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

Dec 29, 2023Angewandte Chemie (International ed. in English)

How Human ALKBH3 Protein Removes a Specific Chemical Mark from RNA

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Abstract

Structures of ALKBH3 complexes reveal that specific residues facilitate recognition and demethylation of modified RNA substrates.

ALKBH3 uses two β-hairpins and an α-helix for binding to single-stranded RNA.
A bubble-like region around Asp194 and Thr133 in the active site are crucial for recognizing mA and 3-methylcytidine-modified substrates.
Mutating Thr133 or deleting Asp194 alters substrate selectivity of ALKBH3 from mA to N-methyladenosine.
Findings contribute to understanding the molecular mechanisms of mA demethylation.

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N-methyladenosine (mA) 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 mA 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 mA- and 3-methylcytidine (mC)-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 mA to N-methyladenosine (mA), as did Asp194 deletion. Our findings provide a molecular basis for understanding the mechanisms of substrate recognition and mA demethylation by ALKBH3. This study is expected to aid structure-guided design of chemical probes for further functional studies and therapeutic applications. 1 1 1 1 3 1 6 6 1

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

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