N6-methyladenosine is a modification found in eukaryotic mRNAs and long non-coding RNAs. Recent research indicates that N6-methyladenosine is part of a controlling mechanism that regulates cellular functions such as the circadian rhythm, meiosis and stem cell development. In prokaryotic DNA, N6-methyladenosine primarily functions in the host defense system. However, until recently the significance of this modification in eukaryotes had been unclear.
DNA methylation is fundamental to epigenetic regulation. The most common DNA modification observed in eukaryotes is 5-methylcytosine (5mC, or m5A), whereas N6-methyladenosine (6mA) is most prevalent in prokaryotes. Because of the widespread distribution of 5mC in mammals and plants, earlier sequencing studies have focused on this methylated nucleoside. The presence of 5mC in eukaryotes has been unclear. However, new research now indicates that adenine bases are also methylated in eukaryotes, including in mammals and humans.
Figure 1: Structural models of 5-methylcytosine (5mC) and N6-methyladenine (6mA).
Figure 2: Structural models of Ythdf1 YTH domain in complex with 5mer 6mA RNA. PDB 4RCJ. The structure provides the molecular basis for the recognition of 6mA by the YTH domain of YTHDF2.
N6-methyladenosine and the regulation of messenger RNA stability
According to Wang et al. (2014) N6-methyladenosine (6mA) is a prevalent internal modification in eukaryotic messenger RNAs. The recent discovery of 6mA demethylases in mammalian cells indicates that this modification may have some important function in cells. Also, its misregulation maybe a cause for diseases as well. Wang et al. were able to show that 6mA is selectively recognized by the human YTH domain family 2 protein (YTHDF2) to regulate mRNA degradation. The YTHDF2 protein family specifically recognizes and binds 6mA-containing RNAs. This protein family is known to regulate the stability of mRNAs and plays a role in the efficiency of mRNA splicing, processing, and stability. The binding of 6mA-containing RNAs results in localization to mRNA decay sites including the processing of P-bodies. Some 6mA methylation marks at the 5’-untranslated region (5’-UTR) promote cap-independent mRNA translation.
Combined recent discoveries of 6mA modifications suggest that this modification is a prevalent internal modification in eukaryotic RNA as part of an essential RNA regulatory mechanism.
Writers, Readers, and Erasers
Similarly to the histone code this methylation mark is written, read, and erased by specific RNA binding proteins. The modification is post-transcriptionally installed, written or added by 6mA methyltransferases (METTL3-METTL14-WTAP complex. “Writer” protein), recognized or “Read” by the YTH domain of YTHDF2 proteins, and erased by 6mA demethylases (FTO and ALKBH5. “Eraser” proteins). Li et al. in 2014 solved the structure of the YTH domain of human YTHDF2 in complex with 6mA mononucleotide. They were also able to show that a 6mA-containing RNA probe sequence derived from mRNA binds to YTH-YTHDF2. The RNA probe used was AUGG(6mA)CUCC.
N6-methyladenosine in eukaryotic DNA
The latest development of highly sensitive methods allowed for the detection of this N6-methyladenosine in eukaryotic DNA such as Chlamydomonas, Tetrahymena, C. elegans, Drosophila, and green algae.
Luo et al. in 2015 discuss recent publications documenting the presence of m6A in Chlamydomonas reinhardtii, Drosophila melanogaster, and Caenorhabditis elegans. This paper considers possible roles for this DNA modification. Reported results imply that m6A takes part in regulating transcription, the activity of transposable elements and transgenerational epigenetic inheritance. Furthermore, Luo et al. propose 6mA as a new epigenetic mark in eukaryotes. However, the prevalence and significance of this modification in eukaryotes had been unclear until recently.
Modern mass spectrometry now allows accurate quantification amounts of methylated DNA and next-generation sequencing (NGS) provides a powerful additional tool for the genome-wide study of DNA modifications. The combination of next-generation sequencing methods with enzymatic methods now allows for the characterization methylated nucleic acid bases in DNA.
Luo et al. in 2015 applied a sensitive restriction enzyme-assisted sequencing method for the study of m6A sites in DNA at single-base resolution. The research group used an approach that uses the restriction enzyme DpnI for the cleavage of methylated adenine sites in duplex DNA together with NGS. Luo et al. found that DpnI recognizes CATC, GATG and GATC sides and cleaves G(m6A)TC sites as well as G9m6A)TG sites.
The resulting sequencing data suggested that the m6A non-GATC sites are highly enriched at promoter regions and form a periodic pattern around transcription start sites.
To validate their results, the researchers used liquid chromatography in tandem with mass spectrometry (LC-MS/MS) to quantify m6A abundance in four candidate organisms. Modern mass spectrometry can detect the very low abundance of nucleotide modifications but can only provide the overall ratio of the modification in total DNA. On the other hand, high-throughput sequencing coupled with bioinformatic analysis offers a powerful tool for the study of m6A pattern and other modifications in eukaryotic DNA.
Reference
http://www.uniprot.org/uniprot/Q9Y5A9
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"N(6)-methyladenosine modulates messenger RNA translation efficiency." Wang X., Zhao B.S., Roundtree I.A., Lu Z., Han D., Ma H., Weng X., Chen K., Shi H., He C. [PubMed] [Europe PMC] [Abstract]
"Structural basis for the discriminative recognition of N6-methyladenosine RNA by the human YT521-B homology domain family of proteins." Xu C., Liu K., Ahmed H., Loppnau P., Schapira M., Min J.
J. Biol. Chem. 290:24902-24913(2015) [PubMed] [Europe PMC] [Abstract] - RNA-BINDING.
"Dynamic m(6)A mRNA methylation directs translational control of heat shock response." Zhou J., Wan J., Gao X., Zhang X., Jaffrey S.R., Qian S.B.; [PubMed] [Europe PMC] [Abstract] - SUBCELLULAR LOCATION, INDUCTION.
"Structure of the YTH domain of human YTHDF2 in complex with an m(6)A mononucleotide reveals an aromatic cage for m(6)A recognition." Li F., Zhao D., Wu J., Shi Y.; Cell Res. 24:1490-1492(2014) [PubMed] [Europe PMC] [Abstract] - X-RAY CRYSTALLOGRAPHY (2.10 ANGSTROMS) OF 408-552 IN COMPLEX WITH N6-METHYLADENOSINE (M6A)-CONTAINING RNA, MUTAGENESIS OF TRP-432; TRP-486 AND TRP-491.
"Crystal structure of the YTH domain of YTHDF2 reveals mechanism for recognition of N6-methyladenosine."
Zhu T., Roundtree I.A., Wang P., Wang X., Wang L., Sun C., Tian Y., Li J., He C., Xu Y.; [PubMed] [Europe PMC] [Abstract] - X-RAY CRYSTALLOGRAPHY (2.12 ANGSTROMS) OF 383-553, FUNCTION, RNA-BINDING, MUTAGENESIS OF ARG-411; LYS-416; TRP-432; ARG-441; TRP-486 AND ARG-527.
Guan-Zheng Luo, Mario Andres Blanco, Eric Lieberman Greer, Chuan He & Yang Shi; DNA N6-methyladenine: a new epigenetic mark in eukaryotes? Nature Reviews Molecular Cell Biology 16, 705–710 (2015) [PMC]