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

Abstract

AbstractN1‐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.