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.

• Klíčová slova
• ADAR, ALKBH5, FTO, RNA editing, adenosine methylation, inosine,
• MeSH
• adenosin * analogy a deriváty   metabolismus   analýza   MeSH
• alfa-ketoglutarát-dependentní dioxygenasa, AlkB homolog 5 genetika   metabolismus   MeSH
• chromatografie kapalinová metody   MeSH
• gen pro FTO genetika   metabolismus   MeSH
• HEK293 buňky MeSH
• inosin * metabolismus   MeSH
• kapalinová chromatografie-hmotnostní spektrometrie MeSH
• lidé MeSH
• messenger RNA * genetika   chemie   metabolismus   MeSH
• metylace MeSH
• posttranskripční úpravy RNA MeSH
• tandemová hmotnostní spektrometrie metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenosin * MeSH
• alfa-ketoglutarát-dependentní dioxygenasa, AlkB homolog 5 MeSH
• ALKBH5 protein, human MeSH Prohlížeč
• FTO protein, human MeSH Prohlížeč
• gen pro FTO MeSH
• inosin * MeSH
• messenger RNA * MeSH
• N-methyladenosine MeSH Prohlížeč

FTO and ALKBH5 proteins are essential erasers of N6-adenosine methylation in RNA. We studied how levels of FTO and ALKBH5 proteins changed during mouse embryonic development, aging, cardiomyogenesis, and neuroectodermal differentiation. We observed that aging in male and female mice was associated with FTO up-regulation in mouse hearts, brains, lungs, and kidneys, while the ALKBH5 level remained stable. FTO and ALKBH5 proteins were up-regulated during experimentally induced cardiomyogenesis, but the level of ALKBH5 protein was not changed when neuroectodermal differentiation was induced. HDAC1 depletion in mouse ES cells caused FTO down-regulation. In these cells, mRNA, carrying information from genes that regulate histone signature, RNA processing, and cell differentiation, was characterized by a reduced level of N6-adenosine methylation in specific gene loci, primarily regulating cell differentiation into neuroectoderm. Together, when we compared both RNA demethylating proteins, the FTO protein level undergoes the most significant changes during cell differentiation and aging. Thus, we conclude that during aging and neuronal differentiation, m6A RNA demethylation is likely regulated by the FTO protein but not via the function of ALKBH5.

• MeSH
• adenosin metabolismus   MeSH
• alfa-ketoglutarát-dependentní dioxygenasa, AlkB homolog 5 * genetika   metabolismus   MeSH
• buněčná diferenciace MeSH
• embryonální vývoj MeSH
• gen pro FTO * genetika   metabolismus   MeSH
• myši MeSH
• RNA metabolismus   MeSH
• stárnutí genetika   MeSH
• upregulace MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenosin MeSH
• alfa-ketoglutarát-dependentní dioxygenasa, AlkB homolog 5 * MeSH
• FTO protein, mouse MeSH Prohlížeč
• gen pro FTO * MeSH
• RNA MeSH

Transfer RNAs acquire a large plethora of chemical modifications. Among those, modifications of the anticodon loop play important roles in translational fidelity and tRNA stability. Four human wobble U-containing tRNAs obtain 5-methoxycarbonylmethyluridine (mcm5U34) or 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U34), which play a role in decoding. This mark involves a cascade of enzymatic activities. The last step is mediated by alkylation repair homolog 8 (ALKBH8). In this study, we performed a transcriptome-wide analysis of the repertoire of ALKBH8 RNA targets. Using a combination of HITS-CLIP and RIP-seq analyses, we uncover ALKBH8-bound RNAs. We show that ALKBH8 targets fully processed and CCA modified tRNAs. Our analyses uncovered the previously known set of wobble U-containing tRNAs. In addition, both our approaches revealed ALKBH8 binding to several other types of noncoding RNAs, in particular C/D box snoRNAs.

• Klíčová slova
• ALKBH8, HITS-CLIP, Trm9, mcm5U, mcm5s2U, wobble uridine modification,
• MeSH
• AlkB homolog 8, tRNA methyltransferasa genetika   MeSH
• antikodon MeSH
• ChiP sekvenování * MeSH
• lidé MeSH
• nekódující RNA genetika   MeSH
• RNA transferová * genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• AlkB homolog 8, tRNA methyltransferasa MeSH
• ALKBH8 protein, human MeSH Prohlížeč
• antikodon MeSH
• nekódující RNA MeSH
• RNA transferová * 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

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

The DNA damage response is mediated by both DNA repair proteins and epigenetic markers. Here, we observe that N6-methyladenosine (m6A), a mark of the epitranscriptome, was common in RNAs accumulated at UV-damaged chromatin; however, inhibitors of RNA polymerases I and II did not affect the m6A RNA level at the irradiated genomic regions. After genome injury, m6A RNAs either diffused to the damaged chromatin or appeared at the lesions enzymatically. DNA damage did not change the levels of METTL3 and METTL14 methyltransferases. In a subset of irradiated cells, only the METTL16 enzyme, responsible for m6A in non-coding RNAs as well as for splicing regulation, was recruited to microirradiated sites. Importantly, the levels of the studied splicing factors were not changed by UVA light. Overall, if the appearance of m6A RNAs at DNA lesions is regulated enzymatically, this process must be mediated via the coregulatory function of METTL-like enzymes. This event is additionally accompanied by radiation-induced depletion of 2,2,7-methylguanosine (m3G/TMG) in RNA. Moreover, UV-irradiation also decreases the global cellular level of N1-methyladenosine (m1A) in RNAs. Based on these results, we prefer a model in which m6A RNAs rapidly respond to radiation-induced stress and diffuse to the damaged sites. The level of both (m1A) RNAs and m3G/TMG in RNAs is reduced as a consequence of DNA damage, recognized by the nucleotide excision repair mechanism.

• Klíčová slova
• DNA repair, METTL-like enzymes, RNA methylation, epigenetics, histones,
• MeSH
• adenosin analogy a deriváty   metabolismus   MeSH
• chromatin metabolismus   MeSH
• demetylace DNA účinky záření   MeSH
• fyziologický stres účinky záření   MeSH
• guanosin analogy a deriváty   metabolismus   MeSH
• metylace DNA genetika   účinky záření   MeSH
• metylace účinky záření   MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nekódující RNA metabolismus   MeSH
• nestabilita genomu účinky záření   MeSH
• poškození DNA MeSH
• RNA metabolismus   MeSH
• ultrafialové záření * MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosin MeSH
• chromatin MeSH
• guanosin MeSH
• N-methyladenosine MeSH Prohlížeč
• N(2),N(2),7-trimethylguanosine MeSH Prohlížeč
• nekódující RNA MeSH
• RNA MeSH

Eukaryotic RNA can carry more than 100 different types of chemical modifications. Early studies have been focused on modifications of highly abundant RNA, such as ribosomal RNA and transfer RNA, but recent technical advances have made it possible to also study messenger RNA (mRNA). Subsequently, mRNA modifications, namely methylation, have emerged as key players in eukaryotic gene expression regulation. The most abundant and widely studied internal mRNA modification is N6 -methyladenosine (m6 A), but the list of mRNA chemical modifications continues to grow as fast as interest in this field. Over the past decade, transcriptome-wide studies combined with advanced biochemistry and the discovery of methylation writers, readers, and erasers revealed roles for mRNA methylation in the regulation of nearly every aspect of the mRNA life cycle and in diverse cellular, developmental, and disease processes. Although large parts of mRNA function are linked to its cytoplasmic stability and regulation of its translation, a number of studies have begun to provide evidence for methylation-regulated nuclear processes. In this review, we summarize the recent advances in RNA methylation research and highlight how these new findings have contributed to our understanding of methylation-dependent RNA processing in the nucleus. This article is categorized under: RNA Processing > RNA Editing and Modification RNA Processing > Splicing Regulation/Alternative Splicing RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.

• Klíčová slova
• RNA demethylase, RNA methylase, RNA processing,
• MeSH
• buněčné jádro metabolismus   MeSH
• epigeneze genetická MeSH
• lidé MeSH
• messenger RNA metabolismus   MeSH
• metylace MeSH
• prekurzory RNA metabolismus   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
• prekurzory RNA MeSH