N(6)-Methyladenosine: a conformational marker that regulates the substrate specificity of human demethylases FTO and ALKBH5.

Shui Zou,Esther C Y Woon,Kendra H Q Wong,Yong-Gui Gao,Wanjin Hong,Joel D W Toh

doi:10.1038/srep25677

Abstract

N6-Methyladenosine (m6A) is currently one of the most intensively studied post-transcriptional modifications in RNA. Due to its critical role in epigenetics and physiological links to several human diseases, it is also of tremendous biological and medical interest. The m6A mark is dynamically reversed by human demethylases FTO and ALKBH5, however the mechanism by which these enzymes selectively recognise their target transcripts remains unclear. Here, we report combined biophysical and biochemical studies on the specificity determinants of m6A demethylases, which led to the identification of an m6A-mediated substrate discrimination mechanism. Our results reveal that m6A itself serves as a ‘conformational marker’, which induces different conformational outcomes in RNAs depending on sequence context. This critically impacts its interactions with several m6A-recognising proteins, including FTO and ALKBH5. Remarkably, through the RNA-remodelling effects of m6A, the demethylases were able to discriminate substrates with very similar nucleotide sequences. Our findings provide novel insights into the biological functions of m6A modifications. The mechanism identified in this work is likely of significance to other m6A-recognising proteins.

Highlights

As METTL3-METTL14-WTAP26,27, and this can be directly reversed by human m6A demethylases FTO28 and ALKBH5 (AlkB homologue 5)[25], both of which are medically-important enzymes belonging to the iron- and 2-oxoglutarate (2OG)-dependent family of AlkB oxygenases (Fig. 1)[18,29]
The FTO and ALKBH5 are highly specific for m6A, with little or no activity for other methylated-nucleotides, such as N1-methyladenosine (m1A, a cytotoxic lesion in DNA), and 5-methylcytosine (m5C, another ubiquitous epigenetic modification found in DNA and RNA)[30,31]
Besides messenger RNA (mRNA), recent studies have identified m6A modifications in non-coding RNAs, such as transfer RNA, ribosomal RNA, small nuclear RNA and long non-coding RNA, where they are not found within the same consensus motifs[45,46,47]

Summary

Introduction

As METTL3-METTL14-WTAP26,27, and this can be directly reversed by human m6A demethylases FTO (fat mass and obesity-associated protein)[28] and ALKBH5 (AlkB homologue 5)[25], both of which are medically-important enzymes belonging to the iron- and 2-oxoglutarate (2OG)-dependent family of AlkB oxygenases (Fig. 1)[18,29]. Recent study investigating the structural profiles of RNA in living cells revealed that m6A-modified sites exhibit specific structural signatures; a loss of m6A modifications (through mettl[3] knockout) was accompanied by a significant loss of these structural signatures[42]. It was found that m6A modification on MALAT1 (Metastasis Associated Lung Adenocarcinoma Transcript 1; a lncRNA) caused significant alteration to its local structure, which likely serve to facilitate the binding of its regulatory protein HNRNPC43,44. Inspired by these interesting observations, we envisaged that m6A-induced conformational change could provide a basis for substrate discrimination by m6A demethylases. The unique recognition strategy identified in this work is likely of significance to other m6A-recognising proteins and, more widely, other RNA-binding proteins

Methods

Results

Conclusion