N6-methyladenosine RNA Modification Research Articles

N6-methyladenosine RNA modification (m6A) is of prognostic value in HPV-dependent vulvar squamous cell carcinoma

BackgroundVulvar squamous cell carcinoma (VSCC) is an uncommon gynecologic malignancy but with an increasing incidence in recent years. Etiologically, VSCC is classified into two subtypes: HPV-dependent and HPV-independent. Localized VSCC is treated surgically and/or with radiation therapy, but for advanced, metastatic or recurrent disease, therapeutic options are still limited.N6-methyladenosine (m6A) is the most prevalent post-transcriptional messenger RNA (mRNA) modification and involved in many physiological processes. The group of m6A proteins can be further divided into: ‚writers’ (METTL3, METTL4, METTL14, WTAP, KIAA1429), ‚erasers’ (FTO, ALKBH5), and ‚readers’ (HNRNPA2B1, HNRNPC, YTHDC1, YTHDF1-3). Dysregulated m6A modification is implicated in carcinogenesis, progression, metastatic spread, and drug resistance across various cancer entities. Up to date, however, only little is known regarding the role of m6A in VSCC.MethodsHere, we comprehensively investigated protein expression levels of a diverse set of m6A writers, readers and erasers by applying immunohistochemical staining in 126 patients with primary VSCC.ResultsIn the entire study cohort, dominated by HPV-independent tumors, m6A protein expression was not associated with clinical outcome. However, we identified enhanced protein expression levels of the ‚writers’ METTL3, METTL14 and the ‚reader’ YTHDC1 as poor prognostic markers in the 23 patients with HPV-dependent VSCC.ConclusionOur study suggests dysregulated m6A modification in HPV-associated VSCC.

Modification of m6A mediates tissue immune microenvironment in calcific aortic valve disease.

BackgroundSeveral human diseases are associated with aberrant expression of regulators involved in N6-methyladenosine (m6A) RNA modification. However, their role in aortic valve calcification (AVC) is largely unknown. The aim of this study was to determine the general expression pattern and potential function of m6A regulators in AVC by bioinformatics methods.MethodsWe obtained AVC datasets from the Gene Expression Omnibus (GEO). The identification of m6A-related differentially expressed genes (DEGs) and the Consensus Clustering method was performed to type AVC individuals based DEGs. Then, we quantified the effect of typing by principal component analysis (PCA). Next, we performed the weighted gene co-expression network analysis (WGCNA) and identified the main modules as well as functional analysis. Additionally, the key genes were screened by protein-protein interaction network (PPIN) analysis and identifying important genes of important modules. We again typed AVC individuals by the same method using key genes. Finally, we evaluated the link between key genes and immune infiltration.ResultsWe discovered that METTL14, ZC3H13, FTO, FMR1, HNRNPA2B1, HNRNPC, LRPPRC, YTHDC1, YTHDC2, and YTHDF1 expression levels decreased considerably in AVC tissues. Based on 10 genes, we typed 240 AVC samples as clusters A and B. We assessed the immune cell content in 240 samples using Cell-type Identification by Estimating Relative Subsets of RNA Transcripts (CIBERSORT) and found that B cell memory, CD8 T cells, T follicular helper cells, monocytes, M0 macrophages, resting dendritic cells (DCs), and interleukin-10 (IL-10) were concentrated in the cluster A group. Additionally, based on the important WGCNA modules, we identified 7 key genes. Next, 240 samples were retyped based on 7 key genes; we found that T cells CD8, T cells CD4 memory activated, T cells follicular helper, and macrophages M1 were significantly increased in gene cluster-1. Finally, we performed functional enrichment of gene cluster-typed samples, showing potential functional differences between different types.ConclusionsOur study provides a review of the m6A regulators’ expression pattern and functional importance in human AVC. The data from this study might serve as a significant resource for future mechanistic and therapeutic investigations into the role of critical m6A regulators in AVC.

A m6A methyltransferase-mediated immune signature determines prognosis, immune landscape and immunotherapy efficacy in patients with lung adenocarcinoma.

As the most abundant modification in mRNA, the N6-methyladenosine (m6A) RNA modification is involved in the occurrence and development of various tumors. However, the underlying functions of this alteration in the immune microenvironment of lung adenocarcinoma (LUAD) remain unknown. We identified m6A-mediated immune genes by performing a correlation analysis. Next, a m6A-mediated immune model was constructed using multiple machine learning algorithms, including univariate, least absolute shrinkage and selection operator, and multivariate Cox regression analyses. The potential of this model to predict the immune landscapes, drug sensitivities, and immunotherapy responses of different LUAD risk groups was studied. A m6A-mediated immune model containing 13 m6A-mediated immune genes was established and found to be an independent predictor of survival time. The prognosis of low-risk patients was significantly better than that of high-risk patients. These two risk groups displayed different immune environments, genomic backgrounds, chemotherapy responses and immunotherapy response tendencies. The low- and high-risk groups strongly corresponded to the immune-hot and immune-cold phenotypes, respectively. The low-risk group was more enriched in immune-related biological processes, and the high-risk group was more enriched in proliferation-related biological processes. Furthermore, low-risk patients responded better to immunotherapy based on the results obtained from the tumor immune dysfunction and exclusion (TIDE) algorithm and subclass mapping algorithm using five external independent immunotherapy cohorts. Our results suggest that the m6A modification participates in regulating the tumor microenvironment. The m6A-mediated immune model may be useful to predict the immunotherapy responses and outcomes of patients with LUAD.

Comprehensive analysis of molecular features, prognostic values, and immune landscape association of m6A-regulated immune-related lncRNAs in smoking-associated lung squamous cell carcinoma.

Lung squamous cell carcinoma (LUSC) is the second most common histopathological subtype of lung cancer, and smoking is the leading cause of this type of cancer. However, the critical factors that directly affect the survival rate and sensitivity to immunotherapy of smoking LUSC patients are still unknown. Previous studies have highlighted the role of N6-methyladenosine (m6A) RNA modification, the most common epigenetic modification in eukaryotic species, together with immune-related long non-coding RNAs (lncRNAs) in promoting the development and progression of tumors. Thus, elucidating m6A-modified immune lncRNAs in LUSC patients with smoking history is vital. In this study, we described the expression and mutation features of the 24 m6A-related regulators in the smoking-associated LUSC cohort from The Cancer Genome Atlas (TCGA) database. Then, two distinct subtypes based on the expression levels of the prognostic m6A-regulated immune lncRNAs were defined, and differentially expressed genes (DEGs) between the subtypes were identified. The distributions of clinical characteristics and the tumor microenvironment (TME) between clusters were analyzed. Finally, we established a lncRNA-associated risk model and exhaustively clarified the clinical features, prognosis, immune landscape, and drug sensitivity on the basis of this scoring system. Our findings give insight into potential mechanisms of LUSC tumorigenesis and development and provide new ideas in offering LUSC patients with individual and effective immunotherapies.

Rational Design and Optimization of m6A-RNA Demethylase FTO Inhibitors as Anticancer Agents.

Aberrant regulation of N6-methyladenosine (m6A) RNA modification has been implicated in the progression of multiple diseases, including cancer. Previously, we identified a small molecule inhibitor of the m6A demethylase fat mass- and obesity-associated protein (FTO), which removes both m6A and N6,2′-O-dimethyladenosine (m6Am) RNA modifications. In this work, we describe the rational design and optimization of a new class of FTO inhibitors derived from our previous lead FTO-04 with nanomolar potency and high selectivity against the homologous m6A RNA demethylase ALKBH5. The oxetanyl class of compounds comprise competitive inhibitors of FTO with potent antiproliferative effects in glioblastoma, acute myeloid leukemia, and gastric cancer models where lead FTO-43 demonstrated potency comparable to clinical chemotherapeutic 5-fluorouracil. Furthermore, FTO-43 increased m6A and m6Am levels in a manner comparable to FTO knockdown in gastric cancer cells and regulated Wnt/PI3K-Akt signaling pathways. The oxetanyl class contains significantly improved anticancer agents with a variety of applications beyond glioblastoma.

N6-Methyladenosine RNA Modification Landscape in the Occurrence and Recurrence of Nasopharyngeal Carcinoma.

BackgroundNasopharyngeal carcinoma (NPC) is a type of squamous head and neck cancer with variable geographic distributions, with the highest incidence in Southeast Asia. Its primary treatment is radiotherapy due to its high radio sensitivity. However, the N6-methyladenosine (m6A) landscape in NPC, including recurrent NPC, has not been reported.MethodsIn this study, m6A RNA immunoprecipitation (RIP) sequencing and microarray sequencing were performed on 12 tissue samples tissues of patients with primary and recurrent NPC. The expression profiles of m6A-related and non-coding RNAs were constructed and explored. Then, function experiments were performed to evaluate the effects of methyltransferase (METTL)3, METTL14 and WT1 associated protein (WTAP) on progressions of NPC. Finally, immunohistochemistry (IHC) and survival analysis were performed to confirm the correlation between METTL3, METTL14 and WTAP and NPC patients’ clinical outcomes.ResultsThis study mapped m6A RNA modification and RNA expression profiles in normal nasopharynx, primary NPC, and recurrent NPC tissues. This study also explored the role of m6A modificators in NPC development and recurrence. METTL3, METTL14, and WTAP could promote invasion and metastasis of NPC, and that these three proteins could induce radiotherapy resistance in NPC cells through DNA repair. Moreover, we found that METTL3, METTL14, and WTAP promoted an increase in exosomes within NPC microenvironment.ConclusionsThis study suggests that the alteration of m6A modification in primary and recurrent NPCs may play an important role in the development and progression of NPC.

Regulation of N6-Methyladenosine after Myocardial Infarction.

Development of heart failure (HF) after myocardial infarction (MI) is responsible for premature death. Complex cellular and molecular mechanisms are involved in this process. A number of studies have linked the epitranscriptomic RNA modification N6-methyladenosine (m6A) with HF, but it remains unknown how m6A affects the risk of developing HF after MI. We addressed the regulation of m6A and its demethylase fat mass and obesity-associated (FTO) after MI and their association with HF. Using liquid chromatography coupled to mass spectrometry, we observed an increase of m6A content in the infarcted area of rat hearts subjected to coronary ligation and a decrease in blood. FTO expression measured by quantitative PCR was downregulated in the infarcted hearts. In whole blood samples collected at the time of reperfusion in MI patients, m6A content was lower in patients who developed HF as attested by a 4-month ejection fraction (EF) of ≤40% as compared to patients who did not develop HF (EF > 50%). M6A content was higher in females. These results show that m6A measured in blood is associated with HF development after MI and motivate further investigation of the potential role of m6A as a novel epitranscriptomics biomarker and therapeutic target of HF.

The Emerging Role of RNA N6-Methyladenosine Modification in Pancreatic Cancer.

Pancreatic cancer (PC) is one of the most common malignant cancers, ranking the seventh highest causes of cancer-related deaths globally. Recently, RNA N6-methyladenosine (m6A) is emerging as one of the most abundant RNA modifications in eukaryote cells, involved in multiple RNA processes including RNA translocation, alternative splicing, maturation, stability, and degradation. As reported, m6A was dynamically and reversibly regulated by its “writers”, “erasers”, and “readers”, Increasing evidence has revealed the vital role of m6A modification in the development of multiple types of cancers including PC. Currently, aberrant m6A modification level has been found in both PC tissues and cell lines. Moreover, abnormal expressions of m6A regulators and m6A-modified genes have been reported to contribute to the malignant development of PC. Here in this review, we will focus on the function and molecular mechanism of m6A-modulated RNAs including coding RNAs as well as non-coding RNAs. Then the m6A regulators will be summarized to reveal their potential applications in the clinical diagnosis, prognosis, and therapeutics of PC.

RNA N6-methyladenosine modification in regulating cancer stem cells and tumor immune microenvironment and its implication for cancer therapy.

Therapy resistance is a well-known phenomenon in cancer treatment. It can be intrinsic or acquired, accountable for frequent tumor relapse and death worldwide. The interplay between cancer cells and their neighboring environment can activate complex signaling mechanisms influencing epigenetic changes and maintain cancer cell survival leading to the malignant phenotype. Cancer stem cells (CSCs) are tumor-initiating cells (TICs) and constitute the primary source of drug resistance and tumor recurrence. Studies have shown that cancer cells exhibit dysregulated RNA N6-methyladenosine (m6A) "writers," "erasers," and "readers" levels after acquiring drug resistance. The present review provides novel insight into the role of m6A modifiers involved in CSC generation, cancer cell proliferation, and therapy resistance. m6A RNA modifications in the cross-talk between CSC and the tumor immune microenvironment (TIME) have also been highlighted. Further, we have discussed the therapeutic potential of targeting m6A machinery for cancer diagnosis and the development of new therapies for cancer treatment.

Augmentation of the RNA m6A reader signature is associated with poor survival by enhancing cell proliferation and EMT across cancer types

N6-Methyladenosine (m6A) RNA modification plays a critical role in the posttranscriptional regulation of gene expression. Alterations in cellular m6A levels and m6A-related genes have been reported in many cancers, but whether they play oncogenic or tumor-suppressive roles is inconsistent across cancer types. We investigated common features of alterations in m6A modification and m6A-related genes during carcinogenesis by analyzing transcriptome data of 11 solid tumors from The Cancer Genome Atlas database and our in-house gastric cancer cohort. We calculated m6A writer (W), eraser (E), and reader (R) signatures based on corresponding gene expression. Alterations in the W and E signatures varied according to the cancer type, with a strong positive correlation between the W and E signatures in all types. When the patients were divided according to m6A levels estimated by the ratio of the W and E signatures, the prognostic effect of m6A was inconsistent according to the cancer type. The R and especially the R2 signatures (based on the expression of IGF2BPs) were upregulated in all cancers. Patients with a high R2 signature exhibited poor prognosis across types, which was attributed to enrichment of cell cycle- and epithelial–mesenchymal transition-related pathways. Our study demonstrates common features of m6A alterations across cancer types and suggests that targeting m6A R proteins is a promising strategy for cancer treatment.

M6A demethylase ALKBH5 is required for antibacterial innate defense by intrinsic motivation of neutrophil migration

Neutrophil migration into the site of infection is necessary for antibacterial innate defense, whereas impaired neutrophil migration may result in excessive inflammation and even sepsis. The neutrophil migration directed by extracellular signals such as chemokines has been extensively studied, yet the intrinsic mechanism for determining neutrophil ability to migrate needs further investigation. N6-methyladenosine (m6A) RNA modification is important in immunity and inflammation, and our preliminary data indicate downregulation of RNA m6A demethylase alkB homolog 5 (ALKBH5) in neutrophils during bacterial infection. Whether m6A modification and ALKBH5 might intrinsically modulate neutrophil innate response remain unknown. Here we report that ALKBH5 is required for antibacterial innate defense by enhancing intrinsic ability of neutrophil migration. We found that deficiency of ALKBH5 increased mortality of mice with polymicrobial sepsis induced by cecal ligation and puncture (CLP), and Alkbh5-deficient CLP mice exhibited higher bacterial burden and massive proinflammatory cytokine production in the peritoneal cavity and blood because of less neutrophil migration. Alkbh5-deficient neutrophils had lower CXCR2 expression, thus exhibiting impaired migration toward chemokine CXCL2. Mechanistically, ALKBH5-mediated m6A demethylation empowered neutrophils with high migration capability through altering the RNA decay, consequently regulating protein expression of its targets, neutrophil migration-related molecules, including increased expression of neutrophil migration-promoting CXCR2 and NLRP12, but decreased expression of neutrophil migration-suppressive PTGER4, TNC, and WNK1. Our findings reveal a previously unknown role of ALKBH5 in imprinting migration-promoting transcriptome signatures in neutrophils and intrinsically promoting neutrophil migration for antibacterial defense, highlighting the potential application of targeting neutrophil m6A modification in controlling bacterial infections.

Gene amplification-driven RNA methyltransferase KIAA1429 promotes tumorigenesis by regulating BTG2 via m6A-YTHDF2-dependent in lung adenocarcinoma.

BackgroundEpigenetic alterations have been shown to contribute immensely to human carcinogenesis. Dynamic and reversible N6‐methyladenosine (m6A) RNA modification regulates gene expression and cell fate. However, the reasons for activation of KIAA1429 (also known as VIRMA, an RNA methyltransferase) and its underlying mechanism in lung adenocarcinoma (LUAD) remain largely unexplored. In this study, we aimed to clarify the oncogenic role of KIAA1429 in the tumorigenesis of LUAD.MethodsWhole‐genome sequencing and transcriptome sequencing of LUAD data were used to analyze the gene amplification of RNA methyltransferase. The in vitro and in vivo functions of KIAA1429 were investigated. Transcriptome sequencing, methylated RNA immunoprecipitation sequencing (MeRIP‐seq), m6A dot blot assays and RNA immunoprecipitation (RIP) were performed to confirm the modified gene mediated by KIAA1429. RNA stability assays were used to detect the half‐life of the target gene.ResultsCopy number amplification drove higher expression of KIAA1429 in LUAD, which was correlated with poor overall survival. Manipulating the expression of KIAA1429 could regulate the proliferation and metastasis of LUAD. Mechanistically, the target genes of KIAA1429‐mediated m6A modification were confirmed by transcriptome sequencing and MeRIP‐seq assays. We also revealed that KIAA1429 could regulate BTG2 expression in an m6A‐dependent manner. Knockdown of KIAA1429 significantly decreased the m6A levels of BTG2 mRNA, leading to enhanced YTH m6A RNA binding protein 2 (YTHDF2, the m6A “reader”)‐dependent BTG2 mRNA stability and promoted the expression of BTG2; thus, participating in the tumorigenesis of LUAD.ConclusionsOur data revealed the activation mechanism and important role of KIAA1429 in LUAD tumorigenesis, which may provide a novel view on the targeted molecular therapy of LUAD.

Abstract 2188: The N6-methyladenosine reader YTHDC1 is essential for TGF-beta-mediated metastasis of triple negative breast cancer

Abstract Metastatic triple negative breast cancer (TNBC) patients have a 5-year survival rate of just 12%. Therefore, there is an urgent need to discover novel molecular mechanisms that contribute to TNBC metastasis such as N6-methyladenosine (m6A) RNA modification. To do this, we conducted a selective CRISPR-cas9 screen of 14 m6A-regulators and identified an m6A reader protein, YTHDC1, as an essential gene for the survival of MDA-MB-231 cells. The Cancer Genome Atlas (TCGA) dataset showed that patients with poorer prognosis had elevated YTHDC1 expression, which supports a tumor-promoting role of YTHDC1. In vitro, genetic depletion of YTHDC1 significantly impaired Transwell migration and invasion of human TNBC (MDA-MB-231 and SUM159) cells. In vivo, YTHDC1 overexpressing TNBC cells engrafted into the mammary fat pad of NSG mice formed greater numbers and larger sizes of lung metastatic nodules compared to control. Conversely, YTHDC1 knockout (KO) cells produced fewer and smaller lung metastatic nodules compared to control. Together, our in vitro and in vivo data demonstrate YTHDC1’s role in promoting TNBC metastasis. To identify high confidence YTHDC1 targets, we conducted three layers of transcriptome-wide sequencing with MDA-MB-231 cells: RNA-seq (profiling differential gene expression and nuclear export), m6A-seq (profiling m6A methylation in mRNA), and RIP-seq (profiling YTHDC1 binding sites in mRNA). Pathway analysis of differentially expressed genes in MDA-MB-231 YTHDC1 KO cells identified pathways related to metastasis and TGF-β signaling, which is consistent with our phenotypic observations. Overlapping the three sequencing datasets identified SMAD3 as a YTHDC1 target mRNA. Mechanistically, YTHDC1 KO impairs the nuclear export of SMAD3 mRNA, resulting in reduced protein levels of this critical effector of TGF-β signaling. YTHDC1 KO significantly reduced Transwell migration and invasion of TGF-β induced TNBC cells and the expression of TGF-β responsive genes such as SNAIL, IL11, FN, and CTGF. Overexpression of SMAD3 was able to rescue the impaired migration and invasion of YTHDC1 KO TNBC cells and restore the expression of TGF-β responsive genes following TGF-β treatment. We also demonstrated that the oncogenic role of YTHDC1 is mediated through its recognition of m6A, as m6A-binding defective mutants of YTHDC1 were unable to rescue impaired Transwell migration and invasion of YTHDC1 KO TNBC cells. In summary, our study reveals that TNBC metastasis is mediated at least in part through YTHDC1’s function in promoting SMAD3 expression to augment the TGF-β signaling cascade. Citation Format: Brandon Tan, Wei Liu, Keren Zhou, Emily Prince, Lu Yang, Yangchan Li, Ying Qing, Li Han, Zhicong Zhao, Rui Su, Chun-Wei Chen, Jianjun Chen. The N6-methyladenosine reader YTHDC1 is essential for TGF-beta-mediated metastasis of triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2188.

Genome-Wide Identification of m6A Writers, Erasers and Readers in Poplar 84K.

N6-methyladenosine (m6A) RNA modification is a conserved mechanism to regulate gene expression that plays vital roles in the development of plants. However, the m6A RNA modification in forest trees remains limited. Here, we performed a complete analysis of m6A writers, erasers and readers in Poplar 84K, including gene location, gene structures, conserved motifs, phylogenetic relationships, promoter analysis, expression profiles and the homology modeling. We have identified 61 m6A pathway genes in Poplar 84K (Populus alba × Populus glandulosa), including 14 m6A writers, 14 m6A erasers and 33 m6A readers. Phylogenetic analysis indicated that the m6A writers and erasers were clustered into four groups and m6A readers were clustered into two groups. Promoter analysis showed that m6A pathway genes were mainly responsive to low oxygen followed by ABA and ethylene. The expression of the identified m6A pathway genes showed tissue-specific expression patterns in leaves, xylem, phloem and roots. Moreover, 17 genes were significantly up-regulated and 13 genes were significantly down-regulated in poplar overexpressing the transcription factor LBD15. Homology modeling and molecular docking results suggested that PagFIP37b was most likely to be regulated by LBD15, and the qPCRshowed that PagFIP37b were up-regulated in the LBD15-oe plants. The results provide insights that aid in the future elucidation of the functions of these m6A pathway genes and the epigenetic regulation mechanism of these genes in Poplar 84K.

One Stone, Two Birds: N6-Methyladenosine RNA Modification in Leukemia Stem Cells and the Tumor Immune Microenvironment in Acute Myeloid Leukemia.

Acute myeloid leukemia is the most common acute leukemia in adults, with accumulation of abundant blasts and impairment of hematogenic function. Despite great advances in diagnosis and therapy, the overall survival of patients with acute myeloid leukemia remains poor. Leukemia stem cells are the root cause of relapse and chemoresistance in acute myeloid leukemia. The tumor immune microenvironment is another trigger to induce recurrence and drug resistance. Understanding the underlying factors influencing leukemia stem cells and the tumor immune microenvironment is an urgent and unmet need. Intriguingly, N6-methyladenosine, the most widespread internal mRNA modification in eukaryotes, is found to regulate both leukemia stem cells and the tumor immune microenvironment. Methyltransferases and demethylases cooperatively make N6-methyladenosine modification reversible and dynamic. Increasing evidence demonstrates that N6-methyladenosine modification extensively participates in tumorigenesis and progression in various cancers, including acute myeloid leukemia. In this review, we summarize the current progress in studies on the functions of N6-methyladenosine modification in acute myeloid leukemia, especially in leukemia stem cells and the tumor immune microenvironment. We generalize the landscape of N6-methyladenosine modification in self-renewal of leukemia stem cells and immune microenvironment regulation, as well as in the initiation, growth, proliferation, differentiation, and apoptosis of leukemia cells. In addition, we further explore the clinical application of N6-methyladenosine modification in diagnosis, prognostic stratification, and effect evaluation. Considering the roles of N6-methyladenosine modification in leukemia stem cells and the tumor immune microenvironment, we propose targeting N6-methyladenosine regulators as one stone to kill two birds for acute myeloid leukemia treatment.

Investigating the structural changes due to adenosine methylation of the Kaposi's sarcoma-associated herpes virus ORF50 transcript.

Kaposi's sarcoma-associated herpes virus (KSHV) is a human oncovirus. KSHV relies on manipulating the host cell N6-methyl adenosine (m6A) RNA modification pathway to enhance virus replication. Methylation within a RNA stem loop of the open reading frame 50 (ORF50) increases transcript stability via the recruitment of the m6A reader, SND1. In this contribution we explore the energy landscapes of the unmethylated and methylated RNA stem loops of ORF50 to investigate the effect of methylation on the structure of the stem loop. We observe a significant shift upon methylation between an open and closed configuration of the top of the stem loop. In the unmethylated stem loop the closed configuration is much lower in energy, and, as a result, exhibits higher occupancy.

METTL3-Mediated N6-Methyladenosine Modification of Trim59 mRNA Protects Against Sepsis-Induced Acute Respiratory Distress Syndrome.

N6-methyladenosine (m6A) RNA modification is a fundamental determinant of mRNA metabolism in eukaryotic cells and is involved in numerous physiological and pathological processes. However, the specific role of m6A modification in sepsis-induced acute respiratory distress syndrome(ARDS) remains unknown. Here, we show that the levels of m6A RNA were significantly decreased in septic lungs and that METTL3 was the main regulator involved in the absence of m6A RNA modification. Pulmonary endothelial barrier damage is a critical process in the pathogenesis of acute lung injury during sepsis. METTL3 regulated endothelial barrier dysfunction and inflammatory responses in sepsis-induced ARDS in vivo and in vitro. Furthermore, we identified tripartite motif-containing (Trim)59 as a key m6A effector and Trim59 deficiency exacerbated lung injury. Mechanistically, METTL3 inhibited endothelial injury in sepsis-induced ARDS through Trim59-associated NF-κB inactivation. Our findings revealed novel insights into epitranscriptional mechanisms in sepsis-induced ARDS via m6A modifications, which has important application value in the diagnosis, prognosis, and molecular-targeted therapy of sepsis-associated lung injury.

Controlling the lytic switch; can m6A-modified RNA be used as an Anti-Viral Target?

KSHV has a biphasic life cycle encompassing a latent state and lytic replication. The KSHV replication and transcription activator viral protein, encoded from open reading frame 50 (ORF50), is the key viral protein which drives the switch between the latent and lytic phases (Guito and Lukac, 2012). We have recently demonstrated that KSHV manipulates the host cell N6-methyl adenosine (m6A) RNA modification pathway to enhance viral gene expression. Specifically, we have shown that the KSHV ORF50 transcript is m6A methylated, allowing the recruitment of the m6A reader protein, Staphylococcal nuclease domain-containing protein 1 (SND1), resulting in the stabilisation of the ORF50 transcript and efficient KSHV lytic replication (Baquero-Perez et al. 2019). Further analysis of the m6A modified site with the ORF50 transcript has identified an RNA stem-loop, termed ORF50-1, which is a m6A-modified 43-mer, essential for SND-1 binding, thought to occur in a secondary structure/ sequence-dependent manner. Generating in silico 3D structures of the ORF50-1 RNA in its native ‘N’ and m6A-modified ‘M’ form confirms that the presence of the m6A-modification has repercussions in the lower part of the stem-loop and predicts a different secondary structure(collab. Pasquale and Roeder). Due to the obvious structural differences anticipated, RNA-binding ligands have been identified using Small Molecule Microarrays (SMMS) (collab. Fullenkamp and Schneekloth). By investigating these ligands in biophysicalexperiments, we have verified RNA-binding and optimised cell-based assays to assess anti-viral properties. It is hoped these experiments will highlight the importance of A versus m6A within the lytic phase of KSHV’s lifecycle.

N6-methyladenosine methyltransferase KIAA1429 elevates colorectal cancer aerobic glycolysis via HK2-dependent manner

ABSTRACT Emerging evidence has emphasized the critical roles played by N6-methyladenosine RNA (m6A) modification in colorectal carcinoma (CRC) initiation and progression. However, the roles and mechanism of m6A and KIAA1429 in CRC progression require further clarification. Here, our research aimed to investigate the functions of KIAA1429 in CRC tumorigenesis. Results indicated that KIAA1429 up-regulation closely correlated to the poor prognosis of CRC patients. Bio-functional assays demonstrated that KIAA1429 promoted the aerobic glycolysis, including glucose uptake, lactate production, ATP generation and extracellular acidification rate (ECAR). Mechanistically, KIAA1429 positively up-regulated HK2 level via increasing its mRNA stability by binding the m6A site of HK2 mRNA via m6A-independent manner. Collectively, our work indicates that KIAA1429 has the potential to promote CRC carcinogenesis by targeting HK2 via m6A-independent manner, providing insight into the critical roles of m6A in CRC.

Comprehensive Analysis of N6-Methyladenosine Regulators in the Subcluster Classification and Drug Candidates Prediction of Severe Obstructive Sleep Apnea.

Background: Obstructive sleep apnea (OSA) is the most common type of sleep apnea that impacts the development or progression of many other disorders. Abnormal expression of N6-methyladenosine (m6A) RNA modification regulators have been found relating to a variety of human diseases. However, it is not yet known if m6A regulators are involved in the occurrence and development of OSA. Herein, we aim to explore the impact of m6A modification in severe OSA. Methods: We detected the differentially expressed m6A regulators in severe OSA microarray dataset GSE135917. The least absolute shrinkage and selection operator (LASSO) and support vector machines (SVM) were used to identify the severe OSA-related m6A regulators. Receiver operating characteristic (ROC) curves were performed to screen and verify the diagnostic markers. Consensus clustering algorithm was used to identify m6A patterns. And then, we explored the character of immune microenvironment, molecular functionals, protein-protein interaction networks and miRNA-TF coregulatory networks for each subcluster. Finally, the Connectivity Map (CMap) tools were used to tailor customized treatment strategies for different severe OSA subclusters. An independent dataset GSE38792 was used for validation. Results: We found that HNRNPA2B1, KIAA1429, ALKBH5, YTHDF2, FMR1, IGF2BP1 and IGF2BP3 were dysregulated in severe OSA patients. Among them, IGF2BP3 has a high diagnostic value in both independent datasets. Furthermore, severe OSA patients can be accurately classified into three m6A patterns (subcluster1, subcluster2, subcluster3). The immune response in subcluster3 was more active because it has high M0 Macrophages and M2 Macrophages infiltration and up-regulated human leukocyte antigens (HLAs) expression. Functional analysis showed that representative genes for each subcluster in severe OSA were assigned to histone methyltransferase, ATP synthesis coupled electron transport, virus replication, RNA catabolic, multiple neurodegeneration diseases pathway, et al. Moreover, our finding demonstrated cyclooxygenase inhibitors, several of adrenergic receptor antagonists and histamine receptor antagonists might have a therapeutic effect on severe OSA. Conclusion: Our study presents an overview of the expression pattern and crucial role of m6A regulators in severe OSA, which may provide critical insights for future research and help guide appropriate prevention and treatment options.