Mechanisms of substrate recognition and N6-methyladenosine demethylation revealed by crystal structures of ALKBH5–RNA complexes

Journal article

AlkB homologue 5 (ALKBH5) is a ferrous iron and 2-oxoglutarate dependent oxygenase that demethylates RNA N⁶-methyladenosine (m⁶A), 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 m⁶A 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 m⁶A demethylation is lacking. We report three crystal structures of ALKBH5 in complex with an m⁶A-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)m⁶AC consensus sequence motif. The results support a mechanism involving formation of an m⁶A hemiaminal intermediate, followed by efficient ALKBH5 catalyzed demethylation, enabled by a proton shuttle network involving Lys132 and Tyr139.

Authors: Kaur, S; Tam, NY; McDonough, MA; Schofield, CJ; Aik, WS

• Publication status: Published
• Peer review status: Peer reviewed
• Publisher: Oxford University Press
• Journal: Nucleic Acids Research
• Volume: 50
• Issue: 7
• Pages: 4148–4160
• Place of publication: England
• Publication date: 2022-03-25
• Acceptance date: 2022-03-14
• DOI: 10.1093/nar/gkac195
• EISSN: 1362-4962
• ISSN: 0305-1048
• Pmid: 35333330
• Language: English
• Keywords: RNA; AlkB homolog 5; alpha-ketoglutarate-dependent dioxygenase FTO; demethylation; RNA demethylase; FFR; adenosine