N6-Methyladenosine: a conformational marker that regulates the substrate specificity of human demethylases FTO and ALKBH5

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.

FTO, m6Am, and the hypothesis of reversible epitranscriptomic mRNA modifications

“…The original studies on FTO-mediated m 6 A demethylation [25], as well as follow-up studies [46], showed that FTO efficiently demethylates m 6 A irrespective of sequence context. The original studies on FTO-mediated m 6 A demethylation [25], as well as follow-up studies [46], showed that FTO efficiently demethylates m 6 A irrespective of sequence context.…”

The m6A‐epitranscriptomic signature in neurobiology: from neurodevelopment to brain plasticity

“…The m6A demethylases: focus on FTO In mammals, two m6A demethylating enzymes have been identified to date, namely the fat mass and obesity-associated (FTO) protein (Jia et al 2011) and Alkbh5 (Zheng et al 2012). Both belong to the AlkB family of the Fe(II)/aketoglutarate-dependent dioxygenase superfamily and possess a similar catalytic core, although they have different substrate preferences (Aik et al 2014;Zou et al 2016) and are differentially expressed in different mammalian organs (Gerken et al 2007;Zheng et al 2012). ALKBH5 is highly expressed in testis and is essential for spermatogenesis (Zheng et al 2012).…”

“…This not only indicates a compensatory mechanism driven by Alkbh5, but also raises the question of whether FTO and Alkbh5 have different substrate specificity. A modeling study of m6A containing RNAs suggests that the conformational change induced by m6A could be a critical factor for FTO or ALKBH5 recruitment (Zou et al 2016).…”

Biochemical Characterization of AP Lyase and m6A Demethylase Activities of Human AlkB Homologue 1 (ALKBH1)

“…15–17 Alkbh3 hydroxylates methyl lesions in single-stranded (ss) DNA as well as m 1 A in mRNA, thereby expanding the substrate range to RNA. 18 Alkbh5 and FTO also act on RNA, 19–22 but these enzymes demethylate 6-methyl adenine (m 6 A) in this substrate in vitro , affecting mRNA stability. 1 In vivo , Alkbh5 plays a role in fertility while FTO is important for controlling body weight in mice and humans.…”