FTO-mediated formation of N6-hydroxymethyladenosine and N6-formyladenosine in mammalian RNA

Abstract

N6-methyladenosine (m6A) is a prevalent internal modification in mRNA and non- coding RNA affecting various cellular pathways. Here we report the discovery of two additional modifications, N6-hydroxymethyladenosine (hm6A) and N6- formyladenosine (f6A), in mammalian mRNA. We show that FeII- and α-ketoglutarate (α-KG)-dependent fat mass and obesity associated (FTO) protein oxidizes m6A to generates hm6A as an intermediate modification and f6A as a further oxidized product. hm6A and f6A have half-life times of ~3 h in aqueous solution under physiological relevant conditions, and are present in isolated mRNA from human cells as well as mouse tissues. These previously unknown modifications derived from the prevalent m6A in mRNA, formed through oxidative RNA demethylation, may dynamically modulate RNA-protein interactions to affect gene expression regulation.

Adenine methylation in eukaryotes: Apprehending the complex evolutionary history and functional potential of an epigenetic modification

“…This suggested that in addition to repair of DNA damaged by alkylating adducts, members of the AlkB family are likely to be key players in m 6 A demethylation in DNA. It remains to be seen whether the metastable intermediates, N 6 hmA and N 6 fA, have any independent role in DNA as proposed for modified RNA 100.…”

Kaposi's Sarcoma-Associated Herpesvirus Utilizes and Manipulates RNA N 6 -Adenosine Methylation To Promote Lytic Replication

“…Three methyltransferases, including methyltransferase-like 3 (METTL3), methyltransferase-like 14 (METTL14), and Wilms' tumor 1-associated protein (WTAP), act as m 6 A writers and catalyze RNA m 6 A at specific sites with the consensus sequence [(G/A)GAC, where the underlined adenosine is the methylation site] (11,12). Two demethylases, fat mass-and obesity-associated protein (FTO) and AlkB homolog 5 (ALKBH5), both of which act as m 6 A erasers, reverse this process (13)(14)(15)(16)(17). Most m 6 A sites are located near the transcription start sites, exonic regions flanking splicing sites, stop codons, and the 3= untranslated region (3= UTR) (1, rabbit anti-m 6 A antibody and control IgG, followed by conversion of the input RNAs and the RNA immunoprecipitation (RIP) products into cDNAs with reverse transcriptase (RT) (Fig.…”

Epitranscriptomics for Biomedical Discovery

“…Unlike Ψ, m 6 A modiications are reversible, suggesting that the modiications are involved in regulatory switches. Methyltransferases (METTL3, METTL14, and WTAP), termed writers, catalyze the methylation of adenosine [21][22][23], whereas demethylases (FTO and ALKBH5), termed erasers, remove the methyl group [24,25]. The m 6 A marks are recognized by YTH domain proteins, termed readers, which regulate mRNA processing and metabolism [26,27].…”