Regulation of m6A (N6-Methyladenosine) methylation modifiers in solid cancers.

Solid cancers constitute a tremendous burden on global healthcare, requiring a deeper understanding of the molecular mechanisms underlying cancer development and progression. Epigenetic changes, notably N6-methyladenosine (m6A) RNA methylation, have emerged as important contributors to the biology of solid tumors in recent years. This epigenetic mark dynamically affects gene expression at the post-transcriptional level and modulates a variety of cellular processes, making it a focus of research in the context of solid tumors. m6A modification patterns are dysregulated in a variety of solid cancers, including ovarian, breast, lung, colorectal, pancreatic, and others. This dysregulated m6A landscape has been shown to induce significant changes in the expression of oncogenes, tumor suppressors, and genes involved in cancer stem cells, metastasis, and treatment resistance. In solid tumors, the interaction of m6A "writers" (e.g., METTL3, METTL14, and others), "erasers" (e.g., ALKBH5, FTO), and "readers" (e.g., members of YTHDF proteins and others) delicately changes the m6A methylome. Targeting m6A regulators as a potential therapeutic method to control gene expression and prevent tumor development seems a novel strategy. To enhance treatment results, advances in this area of research have led to the development of targeted treatments aiming at restoring or altering m6A alteration patterns in solid tumors.

The indispensability of methyltransferase-like 3 in the immune system: from maintaining homeostasis to driving function.

Methyltransferase-like 3(METTL3), recognized as the primary N6-methyladenosine methyltransferase, influences cellular functions such as proliferation, migration, invasion, differentiation, and fate determination by regulating gene expression post-transcriptionally. Recent studies have highlighted the indispensability of METTL3 in various immune cells such as hematopoietic stem/progenitor cells, innate immune cells (monocytes, macrophages, dendritic cells), and adaptive immune cells (thymic epithelial cell, T cells, natural killer cells). However, a comprehensive summary and analysis of these findings to elucidate the relationship between METTL3 and the immune system is yet to be undertaken. Therefore, in this review, we systematically collate reports detailing the mechanism underlying the role of METTL3 in regulating various immune processes and examine the modification of METTL3 and its potential implications. This review suggests that METTL3 plays an essential role in the immune system, ranging from maintaining homeostasis to regulating functions. Collectively, this review provides a comprehensive analysis of the relationship between METTL3 and the immune system, serving convenient researchers to understand the frontiers of immunological research and facilitate future clinical applications.

Decoding Substrate Selectivity of an Archaeal RlmCD-like Methyltransferase Through Its Salient Traits.

5-Methyluridine (m5U) rRNA modifications frequently occur at U747 and U1939 (Escherichia coli numbering) in domains II and IV of the 23S rRNA in Gram-negative bacteria, with the help of S-adenosyl-l-methionine (SAM)-dependent rRNA methyltransferases (MTases), RlmC and RlmD, respectively. In contrast, Gram-positive bacteria utilize a single SAM-dependent rRNA MTase, RlmCD, to modify both corresponding sites. Notably, certain archaea, specifically within the Thermococcales group, have been found to possess two genes encoding SAM-dependent archaeal (tRNA and rRNA) m5U (Arm5U) MTases. Among these, a tRNA-specific Arm5U MTase (PabTrmU54) has already been characterized. This study focused on the structural and functional characterization of the rRNA-specific Arm5U MTase from the hyperthermophilic archaeon Pyrococcus horikoshii (PhRlmCD). An in-depth structural examination revealed a dynamic hinge movement induced by the replacement of the iron-sulfur cluster with disulfide bonds, obstructing the substrate-binding site. It revealed distinctive characteristics of PhRlmCD, including elongated positively charged loops in the central domain and rotational variations in the TRAM domain, which influence substrate selectivity. Additionally, the results suggested that two potential mini-rRNA fragments interact in a similar manner with PhRlmCD at a positively charged cleft at the interface of domains and facilitate dual MTase activities akin to the protein RlmCD. Altogether, these observations showed that Arm5U MTases originated from horizontal gene transfer events, most likely from Gram-positive bacteria.

Methyltransferase METTL3 regulates neuropathic pain through m6A methylation modification of SOCS1.

The mechanisms of neuropathic pain (NP) are considered multifactorial. Alterations in the suppressor of cytokine signaling 1 (SOCS1) play a critical role in neural damage and inflammation. Epigenetic RNA modifications, specifically N6-methyladenosine (m6A) methylation, have increasingly been observed to impact the nervous system. Nevertheless, there is a scarcity of studies investigating the connection between m6A methylation and SOCS1 in the molecular mechanisms of NP. This study investigates the roles and potential mechanisms of the m6A methyltransferase like 3 (METTL3) and SOCS1 in female rats with spinal nerve ligation (SNL)-induced NP. It was found that in NP, both METTL3 and overall m6A levels were downregulated, leading to the activation of pro-inflammatory cytokines, such as interleukin-1ß, interleukin 6, and tumor necrosis factor-α. Notably, The SOCS1 mRNA is significantly enriched with m6A methylation modifications, with the most prevalent m6A methyltransferase METTL3 stabilizing the downregulation of SOCS1 by targeting m6A methylation modifications at positions 151, 164, and 966.Exogenous supplementation of METTL3 improved NP-related neuroinflammation and behavioral dysfunctions, but these effects could be reversed by the absence of SOCS1. Additionally, the depletion of endogenous SOCS1 promoted NP progression by inducing the toll-like receptor 4 (TLR4) signaling pathway. The dysregulation of METTL3 and the resulting m6A modification of SOCS1 form a crucial epigenetic regulatory loop that promotes the progression of NP. Targeting the METTL3/SOCS1 axis might offer new insights into potential therapeutic strategies for NP.

METTL3-Mediated N 6 -Methyladenosine mRNA Modification and cGAS-STING Pathway Activity in Kidney Fibrosis.

Background: Chemical modifications on RNA profoundly affect RNA function and regulation. m6A, the most abundant RNA modification in eukaryotes, plays a pivotal role in diverse cellular processes and disease mechanisms. However, its importance is understudied in human CKD samples regarding its influence on pathological mechanisms. Methods: Liquid chromatography­tandem mass spectrometry and methylated RNA immunoprecipitation sequencing were used to examine alterations in m6A levels and patterns in CKD samples. Overexpression of the m6A writer METTL3 in cultured kidney tubular cells was performed to confirm the effect of m6A in tubular cells and explore the biological functions of m6A modification on target genes. In addition, tubule-specific deletion of Mettl3 (Ksp-Cre Mettl3f/f) mice and antisense oligonucleotides inhibiting Mettl3 expression were used to reduce m6A modification in an animal kidney disease model. Results: By examining 127 human CKD samples, we observed a significant increase in m6A modification and METTL3 expression in diseased kidneys. Epitranscriptomic analysis unveiled an enrichment of m6A modifications in transcripts associated with the activation of inflammatory signaling pathways, particularly the cyclic guanosine monophosphate­AMP synthase (cGAS)-stimulator of IFN genes (STING) pathway. m6A hypermethylation increased mRNA stability in cGAS and STING1 as well as elevated the expression of key proteins within the cGAS-STING pathway. Both the tubule-specific deletion of Mettl3 and the use of antisense oligonucleotides to inhibit Mettl3 expression protected mice from inflammation, reduced cytokine expression, decreased immune cell recruitment, and attenuated kidney fibrosis. Conclusions: Our research revealed heightened METTL3-mediated m6A modification in fibrotic kidneys, particularly enriching the cGAS-STING pathway. This hypermethylation increased mRNA stability for cGAS and STING1, leading to sterile inflammation and fibrosis.

N6-methyladenosine-mediated upregulation of LNCAROD confers radioresistance in esophageal squamous cell carcinoma through stabilizing PARP1.

BACKGROUND: Radiotherapy is a primary therapeutic modality for esophageal squamous cell carcinoma (ESCC), but its effectiveness is still restricted due to the resistance of cancer cells to radiation. Long non-coding RNAs (lncRNAs) and N6-methyladenosine (m6A) have been shown to play significant roles in tumour radioresistance. However, the precise manifestation and role of m6A-modified lncRNAs in ESCC radioresistance remain unclear. METHODS: Bioinformatics analysis was conducted to identify m6A-modified lncRNAs implicated in the radioresistance of ESCC. A series of functional experiments were performed to investigate the function of LNCAROD in ESCC. Methylated RNA immunoprecipitation, chromatin isolation by RNA purification-mass spectrometry, RNA immunoprecipitation, and co-immunoprecipitation experiments were performed to explore the mechanism of m6A-mediated upregulation of LNCAROD expression and the downstream mechanism enhancing the radioresistance of ESCC. The efficacy of LNCAROD in vivo was assessed using murine xenograft models. RESULTS: Herein, we identified LNCAROD as a novel METTL3-mediated lncRNA that enhanced radioresistance in ESCC cells and was post-transcriptionally stabilised by YTHDC1. Moreover, we confirmed that LNCAROD prevented ubiquitin-proteasome degradation of PARP1 protein by facilitating PARP1-NPM1 interaction, thereby contributing to homologous recombination-mediated DNA double-strand breaks repair and enhancing the radiation resistance of ESCC cells. Silencing LNCAROD in a nude mouse model of ESCC in vivo resulted in slower tumour growth and increased radiosensitivity. CONCLUSION: Our findings enhance the understanding of m6A-modified lncRNA-driven machinery in ESCC radioresistance and underscore the significance of LNCAROD in this context, thereby contributing to the development of a potential therapeutic target for ESCC patients.

Stabilization of RRBP1 mRNA via an m6A-dependent manner in prostate cancer constitutes a therapeutic vulnerability amenable to small-peptide inhibition of METTL3.

Mounting evidence has implicated the RNA m6A methylation catalyzed by METTL3 in a wide range of physiological and pathological processes, including tumorigenesis. The detailed m6A landscape and molecular mechanism of METTL3 in prostate cancer (PCa) remains ill-defined. We find that METTL3 is overexpressed in PCa and correlates with worse patient survival. Functional studies establish METTL3 as an oncoprotein dependent on its m6A enzymatic activity in both AR+ and AR- PCa cells. To dissect the regulatory network of m6A pathway in PCa, we map the m6A landscape in clinical tumor samples using m6A-seq and identify genome-wide METTL3-binding transcripts via RIP-seq. Mechanistically, we discover RRBP1 as a direct METTL3 target in which METTL3 stabilizes RRBP1 mRNA in an m6A-dependent manner. RRBP1 positively correlates with METTL3 expression in PCa cohorts and exerts an oncogenic role in aggressive PCa cells. Leveraging the 3D structural protein-protein interaction between METTL3 and METTL14, we successfully develop two potential METTL3 peptide inhibitors (RM3 and RSM3) that effectively suppress cancer cell proliferation in vitro and tumor growth in vivo. Collectively, our study reveals a novel METTL3/m6A/RRBP1 axis in enhancing aggressive traits of PCa, which can be therapeutically targeted by small-peptide METTL3 antagonists.

Intracellular C5aR1 inhibits ferroptosis in glioblastoma through METTL3-dependent m6A methylation of GPX4.

Glioblastoma (GBM) is the most common primary intracranial malignant tumor. Recent literature suggests that induction of programmed death has become a mainstream cancer treatment strategy, with ferroptosis being the most widely studied mode. Complement C5a receptor 1 (C5aR1) is associated with both tumorigenesis and tumor-related immunity. However, knowledge regarding the role of C5aR1 in GBM progression is limited. In the present study, we observed significant upregulation of C5aR1 in glioma tissue. In addition, C5aR1 expression was found to be closely associated with patient prognosis and survival. Subsequent experimental verification demonstrated that C5aR1 promoted the progression of GBM mainly by suppressing ferroptosis induction, inhibiting the accumulation of lipid peroxides, and stabilizing the expression of the core antiferroptotic factor glutathione peroxidase 4 (GPX4). Aberrant N6-methyladenosine (m6A) modification of GPX4 mRNA contributes significantly to epigenetic tumorigenesis, and here, we report that selective methyltransferase-like 3 (METTL3)-dependent m6A methylation of GPX4 plays a key role in C5AR1 knockdown-induced ferroptosis induction. Mechanistically, ERK1/2 signaling pathway activation increases the METTL3 protein abundance in GBM cells. This activation then increases the stability of METTL3-mediated m6A modifications on GPX4, enabling it to fulfill its transcriptional function. More importantly, in an intracranial xenograft mouse model, PMX205, a C5aR1 inhibitor, promoted alterations in ferroptosis in GBM cells and inhibited GBM progression. In conclusion, our findings suggest that C5aR1 inhibits ferroptosis in GBM cells and promotes MettL3-dependent GPX4 expression through ERK1/2, thereby promoting glioma progression. Our study reveals a novel mechanism by which the intracellular complement receptor C5aR1 suppresses ferroptosis induction and promotes GBM progression. These findings may facilitate the identification of a potential therapeutic target for glioma.

Identification of a polyphenol O-methyltransferase with broad substrate flexibility in Streptomyces albidoflavus J1074.

Flavonoids are a large and important group of phytochemicals with a great variety of bioactivities. The addition of methyl groups during biosynthesis of flavonoids and other polyphenols enhances their bioactivities and increases their stability. In a previous study of our research group, we detected a novel flavonoid O-methyltransferase activity in Streptomyces albidoflavus J1074, which led to the heterologous biosynthesis of homohesperetin from hesperetin in feeding cultures. In this study, we identify the O-methyltransferase responsible for the generation of this methylated flavonoid through the construction of a knockout mutant of the gene XNR_0417, which was selected after a blast analysis using the sequence of a caffeic acid 3'-O-methyltransferase from Zea mays against the genome of S. albidoflavus J1074. This mutant strain, S. albidoflavus ∆XNR_0417, was no longer able to produce homohesperetin after hesperetin feeding. Subsequently, we carried out a genetic complementation of the mutant strain in order to confirm that the enzyme encoded by XNR_0417 is responsible for the observed O-methyltransferase activity. This new strain, S. albidoflavus SP43-XNR_0417, was able to produce not only homohesperetin from hesperetin, but also different mono-, di-, tri- and tetra-methylated derivatives on other flavanones, flavones and stilbenes, revealing a broad substrate flexibility. Additionally, in vitro experiments were conducted using the purified enzyme on the substrates previously tested in vivo, demonstrating doubtless the capability of XNR_0417 to generate various methylated derivatives.

25SrRNA Methyltransferase CgBMT5 From Candida glycerinogenes Improves Tolerance and Fermentation Performance of Saccharomyces cerevisiae and Yarrowia lipolytica From Undetoxified Cellulose Hydrolysate.

The hydrolysis of cellulose generates inhibitors like acetate, suppressing fermentation performance. Here, 25SrRNA methyltransferase CgBMT5 from stress-tolerant yeast Candida glycerinogenes was used as an anti-stress gene element in Saccharomyces cerevisiae and Yarrowia lipolytica. Expression of CgBMT5 in S. cerevisiae increased cell tolerance to acetate, high osmolarity, and heat stress and rescued the delay in cell growth under acetate stress. Ethanol productivity was improved from 0.52 g·(L/h) to 0.69 g·(L/h). CgBMT5 improved GFP expression. The transcription factor ARG81 binds to the promoter of CgBMT5. CgBMT5 upregulated HOG1, GPD1, HAA1, and PMA1 and reduced ROS level, thereby improving cell resistance to acetate. CgBMT5 also improved resistance of Y. lipolytica Po1g to multiple-stress. The lipid titer was improved by 37% in the typical medium. Y. lipolytica-CgBMT5 produced 94 mg/L lipid in the undetoxified cellulose hydrolysate.

N6-methyladenosine modification-tuned lipid metabolism controls skin immune homeostasis via regulating neutrophil chemotaxis.

Disrupted N6-methyladenosine (m6A) modification modulates various inflammatory disorders. However, the role of m6A in regulating cutaneous inflammation remains elusive. Here, we reveal that the m6A and its methyltransferase METTL3 are down-regulated in keratinocytes in inflammatory skin diseases. Inducible deletion of Mettl3 in murine keratinocytes results in spontaneous skin inflammation and increases susceptibility to cutaneous inflammation with activation of neutrophil recruitment. Therapeutically, restoration of m6A alleviates the disease phenotypes in mice and suppresses inflammation in human biopsy specimens. We support a model in which m6A modification stabilizes the mRNA of the lipid-metabolizing enzyme ELOVL6 via the m6A reader IGF2BP3, leading to a rewiring of fatty acid metabolism with a reduction in palmitic acid accumulation and, consequently, suppressing neutrophil chemotaxis in cutaneous inflammation. Our findings highlight a previously unrecognized epithelial-intrinsic m6A modification-lipid metabolism pathway that is essential for maintaining epidermal and immune homeostasis and lay the basis for potential therapeutic targeting of m6A modulators to attenuate inflammatory skin diseases.

Structural insights into phosphatidylethanolamine N-methyltransferase PmtA mediating bacterial phosphatidylcholine synthesis.

Phosphatidylethanolamine N-methyltransferase (PmtA) catalyzes the biosynthesis of phosphatidylcholine (PC) from phosphatidylethanolamine (PE). Although PC is one of the major phospholipids constituting bilayer membranes in eukaryotes, certain bacterial species encode PmtA, a membrane-associated methyltransferase, to produce PC, which is correlated with cellular stress responses, adaptability to environmental changes, and symbiosis or virulence with eukaryotic hosts. Depending on the organism, multiple PmtAs may be required for producing monomethyl- and dimethyl-PE derivatives along with PC, whereas in organisms such as Rubellimicrobium thermophilum, a single enzyme is sufficient to direct all three methylation steps. In this study, we present the x-ray crystal structures of PmtA from R. thermophilum in complex with dimethyl-PE and S-adenosyl-l-homocysteine, as well as in its lipid-free form. Moreover, we demonstrate that the enzyme associates with the cellular membrane via electrostatic interactions facilitated by a group of critical basic residues and can successively methylate PE and its methylated derivatives, culminating in the production of PC.

Elucidation of Spartina dimethylsulfoniopropionate synthesis genes enables engineering of stress tolerant plants.

The organosulfur compound dimethylsulfoniopropionate (DMSP) has key roles in stress protection, global carbon and sulfur cycling, chemotaxis, and is a major source of climate-active gases. Saltmarshes are global hotspots for DMSP cycling due to Spartina cordgrasses that produce exceptionally high concentrations of DMSP. Here, in Spartina anglica, we identify the plant genes that underpin high-level DMSP synthesis: methionine S-methyltransferase (MMT), S-methylmethionine decarboxylase (SDC) and DMSP-amine oxidase (DOX). Homologs of these enzymes are common in plants, but differences in expression and catalytic efficiency explain why S. anglica accumulates such high DMSP concentrations and other plants only accumulate low concentrations. Furthermore, DMSP accumulation in S. anglica is consistent with DMSP having a role in oxidative and osmotic stress protection. Importantly, administration of DMSP by root uptake or over-expression of Spartina DMSP synthesis genes confers plant tolerance to salinity and drought offering a route for future bioengineering for sustainable crop production.

METTL3-mediated m6A modification enhances lncRNA H19 stability to promote endothelial cell inflammation and pyroptosis to aggravate atherosclerosis.

This study explored the impact of N6-methyladenosine (m6A) modification on the regulation of long noncoding RNA (lncRNA) and atherosclerosis progression. An atherosclerosis cell model was established by treating human aortic endothelial cells (HAECs) with oxidized low-density lipoprotein. Additionally, an atherosclerotic animal model was developed using ApoE-/- C57BL/6 male mice fed a high-fat diet. Both models were employed to assess the expression changes of proteins associated with m6A modification. First, the effect of m6A modification writer protein methyltransferase-like 3 (METTL3) knockdown on changes in the level of pyroptosis in HAECs was investigated, and bioinformatic analysis confirmed that lncRNA H19 (H19) was the potential target of m6A modification. RNA-binding protein immunoprecipitation assays were subsequently performed to explore the interaction between H19 and the m6A writer protein METTL3, as well as the reader protein recombinant insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2). Finally, the effect of H19 expression on pyroptosis levels in HAECs was evaluated. In the aortas of atherosclerosis mice, overall m6A levels were significantly elevated compared with controls (p < .05), with METTL3 and METTL14 mRNA and protein levels notably increased (p < .05). Similarly, ox-LDL-treated HAECs showed a significant rise in m6A levels, along with increased METTL3 and METTL14 expression (p < .05). METTL3 knockdown in HAECs led to decreased pyroptosis, as evidenced by reduced lactate dehydrogenase release and lower levels of IL-1ß, IL-18, and IL-6 (p < .05). Overexpression of H19 reversed these effects, indicating METTL3's role in promoting atherosclerosis by stabilizing H19 through m6A modification. H19 was the primary target lncRNA molecule of METTL3-mediated m6A modification in the pathogenesis of atherosclerosis. METTL3-mediated m6A modification regulated H19 expression, thereby aggravating atherosclerosis by activating pyroptosis.

Nr-CWS regulates METTL3-mediated m6A modification of CDS2 mRNA in vascular endothelial cells and has prognostic significance.

Metabolic memory (MM) is a major factor in the delayed wound healing observed in diabetic patients. While "Nocardia rubrum cell wall skeleton" (Nr-CWS) is utilized to enhance macrophage proliferation in immune diseases, its impact on MM wounds in diabetes is unclear. This study demonstrates that transient hyperglycemia leads to prolonged damage in vascular endothelial cells by decreasing METTL3 expression, leading to decreased RNA methylation and impaired cellular metabolism. Remarkably, Nr-CWS application increases METTL3 levels in these cells, facilitating the recovery of cell function. Further in vivo and in vitro analyses demonstrate that transient hyperglycemia-induced reduction in METTL3 hinders RNA methylation of the downstream gene Cds2, impacting mitochondrial function and energy metabolism and consequently reducing angiogenic capacity in endothelial cells. This impairment significantly influences diabetic wound healing. Our findings highlight the profound impact of transient hyperglycemia on wound healing, establishing METTL3 as a significant role in vascular complications of diabetes. This study not only elucidates the pathophysiological mechanisms behind MM in diabetic wounds but also suggests Nr-CWS as a potential therapeutic agent, offering a novel approach for treating diabetic wounds.

PSAT1 is upregulated by METTL3 to attenuate high glucose-induced retinal pigment epithelial cell apoptosis and oxidative stress.

BACKGROUND: Diabetic retinopathy (DR) is a major ocular complication of diabetes mellitus, and a significant cause of visual impairment and blindness in adults. Phosphoserine aminotransferase 1 (PSAT1) is an enzyme participating in serine synthesis, which might improve insulin signaling and insulin sensitivity. Furthermore, it has been reported that the m6A methylation in mRNA controls gene expression under many physiological and pathological conditions. Nevertheless, the influences of m6A methylation on PSAT1 expression and DR progression at the molecular level have not been reported. METHODS: High-glucose (HG) was used to treat human retinal pigment epithelial cells (ARPE-19) to construct a cell injury model. PSAT1 and Methyltransferase-like 3 (METTL3) levels were detected by real-time quantitative polymerase chain reaction (RT-qPCR). PSAT1, B-cell lymphoma-2 (Bcl-2), Bcl-2 related X protein (Bax), and METTL3 protein levels were examined by western blot assay. Cell viability and apoptosis were detected by Cell Counting Kit-8 (CCK-8) and TUNEL assays. Reactive oxygen species (ROS), malondialdehyde (MDA), and Glutathione peroxidase (GSH-Px) levels were examined using special assay kits. Interaction between METTL3 and PSAT1 was verified using methylated RNA immunoprecipitation (MeRIP) and dual-luciferase reporter assay. RESULTS: PSAT1 and METTL3 levels were decreased in DR patients and HG-treated ARPE-19 cells. Upregulation of PSAT1 might attenuate HG-induced cell viability inhibition and apoptosis and oxidative stress promotion in ARPE-19 cells. Moreover, PSAT1 was identified as a downstream target of METTL3-mediated m6A modification. METTL3 might improve the stability of PSAT1 mRNA via m6A methylation. CONCLUSION: METTL3 might mitigate HG-induced ARPE-19 cell damage partly by regulating the stability of PSAT1 mRNA, providing a promising therapeutic target for DR.

METTL3 mediates m6A modification of lncRNA CRNDE to promote ATG10 expression and improve brain ischemia/reperfusion injury through YTHDC1.

BACKGROUND: Ischemia/reperfusion (I/R) injury is a severe brain disorder with currently limited effective treatments. This study aims to explore the role of N6-methyladenosine (m6A) modification and associated regulatory factors in I/R to identify potential therapeutic targets. METHODS: We utilized a middle cerebral artery occlusion (MCAO) rat model and SH-SY5Y cells subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) to assess m6A levels and investigate the impact of METTL3 overexpression on long non-coding RNA (lncRNA) CRNDE expression. The effects of silencing lncRNA CRNDE on the interaction between YTHDC1 and ATG10 mRNA, as well as the stability of ATG10 mRNA, were evaluated. Additionally, apoptosis rates, pro-inflammatory and anti-inflammatory factor levels, ATG10 expression, and autophagic activity were analyzed to determine the effects of METTL3. The reverse effects of YTHDC1 overexpression were also examined. RESULTS: MCAO rats and OGD/R-treated SH-SY5Y cells exhibited reduced m6A levels. METTL3 overexpression significantly inhibited lncRNA CRNDE expression. Silencing lncRNA CRNDE mitigated OGD/R-induced apoptosis and inflammation in SH-SY5Y cells, while enhancing autophagy and stabilizing ATG10 mRNA. METTL3 overexpression decreased cell apoptosis, reduced the levels of pro-inflammatory cytokines TNF-α, IL-1ß, IL-6, and increased IL-10 secretion. Furthermore, METTL3 overexpression upregulated ATG10 expression and promoted autophagy. Conversely, lncRNA CRNDE overexpression negated these effects. CONCLUSION: The inhibition of lncRNA CRNDE affects the interaction between YTHDC1 and ATG10 mRNA and stabilizes ATG10 mRNA, mediated by METTL3 overexpression. These findings suggest that targeting lncRNA CRNDE to reduce apoptosis, inhibit inflammation, increase ATG10 expression, and enhance autophagy could offer new therapeutic strategies for I/R injury.

Cap-Specific m6Am Methyltransferase PCIF1/CAPAM Regulates mRNA Stability of RAB23 and CNOT6 through the m6A Methyltransferase Activity.

Chemical modifications of cellular RNAs play key roles in gene expression and host defense. The cap-adjacent N6,2'-O-dimethyladenosine (m6Am) is a prevalent modification of vertebrate and viral mRNAs and is catalyzed by the newly discovered N6 methyltransferase PCIF1. However, its role in gene expression remains unclear due to conflicting reports on its effects on mRNA stability and translation. In this study, we investigated the impact of siRNA-mediated transient suppression of PCIF1 on global mRNA expression in HeLa cells. We identified a subset of differentially expressed genes (DEGs) that exhibited minimal overlap with previously reported DEGs. Subsequent validation revealed that PCIF1 positively and negatively regulates RAB23 and CNOT6 expression, respectively, at both the mRNA and protein levels. Mechanistic analyses demonstrated that PCIF1 regulates the stability of these target mRNAs rather than their transcription, and rescue experiments confirmed the requirement of PCIF1's methyltransferase activity for these regulations. Furthermore, MeRIP-qPCR analysis showed that PCIF1 suppression significantly reduced the m6A levels of RAB23 and CNOT6 mRNAs. These findings suggest that PCIF1 regulates the stability of specific mRNAs in opposite ways through m6A modification, providing new insights into the role of m6Am in the regulation of gene expression.

Regulation of renal ischemia-reperfusion injury and tubular epithelial cell ferroptosis by pparγ m6a methylation: mechanisms and therapeutic implications.

This study aimed to elucidate the role and underlying mechanisms of Peroxisome proliferator-activated receptor gamma (PPARγ) and its m6A methylation in renal ischemia-reperfusion (I/R) injury and ferroptosis of tubular epithelial cells (TECs). High-throughput transcriptome sequencing was performed on renal tissue samples from I/R injury models and sham-operated mice, complemented by in vivo and in vitro experiments focusing on the PPARγ activator Rosiglitazone and the manipulation of METTL14 and IGF2BP2 expression. Key evaluations included renal injury assessment, ferroptosis indicator measurement, and m6A methylation analysis of PPARγ. Our findings highlight the critical role of the PPARγ pathway and ferroptosis in renal I/R injury, with Rosiglitazone ameliorating renal damage and TEC ferroptosis. METTL14-mediated m6A methylation of PPARγ, dependent on IGF2BP2, emerged as a pivotal regulator of PPARγ expression, renal injury, and ferroptosis. This study reveals that PPARγ m6A methylation, orchestrated by METTL14 through an IGF2BP2-dependent mechanism, plays a crucial role in mitigating renal I/R injury and TEC ferroptosis. These insights offer promising avenues for therapeutic strategies targeting acute kidney injury.

The RNA m5C methyltransferase NSUN1 modulates human malaria gene expression during intraerythrocytic development.

Introduction: Plasmodium falciparum is the most damaging malaria pathogen and brings a heavy burden to global health. Host switching and morphological changes in P. falciparum are dependent on an effective gene expression regulatory system. C5 methylation of cytosines is a common RNA modification in eukaryotes, and the NSUN family are essential m5C modification executors. Currently, little is known about this family in Plasmodium spp. In this study, we focus on exploring the function of PfNSUN1 protein. Methods: An efficient CRISPR/Cas9 gene editing technique was applied to construct the PfNSUN1 knockdown strain. The knockdown efficiency was confirmed by growth curves and western blot experiments. The knockdown transcriptome data was acquired to find differentially expressed genes, and target genes of PfNSUN1 protein were identified by RNA immunoprecipitation and high-throughput sequencing experiments. Results: The efficiency of PfNSUN1 protein down-regulated was about 34%. RNA-seq data revealed that differentially expressed genes were mainly down-regulated. And there were 224, 278, 556 genes that were down-regulated with more than 2-fold changes and p-adj<0.05 at ring, trophozoite and schizont stages, respectively. PfNSUN1 protein was significantly enriched on 154 target genes, including 28S ribosomal RNA and pfap2-g5 transcription factor. Discussion: PfNSUN1 is a crucial RNA post-transcriptional modification protein in P. falciparum. It plays a pivotal role in regulating gene expression and parasite growth by targeting 28S ribosomal RNA and pfap2-g5 transcription factor.