Formation and removal of 1,N6-dimethyladenosine in mammalian transfer RNA

RNA molecules harbor diverse modifications that play important regulatory roles in a variety of biological processes. Over 150 modifications have been identified in RNA molecules. N⁶-methyladenosine (m⁶A) and 1-methyladenosine (m¹A) are prevalent modifications occurring in various RNA species of mammals. Apart from the single methylation of adenosine (m⁶A and m¹A), dual methylation modification occurring in the nucleobase of adenosine, such as N⁶,N⁶-dimethyladenosine (m^6,6A), also has been reported to be present in RNA of mammals. Whether there are other forms of dual methylation modification occurring in the nucleobase of adenosine other than m^6,6A remains elusive. Here, we reported the existence of a novel adenosine dual methylation modification, i.e. 1,N⁶-dimethyladenosine (m^1,6A), in tRNAs of living organisms. We confirmed that m^1,6A is located at position 58 of tRNAs and is prevalent in mammalian cells and tissues. The measured level of m^1,6A ranged from 0.0049% to 0.047% in tRNAs. Furthermore, we demonstrated that TRMT6/61A could catalyze the formation of m^1,6A in tRNAs and m^1,6A could be demethylated by ALKBH3. Collectively, the discovery of m^1,6A expands the diversity of RNA modifications and may elicit a new tRNA modification-mediated gene regulation pathway.     

Mapping messenger RNA methylations at single base resolution

The messenger RNA (mRNA) methylations in mammalian cells have been found to contain N⁶-methyladenosine (m⁶A), N⁶-2′-O-dimethyladenosine (m⁶Am), 7-methylguanosine (m⁷G), 1-methyladenosine (m¹A), 5-methylcytosine (m⁵C), and 2′-O-methylation (2′-OMe). Their regulatory functions in control of mRNA fate and gene expression are being increasingly uncovered. To unambiguously understand the critical roles of mRNA methylations in physiological and pathological processes, mapping these methylations at single base resolution is highly required. Here, we will review the progresses made in methylation sequencing methodologies developed mainly in recent two years, with an emphasis on chemical labeling-assisted single base resolution methods, and discuss the problems and prospects as well.     

Adenine methylation in eukaryotes: Apprehending the complex evolutionary history and functional potential of an epigenetic modification

While N⁶‐methyladenosine (m⁶A) is a well‐known epigenetic modification in bacterial DNA, it remained largely unstudied in eukaryotes. Recent studies have brought to fore its potential epigenetic role across diverse eukaryotes with biological consequences, which are distinct and possibly even opposite to the well‐studied 5‐methylcytosine mark. Adenine methyltransferases appear to have been independently acquired by eukaryotes on at least 13 occasions from prokaryotic restriction‐modification and counter‐restriction systems. On at least four to five instances, these methyltransferases were recruited as RNA methylases. Thus, m⁶A marks in eukaryotic DNA and RNA might be more widespread and diversified than previously believed. Several m⁶A‐binding protein domains from prokaryotes were also acquired by eukaryotes, facilitating prediction of potential readers for these marks. Further, multiple lineages of the AlkB family of dioxygenases have been recruited as m⁶A demethylases. Although members of the TET/JBP family of dioxygenases have also been suggested to be m⁶A demethylases, this proposal needs more careful evaluation. Also watch the Video Abstract .     

A dynamic reversible RNA N6-methyladenosine modification: current status and perspectives

N⁶-methyladenosine (m⁶A), as the most abundant RNA epigenetic modifications, has been shown to play critical roles in various biological functions. Research about enzymes that can catalyze and remove m⁶A have revealed its comprehensive roles in messenger RNA (mRNA) metabolism and other physiological processes. The “readers” including YTH domain-containing proteins, hnRNPC, hnRNPG, hnRNPA2B1, IGF2BP1, IGF2BP2, and IGF2BP3, which can affect the fates of mRNA in an m⁶A-dependent manner. In this review, we focus on recent advances in the research of the m⁶A modifications, especially about the latest functions of its writers, erasers, readers in RNA metabolism, cancer, and lipid metabolism. In the end, we provide insights into the underlying molecular mechanisms of m⁶A modifications.     

Regulation of RNA N6-methyladenosine modification and its emerging roles in skeletal muscle development

N⁶-methyladenosine (m⁶A) is one of the most widespread and highly conserved chemical modifications in cellular RNAs of eukaryotic genomes. Owing to the development of high-throughput m⁶A sequencing, the functions and mechanisms of m⁶A modification in development and diseases have been revealed. Recent studies have shown that RNA m⁶A methylation plays a critical role in skeletal muscle development, which regulates myoblast proliferation and differentiation, and muscle regeneration. Exploration of the functions of m⁶A modification and its regulators provides a deeper understanding of the regulatory mechanisms underlying skeletal muscle development. In the present review, we aim to summarize recent breakthroughs concerning the global landscape of m⁶A modification in mammals and examine the biological functions and mechanisms of enzymes regulating m⁶A RNA methylation. We describe the interplay between m⁶A and other epigenetic modifications and highlight the regulatory roles of m⁶A in development, especially that of skeletal muscle. m⁶A and its regulators are expected to be targets for the treatment of human muscle-related diseases and novel epigenetic markers for animal breeding in meat production.     

A Test and Refinement of Folding Free Energy Nearest Neighbor Parameters for RNA Including N6-Methyladenosine

RNA folding free energy change parameters are widely used to predict RNA secondary structure and to design RNA sequences. These parameters include terms for the folding free energies of helices and loops. Although the full set of parameters has only been traditionally available for the four common bases and backbone, it is well known that covalent modifications of nucleotides are widespread in natural RNAs. Covalent modifications are also widely used in engineered sequences. We recently derived a full set of nearest neighbor terms for RNA that includes N⁶-methyladenosine (m⁶A). In this work, we test the model using 98 optical melting experiments, matching duplexes with or without N⁶-methylation of A. Most experiments place RRACH, the consensus site of N⁶-methylation, in a variety of contexts, including helices, bulge loops, internal loops, dangling ends, and terminal mismatches. For matched sets of experiments that include either A or m⁶A in the same context, we find that the parameters for m⁶A are as accurate as those for A. Across all experiments, the root mean squared deviation between estimated and experimental free energy changes is 0.67 kcal/mol. We used the new experimental data to refine the set of nearest neighbor parameter terms for m⁶A. These parameters enable prediction of RNA secondary structures including m⁶A, which can be used to model how N⁶-methylation of A affects RNA structure.     

YTH Domain: A Family of N6-methyladenosine (m6A) Readers

Like protein and DNA, different types of RNA molecules undergo various modifications. Accumulating evidence suggests that these RNA modifications serve as sophisticated codes to mediate RNA behaviors and many important biological functions. N⁶-methyladenosine (m⁶A) is the most abundant internal RNA modification found in a variety of eukaryotic RNAs, including but not limited to mRNAs, tRNAs, rRNAs, and long non-coding RNAs (lncRNAs). In mammalian cells, m⁶A can be incorporated by a methyltransferase complex and removed by demethylases, which ensures that the m⁶A modification is reversible and dynamic. Moreover, m⁶A is recognized by the YT521-B homology (YTH) domain-containing proteins, which subsequently direct different complexes to regulate RNA signaling pathways, such as RNA metabolism, RNA splicing, RNA folding, and protein translation. Herein, we summarize the recent progresses made in understanding the molecular mechanisms underlying the m⁶A recognition by YTH domain-containing proteins, which would shed new light on m⁶A-specific recognition and provide clues to the future identification of reader proteins of many other RNA modifications.     

The thermodynamic stability of RNA duplexes and hairpins containing N6-alkyladenosines and 2-methylthio-N6-alkyladenosines

The N⁶-alkyladenosines and 2-methylthio-N⁶-alkyladenosines make up over half of the population of all naturally modified adenosines and they are present in the transfer ribonucleic acids (tRNA) at position 37. We measured effects of N⁶-alkyladenosines and 2-methylthio-N⁶-alkyladenosines on the thermodynamic stability of RNA duplexes containing a U-A^Mod base pair at internal and terminal duplex positions, as well as containing modified adenosines as a 3′-terminal unpaired nucleotide. Beside naturally modified adenosines such as N⁶-isopentenyladenosine (i⁶A), N⁶-methyladenosine (m⁶A), 2-methylthio-N⁶-isopentenyladenosine (ms²i⁶A) and 2-methylthio-N⁶-methyladenosine (ms²m⁶A), we studied several artificial modifications to evaluate the steric and electronic effects of N⁶-alkyl substituents. Moreover, some N⁶-alkyladenosines and 2-methylthio-N⁶-alkyladenosines were placed in hairpins at positions corresponding to nucleotide 37 of the tRNA anticodon arm, and the thermodynamic stability of those hairpins was studied. The stability of the modified RNA hairpins was measured in standard melting buffer containing 1 M sodium chloride as well as in physiological buffer containing 10 mM magnesium chloride and 150 mM potassium chloride. The results obtained indicate that the nature of the adenosine modification and the position of U-A^Mod base pairs within the duplex influence the thermodynamic stability of RNA duplexes. For most of the modification, the destabilization of duplexes was observed. Moreover, we found that the buffer composition and the structure of the modified adenosine very significantly affect the thermodynamic stability of RNA.