m6A Methylation Levels Research Articles

Genome-wide characterization of the AlkB homolog (ALKBH) gene family in Litopenaeus vannamei identifies LvALKBH1 and LvALKBH8 as promising crustacean m6A demethylases involved in molting regulation and ammonia stress response.

N6-methyladenosine (m6A) is the most abundant chemical modification on eukaryotic mRNAs. In crustaceans, the absence of canonical m6A demethylases, namely fat mass and obesity-associated protein (FTO) and AlkB Homolog 5 (ALKBH5), poses an unresolved puzzle about the m6A demethylation machinery of these invertebrates. Here, a genome-wide search for potential ALKBH gene family members in the whiteleg shrimp Litopenaeus vannamei was conducted. Six homologues of the ALKBH family were identified from the genome of L. vannamei, and comparative genomics analysis indicated that ALKBH3 and ALKBH5 are likely to exist in the common ancestor of arthropods and molluscs but are then lost in arthropods. Except for LvALKBH4 and LvALKBH7, all LvALKBH proteins possessed a typical 2OG-Fe(II)_Oxy_2 functional domain. Functional experiments revealed that LvALKBH1 and LvALKBH8 possessed significant m6A demethylation activity. Moreover, LvALKBH1 and LvALKBH8 significantly affected the m6A methylation and expression levels of the ecdysone receptor (EcR), retinoid X receptor (RXR), aspartate aminotransferase (AST), and glutamine synthetase (GS), implying their potential roles in regulating shrimp molting and ammonia toxicity resistance. The study provides important baseline information on the molecular characteristics of the ALKBH gene family in shrimp, and fills a current research gap concerning the m6A demethylation toolkit of crustaceans.

Epitranscriptome profiles reveal participation of the RNA methyltransferase gene OsMTA1 in rice seed germination and salt stress response

BackgroundRNA m6A methylation installed by RNA methyltransferases plays a crucial role in regulating plant growth and development and environmental stress responses. However, the underlying molecular mechanisms of m6A methylation involved in seed germination and stress responses are largely unknown. In the present study, we surveyed global m6A methylation in rice seed germination under salt stress and the control (no stress) using an osmta1 mutant and its wild type.ResultsThe knockout of OsMTA1 resulted in a decreased level of m6A methylation and delayed seed germination, together with increased oxidative damage in the osmta1-1 mutant, especially under salt stress, indicating that OsMTA1 performs a crucial function in rice seed germination and salt stress response. Comparative analysis of m6A profiling using methylated RNA immunoprecipitation sequencing revealed that a unique set of genes that functioned in seed germination, cell growth, and development, including OsbZIP78 and OsA8, were hypomethylated in osmta1-1 embryos and germinating seeds. Numerous genes involved in plant growth and stress response were hypomethylated in the osmta1-1 mutant during seed germination under salt stress. Further combined analysis of the m6A methylome and transcriptome revealed that the loss of function of OsMTA1 had a more complex impact on gene expression in osmta1-1. Several hypomethylated genes with a negative role in growth and development, such as OsHsfA7 and OsHDAC3, were highly up-regulated in the osmta1-1 mutant under the control condition. In contrast, several hypomethylated genes positively associated with stress response were down-regulated, whereas a different set of hypomethylated genes that functioned as negative regulators of growth and stress response were up-regulated in the osmta1-1 mutant under salt stress. These results further demonstrated that OsMTA1-mediated m6A methylation modulated rice seed germination and salt stress response by regulating transcription of a unique set of genes with diverse functions.ConclusionOur results reveal a crucial role for the m6A methyltransferase gene OsMTA1 in regulating rice seed germination and salt stress response, and provide candidate genes to assist in breeding new stress-tolerant rice varieties.

Semaphorin-4D signaling in recruiting dental stem cells for vascular stabilization

BackgroundAchieving a stable vasculature is crucial for tissue regeneration. Endothelial cells initiate vascular morphogenesis, followed by mural cells that stabilize new vessels. This study investigated the in vivo effects of Sema4D-Plexin-B1 signaling on stem cells from human exfoliated deciduous teeth (SHED)-supported angiogenesis, focusing on its mechanism in PDGF-BB secretion. We also explored macrophages as an endogenous source of Sema4D for vascular stabilization.MethodsThe in vivo Matrigel plug angiogenesis assay was conducted to examine the impact of Sema4D on vessel formation and stabilization supported by SHED. Knockdown of Plexin-B1 in human umbilical vein endothelial cells (HUVECs) and PDGFR-β inhibitors were utilized to explore the fundamental regulatory mechanisms. Furthermore, the m6A methylation levels of total RNA and the expression of Methyltransferase-like 3 (METTL3) were assessed under conditions of Sema4D treatment in vitro. An ELISA was employed to measure the levels of Sema4D in the supernatants derived from THP-1 cell-mediated macrophages. Additionally, a three-dimensional vasculature-on-a-chip microfluidic device was used to investigate the role of M2c macrophage-derived Sema4D in the stabilization of vascular structures.ResultsSema4D induced the formation of a greater number of perfused vessels by HUVECs and enhanced the coverage of these vessels by SM22α-positive SHED (SM22α+SHED). Conversely, the knockdown of the Plexin-B1 receptor in HUVECs or inhibition of PDGFR-β reversed the Sema4D-induced vascular stabilization, thereby confirming the regulatory role of the Plexin-B1/PDGF-BB axis in the recruitment of mural cells mediated by Sema4D. Mechanistically, Sema4D was found to upregulate the expression of methyltransferases, specifically METTL3, and to elevate the level of m6A modification in HUVECs. This modification was determined to be critical for enhancing PDGF-BB secretion, suggesting that Sema4D activates an epigenetic regulatory mechanism. Additionally, we investigated the secretion of Sema4D by various macrophage phenotypes, identifying that M2c macrophages secrete significant levels of Sema4D. This secretion recruited SM22α+SHED as mural cells by inducing endothelial PDGF production on a vasculature-on-a-chip platform, indicating a potential role for macrophages in facilitating vascular stabilization.ConclusionsSema4D acts on Plexin-B1, inducing METTL3-mediated PDGF-BB secretion to recruit SHED to stabilize vessels. Macrophages could be a key source of Sema4D for vascular stabilization.

METTL3 and FTO Regulate Heat Stress Response in Hu Sheep Through Lipid Metabolism via m6A Modification.

In an established hepatocyte lipid deposition heat stress model, the expression levels of METTL3 and FTO were significantly upregulated (p < 0.05), indicating that METTL3 and FTO play important roles in the process of lipid deposition heat stress in hepatocytes. Transcriptome and metabolome analyses showed that lipid deposition heat stress had significant effects on the linoleic acid, linolenic acid, glycerophospholipid, and arachidonic acid metabolic pathways in hepatocytes. After METTL3 knockdown, the m6A methylation level decreased, but the difference was not significant (p > 0.05), the FABP4 and Accα expression levels increased, and the HSP60, HSP70, and HSP110 expression levels decreased significantly. After METTL3 overexpression, the m6A methylation level increased significantly and the expression levels of FABP4, ATGL, Accα, HSP60, HSP70, HSP90, and HSP110 decreased significantly, indicating that the overexpression of METTL3 reduced the expression of heat shock genes by inhibiting the lipid-deposition-related gene expression in an m6A-dependent manner. The m6A methylation level increased significantly after FTO knockdown, while HSP60, HSP110, FABP4, ATGL, and Accα expression levels were significantly reduced. Following FTO overexpression, the m6A methylation level and HSP60, HSP90, and HSP110 expression levels significantly decreased, while the ATGL and Accα expression levels significantly increased. This indicates that the overexpression of FTO promoted the expression of lipid-deposition-related genes in an m6A-dependent manner to reduce the expression of heat shock genes. Transcriptome and metabolome sequencing screened a large number of differential genes and metabolites, and a KEGG enrichment analysis showed that m6A methylation mainly regulated heat stress by affecting the TNF, cAMP, MAPK, lipolysis, and synthesis pathways in hepatocytes. In the lipid deposition heat stress model of preadipocytes, the regulation of gene expression was similar to that in hepatocytes.

ALKBH5, an m6A demethylase, attenuates tumor growth and inhibits metastasis in papillary thyroid carcinoma

The significance of ALKBH5 in erasing mRNA methylation in mRNA biogenesis, decay, and translation control has emerged as a prominent research focus. Additionally, ALKBH5 is associated with the development of numerous human cancers. However, it remains unclear whether ALKBH5 regulates the growth and metastasis of papillary thyroid carcinoma (PTC). Here, we compared cancer tissues and paracancerous tissues from PTC patients, along with cultured cells expressing ALKBH5 (overexpression, silent gene expression, normal stable expression). Our primary objective was to investigate the impact of ALKBH5 on PTC. Selected 30 cases of PTC tissues and their adjacent noncancerous tissues to compare the protein expression levels of ALKBH5 between the two groups using immunohistochemical analysis. qRT-PCR and western blot were used to detect the expression of ALKBH5 in normal thyroid follicular epithelial cells (Nthy-ori3-1) and 4 PTC cell lines (human PTC cell lines K1, BCPAP, IHH4, and TPC1). Appropriate cell lines were screened for subsequent experiments. Immunofluorescence staining was used to localize the high accumulation of ALKBH5 in cells. Construct the ALKBH5 knockdown vector and ALKBH5 overexpression vector separately, and construct the overexpression ALKBH5-mut vector with m6A domain mutation. The impact of different levels of ALKBH5 in the three cell lines on RNA m6A methylation levels was compared using qRT-PCR and western blot methods. Furthermore, cell viability was assessed using the CCK-8 assay, while the impact on cell proliferation was examined using plate colony formation assay. Cell invasion was evaluated using the Transwell assay. Immunohistochemical staining results showed that the expression of ALKBH5 protein in PTC cancer tissue was significantly lower than in adjacent non-cancerous tissue (P < 0.05). Lymph node metastasis in PTC patients may have been linked to ALKBH5 protein levels in their cancerous tissues (P = 0.034). The expression of ALKBH5 in PTC cell lines BCPAP, IHH4, and TPC1 was significantly lower than Nthy-ori3-1 (P < 0.05). IHH4 and TPC1 cell lines were selected for subsequent experiments. Immunofluorescence single staining results showed a high accumulation of ALKBH5 protein in the cell nucleus. Cell viability results suggested that compared to the overexpression-negative control group, cell proliferation, and invasion were significantly decreased in the ALKBH5 overexpression group (P < 0.05) and the mut-ALKBH5 overexpression group (P < 0.05). Additionally, compared to the ALKBH5 overexpression group, cell proliferation and invasion were significantly more decreased in the mut-ALKBH5 overexpression group (P < 0.05). However, compared to the interference-negative control group, cell proliferation and invasion were significantly increased in the ALKBH5 interference group (P < 0.05). The presented findings suggested that m6A demethylase ALKBH5 inhibits tumor growth and metastasis in PTC. Moreover, effective inhibition of m6A modification of ALKBH5 might constitute a potential treatment strategy for PTC.

ALKBH5 alleviates lower extremity arteriosclerosis by regulating ITGB1 demethylation and influencing macrophage polarization.

M6A methylation-regulated macrophages play an important role in the occurrence and development of arteriosclerosis. However, their role in lower extremity arteriosclerosis remains unclear. Therefore, this study aims to explore the key factors that regulate arteriosclerosis methylation in the lower extremities and the mechanism by which they affect arteriosclerosis by influencing macrophage polarization. The m6A methylation levels in peripheral blood mononuclear cells (PBMCs) of patients with lower extremity atherosclerosis was investigated using the Dot blot method. Additionally, the expression levels of RNA methyltransferases and demethylases were examined using ELISA and Western blotting. Inflammatory macrophages were established using RAW264.7cells stimulated with LPS (100ng/mL), and the expression of ALKBH5 and ITGB1 was evaluated using Western blotting. Immunofluorescence staining was conducted to assess the expression of M1 macrophage markers (F4/80+CD86) and M2 macrophage markers (F4/80+CD206) in renal tissue. ELISA was employed to measure the levels of cytokines (IL-6, IL-1β, TNF-a, IL-10, and TGF-β) and plasma lipid levels in mice. An atherosclerosis model was established in ApoE-/- mice through balloon pull surgery and high-fat feeding. Oil Red O staining and hematoxylin and eosin (HE) staining were performed to measure the area of atherosclerotic plaques and the size of the necrotic core in mouse femoral artery, respectively. Additionally, Starbase2.0 was used for downstream target gene prediction of ALKBH5. The half-life of ITGB1 was evaluated using RT-qPCR. The m6A methylation levels were significantly increased in PBMCs of patients with lower extremity atherosclerosis. Among them, the expression of the RNA demethylase ALKBH5 was the lowest in PBMCs of patients with lower extremity atherosclerosis. Further analysis revealed that ALKBH5 can alleviate the progression of lower extremity atherosclerosis and promote the polarization of M2 macrophages. ALKBH5 can reduce the stability of ITGB1 through demethylation. Mechanistically, ALKBH5 influences macrophage polarization and mitigates lower extremity atherosclerosis by affecting the demethylation of ITGB1. ALKBH5 promotes M2 macrophage polarization and alleviates lower extremity atherosclerosis by regulating the demethylation of ITGB1. A deeper understanding of this process not only helps elucidate the molecular mechanisms of atherosclerosis but also provides possibilities for the development of new therapeutic strategies.

Extractable organic matter from PM2.5 inhibits cardiomyocyte differentiation via AHR-mediated m6A RNA methylation.

An ever-increasing body of research has established a link between maternal PM2.5 exposure and congenital heart diseases in the offspring, but the underlying mechanisms remain elusive. We recently reported that activation of the aryl hydrocarbon receptor (AHR) by PM2.5 causes aberrant m6A RNA methylation, leading to cardiac malformations in zebrafish embryos. We hypothesized that PM2.5 can disrupt heart development by inducing m6A methylation changes through AHR in mammals. In this study, we observed that extractable organic matters (EOM) from PM2.5 significantly impaired cardiomyocyte differentiation in embryonic rat cardiomyoblasts H9c2. Importantly, EOM exposure reduced global m6A methylation levels, which was reversed by AHR inhibition. Moreover, AHR, activated by EOM directly promoted the transcription of the demethylase, FTO, leading to global m6A hypomethylation. Specifically, AHR-induced FTO overexpression decreased the m6A methylation levels of Nox4 mRNA, resulting in NOX4 overexpression and subsequent oxidative stress in EOM samples. We then demonstrated that oxidative stress contributes to the inhibition of cardiomyocyte differentiation by EOM through suppression of Wnt/β-catenin signaling. In summary, our findings indicate that AHR activation by PM2.5 directly enhances the expression of the demethylase, FTO, which increases NOX4 expression by reducing its m6A methylation. The oxidative stress caused by NOX4 overexpression inhibits Wnt/β-catenin signaling, thereby compromising cardiomyocyte differentiation.

METTL3 mediated WISP1 m6A modification promotes epithelial-mesenchymal transition and tumorigenesis in laryngeal squamous cell carcinoma via m6A reader IGF2BP1.

N6-methyladenosine (m6A), is well known as the most abundant epigenetic modification in messenger RNA, but its influence on laryngeal squamous cell carcinoma (LSCC) remains largely unexplored and poorly understood. This study was designed to explore the effects of m6A on WISP1-mediated epithelial-mesenchymal transition (EMT) and tumorigenesis in LSCC. m6A methylated and expression levels of WISP1 in LSCC tumor tissues and cells were measured by MeRIP-qPCR, qRT-PCR, and western blotting. The regulatory mechanism of m6A modification of WISP1 in LSCC was determined using MeRIP-qPCR, RIP, dual luciferase reporter assay, and RNA stability assay. Cell viability was assessed utilizing MTT method. The invasion and migration ability of LSCC cells were determined by transwell and wound healing method, respectively. Tumor xenograft models were used for the in vivo experiments. The m6A methylation level of WISP1 was significantly enhanced in LSCC patients and LSCC cell lines. Overexpression of the m6A methyltransferase METTL3 significantly upregulated WISP1 expression by promoting its m6A methylation level, whereas METTL3 inhibition exhibited the opposite effect in LSCC cells. Functionally, we found that METTL3 accelerated the viability, invasion, migration, and EMT of LSCC cells by upregulating WISP1. Additionally, overexpression of METTL3 increased WISP1 expression and tumorigenesis were verified in in vivo experiments. Mechanistically, m6A-modified WISP1 was recognized by IGF2BP1, which enhanced the stability of WISP1 mRNA. Our findings indicate that the m6A modification of WISP1 promotes EMT in LSCC by enhancing WISP1 mRNA stability via an IGF2BP1-dependent manner, which may highlight an m6A methylation-based approach for LSCC diagnosis and therapy.

RBM15-dependent m6A modification mediates progression of non-small cell lung cancer cells

Abstract Background Non-small cell lung cancer (NSCLC) is the predominant form of lung cancer, contributing significantly to global health and economic challenges. This study elucidated the role of RBM15 in NSCLC progression through its involvement in m6A modifications. Methods RBM15 levels in NSCLC tissues and cells were assessed via RT-qPCR and Western blotting. The impact of RBM15 knockdown on NSCLC proliferation, invasion, and migration was evaluated using CCK-8, colony formation, and Transwell assays. Expression levels of KLF1, TRIM13, and ANXA8 were determined by RT-qPCR and Western blot. m6A methylation levels were analyzed, while RIP and MeRIP assays were employed to explore the interaction between YTHDF1/YTHDF2/m6A and KLF1/TRIM13, as well as KLF1 binding to the ANXA8 promoter. The ubiquitination of ANXA8 was examined through ubiquitination assays. Xenograft and metastasis models were utilized to assess RBM15’s role in vivo. Results RBM15 was found to be overexpressed in NSCLC. Silencing RBM15 led to decreased cell proliferation, invasion, and migration of NSCLC cells. RBM15 upregulated KLF1 and downregulated TRIM13 via YTHDF1/YTHDF2, resulting in the promotion of ANXA8 expression. KLF1 overexpression or TRIM13 downregulation partially reversed the suppressive effects of RBM15 knockdown on NSCLC cell proliferation. ANXA8, upregulated in NSCLC, mitigated the inhibitory effects of RBM15 silencing on malignant behaviors. In vivo, RBM15 downregulation hindered NSCLC cell proliferation and metastasis by modulating the KLF1-TRIM13/ANXA8 axis. Conclusion RBM15-mediated m6A methylation enhances KLF1 expression and suppresses TRIM13 via YTHDF1/YTHDF2, thereby promoting ANXA8 and facilitating NSCLC progression. These findings provide novel insights and potential therapeutic targets for NSCLC treatment.

Mettl3-Mediated m6A Modification is Essential for Visual Function and Retinal Photoreceptor Survival.

N6-methyladenosine (m6A) modification, one of the most common epigenetic modifications in eukaryotic mRNA, has been shown to play a role in the development and function of the mammalian nervous system by regulating the biological fate of mRNA. METTL3, the catalytically active component of the m6A methyltransferase complex, has been shown to be essential in development of in the retina. However, its role in the mature retina remains elusive. In this study we aim to investigate the in vivo function of Mettl3 in the photoreceptor cells using a conditional knockout allele of Mettl3. Deletion of Mettl3 in rod cells led to progressive retinal degeneration, including progressive retinal thinning, impaired visual function, shortened photoreceptor outer segments (OS), and reduced expression of disk membrane proteins. Similarly, Mettl3 deficiency in cone cells led to the gradual degeneration of cone opsins. Additionally, Mettl3 knockout significantly decreased the expression of the METTL14 subunit and overall m6A methylation levels in the retina. Multi-omics analyses revealed that Mettl3 deletion led to the downregulation of mRNA and protein levels of 10 key target genes in rod cells, ultimately resulting in the progressive death of photoreceptors. Mettl3 controls expression of its target genes by regulating their m6A modification, ultimately leading to rod cell death. These findings highlight critical roles of METTL3 in maintaining retinal photoreceptor function and further elucidate the mechanisms of m6A modification in photoreceptors.

M6A-Methylated NUTM2B-AS1 Promotes Hepatocellular Carcinoma Stemness Feature via Epigenetically Activating BMPR1A Transcription.

Hepatocellular carcinoma (HCC) is one of the most lethal malignancies in the world. Oncofetal proteins are the optimal diagnostic biomarkers and therapeutic targets for HCC. As the most abundant modification in RNA, N6-methyladenosine (m6A) has been reported to be involved in HCC initiation and progression. However, whether m6A has oncofetal characteristics remains unknown. Gene expression in HCC tissues and cells was detected using qPCR. The level of m6A methylation was determined using methylated RNA immunoprecipitation assay. The biological roles of NUTM2B-AS1 in HCC were detected using Cell Counting Kit-8, 5-ethynyl-2'-deoxyuridine incorporation, and spheroid formation assays. The mechanisms underlying the roles of NUTM2B-AS1 were explored using RNA immunoprecipitation (RIP), chromatin isolation by RNA purification (ChIRP), chromatin immunoprecipitation (ChIP), and assay for transposase-accessible chromatin (ATAC). NUTM2B-AS1 was identified as a novel oncofetal long noncoding RNA that was upregulated in the fetal liver and HCC and silenced in adult liver tissues. METTL3 and METTL16 induce m6A hypermethylation of NUTM2B-AS1. The m6A methylation levels of NUTM2B-AS1 exhibit oncofetal characteristics. m6A methylation upregulates NUTM2B-AS1 expression by increasing NUTM2B-AS1 transcript stability. m6A-methylated NUTM2B-AS1 promotes HCC cell proliferation and stemness via epigenetically activating BMPR1A expression. NUTM2B-AS1 specifically binds to BMPR1A promoter. m6A-methylated NUTM2B-AS1 is recognized by the m6A reader YTHDC2, which further binds to the H3K4 methyltransferase MLL1. m6A-methylated NUTM2B-AS1 recruits YTHDC2 and MLL1 to BMPR1A promoter, leading to increased H3K4me3 and chromatin accessibility at BMPR1A promoter. Functional rescue assays suggest that BMPR1A is a critical mediator of the oncogenic role of m6A-methylated NUTM2B-AS1 in HCC. METTL3- and METTL16-mediated m6A methylation of NUTM2B-AS1 is a novel oncofetal molecular event in HCC that promotes HCC stemness via epigenetically activating BMPR1A transcription.

Transcriptome-wide identification and analysis reveals m6A regulation of metabolic reprogramming in shrimp (Marsupenaeus japonicus) under virus infection.

It has been reported that the most common post-transcriptional modification of eukaryotic RNA is N6-methyladenosine (m6A). Previous studies show m6A is a key regulator for viral infection and immune response. However, whether there is a pathogen stimulus-dependent m6A regulation in invertebrate shrimp has not been studied. In this study, we performed a transcriptome-wide profiling of mRNA m6A methylation in shrimp (Marsupenaeus japonicus) after white spot syndrome virus (WSSV) infection by methylated RNA immunoprecipitation sequencing (MeRIP-seq). A total of 15,436 m6A peaks were identified in the shrimp, distributed in 8,108 genes, mainly enriched in the CDS, 3' UTR region and near the stop codon. After WSSV infection, we identified 2,260 m6A peaks with significantly changes, of which 1,973 peaks were significantly up-regulated and 287 peaks were significantly down-regulated. 1,795 genes were identified as differentially methylated genes. GO and KEGG analysis showed that hyper-methylated genes or hypo-methylated genes were highly associated with innate immune process and related to metabolic pathways including HIF-1 signaling pathway, lysine degradation and Wnt signaling pathway. Combined analysis showed a positive correlation between m6A methylation levels and mRNA expression levels. In addition, computational predictions of protein-protein interaction indicated that genes with altered levels of m6A methylation and mRNA expression clustered in metabolism, DNA replication, and protein ubiquitination. ZC3H12A and HIF-1 were two hub genes in protein-protein interaction (PPI) network that involved in immune and metabolism processes, respectively. Our study explored the m6A methylation pattern of mRNA in shrimp after WSSV infection, exhibited the first m6A map of shrimp at the stage of WSSV induced metabolic reprogramming. These findings may reveal the possible mechanisms of m6A-mediated innate immune response in invertebrates.

ZnO nanoparticles enhances cadmium tolerance by modulating N6-methyladenosine (m6A) level of stress-responsive genes NRT1 and GM35E in vegetable soybean

Cadmium (Cd) is a hazardous heavy metal pollutant that poses significant risks to agricultural production and human health. Nanoparticles (NPs) can alleviate the effects of cadmium on crops by regulating the expression of stress-responsive genes, however, the mechanism of regulation is unknown. N6-methyladenosine (m6A) is a prevalent RNA modification, which determines the expression level of RNA. In this study, we performed m6A methylome analysis and gene editing to investigate the regulation of stress-responsive genes by m6A under Cd stress with ZnO nanoparticles (ZnO NPs). Firstly, we identified 16 differentially expressed genes (DEGs) with differential m6A-modification by m6A methylome seq, which included 6 stress-responsive genes. ZnO NPs treatment reduced the m6A level and stabilized the mRNAs of these stress-responsive genes. Then, we utilized the in vivo m6A modification tool, Plant m6A Editors (PMEs), specifically reduced the m6A level of NRT1 and GM35E in transgenic plants. The expression of NRT1 and GM35E was increased, and plants carrying PMEs-NRT1 and PMEs-GM35E showed stronger Cd tolerance than control. Therefore, we can conclude that NPs enhance Cd tolerance in plants by regulating the m6A methylation level of stress-responsive genes such as NRT1 and GM35E, which ultimately affects the expression of these genes. This study proposed a post-transcriptional regulatory network in vegetable soybean roots responding to Cd with ZnO NPs, potentially offering new insights into gene manipulation for controlling low Cd accumulation in crops.

A bone-targeting delivery platform based on mesoporous silica loaded with piR7472 for the treatment of osteoporosis

Promoting osteogenic differentiation and inhibiting osteoclast formation remain significant challenges in the treatment of osteoporosis. With the growing understanding of osteoporosis, increasing literature has highlighted the regulatory role of m6A methylation in this condition. However, there is currently no reliable method to stably regulate cellular m6A methylation levels. Here, we report a novel approach utilizing alendronate (aln)-modified mesoporous silica nanoparticles (MSNs) to deliver sodium bicarbonate and piR7472, modulating cellular behavior. Our experimental results demonstrate that Aln modification enables the nanoparticles to stably target hydroxyapatite, thereby accumulating in osteoporotic regions. Sodium bicarbonate suppresses osteoclastogenesis, while piR7472 enhances m6A methylation, promoting osteogenic differentiation of bone marrow stromal cells (BMSCs). Computed tomography (CT) and hematoxylin and eosin (HE) staining showed that after 2 weeks of treatment with MSNs-Na@piR7472, cortical bone thickened, trabecular bone density increased, collagen fiber thickness improved, and both the number and staining area of osteoclasts were significantly reduced. These findings indicate a marked improvement in osteoporosis.

T-2 toxin induces chondrocyte extracellular matrix degradation by regulating the METTL3-mediated Ctsk m6A modification

T-2 toxin is a major cause of Kashin-Beck disease (KBD), which is characterised by cartilage damage. N6-adenosine-methyltransferase-like 3 (METTL3) regulates cartilage injury; however, its role in T-2 toxin-induced cartilage injury remains elusive. Herein, we investigated the involvement of METTL3-mediated m6A modification in T-2 toxin-induced cartilage damage. METTL3-mediated m6A methylation levels were correlated with cartilage extracellular matrix (ECM) degradation, which was exacerbated following METTL3 silencing. Cathepsin K (Ctsk) was identified as a downstream target of METTL3 using m6A-methylated RNA immunoprecipitation（MeRIP）sequencing and RNA sequencing. Silencing Ctsk aggravated HT-2 toxin-induced ECM degradation. Increasing the m6A methylation levels in vivo via dietary methionine supplementation mitigated cartilage damage. In summary, HT-2 toxin induced cartilage ECM degradation by regulating the METTL3-mediated m6A modification of Ctsk. These findings highlight the METTL3/m6A/Ctsk axis as a potential therapeutic target for the treatment of KBD and other cartilage-associated diseases.

Dynamic landscape of m6A modifications and related post-transcriptional events in muscle-invasive bladder cancer

BackgroundMuscle-invasive bladder carcinoma (MIBC) is a serious and more advanced stage of bladder carcinoma. N6-Methyladenosine (m6A) is a dynamic and reversible modifications that primarily affects RNA stability and alternative splicing. The dysregulation of m6A in MIBC can be potential target for clinical interventions, but there have been limited studies on m6A modifications in MIBC and their associations with post-transcriptional regulatory processes.MethodsPaired tumor and adjacent-normal tissues were obtained from three patients with MIBC following radical cystectomy. The additional paired tissues for validation were obtained from patients underwent transurethral resection. Utilizing Nanopore direct-RNA sequencing, we characterized the m6A RNA methylation landscape in MIBC, with a focus on identifying post-transcriptional events potentially affected by changes in m6A sites. This included an examination of differential transcript usage, polyadenylation signal sites, and variations in poly(A) tail length, providing insights into the broader impact of m6A alterations on RNA processing in MIBC.ResultsThe prognostic-related m6A genes and m6A-risk model constructed by machine learning enables the stratification of high and low-risk patients with precision. A novel m6A modification site in the 3’ untranslated region (3’UTR) of IGLL5 gene were identified, characterized by a lower m6A methylation ratio, elongated poly(A) tails, and a notable bias in transcript usage. Furthermore, we discovered two particular transcripts, VWA1-203 and CEBPB-201. VWA1-203 displayed diminished m6A methylation levels, a truncated 3’UTR, and an elongated poly(A) tail, whereas CEBPB-201 showed opposite trends, highlighting the complex interplay between m6A modifications and RNA processing. Source code was provided on GitHub (https://github.com/lelelililele/Nanopore-m6A-analysis).ConclusionsThe state-of-the-art Nanopore direct-RNA sequencing and machine learning techniques enables comprehensive identification of m6A modification and provided insights into the potential post-transcriptional regulation mechanisms on the development and progression in MIBC.

Evidence for the potential role of m6A modification in regulating autophagy in models of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease. Research indicates that N6-methyladenosine (m6A) modification plays a crucial role in cellular autophagy during ALS development. This study investigates the role of autophagy in ALS, with a focus on the effect of messenger ribonucleic acid m6A methylation modification on disease progression. We compared m6A levels and regulatory molecule expressions in transgenic superoxide dismutase (SOD1)-G93A and non-transgenic mice, categorized into end-stage and control groups, using quantitative polymerase chain reaction and Western blotting. The NSC-34 cell line, which was modified to model ALS, enabled the investigation of apoptosis, autophagy, and autophagy disruption through terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling assays, Western blotting, and fluorescent staining. Our findings indicate significantly elevated m6A methylation levels in ALS mice (0.262 ± 0.005) compared with the controls (0.231 ± 0.003) and in the ALS model cells (0.242±0.005) relative to those belonging to the wild-type control group (0.183 ± 0.007). Furthermore, the proteins involved in m6A RNA modification differed between groups, which suggest impaired autophagy flux in the ALS models. These results suggest that m6A methylation may accelerate ALS progression through the disruption of autophagic processes. Our study underscores the role of m6A methylation in the pathology of ALS and proposes the targeting of m6A methylation as a potential therapeutic strategy for disease treatment. Although this study primarily used transgenic SOD1-G93A mice and NSC-34 cell models to investigate ALS pathology, potential differences in disease mechanisms between animal models and humans must be considered. Although a correlation was detected between m6A methylation levels and autophagy disruption in ALS, the study primarily established an association rather than provided detailed mechanistic insights.

STM2457 Inhibits METTL3-Mediated m6A Modification of miR-30c to Alleviate Spinal Cord Injury by Inducing the ATG5-Mediated Autophagy.

The study aimed to investigate the role of N6-methyladenosine (m6A) modification in spinal cord injury (SCI) and its underlying mechanism, focusing on the interplay between m6A methyltransferase-like 3 (METTL3), miR-30c, and autophagy-related proteins. An SCI model was established in rats, and changes in autophagy-related proteins, m6A methylation levels, and miR-30c levels were analyzed. Hydrogen peroxide (H2O2)-stimulated spinal cord neuron cells (SCNCs) were used to assess the impact of METTL3 overexpression. The effects of STM2457, an antagonist of METTL3, were evaluated on cell viability, apoptosis, and autophagy markers in H2O2-stimulated SCNCs. In the SCI model, decreased levels of autophagy markers and increased m6A methylation, miR-30c levels, and METTL3 were observed. Overexpression of METTL3 in SCNCs led to reduced cell viability, increased apoptosis, and suppressed autophagy. Conversely, co-overexpression of autophagy-related protein 5 (ATG5) or miR-30c inhibition reversed these effects. Knocking out METTL3 yielded opposite results. STM2457 treatment improved cell viability, reduced apoptosis, and upregulated autophagy markers in SCNCs, which also enhanced functional recovery in rats as measured by the Basso-Beattie-Bresnahan score and inclined plate test. STM2457 alleviated SCI by suppressing METTL3-mediated m6A modification of miR-30c, which in turn induces ATG5-mediated autophagy. This study provides insights into the role of m6A modification in SCI and suggests a potential therapeutic approach through targeting METTL3.

RNA m6A methylation regulatory mechanism of resveratrol in premature senescence cells.

Resveratrol, a natural compound found in various plants, is known for its anti-inflammatory, antioxidant, and senescence-delaying properties. RNA N6-methyladenosine (m6A) methylation plays a crucial role in oxidative stress and premature cellular senescence processes and is closely associated with age-related disorders. However, the anti-premature senescence via RNA m6A methylation mechanism of resveratrol is still not fully understood. In this study, based on premature senescence model of human embryonic lung fibroblasts (HEFs) induced by hydrogen peroxide (H2O2), a widely accepted model of premature senescence caused by oxidative stress, we explored the anti-aging regulatory effects of resveratrol at the RNA m6A methylation level. Our data suggested that resveratrol significantly delayed premature senescence by increasing cell viability, reducing SA-β-gal blue staining rate, ROS levels, and senescence-associated secretory phenotypes (SASP) expression in HEFs. Meanwhile, resveratrol increased the whole RNA methyltransferases activity and the overall m6A level during senescence. Furthermore, three genes CCND2, E2F1, and GADD45B have been identified as the main ones regulating premature by resveratrol. Specifically, it decreased E2F1, GADD45B RNA m6A methylation level, and increased CCND2 level in premature cells. Our study provided new clues for exploring the mechanism and application of resveratrol in the field of premature aging.

Methyltransferase-Like 3-Mediated N6-Methyladenosine RNA Methylation Regulates Hypoxia-Induced Pulmonary Arterial Smooth Muscle Cell Pyroptosis by Targeting PTEN.

Pulmonary hypertension is a rare, progressive disorder that can lead to right ventricular hypertrophy, right heart failure, and even sudden death. N6-methyladenosine modification and the main methyltransferase that mediates it, methyltransferase-like (METTL) 3, exert important effects on many biological and pathophysiological processes. However, the role of METTL3 in pyroptosis remains unclear. Here, we characterized the role of METTL3 and the underlying cellular and molecular mechanisms of pyroptosis, which is involved in pulmonary hypertension. METTL3 was downregulated in a pulmonary hypertension mouse model and in hypoxia-exposed pulmonary artery smooth muscle cell. The small interfering RNA-induced silencing of METTL3 decreased the m6A methylation levels and promoted pulmonary artery smooth muscle cell pyroptosis, mimicking the effects of hypoxia. In contrast, overexpression of METTL3 suppressed hypoxia-induced pulmonary artery smooth muscle cell pyroptosis. Mechanistically, we identified the phosphate and tension homology deleted on chromosome 10 (PTEN) gene as a target of METTL3-mediated m6A modification, and methylated phosphate and tension homology deleted on chromosome 10 mRNA was subsequently recognized by the m6A "reader" protein insulin-like growth factor 2 mRNA-binding protein 2, which directly bound to the m6A site on phosphate and tension homology deleted on chromosome 10 mRNA and enhanced its stability. These results identify a new signaling pathway, the METTL3/phosphate and tension homology deleted on chromosome 10/insulin-like growth factor 2 mRNA-binding protein 2 axis, that participates in the regulation of hypoxia-induced pyroptosis.