mRNA Modification Research Articles

MRNA ADENOSINE METHYLASE promotes drought tolerance through N6-methyladenosine-dependent and independent impacts on mRNA regulation in Arabidopsis.

Among many mRNA modifications, adenine methylation at the N6 position (N6-methyladenosine, m6A) is known to affect mRNA biology extensively. The influence of m6A has yet to be assessed under drought, one of the most impactful abiotic stresses. We show that Arabidopsis thaliana (L.) Heynh. (Arabidopsis) plants lacking mRNA ADENOSINE METHYLASE (MTA) are drought-sensitive. Subsequently, we comprehensively assess the impacts of MTA-dependent m6A changes during drought on mRNA abundance, stability, and translation in Arabidopsis. During drought, there is a global trend toward hypermethylation of many protein-coding transcripts that does not occur in mta. We also observe complex regulation of m6A at a transcript-specific level, possibly reflecting compensation by other m6A components. Importantly, a subset of transcripts that are hypermethylated in an MTA-dependent manner exhibited reduced turnover and translation in mta, compared with wild-type (WT) plants, during drought. Additionally, MTA impacts transcript stability and translation independently of m6A. We also correlate drought-associated deposition of m6A with increased translation of modulators of drought response, such as RD29A, COR47, COR413, ALDH2B, ERD7, and ABF4 in WT, which is impaired in mta. m6A is dynamic during drought and, alongside MTA, promotes tolerance by regulating drought-responsive changes in transcript turnover and translation.

FSP1 Acts in Parallel with GPX4 to Inhibit Ferroptosis in COPD.

Glutathione peroxidase 4 (GPX4) has recently been reported to play an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD). Ferroptosis suppressor protein-1 (FSP1) is a protein that defends against ferroptosis in parallel with GPX4, but its role in the pathogenesis of COPD remains unexplored, and further research is needed. Normal and COPD lung tissues were obtained from lobectomy and lung transplant specimens, respectively. FSP1-overexpressing mice were established by monthly transfection with AAV9-FSP1 through modified intranasal administration. The expression of FSP1, GPX4, and prostaglandin-endoperoxide synthase 2 (PTGS2) was measured by Western blotting, immunohistochemistry and other methods. The correlation between FSP1 and ferroptosis and the role of FSP1 in COPD were explored by screening the expression of ferroptosis-related genes in a COPD cell model after the inhibition and overexpression of FSP1. We then explored the underlying mechanism of low FSP1 expression in patients with COPD in vitro by methylated RNA immunoprecipitation (MeRIP)-qPCR. We found that cigarette smoke exposure can lead to an increase in lipid peroxide production and ultimately ferroptosis, which is negatively regulated by FSP1 activity. FSP1 overexpression can prevent ferroptosis and alleviate emphysema. Next, we found that decreased FSP1 expression was caused by increased m6A modification of FSP1 mRNA. Moreover, the level of FSP1 decreased in a YTHDF2-dependent manner. These results indicate that METTL3-induced FSP1 mRNA methylation leading to low FSP1 expression is a potential therapeutic target for COPD. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).

METTL14 Promotes Ischemic Stroke-induced Brain Injury by Stabilizing HDAC3 Expression in an m6A-IGF2BP3 Mechanism.

N6-methyladenosine (m6A) modification is the most abundant post-transcriptional modification of mRNAs and has been identified to play critical roles in ischemic stroke (IS). Herein, this study aimed to investigate the function and mechanism of Methyltransferase-like 14 (METTL14) methylase in cerebral IS. Murine BV-2 microglial cell OGD/R models and rat middle cerebral artery occlusion (MCAO) models were established to mimic IS-induced neuronal damage in vitro and brain injury in vivo. Levels of METTL14, Histone Deacetylase 3 (HDAC3) and cGAS-STING axis-related proteins were detected using qRT-PCR or western blotting. Cell proliferation and inflammation were assessed by CCK-8 assay, EdU assay and ELISA. Flow cytometry detected microglia polarization. Cell pyroptosis was analyzed by detecting related-protein markers by western blotting. The m6A modification was determined by methylated RNA immunoprecipitation assay. Brain injury was analyzed by evaluating infarct volume and neurologic score. METTL14 levels were higher in OGD/R-induced microglial cells, primary microglia and infarct brain tissues of rat MCAO models. Functionally, METTL14 silencing reversed OGD/R-induced proliferation inhibition, inflammation and pyroptosis in microglial cells and primary microglia in vitro, and ameliorated cerebral ischemic injury in rat MCAO models. Mechanistically, METTL14 induced HDAC3 m6A modification in an IGF2BP3-dependent manner, and could activate cGAS-STING pathway through HDAC3. Moreover, HDAC3 overexpression reversed the neuroprotective effects of METTL14 silencing. METTL14 silencing reversed ischemic stroke-induced brain injury by inducing HDAC3 m6A modification in an IGF2BP3-dependent mechanism, recommending a novel insight for ameliorating cerebral ischemic stroke.

The deficiency of ALKBH5 contributes to hepatic lipid deposition by impairing VPS11-dependent autophagic flux.

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Hepatic lipid deposition is a key factor in the development of NAFLD. N6-methyladenosine (m6A) modification, the most prevalent mRNA modification in eukaryotic cells, plays an important role in regulating hepatic lipid metabolism. However, its potential role in hepatic lipid deposition remains poorly understood. Histological and immunohistochemistry studies were used to investigate lipid deposition in free fatty acids (FFAs)-incubated LO2 cells, high-fat diet-fed mice models and clinical samples. Stable overexpression and knockdown of AlkB homolog 5 (ALKBH5) was manipulated to investigate the effects of ALKBH5 on m6A methylation and lipid metabolism in hepatocytes. RNA-sequencing transcriptome analysis and methylated RNA immunoprecipitation-quantitative-PCR analysis were used to reveal the potential downstream molecular targets of ALKBH5. ALKBH5 was down-regulated in fatty liver compared to normal liver in both humans and mice. Overexpression of ALKBH5 significantly improved FFA-induced lipid accumulation and promoted autophagosome-lysosome fusion in hepatocytes. Meanwhile, knockdown of ALKBH5 significantly increased the expression of microtubule-associated protein 1A/1B-light chain 3B and Sequestosome 1, leading to impaired autophagic flux and further lipid deposition in hepatocytes under FFA incubation. Overexpression of vacuolar protein sorting 11 (VPS11) reversed FFA-induced lipid accumulation in ALKBH5-silenced hepatocytes. Mechanistically, ALKBH5 alleviated hepatic lipid deposition and impaired autophagic flux by removing the m6A modification on VPS11 mRNA to promote its translation. Collectively, our findings revealed an epigenetic mechanism by which ALKBH5 alleviates hepatic lipid deposition by restoring VPS11-dependent autophagic flux, providing a potential target to counteract NAFLD.

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.

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

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

ALKBH5 modulation of ferroptosis in recurrent miscarriage: implications in cytotrophoblast dysfunction.

As one of the most common and abundant internal modifications of eukaryotic mRNA, N6-methyladenosine (m6A) modifications are closely related to placental development. Ferroptosis is a newly discovered form of programmed cell death. During placental development, placental trophoblasts are susceptible to ferroptosis. However, the interactions of m6A and ferroptosis in trophoblast physiology and injury are unclear. Recurrent miscarriage (RM) was selected as the main gestational disease in this study. Published data (GSE76862) were used to analyze the gene expression profiles in patients with RM. The extent of m6A modification in total RNA of villous tissues between patients with RM and healthy controls (HC) was compared. ALKBH5 (encoding AlkB homolog 5, RNA demethylase) was selected as the candidate gene for further research. Quantitative real-time reverse transcription PCR, western blotting, and immunohistochemistry (IHC) confirmed the elevated expression of ALKBH5 in the cytotrophoblasts of patients with RM. Then, cell counting kit-8 assays, glutathione disulfide/glutathione quantification, 2',7'-dichlorfluorescein-diacetate staining, and malonaldehyde assays were used to explore the alterations of ferroptosis-related characteristics following RAS-selective lethal (RSL3) stimulation after overexpression of ALKBH5. Thereafter, we re-analyzed the published RNA sequencing data upon knockdown of ALKBH5, combined with published tissue RNA-seq data, and FTL (encoding ferritin light chain) was identified as the ferroptosis-related gene in cytotrophoblasts of patients with RM that is regulated by ALKBH5. Finally, western blotting and IHC confirmed the increased expression of FTL in the cytotrophoblasts from patients with RM. Total m6A levels were decreased in patients with RM. The most significant differentially m6A-related gene was ALKBH5, which was increased in patients with RM. In vitro cell experiments showed that treatment with RSL3 resulted in increased cell death and upregulated ALKBH5 expression. Overexpression of ALKBH5 alleviated RSL3-induced HTR8 cell death and caused decreased levels of intracellular oxidation products. Published transcriptome sequencing revealed that FTL was the major ferroptosis-related gene regulated by ALKBH5 in the villous tissues of patients with RM. Consistent with the expression of ALKBH5, FTL was increased by RSL3-induction and increased in patients with RM. Elevated ALKBH5 alleviated RSL3-induced cytotrophoblast cell death by promoting the expression of FTL in patients with RM. Our results supported the view that ALKBH5 is an important regulator of the ferroptosis-related etiology of RM and suggested that ALKBH5 could be responsible for epigenetic aberrations in RM pathogenesis.

The landscape of N1-methyladenosine (m1A) modification in mRNA of the decidua in severe preeclampsia.

Recent discoveries in mRNA modification have highlighted N1-methyladenosine (m1A), but its role in preeclampsia (PE) pathogenesis remains unclear. In this study, we utilized methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) to identify m1A peaks and the expression profile of mRNA in the decidua of humans with early-onset PE (EPE), late-onset PE (LPE), and normal pregnancy (NP). We assessed the m1A modification patterns in preeclamptic decidua using 10 m1A modulators. Our bioinformatic analysis focused on differentially methylated mRNAs (DMGs) and differentially expressed mRNAs (DEGs) in pairwise comparisons of EPE vs. NP, LPE vs. NP, and EPE vs. LPE, as well as m1A-related DEGs. The comparisons of EPE vs. NP, LPE vs. NP, and EPE vs. LPE identified 3110, 2801, and 2818 DMGs, respectively. We discerned three different m1A modification patterns from this data. Further analysis revealed that key PE-related DMGs and m1A-related DEGs predominantly influence signaling pathways critical for decidualization, including cAMP, MAPK, PI3K-Akt, Notch, and TGF-β pathways. Additionally, these modifications impact pathways related to vascular smooth muscle contraction, estrogen signaling, and relaxin signaling, contributing to vascular dysfunction. Our findings demonstrate that preeclamptic decidua exhibits unique mRNA m1A modification patterns and gene expression profiles that significantly alter signaling pathways essential for both decidualization and vascular dysfunction. These differences in m1A modification patterns provide valuable insights into the molecular mechanisms influencing the decidualization process and vascular function in the pathogenesis of PE. These m1A modification regulators could potentially serve as potent biomarkers or therapeutic targets for PE, warranting further investigation.

Self‐Initiated Nano‐Micelles Mediated Covalent Modification of mRNA for Labeling and Treatment of Tumors

AbstractAs a promising gene therapy strategy, controllable small molecule‐mRNA covalent modification in tumor cells could be initiated by singlet oxygen (1O2) to complete the modification process. However, in vivo generation of 1O2 is usually dependent on excitation of external light, and the limited light penetration of tissues greatly interferes the development of deep tumor photo therapy. Here, we constructed a tumor‐targeting nano‐micelle for the spontaneous intracellular generation of 1O2 without the need for external light, and inducing a high level of covalent modification of mRNA in tumor cells. Luminol and Ce6 were chemically bonded to produce 1O2 by chemiluminescence resonance energy transfer (CRET) triggered by high levels of hydrogen peroxide (H2O2) in the tumor microenvironment (TME). The sufficient 1O2 oxidized the loaded furan to highly reactive dicarbonyl moiety, which underwent cycloaddition reaction with adenine (A), cytosine (C) or guanine (G) on the mRNA for interfering with the tumor cell protein expression, thereby inhibiting tumor progression. In vitro and in vivo experiments demonstrated that this self‐initiated gene therapy nano‐micelle could induce covalent modification of mRNA by 1O2 without external light, and the process could be monitored in real time by fluorescence imaging, which provided an effective strategy for RNA‐based tumor gene therapy.

METTL14-mediated m6A modification upregulated SOCS3 expression alleviates thyroid cancer progression by regulating the JAK2/STAT3 pathway

Thyroid cancer (TC) is the most common malignant tumor of the head and neck. As a common epigenetic modification in mRNAs, N6-methyladenosine (m6A) modification plays critical roles in biological process of cancers. However, m6A methyltransferase methyltransferase-like 14 (METTL14)-mediated m6A modification and its potential regulatory mechanisms in TC are not fully elucidated. In our study, we observed that METTL14 was decreased in TC tissues and cells. And upregulation of METTL14 induced apoptotic cell death and hampered cell proliferation, epithelial mesenchymal transition (EMT) and tumor growth in vitro and in vivo. Mechanistically, METTL14 increased the expression of suppressor of cytokine signaling 3 (SOCS3) through m6A methylation modification, and knockdown of SOCS3 reversed the inhibitory effect of overexpressing METTL14 on TC tumorigenesis. In addition, METTL14-mediated m6A modification of SOCS3 inactivated the janus kinase 2 (JAK2)-signal transducer and activator of transcription 3 (STAT3) pathway, and in the METTL14-overexpressing TC cells, silencing SOCS3-induced upregulation of cell proliferation, EMT and suppression of apoptosis was reversed by JAK2/STAT3 inhibitor AG490 and WP1066. Together, we indicated that METTL14/m6A/SOCS3/ JAK2/STAT3 axis play an important role in the progression of TC.

Assessing substrate scope of the cyclodehydratase LynD by mRNA display-enabled machine learning models.

Many of the biosynthetic pathways for ribosomal synthesized and post-translationally modified peptide (RiPP) natural products make use of multi-domain enzymes with separate recruitment and catalysis domains that separately bind and modify peptide substrates. This "division of labor" allows RiPP enzymes to use relatively open and promiscuous active sites to perform chemistry at multiple residues within a peptide substrate seemingly regardless of the surrounding context. Defining, measuring, and predicting the seemingly broad substrate promiscuity of RiPPs necessitates high throughput assays, capable of assessing activity against very large libraries of peptides. Using mRNA display, a high throughput peptide display technology, we examine the substrate promiscuity of the RiPP cyclodehydratase, LynD. The vast substrate profiling that can be done with mRNA display enables the construction of deep learning models for accurate prediction of substrate processing by LynD. These models further inform on epistatic interactions involved in enzymatic processing. This work will facilitate the further elucidation of other RiPP enzymes and enable their use in the modification of mRNA display libraries for selection of modified peptide-based inhibitors and therapeutics.

RBM15 drives the progression of lung adenocarcinoma by regulating N6-methyladenosine-mediated LDHA mRNA stability

Abnormal N6-methyladenosine (m6A) methylation in RNA plays a pivotal role in the pathogenesis of many types of tumors by influencing mRNA metabolism, alternative splicing, translocation, stability and translation. However, the specific regulators and underlying mechanisms of m6A modification in the progression of lung adenocarcinoma are not well understood. In this study, we analyzed the RNA-seq transcriptome data downloaded from The Cancer Genome Atlas (TCGA) database, and identified “m6A writer” RNA binding motif protein 15 (RBM15) expression was significantly elevated in lung adenocarcinoma (LUAD) biopsies, and the higher RBM15 levels were correlated with the poorer overall survival (OS) of LUAD patients. Further study confirmed RBM15 was prominently expressed in LUAD tissues and cell lines. Moreover, silencing RBM15 in PC9 and H1299 cells reduced cell proliferation both in vitro and in vivo, while overexpression of RBM15 in A549 cells promoted cell growth. Mechanistically, lactate dehydrogenase A (LDHA) acted as a downstream target of RBM15. RBM15-mediated m6A modification of LDHA mRNA enhanced its stability to exert an oncogenic role in LUAD. Taken together, our findings suggest that the RBM15/LDHA axis might be a novel and promising therapeutic target for LUAD.

Genome-Wide Identification and Expression Analysis of the ALKB Homolog Gene Family in Potato (Solanum tuberosum L.).

N6-methyladenosine (m6A) is an abundant and pervasive post-transcriptional modification in eukaryotic mRNAs. AlkB homolog (ALKBH) proteins play crucial roles in RNA metabolism and translation, participating in m6A methylation modification to regulate plant development. However, no comprehensive investigations have been conducted on ALKBH in potato. Here, 11 StALKBH family genes were identified in potato and renamed according to BLASTP and phylogenetic analyses following the Arabidopsis genome. The characteristics, sequence structures, motif compositions, phylogenetics, chromosomal locations, synteny, and promoter cis-acting element predictions were analyzed, revealing distinct evolutionary relationships between potato and other species (tomato and Arabidopsis). Homologous proteins were classified into seven groups depending on similar conserved domains, which implies that they possess a potentially comparable function. Moreover, the StALKBHs were ubiquitous, and their expression was examined in the various tissues of a whole potato, in which the StALKBH genes, except for StALKBH2, were most highly expressed in the stolon and flower. Multiple hormone and stress-response elements were found to be located in the promoters of the StALKBH genes. Further qRT-PCR results suggest that they may be significantly upregulated in response to phytohormones and abiotic stress (except for cold), and the expression of most of the StALKBH genes exhibited positively modulated trends. Overall, this study is the first to report a genome-wide assessment of the ALKBH family in potato, providing valuable insights into candidate gene selection and facilitating in-depth functional analyses of ALKBH-mediated m6A methylation mechanisms in potato.

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.

M6A-driven NAT10 translation facilitates fatty acid metabolic rewiring to suppress ferroptosis and promote ovarian tumorigenesis through enhancing ACOT7 mRNA acetylation.

RNA epigenetic modifications have been implicated in cancer progression. However, the interplay between distinct RNA modifications and its role in cancer metabolism remain largely unexplored. Our study demonstrates that N-acetyltransferase 10 (NAT10) is notably upregulated in ovarian cancer (OC), correlating with poor patient prognosis. IGF2BP1 enhances the translation of NAT10 mRNA in an m6A-dependent manner in OC cells. NAT10 drives tumorigenesis by mediating N4-acetylcytidine (ac4C) modification of ACOT7 mRNA, thereby augmenting its stability and translation. This NAT10-ACOT7 axis modulates fatty acid metabolism in cancer cells and promotes tumor progression by suppressing ferroptosis. Additionally, our research identifies fludarabine as a small molecule inhibitor targeting NAT10, inhibits the ac4C modification and expression of ACOT7 mRNA. By using cell derived xenograft model and patient derived organoid model, we show that fludarabine effectively suppresses ovarian tumorigenesis. Overall, our study highlights the pivotal role of the NAT10-ACOT7 axis in the malignant cancer progression, underscoring the potential of targeting NAT10-mediated ac4C modification as a viable therapeutic strategy for this disease.

RNA m6A methyltransferase activator affects anxiety-related behaviours, monoamines and striatal gene expression in the rat.

Modification of mRNA by methylation is involved in post-transcriptional regulation of gene expression by affecting the splicing, transport, stability and translation of mRNA. Methylation of adenosine at N6 (m6A) is one of the most common and important cellular modification occurring in the mRNA of eukaryotes. Evidence that m6A mRNA methylation is involved in regulation of stress response and that its dysregulation may contribute to the pathogenesis of neuropsychiatric disorders is accumulating. We have examined the acute and subchronic (up to 18 days once per day intraperitoneally) effect of the first METTL3/METTL14 activator compound CHMA1004 (methyl-piperazine-2-carboxylate) at two doses (1 and 5 mg/kg) in male and female rats. CHMA1004 had a locomotor activating and anxiolytic-like profile in open field and elevated zero-maze tests. In female rats sucrose consumption and swimming in Porsolt's test were increased. Nevertheless, CHMA1004 did not exhibit strong psychostimulant-like properties: CHMA1004 had no effect on 50-kHz ultrasonic vocalizations except that it reduced the baseline difference between male and female animals, and acute drug treatment had no effect on extracellular dopamine levels in striatum. Subchronic CHMA1004 altered ex vivo catecholamine levels in several brain regions. RNA sequencing of female rat striata after subchronic CHMA1004 treatment revealed changes in the expression of a number of genes linked to dopamine neuron viability, neurodegeneration, depression, anxiety and stress response. Conclusively, the first-in-class METTL3/METTL14 activator compound CHMA1004 increased locomotor activity and elicited anxiolytic-like effects after systemic administration, demonstrating that pharmacological activation of RNA m6A methylation has potential for neuropsychiatric drug development.

Knockdown of SlYTHDF2 Accelerates Dark-Induced Tomato Leaf Senescence by Affecting the ABA Pathway.

N6-methyladenosine (m6A) is a widespread post-transcriptional modification in eukaryotic mRNAs. Proteins with the YTH structural domain act as m6A-binding proteins by recognizing the m6A modification and regulating mRNA through this recognition. In this study, SlYTHDF2, a prototypical m6A -binding protein gene in the YTH family was expressed in various tissues, and subcellular localization analyses indicated that the SlYTHDF2 protein was localized in the nucleus and cytoplasm. SlYTHDF2 knockout lines were obtained using CRISPR/Cas9 technology and showed the senesced leaves prematurely increased endogenous ABA accumulation compared with the wild type. Moreover, we found that dark promoted leaf senescence in SlYTHDF2 knockout lines and exogenous ABA further accelerated leaf senescence under dark conditions. The qRT-PCR analysis revealed significant alterations in the expression of genes associated with the ABA pathway. Relative to the wild type, the CR-slythdf2 plants exhibited reduced levels of photosynthetic pigments, higher accumulation of reactive oxygen species, and increased damage to cell membranes. Additionally, we discovered that SlYTHDF2 interacts with the chloroplast-binding protein SlRBCS3 through yeast two-hybrid and BiFC experiments. Overall, our data suggest the important role of SlYTHDF2 in regulating tomato leaf senescence.

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.

9250 carRNA m6A methylation regulates transcription state and chromatin accessibility in human islets in normal physiology and type 2 diabetes

Abstract Disclosure: N. Shukla: None. C. Ju: None. H. Li: None. C. He: None. R. Kulkarni: Advisory Board Member; Self; Novo Nordisk, Biomea Fusion Inc, REDD Pharma. Grant Recipient; Self; Inversago. Chromatin-associated regulatory RNAs (carRNA), which include promoter- and enhancer-associated RNAs as well as several non-coding repeat elements have been established in the literature as important regulators of global gene transcription. However, the regulation of carRNAs themselves has been an enigma. Recent publications from the He Lab at UChicago have shown the role of RNA N6-methyladenosine (m6A) in regulating carRNA stability and function. m6A is among the most abundant mRNA modifications in mammals. Seminal work from our lab has reported that the “writers” of m6A (e.g. methyltransferase-like [METTL3] and METTL14) are significantly downregulated in islets of humans with type 2 diabetes (T2D). Consequently, the global m6A landscape in the islets segregates the T2D from the control group significantly better than the transcriptome (n=7 for Control and n=8 for T2D islets). Hypomethylation of key transcripts in the insulin/IGF-1-AKT-PDX1 pathway led to impaired insulin secretion and decreased β-cell proliferation. We now show this decreased m6A on RNA is not limited to the mRNA but also affects chromatin-associated regulatory RNAs (carRNA). Intriguingly, downregulation of METTL3 and METTL14 in human β-cell line EndoC-βH1 differentially regulates these regulatory versus protein-coding transcripts. We observe that while METTL14 KD causes global downregulation of transcripts belonging to pathways important for β-cell identity and function (including calcium signaling, PI3K-AKT/MAPK signaling pathway, pancreatic secretion etc.), it leads to an upregulation of pathways involved in RNA processing and global gene transcription (Transcription by RNA Pol II, metabolism and splicing of mRNA, chromatin organization and modification etc.) {false discovery rate (FDR) < 0.05}. Considering carRNAs have been previously reported to regulate global chromatin accessibility and transcription, our observations on their ability to get differentially regulated m6A writer proteins provide novel insights into the epigenetic regulation of human islet cells. These findings provide opportunities to develop therapeutic approaches to regulate β-cell identity and/or insulin secretion in T2D. Presentation: 6/3/2024

7624 m6A mRNA Methylation in Brown Adipose Tissue Regulates Systemic Insulin Sensitivity via an Inter-Organ Prostaglandin Signaling Axis

Abstract Disclosure: L. Xiao: None. D. F De Jesus: None. C. Ju: None. J. Wei: None. J. Hu: None. A. DiStefano-Forti: None. T. Tsuji: None. C. Cero: None. V. Männistö: None. S.M. Manninen: None. S. Wei: None. O. Ijaduola: None. M. Blüher: Consulting Fee; Self; Amgen Inc, AstraZeneca, Bayer, Inc., Boehringer Ingelheim, Lilly USA, LLC, Novo Nordisk, Pfizer, Inc., Sanofi. A.M. Cypess: None. J. Pihlajamäki: None. Y. Tseng: None. C. He: Advisory Board Member; Self; Accent Therapeutics. R.N. Kulkarni: Advisory Board Member; Self; Novo Nordisk, Biomea, Inversago, REDD. Brown adipose tissue (BAT) has the capacity to regulate systemic metabolism through the secretion of “Batokines” including signaling lipids. N6-methyladenosine (m6A) is the most prevalent and abundant post-transcriptional mRNA modification and has been reported to regulate BAT adipogenesis and energy expenditure. Here, we demonstrate that the absence of the m6A writer, methyltransferase-like 14 (METTL14), modifies the BAT secretome to initiate inter-organ communication to improve systemic insulin sensitivity. Importantly, these phenotypes are independent of UCP1-mediated energy expenditure and thermogenesis. Using lipidomics, we identified prostaglandin E2 (PGE2) and prostaglandin F2a (PGF2a) as M14KO-BAT-secreted insulin sensitizers. Notably, circulatory PGE2 and PGF2a levels are inversely correlated with insulin sensitivity in humans. Furthermore, in vivo administration of PGE2 and PGF2a in high-fat diet-induced insulin-resistant obese mice recapitulates the phenotypes of Mettl14-deficient animals. PGE2 or PGF2a improves insulin signaling by suppressing the expression of specific AKT phosphatases. Mechanistically, METTL14-mediated m6A installation promotes decay of transcripts encoding prostaglandin synthases and their regulators in human and mouse brown adipocytes in a YTHDF2/3-dependent manner. Collectively, these findings reveal a novel biological mechanism in the m6A-dependent regulation of the BAT secretome in modulating systemic insulin sensitivity in both mice and humans. Presentation: 6/1/2024