Mechanisms of substrate recognition and N6-methyladenosine demethylation revealed by crystal structures of ALKBH5–RNA complexes. Issue 7 (25th March 2022)

• Record Type: Journal Article
• Authors: Kaur, Simranjeet; Tam, Nok Yin; McDonough, Michael A; Schofield, Christopher J; Aik, Wei Shen
• Is Part Of: Nucleic acids research. Volume 50:Issue 7(2022)
• Page Start: 4148
• Page End: 4160
• Publication Date: 2022-03-25
• Subjects: Nucleic acids -- Periodicals; Molecular biology -- Periodicals; 572.805
• DOI: 10.1093/nar/gkac195
• Languages: English
• ISSNs: 0305-1048

Abstract

Abstract: AlkB homologue 5 (ALKBH5) is a ferrous iron and 2-oxoglutarate dependent oxygenase that demethylates RNA N 6 -methyladenosine (m 6 A), a post-transcriptional RNA modification with an emerging set of regulatory roles. Along with the fat mass and obesity-associated protein (FTO), ALKBH5 is one of only two identified human m 6 A RNA oxidizing enzymes and is a potential target for cancer treatment. Unlike FTO, ALKBH5 efficiently catalyzes fragmentation of its proposed nascent hemiaminal intermediate to give formaldehyde and a demethylated nucleoside. A detailed analysis of the molecular mechanisms used by ALKBH5 for substrate recognition and m 6 A demethylation is lacking. We report three crystal structures of ALKBH5 in complex with an m 6 A-ssRNA 8-mer substrate and supporting biochemical analyses. Strikingly, the single-stranded RNA substrate binds to the active site of ALKBH5 in a 5′-3′ orientation that is opposite to single-stranded or double-stranded DNA substrates observed for other AlkB subfamily members, including single-stranded DNA bound to FTO. The combined structural and biochemical results provide insight into the preference of ALKBH5 for substrates containing a (A/G)m 6 AC consensus sequence motif. The results support a mechanism involving formation of an m 6 A hemiaminal intermediate, followed by efficient ALKBH5 catalyzed demethylation, enabled by a proton shuttle network involving Lys132 and Tyr139. Graphical Abstract: