N6-methyladenosine
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BACKGROUND: Epitranscriptomics, the study of RNA modifications such as N6-methyladenosine (m6A), provides a novel layer of gene expression regulation with implications for numerous biological processes, including cellular adaptation to hypoxia. Hypoxia-inducible factor-1 (HIF-1), a master regulator of the cellular response to low oxygen, plays a critical role in adaptive and pathological processes, including cancer, ischemic heart disease, and metabolic disorders. Recent discoveries accent the dynamic interplay between m6A modifications and HIF-1 signaling, revealing a complex bidirectional regulatory network. While the roles of other RNA modifications in HIF-1 regulation remain largely unexplored, emerging evidence suggests their potential significance. MAIN BODY: This review examines the reciprocal regulation between HIF-1 and epitranscriptomic machinery, including m6A writers, readers, and erasers. HIF-1 modulates the expression of key m6A components, while its own mRNA is regulated by m6A modifications, positioning HIF-1 as both a regulator and a target in this system. This interaction enhances our understanding of cellular hypoxic responses and opens avenues for clinical applications in treating conditions like cancer and ischemic heart disease. Promising progress has been made in developing selective inhibitors targeting the m6A-HIF-1 regulatory axis. However, challenges such as off-target effects and the complexity of RNA modification dynamics remain significant barriers to clinical translation. CONCLUSION: The intricate interplay between m6A and HIF-1 highlights the critical role of epitranscriptomics in hypoxia-driven processes. Further research into these regulatory networks could drive therapeutic innovation in cancer, ischemic heart disease, and other hypoxia-related conditions. Overcoming challenges in specificity and off-target effects will be essential for realizing the potential of these emerging therapies.
- MeSH
- adenosin analogy a deriváty metabolismus MeSH
- epigeneze genetická * MeSH
- faktor 1 indukovatelný hypoxií * metabolismus genetika MeSH
- lidé MeSH
- posttranskripční úpravy RNA MeSH
- regulace genové exprese MeSH
- signální transdukce MeSH
- transkriptom MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
The precise and unambiguous detection and quantification of internal RNA modifications represents a critical step for understanding their physiological functions. The methods of direct RNA sequencing are quickly developing allowing for the precise location of internal RNA marks. This detection is, however, not quantitative and still presents detection limits. One of the biggest remaining challenges in the field is still the detection and quantification of m6A, m6Am, inosine, and m1A modifications of adenosine. The second intriguing and timely question remaining to be addressed is the extent to which individual marks are coregulated or potentially can affect each other. Here, we present a methodological approach to detect and quantify several key mRNA modifications in human total RNA and in mRNA, which is difficult to purify away from contaminating tRNA. We show that the adenosine demethylase FTO primarily targets m6Am marks in noncoding RNAs in HEK293T cells. Surprisingly, we observe little effect of FTO or ALKBH5 depletion on the m6A mRNA levels. Interestingly, the upregulation of ALKBH5 is accompanied by an increase in inosine level in overall mRNA.
- MeSH
- adenosin * analogy a deriváty metabolismus genetika analýza MeSH
- alfa-ketoglutarát-dependentní dioxygenasa, AlkB homolog 5 * metabolismus genetika MeSH
- chromatografie kapalinová metody MeSH
- gen pro FTO * metabolismus genetika MeSH
- HEK293 buňky MeSH
- inosin * metabolismus genetika MeSH
- kapalinová chromatografie-hmotnostní spektrometrie MeSH
- lidé MeSH
- messenger RNA * genetika metabolismus MeSH
- posttranskripční úpravy RNA MeSH
- tandemová hmotnostní spektrometrie * metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Nonalcoholic fatty liver disease (NAFLD) is characterized by elevated hepatic lipids caused by nonalcoholic factors, where histone lactylation is lately discovered as a modification driving disease progression. This research aimed to explore the role of histone 3 lysine 18 lactylation (H3K18lac) in NAFLD progression using a high-fat diet (HFD)-treated mouse model and free fatty acids (FFA)-treated L-02 cell lines. Lipids accumulation was screened via Oil Red O staining, real-time quantitative polymerase chain reaction (RT-qPCR), western blotting, and commercially available kits. Similarly, molecular mechanism was analyzed using immunoprecipitation (IP), dual-luciferase reporter assay, and RNA decay assay. Results indicated that FFA upregulated lactate dehydrogenase A (LDHA) and H3K18lac levels in L-02 cells. Besides, LDHA-mediated H3K18lac was enriched on the proximal promoter of methyltransferase 3 (METTL3), translating into an increased expression. Moreover, METTL3 or LDHA knockdown relieved lipid accumulation, decreased total cholesterol (TC) and triglyceride (TG) levels, and downregulated lipogenesis-related proteins in FFA-treated L-02 cell lines, in addition to enhancing the m6A and mRNA levels of stearoyl-coenzyme A desaturase 1 (SCD1). The m6A modification of SCD1 was recognized by YTH N6-methyladenosine RNA binding protein F1 (YTHDF1), resulting in enhanced mRNA stability. LDHA was found to be highly expressed in HFD-treated mice, where knocking down LDHA attenuated HFD-induced hepatic steatosis. These findings demonstrated that LDHA-induced H3K18lac promoted NAFLD progression, where LDHA-induced H3K18lac in METTL3 promoter elevated METTL3 expression, thereby promoting m6A methylation and stabilizing SCD1 via a YTHDF1-dependent manner. Keywords: Nonalcoholic fatty liver disease, LDHA, METTL3, YTHDF1, Histone lactylation.
- MeSH
- adenosin * metabolismus analogy a deriváty MeSH
- dieta s vysokým obsahem tuků škodlivé účinky MeSH
- histony * metabolismus MeSH
- L-laktátdehydrogenasa metabolismus MeSH
- lidé MeSH
- methyltransferasy * metabolismus genetika MeSH
- myši inbrední C57BL * MeSH
- myši MeSH
- nealkoholová steatóza jater * metabolismus patologie MeSH
- progrese nemoci * MeSH
- proteiny vázající RNA * metabolismus genetika MeSH
- stearyl-CoA-desaturasa * metabolismus genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
N6-Methyladenosine (m6A) is the predominant internal RNA modification in eukaryotic messenger RNAs (mRNAs) and plays a crucial role in mRNA stability. Here, using human cells, we reveal that m6A sites in the coding sequence (CDS) trigger CDS-m6A decay (CMD), a pathway that is distinct from previously reported m6A-dependent degradation mechanisms. Importantly, CDS m6A sites act considerably faster and more efficiently than those in the 3' untranslated region, which to date have been considered the main effectors. Mechanistically, CMD depends on translation, whereby m6A deposition in the CDS triggers ribosome pausing and transcript destabilization. The subsequent decay involves the translocation of the CMD target transcripts to processing bodies (P-bodies) and recruitment of the m6A reader protein YT521-B homology domain family protein 2 (YTHDF2). Our findings highlight CMD as a previously unknown pathway, which is particularly important for controlling the expression of developmental regulators and retrogenes.
- MeSH
- 3' nepřekládaná oblast MeSH
- adenosin * analogy a deriváty metabolismus genetika MeSH
- HEK293 buňky MeSH
- HeLa buňky MeSH
- lidé MeSH
- messenger RNA * genetika metabolismus MeSH
- otevřené čtecí rámce * MeSH
- proteiny vázající RNA * genetika metabolismus MeSH
- proteosyntéza * MeSH
- ribozomy metabolismus genetika MeSH
- stabilita RNA * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Myelodysplastic syndromes (MDS) are myeloid malignancies with heterogeneous genotypes and phenotypes, characterized by ineffective haematopoiesis and a high risk of progression towards acute myeloid leukaemia (AML). Prognosis for patients treated with hypomethylating agents (HMAs), as is azacytidine, the main drug used as frontline therapy for MDS is mostly based on cytogenetics and next generation sequencing (NGS) of the initial myeloid clone. Although the critical influence of the epigenetic landscape upon cancer cells survival and development as well on tumour environment establishment is currently recognized and approached within current clinical practice in MDS, the heterogenous response of the patients to epigenetic therapy is suggesting a more complex mechanism of action, as is the case of RNA methylation. In this sense, the newly emerging field of epitranscriptomics could provide a more comprehensive perspective upon the modulation of gene expression in malignancies, as is the proof-of-concept of MDS. We initially did RNA methylation sequencing on MDS patients (n = 6) treated with azacytidine and compared responders with non-responders. Afterwards, the genes identified were assessed in vitro and afterwards validated on a larger cohort of MDS patients treated with azacytidine (n = 58). Our data show that a more accurate prognosis could be based on analysing the methylome and thus we used methylation sequencing to differentially split high-grade MDS patients with identical demographical and cytogenetic features, between azacytidine responders and non-responders.
- MeSH
- azacytidin * farmakologie terapeutické užití MeSH
- epigeneze genetická účinky léků MeSH
- lidé středního věku MeSH
- lidé MeSH
- methylace RNA MeSH
- metylace DNA * účinky léků MeSH
- myelodysplastické syndromy * genetika farmakoterapie patologie MeSH
- prognóza MeSH
- protinádorové antimetabolity terapeutické užití farmakologie MeSH
- sekvenční analýza RNA MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- stanovení celkové genové exprese MeSH
- transkriptom genetika účinky léků MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
N6-methyladenosine (m6A) is the most abundant epitranscriptomic mark that regulates the fate of RNA molecules. Recent studies have revealed a bidirectional interaction between m6A modification and the circadian clock. However, the precise temporal dynamics of m6A global enrichment in the central circadian pacemaker have not been fully elucidated. Our study investigates the relationship between FTO demethylase and molecular clocks in primary cells of the suprachiasmatic nucleus (SCN). In addition, we examined the effects of lipopolysaccharide (LPS) on Fto expression and the role of FTO in LPS-induced reactive oxygen species (ROS) production in primary SCN cell culture. We observed circadian rhythmicity in the global m6A levels, which mirrored the rhythmic expression of the Fto demethylase. Silencing FTO using siRNA reduced the mesor of Per2 rhythmicity in SCN primary cells and extended the period of the PER2 rhythm in SCN primary cell cultures from PER2::LUC mice. When examining the immune response, we discovered that exposure to LPS upregulated global m6A levels while downregulating Fto expression in SCN primary cell cultures. Interestingly, we found a loss of circadian rhythmicity in Fto expression following LPS treatment, indicating that the decrease of FTO levels may contribute to m6A upregulation without directly regulating its circadian rhythm. To explore potential protective mechanisms against neurotoxic inflammation, we examined ROS production following LPS treatment in SCN primary cell cultures pretreated with FTO siRNA. We observed a time-dependent pattern of ROS induction, with significant peak at 32 h but not at 20 h after synchronization. Silencing the FTO demethylase abolished ROS induction following LPS exposure, supporting the hypothesis that FTO downregulation serves as a protective mechanism during LPS-induced neuroinflammation in SCN primary cell cultures.
- MeSH
- adenosin * analogy a deriváty metabolismus MeSH
- cirkadiánní hodiny * účinky léků fyziologie genetika MeSH
- cirkadiánní proteiny Period metabolismus genetika MeSH
- cirkadiánní rytmus účinky léků fyziologie MeSH
- gen pro FTO * metabolismus genetika MeSH
- kultivované buňky MeSH
- lipopolysacharidy * farmakologie MeSH
- methylace RNA MeSH
- metylace účinky léků MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- neurozánětlivé nemoci metabolismus MeSH
- nucleus suprachiasmaticus * metabolismus účinky léků MeSH
- reaktivní formy kyslíku metabolismus MeSH
- RNA genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
RNA modifications affect key stages of the RNA life cycle, including splicing, export, decay, and translation. Epitranscriptomic regulations therefore significantly influence cellular physiology and pathophysiology. Here, we selected some of the most abundant modifications and reviewed their roles in the heart and in cardiovascular diseases: N6-methyladenosine (m6A), N6,2'-O-dimethyladenosine (m6Am), N1-methyladenosine (m1A), pseudouridine (?), 5 methylcytidine (m5C), and inosine (I). Dysregulation of epitranscriptomic machinery affecting these modifications vastly changes the cardiac phenotype and is linked with many cardiovascular diseases such as myocardial infarction, cardiomyopathies, or heart failure. Thus, a deeper understanding of these epitranscriptomic changes and their regulatory mechanisms can enhance our knowledge of the molecular underpinnings of prevalent cardiac diseases, potentially paving the way for novel therapeutic strategies. Keywords: Epitranscriptomics, RNA modifications, Epigenetics, m6A, RNA, Heart.
- MeSH
- adenosin analogy a deriváty metabolismus MeSH
- epigeneze genetická * MeSH
- lidé MeSH
- myokard metabolismus MeSH
- posttranskripční úpravy RNA MeSH
- transkriptom MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
This work reports the first electrochemical bioplatforms developed for the determination of the total contents of either target miRNA or methylated target miRNA. The bioplatforms are based on the hybridization of the target miRNA with a synthetic biotinylated DNA probe, the capture of the formed DNA/miRNA heterohybrids on the surface of magnetic microcarriers, and their recognition with an antibody selective to these heterohybrids or to the N6-methyladenosine (m6A) epimark. The determination of the total or methylated target miRNA was accomplished by labeling such secondary antibodies with the horseradish peroxidase (HRP) enzyme. In both cases, amperometric transduction was performed on the surface of disposable electrodes after capturing the resulting HRP-tagged magnetic bioconjugates. Because of their increasing relevance in colorectal cancer (CRC) diagnosis and prognosis, miRNA let-7a and m6A methylation were selected. The proposed electrochemical bioplatforms showed attractive analytical and operational characteristics for the determination of the total and m6A-methylated target miRNA in less than 75 min. These bioplatforms, innovative in design and application, were applied to the analysis of total RNA samples extracted from cultured cancer cells with different metastatic profiles and from paired healthy and tumor tissues of patients diagnosed with CRC at different stages. The obtained results demonstrated, for the first time using electrochemical platforms, the potential of interrogating the target miRNA methylation level to discriminate the metastatic capacities of cancer cells and to identify tumor tissues and, in a pioneering way, the potential of the m6A methylation in miRNA let-7a to serve as a prognostic biomarker for CRC.
Cardiac tolerance to ischaemia can be increased by dietary interventions such as fasting, which is associated with significant changes in myocardial gene expression. Among the possible mechanisms of how gene expression may be altered are epigenetic modifications of RNA - epitranscriptomics. N6-methyladenosine (m6A) and N6,2'-O-dimethyladenosine (m6Am) are two of the most prevalent modifications in mRNA. These methylations are reversible and regulated by proteins called writers, erasers, readers, and m6A-repelled proteins. We analysed 33 of these epitranscriptomic regulators in rat hearts after cardioprotective 3-day fasting using RT-qPCR, Western blot, and targeted proteomic analysis. We found that the most of these regulators were changed on mRNA or protein levels in fasting hearts, including up-regulation of both demethylases - FTO and ALKBH5. In accordance, decreased methylation (m6A+m6Am) levels were detected in cardiac total RNA after fasting. We also identified altered methylation levels in Nox4 and Hdac1 transcripts, both of which play a role in the cytoprotective action of ketone bodies produced during fasting. Furthermore, we investigated the impact of inhibiting demethylases ALKBH5 and FTO in adult rat primary cardiomyocytes (AVCMs). Our findings indicate that inhibiting these demethylases reduced the hypoxic tolerance of AVCMs isolated from fasting rats. This study showed that the complex epitranscriptomic machinery around m6A and m6Am modifications is regulated in the fasting hearts and might play an important role in cardiac adaptation to fasting, a well-known cardioprotective intervention.
- MeSH
- adenosin * genetika metabolismus MeSH
- krysa rodu rattus MeSH
- messenger RNA genetika MeSH
- omezení příjmu potravy MeSH
- proteomika * MeSH
- RNA metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The rapidly developing research field of epitranscriptomics has recently emerged into the spotlight of researchers due to its vast regulatory effects on gene expression and thereby cellular physiology and pathophysiology. N6-methyladenosine (m6A) and N6,2'-O-dimethyladenosine (m6Am) are among the most prevalent and well-characterized modified nucleosides in eukaryotic RNA. Both of these modifications are dynamically regulated by a complex set of epitranscriptomic regulators called writers, readers, and erasers. Altered levels of m6A and also several regulatory proteins were already associated with diabetic tissues. This review summarizes the current knowledge and gaps about m6A and m6Am modifications and their respective regulators in the pathophysiology of diabetes mellitus. It focuses mainly on the more prevalent type 2 diabetes mellitus (T2DM) and its treatment by metformin, the first-line antidiabetic agent. A better understanding of epitranscriptomic modifications in this highly prevalent disease deserves further investigation and might reveal clinically relevant discoveries in the future.
