Epitranscriptomic modifications have recently emerged into the spotlight of researchers due to their vast regulatory effects on gene expression and thereby cellular physiology and pathophysiology. N6,2'-O-dimethyladenosine (m6Am) is one of the most prevalent chemical marks on RNA and is dynamically regulated by writers (PCIF1, METTL4) and erasers (FTO). The presence or absence of m6Am in RNA affects mRNA stability, regulates transcription, and modulates pre-mRNA splicing. Nevertheless, its functions in the heart are poorly known. This review summarizes the current knowledge and gaps about m6Am modification and its regulators in cardiac biology. It also points out technical challenges and lists the currently available techniques to measure m6Am. A better understanding of epitranscriptomic modifications is needed to improve our knowledge of the molecular regulations in the heart which may lead to novel cardioprotective strategies.

• Klíčová slova
• N6,2‘-O-dimethyladenosine, N6-methyladenosine, epitranscriptomics, heart, m6A, m6Am,
• MeSH
• adenosin * metabolismus   MeSH
• biologie MeSH
• messenger RNA genetika   MeSH
• metylace DNA * MeSH
• RNA metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• adenosin * MeSH
• messenger RNA MeSH
• RNA 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.

• Klíčová slova
• RNA, T2DM, diabetes, epigenetics, epitranscriptomics, m6A, m6Am, type 2 diabetes mellitus,
• MeSH
• adenosin metabolismus   MeSH
• diabetes mellitus 2. typu * genetika   MeSH
• lidé MeSH
• messenger RNA metabolismus   MeSH
• posttranskripční úpravy RNA MeSH
• RNA genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• adenosin MeSH
• messenger RNA MeSH
• RNA MeSH

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.

• Klíčová slova
• ALKBH5, FTO, Fasting, epitranscriptomics, heart, m6A, m6Am,
• 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
• Názvy látek
• adenosin * MeSH
• messenger RNA MeSH
• RNA MeSH

Eukaryotic mRNAs are modified by several chemical marks which have significant impacts on mRNA biology, gene expression, and cellular metabolism as well as on the survival and development of the whole organism. The most abundant and well-studied mRNA base modifications are m6A and ADAR RNA editing. Recent studies have also identified additional mRNA marks such as m6Am, m5C, m1A and Ψ and studied their roles. Each type of modification is deposited by a specific writer, many types of modification are recognized and interpreted by several different readers and some types of modifications can be removed by eraser enzymes. Several works have addressed the functional relationships between some of the modifications. In this review we provide an overview on the current status of research on the different types of mRNA modifications and about the crosstalk between different marks and its functional consequences.

• Klíčová slova
• ADAR, Inosine, epitranscriptome, m1A, m5C, m6A, m6Am, pseudouridine,
• MeSH
• epigeneze genetická * MeSH
• epigenomika metody   MeSH
• lidé MeSH
• messenger RNA genetika   metabolismus   MeSH
• posttranskripční úpravy RNA * MeSH
• transkriptom * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• messenger RNA MeSH

Fat mass and obesity-associated (FTO) protein, a key enzyme integral to the dynamic regulation of epitranscriptomic modifications in RNAs, significantly influences crucial RNA lifecycle processes, including splicing, export, decay, and translation. The role of FTO in altering the epitranscriptome manifests across a spectrum of physiological and pathological conditions. This review aims to consolidate current understanding regarding the implications of FTO in health and disease, with a special emphasis on its involvement in obesity and non-communicable diseases associated with obesity, such as diabetes, cardiovascular disease, and cancer. It also summarizes the established molecules with FTO-inhibiting activity. Given the extensive impact of FTO on both physiology and pathophysiology, this overview provides illustrative insights into its roles, rather than an exhaustive account. A proper understanding of FTO function in human diseases could lead to new treatment approaches, potentially unlocking novel avenues for addressing both metabolic disorders and malignancies. The evolving insights into FTO's regulatory mechanisms hold great promise for future advancements in disease treatment and prevention.

• Klíčová slova
• FTO, cancer, cardiovascular disease, diabetes, m6A, m6Am, obesity,
• 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 way 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, upregulation of ALKBH5 is accompanied by an increase in inosine level in overall mRNA.

• Klíčová slova
• ALKBH5, FTO ADAR, RNA editing, adenosine methylation, inosine,
• 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
• Názvy látek
• adenosin MeSH

N6-methyladenosine (m6A) and N6,2'-O-dimethyladenosine (m6Am) are two abundant modifications found in mRNAs and ncRNAs that can regulate multiple aspects of RNA biology. They function mainly by regulating interactions with specific RNA-binding proteins. Both modifications are linked to development, disease and stress response. To date, three methyltransferases and two demethylases have been identified that modify adenosines in mammalian mRNAs. Here, we present a comprehensive analysis of the interactomes of these enzymes. PCIF1 protein network comprises mostly factors involved in nascent RNA synthesis by RNA polymerase II, whereas ALKBH5 is closely linked with most aspects of pre-mRNA processing and mRNA export to the cytoplasm. METTL16 resides in subcellular compartments co-inhabited by several other RNA modifiers and processing factors. FTO interactome positions this demethylase at a crossroad between RNA transcription, RNA processing and DNA replication and repair. Altogether, these enzymes share limited spatial interactomes, pointing to specific molecular mechanisms of their regulation.

• MeSH
• adaptorové proteiny signální transdukční genetika   metabolismus   MeSH
• adenosin analogy a deriváty   metabolismus   MeSH
• alfa-ketoglutarát-dependentní dioxygenasa, AlkB homolog 5 genetika   metabolismus   MeSH
• anotace sekvence MeSH
• gen pro FTO genetika   metabolismus   MeSH
• genetická transkripce MeSH
• genová ontologie MeSH
• HEK293 buňky MeSH
• jaderné proteiny genetika   metabolismus   MeSH
• lidé MeSH
• mapování interakce mezi proteiny MeSH
• messenger RNA genetika   metabolismus   MeSH
• methyltransferasy genetika   metabolismus   MeSH
• N-demethylasy genetika   metabolismus   MeSH
• nekódující RNA genetika   metabolismus   MeSH
• oprava DNA MeSH
• protein - isoformy genetika   metabolismus   MeSH
• replikace DNA MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adaptorové proteiny signální transdukční MeSH
• adenosin MeSH
• alfa-ketoglutarát-dependentní dioxygenasa, AlkB homolog 5 MeSH
• ALKBH5 protein, human MeSH Prohlížeč
• FTO protein, human MeSH Prohlížeč
• gen pro FTO MeSH
• jaderné proteiny MeSH
• messenger RNA MeSH
• methyltransferasy MeSH
• METTL16 protein, human MeSH Prohlížeč
• N-demethylasy MeSH
• N-methyladenosine MeSH Prohlížeč
• N(6),N(6)-dimethyladenosine MeSH Prohlížeč
• nekódující RNA MeSH
• PCIF1 protein, human MeSH Prohlížeč
• protein - isoformy MeSH

RNA modifications are being recognized as an essential factor in gene expression regulation. They play essential roles in germ line development, differentiation and disease. In eukaryotic mRNAs, N6-adenosine methylation (m6A) is the most prevalent internal chemical modification identified to date. The m6A pathway involves factors called writers, readers and erasers. m6A thus offers an interesting concept of dynamic reversible modification with implications in fine-tuning the cellular metabolism. In mammals, FTO and ALKBH5 have been initially identified as m6A erasers. Recently, FTO m6A specificity has been debated as new reports identify FTO targeting N6,2'-O-dimethyladenosine (m6Am). The two adenosine demethylases have diverse roles in the metabolism of mRNAs and their activity is involved in key processes, such as embryogenesis, disease or infection. In this article, we review the current knowledge of their function and mechanisms and discuss the existing contradictions in the field. This article is part of a Special Issue entitled: mRNA modifications in gene expression control edited by Dr. Soller Matthias and Dr. Fray Rupert.

• Klíčová slova
• ALKBH5, FTO, RNA demethylase, RNA modification, m(6)A, m(6)A(m),
• MeSH
• adenin analogy a deriváty   metabolismus   MeSH
• alfa-ketoglutarát-dependentní dioxygenasa, AlkB homolog 5 metabolismus   MeSH
• lidé MeSH
• posttranskripční úpravy RNA * MeSH
• RNA genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• 6-methyladenine MeSH Prohlížeč
• adenin MeSH
• alfa-ketoglutarát-dependentní dioxygenasa, AlkB homolog 5 MeSH
• RNA MeSH