The Molecular Basis of Human ALKBH3 Mediated RNA N1 -methyladenosine (m1 A) Demethylation

Angew Chem Int Ed Engl. 2024 Feb 12;63(7):e202313900. doi: 10.1002/anie.202313900. Epub 2024 Jan 11.

Abstract

N1 -methyladenosine (m1 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)/α-ketoglutarate-dependent dioxygenase family member ALKBH3 is the only known mRNA m1 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 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 m1 A- and 3-methylcytidine (m3 C)-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 m1 A to N6 -methyladenosine (m6 A), as did Asp194 deletion. Our findings provide a molecular basis for understanding the mechanisms of substrate recognition and m1 A demethylation by ALKBH3. This study is expected to aid structure-guided design of chemical probes for further functional studies and therapeutic applications.

Keywords: ALKBH3; Demethylation; Enzyme Catalysis; N1-Methyladenosine (m1A); RNA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase / metabolism
  • Alpha-Ketoglutarate-Dependent Dioxygenase FTO* / metabolism
  • Demethylation
  • Ferrous Compounds
  • Humans
  • RNA* / chemistry
  • RNA, Messenger / metabolism

Substances

  • RNA
  • Alpha-Ketoglutarate-Dependent Dioxygenase FTO
  • RNA, Messenger
  • Ferrous Compounds
  • ALKBH3 protein, human
  • AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase
  • FTO protein, human