported as an oncogene in non-small cell lung cancer (NSCLC) tumorigenesis and development. However, the regulatory mechanism of XBP1 expression in NSCLC progression was less reported. N6-methyladenosine (m6A) RNA modification is an emerging epigenetic regulatory mechanism for gene expression. This study aimed to investigate the regulatory role of the m6A modification in XBP1 expression in NSCLC. We identified XBP1 as a downstream target of ALKBH5-mediated m6A modification in A549 and PC9 cells. Knockdown of ALKBH5 increased the m6A modification and the stability of XBP1 mRNA, while overexpression of ALKBH5 had the opposite effect. Furthermore, IGF2BP3 was confirmed to be a reader of XBP1 m6A methylation and to enhance the stability of XBP1 mRNA. Additionally, IGF2BP3 knockdown significantly reversed the increase in XBP1 stability mediated by ALKBH5 depletion. In vivo and in vitro experiments demonstrated that ALKBH5/IGF2BP3 promotes the proliferation, migration, and invasion of NSCLC cells by upregulating XBP1 expression. In addition, we also showed that XBP1 promoted NSCLC cell proliferation, migration, and invasion by activating IL-6-JAK-STAT3 signaling. Our research suggested that ALKBH5-mediated m6A modification of XBP1 facilitates NSCLC progression through the IL-6-JAK-STAT3 pathway.

Epitranscriptomic m6A modifications during reactivation of HIV-1 latency in CD4+ T cells.

RNA m6A modification is important for regulating gene expression and innate immune responses to HIV-1 infection. However, the functional significance of m6A modification during HIV-1 latency reactivation is unknown. To address this important question, in this study, we used established cellular models of HIV-1 latency, m6A-specific sequencing at single-base resolution, and functional assays. We demonstrate that HIV-1 latency reversal leads to increased levels of cellular m6A modification, correlates with cellular m6A levels, and is dependent on the catalytic activity of the m6A methyltransferase enzyme. We also identified cellular genes that are differentially m6A-modified during HIV-1 reactivation, as well as the sites of m6A within HIV-1 RNA. Our novel findings point toward a significant role for m6A modification in HIV-1 latency reversal.