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

N-1-methyladenosine (m(1)A) 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)/alpha-ketoglutarate-dependent dioxygenase family member ALKBH3 is the only known mRNA m(1)A 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 beta-hairpins (beta 4-loop-beta 5 and beta '-loop-beta '') and the alpha 2 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 m(1)A- and 3-methylcytidine (m(3)C)-modified substrates. Mutation of Thr133 to the corresponding residue in the AlkB Fe(II)/alpha-ketoglutarate-dependent dioxygenase family members FTO or ALKBH5 converted ALKBH3 substrate selectivity from m(1)A to N-6-methyladenosine (m(6)A), as did Asp194 deletion. Our findings provide a molecular basis for understanding the mechanisms of substrate recognition and m(1)A demethylation by ALKBH3. This study is expected to aid structure-guided design of chemical probes for further functional studies and therapeutic applications.