m6A functions and distribution

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m6A – an overview

Recent advances in sequencing technology have resulted in sensitive techniques which 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. These same advances in sequencing methods and technology have also allowed us to determine the readers, writers, and erasers responsible for m6A's functions, methylation, and demethylation. Here in this pathway card, we cover these essential proteins responsible for m6A addition and removal on RNA, and we go into further detail of some of the crucial readers of m6A and the epigenetic functions these serve.

m6A methylation and demethylation

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 METTL3^1,2 and METTL14, the METTL3 adapter WTAP³, and other associated proteins including KIAA1429⁴ and RBM15/15B⁵. The eraser of m6A is known to be carried out by two different enzymes: FTO and ALKBH5^6,7. 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. 

m6A readers and functions

Knowledge of m6A readers and their functions is rapidly increasing. More and more studies are revealing new m6A-binding proteins with new 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 binders8. These different YTH domain proteins have since been shown to have a broad spectrum of roles associated with their ability to bind m6A. YTHDF2 bound m6A is thought to play a role in mRNA stability⁹. YTHDF1 is known to promote translation efficiency¹⁰. YTHDC1 is implicated to be important in m6A mediated alternative splicing¹¹. Not all m6A binding proteins are YTH containing. eIF3 plays a role in translation preinitiation, and this protein has also been confirmed to bind preferentially to m6A within the 5'UTR of mRNA leading to enhanced translation¹². Download our m6A pathway poster to learn more about m6A readers and their functions. 

m6A sequencing methods

Various methods have been developed to map m6A throughout the transcriptome effectively. Initial studies have used immunoprecipitation methods using highly specific m6A antibodies followed by next-generation sequencing¹³. 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 ab151230¹⁴. miCLIP has since been used to map m6A in many different cell types and organisms revealing new potential functions for m6A. 

References

1) Bokar JA, Shambaugh ME, Polayes D, Matera AG, Rottman FM. 1997. Purification and cDNA cloning of the AdoMet-binding subunit of the human mRNA (N6-adenosine)-methyltransferase. RNA 3:1233–47

2) Geula S, Moshitch-Moshkovitz S, Dominissini D, Mansour AA, Kol N, et al. 2015. m6A mRNA methylation facilitates resolution of naıve pluripotency toward differentiation. Science 347:1002–6

3) Agarwala SD, Blitzblau HG, Hochwagen A, Fink GR. 2012. RNA methylation by the MIS complex regulates a cell fate decision in yeast. PLOS Genet. 8:e1002732

4) Horiuchi K, Kawamura T, Iwanari H, Ohashi R, Naito M, et al. 2013. Identification of Wilms’ tumor 1–associating protein complex and its role in alternative splicing and the cell cycle. J. Biol. Chem. 288:33292–302

5) Patil DP, Chen CK, Pickering BF, Chow A, Jackson C, et al. 2016. m6A RNA methylation promotes XISTmediated transcriptional repression. Nature 537:369–73

6) Jia G, Fu Y, Zhao X, Dai Q, Zheng G, et al. 2011. N6-Methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO. Nat. Chem. Biol. 7:885–87

7) Zheng G, Dahl JA, Niu Y, Fedorcsak P, Huang CM, et al. 2013. ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility. Mol. Cell 49:18–29

8) Dominissini D, Moshitch-Moshkovitz S, Schwartz S, Salmon-Divon M, Ungar L, et al. 2012. Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature 485:201–6

9) Wang X, Lu Z, Gomez A, Hon GC, Yue Y, et al. 2014. N6-Methyladenosine-dependent regulation of messenger RNA stability. Nature 505:117–20

10) Wang X, Zhao BS, Roundtree IA, Lu Z, Han D, et al. 2015. N6-Methyladenosine modulates messenger RNA translation efficiency. Cell 161:1388–99

11) Xiao W, Adhikari S, Dahal U, Chen YS, Hao YJ, et al. 2016. Nuclear m6A reader YTHDC1 regulates mRNA splicing. Mol. Cell 61:507–19

12) Meyer KD, Patil DP, Zhou J, Zinoviev A, Skabkin MA, et al. 2015. 5' UTR m6A promotes cap-independent translation. Cell 163:999–1010

13) Meyer KD, Saletore Y, Zumbo P, Elemento O, Mason CE, Jaffrey SR. 2012. Comprehensive analysis of mRNA methylation reveals enrichment in 3' UTRs and near stop codons. Cell 149:1635–46

14) Linder B, Grozhik AV, Olarerin-George AO, Meydan C, Mason CE, Jaffrey SR. 2015. Single-nucleotide resolution mapping of m6A and m6Am throughout the transcriptome. Nat. Methods 12:767–72