N6-methyladenosine (m6A) is a highly prevalent RNA modification in mRNA and non-coding RNAs that affects RNA splicing, translation, and stability, as well as the epigenetic effects of certain non-coding RNAs¹.

Recent advances in sequencing technology have resulted in sensitive techniques that can be used to map m6A to the transcriptome of various model systems and cell types. Knowing the transcriptome-wide location of m6A has given a clearer indication of this marks many functions. We now have a much greater understanding of the biological and regulatory roles being played by this epigenetic mark, although it is also clear that there is still much left to uncover. The same advances in sequencing methods and technology have also allowed us to determine the readers (m6A-binding proteins), writers (methyltransferases), and erasers (demethylases) responsible for m6A's functions, methylation, and demethylation.

Here, we cover the essential m6A writers and erasers as well as some of the crucial readers of m6A and the epigenetic functions they serve.

m6A writers and erasers

m6A is dynamically regulated within the nucleus by a methyltransferase writer complex, which deposits m6A on mRNA, and m6A demethylases, which will remove the mark.

The writer complex is known to contain the methyltransferases METTL3^2,3 and METTL14, the METTL3 adapter WTAP⁴, and other associated proteins including KIAA1429⁵, RBM15/15B⁶, and ZC3H13⁷. In the METTL3-METTL14 methyltransferase domain complex, METTL3 acts as the catalytic subunit, while METTL14 offers an RNA-binding scaffold, activating and enhancing the catalytic activity of METTL3⁷. WTAP exhibits no catalytic activity but interacts with METTL3 and METTL14 to modulate the m6A levels of RNA transcripts.⁷

Both METTL3 and METTL14 play essential roles in diverse biological processes including embryonic development and neurogenesis. METTL3 has been also shown to regulate cell reprogramming, spermatogenesis, T cell homeostasis, and endothelial-to-hematopoietic transition. METTL14 has been reported to act as a tumor suppressor in glioblastoma and play an oncogenic role in the development and maintenance of acute myeloid leukemia.

Erasers

The removal of m6A is known to be carried out by two different enzymes: FTO and ALKBH5^8,9. However, FTO has been found to preferentially demethylate m6A_m, an m6A-related, highly abundant nucleotide in mRNA¹⁰. This finding suggests that ALKBH5 may serve as the major m6A demethylase.

​The idea that m6A can be added to mRNA and subsequently removed will be an important consideration for future m6A research. Dynamic changes in m6A levels across the transcriptome may point to new functions for this epigenetic mark.

Recombinant monoclonal RabMab® antibodies for key m6A writers and erasers

Achieve highly reproducible results in your epigenetic research with our recombinant monoclonal antibodies for key m6A writers and erasers. All of these antibodies are knockout validated to ensure their specificity.

m6A readers and functions

m6A exerts its effects on RNA by recruiting m6A-binding proteins called readers. Knowledge of m6A readers and their functions is rapidly increasing. More and more studies are revealing new m6A readers with specific roles for m6A in different cell types and model systems.

m6A-binding proteins will commonly contain a YTH (YT521-B homology) domain. RNA pulldown has shown that proteins containing a YTH domain are commonly m6A binders¹¹. These different YTH domain proteins have since been shown to have a broad spectrum of roles associated with their ability to bind m6A. YTHDF1, working together with YTHDF3, is known to promote the translation efficiency of its target RNAs^7,12. YTHDF2 is thought to play a role in mRNA stability¹³. YTHDC1 is implicated in m6A-mediated alternative splicing¹⁴. YTHDC2 has been shown to enhance the translation efficiency of its targets while decreasing target abundance.¹⁵

Not all m6A readers are YTH containing; they also include eukaryotic initiation factor 3 (eIF3), heterogeneous nuclear ribonucleoproteins (HNRNPs), and insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs). eIF3 plays a role in translation preinitiation and has also been confirmed to bind preferentially to m6A within the 5'UTR of mRNA leading to enhanced translation¹⁶. Two HNRNP proteins, HNRNPC and HNRNPG, regulate the processing of m6A-containing RNA transcripts, harboring m6A as a structural switch to make those transcripts more accessible for binding⁷. IGF2BPs have been reported to promote the stability and storage of their target mRNAs in an m6A-dependent manner¹⁷.

Apart from being involved in RNA metabolism, m6A readers also participate in different biological processes, including tumorigenesis, hematopoiesis, virus replication, immune response, and adipogenesis.¹⁸

Recombinant monoclonal RabMab® antibodies for key m6A writers and erasers

Achieve highly reproducible results in your epigenetic research with our recombinant monoclonal antibodies for key m6A writers and erasers. All of these antibodies are knockout validated to ensure their specificity.

m6A sequencing methods

Various methods have been developed to map m6A throughout the transcriptome effectively. Initial studies have used immunoprecipitation methods with highly specific m6A antibodies followed by next-generation sequencing¹⁹. These methods are known as MeRIP-seq. MeRIP has since advanced, and it is now possible to carry out single-nucleotide resolution m6A mapping. This can be achieved using very small fragments of RNA (100–200 nts) in combination with immunoprecipitation. This method is known as miCLIP (m6A individual-nucleotide-resolution-crosslinking and immunoprecipitation), and it was first described in Linder et al. 2015 using the Abcam m6A antibody ab151230²⁰. miCLIP has since been used to map m6A in many different cell types and organisms revealing new potential functions for m6A.

If you want to learn more about key techniques used to study RNA modifications, refer to our RNA modifications guide. If you are interested in the miCLIP method, check out our miCLIP protocol.

References

1. Meyers, K. D. & , Jaffrey, S. R. Rethinking m6A readers, writers, and erasers. Annu Rev Cell Dev Biol  33 ,319-342 (2017)
2. Bokar, J.A.,, Shambaugh, M.E., , Polayes, D., , Matera, A.G.,, & Rottman, F.M. Purification and cDNA cloning of the AdoMet-binding subunit of the human mRNA (N6-adenosine)-methyltransferase. 3, 1233–47 (1997). RNA  3 ,1233-1247 (1997)
3. Guela, S., et al. m6A mRNA methylation facilitates resolution of naıve pluripotency toward differentiation. Science  347, ,1002–1006 (2015)
4. Agarwala, S.D., , Blitzblau, H.G., , Hochwagen, A.,, & Fink, G.R. RNA methylation by the MIS complex regulates a cell fate decision in yeast. PLOS Genet.  8 , (2012)
5. Horiuchi, K., et al. Identification of Wilms’ tumor 1–associating protein complex and its role in alternative splicing and the cell cycle. J. Biol. Chem.  288 ,33292–302 (2013)
6. Patil, D.P., et al. m6A RNA methylation promotes XISTmediated transcriptional repression. 537, 369–373 (2016). Nature  537 ,369-373 (2016)
7. Yang, Y., , Hsu, P.J., , Chen, Y.S., , & Yang, Y.G. Dynamic transcriptomic m6A decoration: writers, erasers, readers and functions in RNA metabolism. Cell Res.  28, ,616-624 (2018)
8. Jia, G., et al. N6-Methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO. Nat. Chem. Biol.  7, ,885–887 (2011)
9. Zheng, G., et al. ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility. Mol. Cell  49 ,18-29 (2013)
10. Mauer, J., et al. Reversible methylation of m6Am in the 5´ cap controls mRNA stability. Nature  541 ,371–375 (2017)
11. Dominissini, D., et al. Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature 485, 201–206 (2012). Nature  485 ,201-206 (2012)
12. Wang, X. et al. N6-Methyladenosine-dependent regulation of messenger RNA stability. Nature  505 ,117-120 (2014)
13. Wang, X. et al. N6-Methyladenosine modulates messenger RNA translation efficiency. Cell  161 ,1388-1399 (2015)
14. Hsu, P.J., et al. Ythdc2 is an N6-methyladenosine binding protein that regulates mammalian spermatogenesis. 27, 1115-1127 (2017). Cell Res.  27 ,1115-1127 (2017)
15. Meyer, K.D., al. 5' UTR m6A promotes cap-independent translation Cell  163 ,999-1010 (2015)
16. Huang, H. et al. Recognition of RNA N6-methyladenosine by IGF2BP proteins enhances mRNA stability and translation. Nat. Cell. Biol.  20 ,285-295 (2018)
17. Zhao, Y., et al. m6A-binding proteins: the emerging crucial performers in epigenetics. 13, 35 (2020). J Hematol Oncol  13 ,35 (2020)
18. Meyer, K. D. et al. Comprehensive analysis of mRNA methylation reveals enrichment in 3' UTRs and near stop codons. Cell  149 ,1635-1646 (2012)
19. Linder, B. et al. Single-nucleotide resolution mapping of m6A and m6Am throughout the transcriptome. Nat. Methods 12, 767–772 (2015). Nat. Methods  12 ,767-772 (2015)