Methyladenosine Research Articles

Novel Regulatory Mechanisms for Expression of Drug Metabolism-related Factors

Interindividual differences in the expression and activity of drug-metabolizing enzymes, including cytochrome P450, UDP-glucuronosyltransferase, and esterases, cause variability of therapeutic effectiveness and side effects during drug treatment. Conventional research has focused on transcriptional regulation by transcription factors and nuclear receptors such as aryl hydrocarbon receptor, pregnane X receptor (PXR), constitutive androstane receptor, and hepatocyte nuclear factor 4α, as the major mechanisms causing the differences in the expression of drug-metabolizing enzymes. Recently, we have revealed that adenosine-to-inosine RNA editing and methylation of adenosine at the N6 position on RNA, two major types of posttranscriptional modification, play a pivotal role in the regulation of drug metabolism. In addition, switch/sucrose non-fermentable complex, a chromatin remodeler, is required for PXR-mediated transcriptional regulation of drug-metabolizing enzymes. This review article introduces the significance of these epitranscriptomic and epigenetic regulations as factors in determining drug metabolism potency. Further research on this link is expected to lead to a deeper understanding of interindividual differences in the therapeutic effectiveness and side effects of medicines.

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

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

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.

Gammaherpesvirus Infection Stimulates Lung Tumor-Promoting Inflammation.

Lung tumor-promoting environmental exposures and γherpesvirus infections are associated with Type 17 inflammation. To test the effect of γherpesvirus infection in promoting lung tumorigenesis, we infected mutant K-Ras-expressing (K-RasLA1) mice with the murine γherpesvirus MHV68 via oropharyngeal aspiration. After 7 weeks, the infected mice displayed a more than 2-fold increase in lung tumors relative to their K-RasLA1 uninfected littermates. Assessment of cytokines in the lung revealed that expression of Type 17 cytokines (Il-6, Cxcl1, Csf3) peaked at day 7 post-infection. These observations correlated with the post-infection appearance of known immune mediators of tumor promotion via IL-17A in the lungs of tumor-bearing mice. Surprisingly, Cd84, an immune cell marker mRNA, did not increase in MHV68-infected wild-type mice lacking lung tumors. Csf3 and Cxcl1 protein levels increased more in the lungs of infected K-RasLA1 mice relative to infected wild-type littermates. Flow cytometric and transcriptomic analyses indicated that the infected K-RasLA1 mice had increased Ly6Gdim/Ly6Chi immune cells in the lung relative to levels seen in uninfected control K-RasLA1 mice. Selective methylation of adenosines (m6A modification) in immune-cell-enriched mRNAs appeared to correlate with inflammatory infiltrates in the lung. These observations implicate γherpesvirus infection in lung tumor promotion and selective accumulation of immune cells in the lung that appears to be associated with m6A modification of mRNAs in those cells.

Accurate Quantification of Ten Methylated Purine Nucleosides by Highly Sensitive and Stable Isotope-Diluted UHPLC-MS/MS.

The dynamic landscape of cellular nucleotides/nucleosides associated with RNA metabolism, particularly in diseases like cancer, has spurred intensive interest. Here, we report a robust stable isotope-diluted UHPLC-ESI-MS/MS method for accurate quantification of 12 purine ribonucleosides, including 10 methylated purine nucleosides. By the use of thermally decomposable ammonium bicarbonate (NH4HCO3) as a mobile phase additive for UHPLC-MS/MS detection, the ESI-MS/MS signal responses of these target compounds were enhanced by 1.7-24.5 folds. Noteworthily, three methylated guanosine isomers (m1G, m2G, and m7G) and two methylated adenosine isomers (m1A and m6A) that are indistinguishable directly by mass spectrometry were well resolved with optimal UHPLC separation. Combined with methanol extraction and solid-phase extraction (SPE) pretreatment, the method quantified intracellular concentrations of three modified nucleosides (Gm, m1G, and m2G), which would otherwise be undetectable because of significant suppression of their signals by the interfering cellular matrix. Nine purine nucleosides were simultaneously quantified in 293T cells, and their concentrations ranged by 4 orders of magnitude. Overall, the method presents high recovery rates over 90% for endogenous modified purine nucleosides in cultured cells, along with good precision, linearity, and LOD ranging from 0.30 fmol to 0.37 pmol per 5 × 105 cells. The developed UHPLC-MS/MS method holds potential for screening purine nucleosides as diagnostic and prognostic biomarkers and for quantifying purine epigenetic nucleosides post-cell metabolome analysis, thereby providing a valuable analytical tool for intracellular nucleoside quantification in future clinical research.

Stroke triggers dynamic m6A reprogramming of cerebral circular RNAs

We previously showed that stroke alters circular RNA (circRNA) expression profiles. Many circRNAs undergo epitranscriptomic modifications, particularly methylation of adenosine to form N6-methyladenosine (m6A). This modification significantly influences the circRNA metabolism and functionality. Hence, we currently evaluated if transient focal ischemia in adult C57BL/6J mice alters the m6A methylation of circRNAs. Changes in m6A were profiled in the peri-infarct cortex following immunoprecipitation coupled with microarrays. Correlation and gene ontology analyses were performed to understand the association of m6A changes with circRNA regulation and functional implications after stroke. Many circRNAs showed differential regulation (up or down) after stroke, and this change was highest at 24h of reperfusion. Notably, most circRNAs differentially regulated after stroke also exhibited temporal changes in m6A modification patterns. The majority of circRNAs that showed post-stroke differential m6A modifications were derived from protein-coding genes. Hyper-than hypomethylation of circRNAs was most prevalent after stroke. Gene ontology analysis of the host genes suggested that m6A-modified circRNAs might regulate functions such as synapse-related processes, indicating that m6A epitranscriptomic modification in circRNAs could potentially influence post-stroke synaptic pathophysiology.

M6A Methylation of Transcription Leader Sequence of SARS-CoV-2 Impacts Discontinuous Transcription of Subgenomic mRNAs.

The SARS-CoV-2 genome has been shown to be m6A methylated at several positions in vivo. Strikingly, a DRACH motif, the recognition motif for adenosine methylation, resides in the core of the transcriptional regulatory leader sequence (TRS-L) at position A74, which is highly conserved and essential for viral discontinuous transcription. Methylation at position A74 correlates with viral pathogenicity. Discontinuous transcription produces a set of subgenomic mRNAs that function as templates for translation of all structural and accessory proteins. A74 is base-paired in the short stem-loop structure 5'SL3 that opens during discontinuous transcription to form long-range RNA-RNA interactions with nascent (-)-strand transcripts at complementary TRS-body sequences. A74 can be methylated by the human METTL3/METTL14 complex in vitro. Here, we investigate its impact on the structural stability of 5'SL3 and the long-range TRS-leader:TRS-body duplex formation necessary for synthesis of subgenomic mRNAs of all four viral structural proteins. Methylation uniformly destabilizes 5'SL3 and long-range duplexes and alters their relative equilibrium populations, suggesting that the m6A74 modification acts as a regulator for the abundance of viral structural proteins due to this destabilization.

CYP19A1 Expression Is Controlled by mRNA Stability of the Upstream Transcription Factor AP-2γ in Placental JEG3 Cells.

CYP19A1 encodes aromatase, which converts testosterone to estrogen, and is induced during placental maturation. To elucidate the molecular mechanism underlying this function, histone methylation was analyzed using the placental cytotrophoblast cell line, JEG3. Treatment of JEG3 cells with 3-deazaneplanocin A, an inhibitor of several methyltransferases, resulted in increased CYP19A1 expression, accompanied by removal of the repressive mark H3K27me3 from the CYP19A1 promoter. However, this increase was not observed in cells treated with GSK126, another specific inhibitor for H3K27me3 methylation. Expression of TFAP2C, which encodes AP-2γ, a transcription factor that regulates CYP19A1, was also elevated on 3-deazaneplanocin A treatment. Interestingly, TFAP2C messenger RNA (mRNA) was readily degraded in JEG3 cells but protected from degradation in the presence of 3-deazaneplanocin A. TFAP2C mRNA contained N6-methyladenosines, which were reduced on drug treatment. These observations indicate that the TFAP2C mRNA undergoes adenosine methylation and rapid degradation, whereas 3-deazaneplanocin A suppresses methylation, resulting in an increase in AP-2γ levels. We conclude that the increase in AP-2γ expression via stabilization of the TFAP2C mRNA is likely to underlie the increased CYP19A1 expression.

Abstract 3231: Pharmacological inhibition of the m6A RNA reader, YTHDC1, as a novel approach to targeting biomolecular condensates in cancer

Abstract Epigenetic dysregulation is a hallmark of cancer, and tumors have evolved complex mechanisms to fine-tune oncogenic gene expression. In acute myeloid leukemia (AML), one such regulatory mechanism involves post transcriptional modification of RNAs. The most abundant post transcriptional modification is methylation of adenosine at the N6 position (m6A). This modification is catalyzed by the m6A “writer”, METTL3 and METTL14 complex, and is removed by “erasers” such as FTO and ALKBH5. m6A RNAs can regulate a variety of cellular processes which can impact tumor phenotypes, including RNA stability, splicing, and nuclear export; and METTL3 catalytic inhibitors are currently in clinical development for AML and other cancers (NCT05584111).The m6A “reader”, YTHDC1, has been recognized as a critical contributor to the proliferation and survival of AML cells. As the primary nuclear m6A reader, YTHDC1 localizes with m6A marked RNAs in phase-separated assemblies, termed biomolecular condensates, helping to maintain the stability of oncogenic transcripts such as MYC. Here, we have employed our proprietary microfluidics based screening platform (termed PhaseScanTM) to identify modulators of YTHDC1 biomolecular condensates. Evaluation of early screening hits helped identify that disruption of the interaction between YTHDC1 and m6A RNA is a functionally relevant mechanism of action (MoA) that results in dissolution of YTHDC1 condensates. Using computational and knowledge based approaches, we have identified other structurally distinct inhibitors that occupy the RNA binding pocket of YTHDC1 and disrupt its interaction with m6A RNA at nanomolar potencies. These compounds disrupt cellular YTHDC1 condensates, suppress oncogenic gene expression, and result in anti proliferative and pro-differentiation phenotypes in AML cell lines. Compounds are selective for YTHDC1 over other YTH family members and are also selective in dissolving YTHDC1 condensates over other nuclear and cytosolic condensate systems. Our optimized chemical matter has suitable ADME properties to support in vivo studies. Thus, targeting YTHDC1 biomolecular condensates represents a novel approach to the treatment of AML. Citation Format: Richard C. Centore, Mark Charles, Mansi Arora, Yiwen Chen, Matthew Watson, Charlotte Kelley, Magdalena Czekalska, Marius Rebmann, Mahmoud Ghandi, Jerome Cattin, Nagakumar Bharatham, Prathima Radhakrishnan, Alex Howarth, Gale Rudlaff, William E. Arter, Seema Qamar, Kadi Saar, Douglas Williamson, Andrew Seeber, Tuomas Knowles, Shilpi Arora. Pharmacological inhibition of the m6A RNA reader, YTHDC1, as a novel approach to targeting biomolecular condensates in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3231.

Comprehensive analysis of m6A methylome alterations after azacytidine plus venetoclax treatment for acute myeloid leukemia by nanopore sequencing

N6 adenosine methylation (m6A), one of the most prevalent internal modifications on mammalian RNAs, regulates RNA transcription, stabilization, and splicing. Growing evidence has focused on the functional role of m6A regulators on acute myeloid leukemia (AML). However, the global m6A levels after azacytidine (AZA) plus venetoclax (VEN) treatment in AML patients remain unclear. In our present study, bone marrow (BM) sample pairs (including pre-treatment [AML] and post-treatment [complete remission (CR)] samples) were harvested from three AML patients who had achieved CR after AZA plus VEN treatment for Nanopore direct RNA sequencing. Notably, the amount of m6A sites and the m6A levels in CR BMs was significantly lower than those in the AML BMs. Such a significant reduction in the m6A levels was also detected in AZA-treated HL-60 cells. Thirteen genes with decreased m6A and expression levels were identified, among which three genes (HPRT1, SNRPC, and ANP32B) were closely related to the prognosis of AML. Finally, we speculated the mechanism via which m6A modifications affected the mRNA stability of these three genes. In conclusion, we illustrated for the first time the global landscape of m6A levels in AZA plus VEN treated AML (CR) patients and revealed that AZA had a significant demethylation effect at the RNA level in AML patients. In addition, we identified new biomarkers for AZA plus VEN-treated AML via Nanopore sequencing technology in RNA epigenetics.

Silencing of METTL3 inhibits m6A methylation of NEK7 to suppress pyrolysis in an HT-22 cell-based model of intracerebral hemorrhage

Intracerebral hemorrhage (ICH) induces severe neurological damage, and its progression is driven by METTL3. This study aimed to investigate the role of METTL3 in ICH via in vitro experiments. For this purpose, HT-22 cells were treated with hemin to mimic ICH in vitro, followed by evaluating cell pyroptosis using flow cytometry, lactic dehydrogenase release analysis, enzyme-linked immunosorbent assay, and western blotting. Moreover, N6-methyl adenosine (m6A) methylation of NEK7 was assessed using methylated RNA immunoprecipitation, RNA immunoprecipitation, dual-luciferase reporter assay, and quantitative real-time polymerase chain reaction. Results indicated that knockdown of METTL3 inhibited hemin-induced pyroptosis and suppressed m6A methylation of NEK7 due to METTL3 downregulation, reducing NEK7 mRNA stability. The effects on METTL3-induced cell pyroptosis were abrogated by overexpressing NEK7, while IGF2BP2 increased NEK7 expression. Similarly, IGF2BP2 silence downregulated NEK7 expression mediated by METTL3. In conclusion, silencing of METTL3 inhibited hemin-induced HT-22 cell pyroptosis by suppressing m6A methylation of NEK7, which was recognized by IGF2BP2. These findings are envisaged to identify a novel therapeutic strategy for ICH.

The role of the m6A/m demethylase FTO in memory is both task and sex-dependent in mice

Formation of long-term memories requires learning-induced changes in both transcription and translation. Epitranscriptomic modifications of RNA recently emerged as critical regulators of RNA dynamics, whereby adenosine methylation (m6A) regulates translation, mRNA stability, mRNA localization, and memory formation. Prior work demonstrated a pro-memory phenotype of m6A, as loss of m6A impairs and loss of the m6A/m demethylase FTO improves memory formation. Critically, these experiments focused exclusively on aversive memory tasks and were only performed in male mice. Here we show that the task type and sex of the animal alter effects of m6A on memory, whereby FTO-depletion impaired object location memory in male mice, in contrast to the previously reported beneficial effects of FTO depletion on aversive memory. Additionally, we show that female mice have no change in performance after FTO depletion, demonstrating that sex of the mouse is a critical variable for understanding how m6A contributes to memory formation. Our study provides the first evidence for FTO regulation of non-aversive spatial memory and sexspecific effects of m6A, suggesting that identification of differentially methylated targets in each sex and task will be critical for understanding how epitranscriptomic modifications regulate memory.

Tet-dependent 5-hydroxymethyl-Cytosine modification of mRNA regulates axon guidance genes in Drosophila.

Modifications of mRNA, especially methylation of adenosine, have recently drawn much attention. The much rarer modification, 5-hydroxymethylation of cytosine (5hmC), is not well understood and is the subject of this study. Vertebrate Tet proteins are 5-methylcytosine (5mC) hydroxylases and catalyze the transition of 5mC to 5hmC in DNA. These enzymes have recently been shown to have the same function in messenger RNAs in both vertebrates and in Drosophila. The Tet gene is essential in Drosophila as Tet knock-out animals do not reach adulthood. We describe the identification of Tet-target genes in the embryo and larval brain by mapping one, Tet DNA-binding sites throughout the genome and two, the Tet-dependent 5hmrC modifications transcriptome-wide. 5hmrC modifications are distributed along the entire transcript, while Tet DNA-binding sites are preferentially located at the promoter where they overlap with histone H3K4me3 peaks. The identified mRNAs are preferentially involved in neuron and axon development and Tet knock-out led to a reduction of 5hmrC marks on specific mRNAs. Among the Tet-target genes were the robo2 receptor and its slit ligand that function in axon guidance in Drosophila and in vertebrates. Tet knock-out embryos show overlapping phenotypes with robo2 and both Robo2 and Slit protein levels were markedly reduced in Tet KO larval brains. Our results establish a role for Tet-dependent 5hmrC in facilitating the translation of modified mRNAs primarily in cells of the nervous system.

Abstract TP302: Circular RNAs Methylation Patterns Positively Correlated With Their Expression Level in the Post-Stroke Brain

Circular RNAs (circRNAs), a novel subgroup of noncoding RNAs derived from reverse splicing of various genes, have emerged as potential regulators of transcription and translation. Recent findings indicate that methylation of adenosine (m 6 A; N6-methyladenosine) in various RNA types might govern their levels by modulating their degradation as well as functional roles. This study investigated the spatiotemporal correlation of methylation patterns of circRNAs with their abundance in the post-stroke brain. Adult male C57BL/6J mice were subjected to transient middle cerebral artery occlusion, and m 6 A methylation in circRNAs was profiled using immunoprecipitated methylated RNAs with microarrays in the peri-infarct cortex at 6h, 12h, and 24h of post-reperfusion. Computational analysis was employed to identify the change in m 6 A levels and evaluate the correlation between expression and methylation patterns. We noted that most circRNAs that alter their levels following ischemic stroke are transcribed from the exonic regions of all chromosomes. While most genes generate a single circRNA, a unique situation occurs with the gene Rn45s, giving rise to a minimum of 30 circRNAs within the brain. Interestingly, following the stroke, the overall m 6 A levels of circRNAs displayed notable alterations. Moreover, the percentage shift in circRNA m 6 A levels increased from 6h to 24h of reperfusion, with some circRNAs, including mmu_circRNA_32433, demonstrating an increase of >40% at the 24h reperfusion. More significantly, many cerebral circRNAs that displayed increased expression also showed elevated m 6 A methylation following stroke. The increased m 6 A methylation suggests potential stabilization and altered function of circRNAs in the post-stroke brain.

Reading the epitranscriptome of the human malaria parasite

Epigenetic machinery has emerged as a central player in gene regulation and chromatin organization in Plasmodium spp. Epigenetic modifications on histones and their role in antigenic variation in P. falciparum are widely studied. Recent discoveries on nucleic acid methylome are exciting and provide a new dimension to the apicomplexan protozoan parasite's gene regulatory process. Reports have confirmed that N6-methyl adenosine (m6A) methylation plays a crucial role in the translational plasticity of the human malaria parasite during its development in RBC. The YTH domain (YT521-B Homology) protein in P. falciparum binds to m6A epitranscriptome modifications on the mRNA and regulates protein translation. The binding of the PfYTH domain protein to the m6A-modified mRNA is mediated through a binding pocket formed by aromatic amino acids. The P. falciparum genome encodes two members of YTH domain proteins, i.e., YTH1 and YTH2, and both have distinct roles in dictating the epitranscriptome in human malaria parasites. This review highlights recent advancements in the functions and mechanisms of YTH domain protein's role in translational plasticity in the various developmental stages of the parasite.

Identifying Potent Fat Mass and Obesity-Associated Protein Inhibitors Using Deep Learning-Based Hybrid Procedures

The fat mass and obesity-associated (FTO) protein catalyzes metal-dependent modifications of nucleic acids, namely the demethylation of methyl adenosine inside mRNA molecules. The FTO protein has been identified as a potential target for developing anticancer therapies. Identifying a suitable ligand-targeting FTO protein is crucial to developing chemotherapeutic medicines to combat obesity and cancer. Scientists worldwide have employed many methodologies to discover a potent inhibitor for the FTO protein. This study uses deep learning-based methods and molecular docking techniques to investigate the FTO protein as a target. Our strategy involves systematically screening a database of small chemical compounds. By utilizing the crystal structures of the FTO complexed with ligands, we successfully identified three small-molecule chemical compounds (ZINC000003643476, ZINC000000517415, and ZINC000001562130) as inhibitors of the FTO protein. The identification process was accomplished by employing a combination of screening techniques, specifically deep learning (DeepBindGCN) and Autodock vina, on the ZINC database. These compounds were subjected to comprehensive analysis using 100 nanoseconds of molecular dynamics and binding free energy calculations. The findings of our study indicate the identification of three candidate inhibitors that might effectively target the human fat mass and obesity protein. The results of this study have the potential to facilitate the exploration of other chemicals that can interact with FTO. Conducting biochemical studies to evaluate these compounds’ effectiveness may contribute to improving fat mass and obesity treatment strategies.

Bioinformatic identification reveals a m6A-binding protein, IGF2BP2, as a novel tumor-promoting gene signature in thyroid carcinoma.

N6-methyladenosine (m6A) modification plays a crucial role in thyroid carcinoma (THCA). Insulin-like growth factor 2 binding protein 2 (IGF2BP2) is a m6A-binding protein. We aimed to explore the effect of IGF2BP2 on the development of THCA. Differentially expressed genes (DEGs) were screened from GSE50901 and GSE60542 datasets. LinkedOmics, Genebank, and Sequence-based RNA Adenosine Methylation Site Predictor databases were employed to find potential m6A modification sites. Protein-protein interaction network and receiver-operating characteristic curves were applied to determine hub genes of THCA. ESTIMATE revealed the effect of IGF2BP2 on tumor immunity. The mRNA expression of IGF2BP2 was detected using real-time quantitative polymerase chain reaction. The viability, migration, and invasion were assessed by Cell Counting Kit-8, wound healing, and transwell assays. A total of 166 common DEGs were identified from GSE50901 and GSE60542 datasets. One m6A-related gene, IGF2BP2, was differentially expressed in THCA and selected as the research target. The hub genes (CD44, DCN, CXCL12, ICAM1, SDC4, KIT, CTGF, and FMOD) were identified with high prediction values for THCA. Subsequently, the target genes of IGF2BP2, SDC4, and ICAM1, which had potential m6A modification sites, were screened out based on the hub genes. IGF2BP2 was upregulated in THCA and IGF2BP2 expression was positively correlated with immune infiltration in THCA. Additionally, knockdown of IGF2BP2 inhibited the proliferation, invasion, and migration of THCA cells. IGF2BP2 has a contributory effect on the progression of THCA, which is a novel biomarker and a therapeutic target for THCA.

AlkB-Facilitated Demethylation Enables Quantitative and Site-Specific Detection of Dual Methylation of Adenosine in RNA.

RNA molecules undergo various chemical modifications that play critical roles in a wide range of biological processes. N6,N6-Dimethyladenosine (m6,6A) is a conserved RNA modification and is essential for the processing of rRNA. To gain a deeper understanding of the functions of m6,6A, site-specific and accurate quantification of this modification in RNA is indispensable. In this study, we developed an AlkB-facilitated demethylation (AD-m6,6A) method for the site-specific detection and quantification of m6,6A in RNA. The N6,N6-dimethyl groups in m6,6A can cause reverse transcription to stall at the m6,6A site, resulting in truncated cDNA. However, we found that Escherichia coli AlkB demethylase can effectively demethylate m6,6A in RNA, generating full-length cDNA from AlkB-treated RNA. By quantifying the amount of full-length cDNA produced using quantitative real-time PCR, we were able to achieve site-specific detection and quantification of m6,6A in RNA. Using the AD-m6,6A method, we successfully detected and quantified m6,6A at position 1851 of 18S rRNA and position 937 of mitochondrial 12S rRNA in human cells. Additionally, we found that the level of m6,6A at position 1007 of mitochondrial 12S rRNA was significantly reduced in lung tissues from sleep-deprived mice compared with control mice. Overall, the AD-m6,6A method provides a valuable tool for easy, accurate, quantitative, and site-specific detection of m6,6A in RNA, which can aid in uncovering the functions of m6,6A in human diseases.

M6A demethylase FTO and osteoporosis: potential therapeutic interventions.

Osteoporosis is a common bone disease, characterized by a descent in bone mass due to the dysregulation of bone homeostasis. Although different studies have identified an association between osteoporosis and epigenetic alterations in osteogenic genes, the mechanisms of osteoporosis remain unclear. N6-methyladenosine (m6A) modification is a methylated adenosine nucleotide, which regulates the translocation, exporting, translation, and decay of RNA. FTO is the first identified m6A demethylase, which eliminates m6A modifications from RNAs. Variation in FTO disturbs m6A methylation in RNAs to regulate cell proliferation, differentiation, and apoptosis. Besides, FTO as an obesity-associated gene, also affects osteogenesis by regulating adipogenesis. Pharmacological inhibition of FTO markedly altered bone mass, bone mineral density and the distribution of adipose tissue. Small molecules which modulate FTO function are potentially novel remedies to the treatment of osteoporosis by adjusting the m6A levels. This article reviews the roles of m6A demethylase FTO in regulating bone metabolism and osteoporosis.

Identification of hepatoblastoma susceptibility loci in the TRMT6 gene from a seven-center case-control study.

Hepatoblastoma, the most frequently diagnosed primary paediatric liver tumour, bears the lowest somatic mutation burden among paediatric neoplasms. Therefore, it is essential to identify pathogenic germline genetic variants, especially those in oncogenic genes, for this disease. The tRNA methyltransferase 6 noncatalytic subunit (TRMT6) forms a tRNA methyltransferase complex with TRMT61A to catalyse adenosine methylation at position N1 of RNAs. TRMT6 has displayed tumour-promoting functions in several cancer types. However, the contribution of its genetic variants to hepatoblastoma remains unclear. In this study, we investigated the association between four TRMT6 polymorphisms (rs236170 A > G, rs451571 T > C, rs236188 G > A and rs236110 C > A) and the risk of hepatoblastoma in a cohort of 313 cases and 1446 healthy controls. Germline DNA was subjected to polymorphism genotyping via the TaqMan qPCR method. Odds ratio (OR) and 95% confidence interval (CI) were used to determine hepatoblastoma susceptibility variants. The rs236170 A > G, rs236188 G > A and rs236110 C > A polymorphisms were significantly associated with hepatoblastoma risk. Combination analysis of the four polymorphisms revealed that children bearing 1-4 risk genotypes were at significantly enhanced hepatoblastoma risk compared to those without risk genotype (adjusted OR = 1.52, 95% CI = 1.19-1.95, p = 0.0008). We also conducted stratification analyses by age, sex and clinical stage. Ultimately, we found that the rs236110 C > A was significantly associated with the downregulation of MCM8, a neighbouring gene of TRMT6. In conclusion, we identified three susceptibility loci in the TRMT6 gene for hepatoblastoma. Our findings warrant further validation by extensive case-control studies across different ethnicities.