Structural insights into FTO’s catalytic mechanism for the demethylation of multiple RNA substrates

Abstract

FTO demethylates internal N6-methyladenosine (m6A) and N6,2′-O-dimethyladenosine (m6Am; at the cap +1 position) in mRNA, m6A and m6Am in snRNA, and N1-methyladenosine (m1A) in tRNA in vivo, and in vitro evidence supports that it can also demethylate N6-methyldeoxyadenosine (6mA), 3-methylthymine (3mT), and 3-methyluracil (m3U). However, it remains unclear how FTO variously recognizes and catalyzes these diverse substrates. Here we demonstrate—in vitro and in vivo—that FTO has extensive demethylation enzymatic activity on both internal m6A and cap m6Am. Considering that 6mA, m6A, and m6Am all share the same nucleobase, we present a crystal structure of human FTO bound to 6mA-modified ssDNA, revealing the molecular basis of the catalytic demethylation of FTO toward multiple RNA substrates. We discovered that (i) N6-methyladenine is the most favorable nucleobase substrate of FTO, (ii) FTO displays the same demethylation activity toward internal m6A and m6Am in the same RNA sequence, suggesting that the substrate specificity of FTO primarily results from the interaction of residues in the catalytic pocket with the nucleobase (rather than the ribose ring), and (iii) the sequence and the tertiary structure of RNA can affect the catalytic activity of FTO. Our findings provide a structural basis for understanding the catalytic mechanism through which FTO demethylates its multiple substrates and pave the way forward for the structure-guided design of selective chemicals for functional studies and potential therapeutic applications.

Mammalian ALKBH1 serves as an N6-mA demethylase of unpairing DNA

“…Substrate-binding pocket of ALKBH1 Substrate-binding pocket of AlkB family members is formed by residues from NRL subdomain and DSBH fold, in which a target base is inserted, with its alkyl group pointing to the active site. 31,32,41,42 Substrate-binding pocket of ALKBH1, compared to those of its homologs, was shown to be more similar to that of AlkB: comprising residues from DSBH fold (βI-III, βVIII and a loop between βII-III) assume nearly identical conformation in both proteins. Major differences come from the NRL subdomain, with a shorter Flip2 from ALKBH1 showing a "back-drawing" conformation, and its "stretch-out" Flip1 resulting in a more exposed pocket (Figs.…”

“…31,37 Unlike the highly conserved catalytic core, the NRL subdomain is structurally variable among AlkB members and has been shown to play a critical role in substrate interaction. [31][32][33][34][35][37][38][39]41 In most ALKBH1 homologs, [31][32][33]37,41,42 the Flip1 motif adopts a bending conformation covering the active center (Supplementary information, Fig. S6), and forms a positively charged groove for nucleic acid binding (Fig.…”

FTO-mediated cytoplasmic m6Amdemethylation adjusts stem-like properties in colorectal cancer cell

“…To better comprehend the biological function of FTO, extensive efforts have been made to identify relevant RNA substrate(s). No clear consensus emerges from recently published studies [29, 41, 51, 52]. Following the identification of FTO as the first m 6 A mRNA demethylase [17], several reports connected demethylation of internal m 6 A nucleotides with a wide range of biological processes such as viral infection, stress- and DNA UV damage-responses [53-55].…”

It’s the Little Things (in Viral RNA)

“…The N 6 -methylation of adenosine is a very dynamic modification. It is added to the RNA by a methyltransferase complex comprising two catalytic subunits (METTL3 and METTL14), a novel protein (KIAA1429), a splicing factor (Wilms’ tumor associated protein [WTAP]), and two other as-yet-unidentified subunits ( 41 ) The modification can then be removed by two demethylases—the fat mass and obesity-associated protein (FTO) and AlkBH5 ( 42 )—in a process that regulates RNA metabolism, stability, localization, and protein interactions, as well as transport and splicing ( 43 – 45 ). The methylation is preferentially located at translational start sites, stop codons, and the 3′ UTR ( 20 ).…”