The Molecular Basis of Human ALKBH3 Mediated RNA N1-methyladenosine (m1A) Demethylation

Lin Zhang, Hong-Chao Duan, Marcin Paduch, Jingyan Hu, Chi Zhang, Yajuan Mu, Houwen Lin, Chuan He, Anthony A. Kossiakoff, Guifang Jia, Liang Zhang

Zitierweise: Angew. Chem. Int. Ed. 2024, 63, e202313900

PMID: 38158383 DOI: 10.1002/ange.202313900

N1-methyladenosine (m1A) is a prevalent post-transcriptional RNA modification, and the distribution and dynamics of the modification play key epitranscriptomic roles in cell development. At present, the human AlkB Fe(II)/α-ketoglutarate-dependent dioxygenase family member ALKBH3 is the only known mRNA m1A demethylase, but its catalytic mechanism remains unclear. Here, we present the structures of ALKBH3-oligo crosslinked complexes obtained with the assistance of a synthetic antibody crystallization chaperone. Structural and biochemical results showed that ALKBH3 utilized two β-hairpins (β4-loop-β5 and β′-loop-β′′) and the α2 helix to facilitate single-stranded substrate binding. Moreover, a bubble-like region around Asp194 and a key residue inside the active pocket (Thr133) enabled specific recognition and demethylation of m1A- and 3-methylcytidine (m3C)-modified substrates. Mutation of Thr133 to the corresponding residue in the AlkB Fe(II)/α-ketoglutarate-dependent dioxygenase family members FTO or ALKBH5 converted ALKBH3 substrate selectivity from m1A to N6-methyladenosine (m6A), as did Asp194 deletion. Our findings provide a molecular basis for understanding the mechanisms of substrate recognition and m1A demethylation by ALKBH3. This study is expected to aid structure-guided design of chemical probes for further functional studies and therapeutic applications.