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Structural and biochemical characterization of mRNA demethylase enzyme FTO: understanding FTO's diverse mechanisms
Date
2025
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
University of Delaware
Abstract
The RNA demethylase FTO acts as a methyl ‘eraser’ to remove either internal N6- methyladenosine (m6A) or 5′ end N6-2′-O-dimethyladenosine (m6Am) modifications on mRNA by way of a hemiaminal intermediate. FTO and its demethylation activity have strong links to human cancers, where FTO is highly expressed and promotes oncogenesis. FTO has an intrinsic preference and significantly faster demethylation rates in vitro for m6Am modifications located at the 5′ mRNA cap structure, but the structural basis for FTO’s ability to discriminate m6A versus m6Am modifications has remained unknown. In addition, the current studies and methods to map changing m6A marks on RNA are not truly capturing and mapping demethylation events. ☐ Here we utilize molecular dynamics simulations, mutagenesis, and enzymology of FTO-RNA cap complexes to identify conserved aromatic residues on the surface of FTO involved in 5′ cap recognition and validate the specificity of these residues in engaging the 5′ cap structure to promote m6Am demethylation. We also begin to test various chemistries to trap FTO demethylation intermediates to be able to fully map demethylation events on mRNA. Moreover, we use proteomics and enzymology to understand the importance of FTO’s self-hydroxylation and how that links to FTO’s catalytic mechanisms. This work provides the first structural insights into how FTO selectively catalyzes m6Am versus m6A demethylation on mRNA and furthers our understanding of how FTO activity is regulated by diverse mechanisms to help control the epitranscriptome.
Description
"At the request of the author or degree granting institution, this graduate work is not available to view or purchase until August 13 2026."--ProQuest abstract/details page.
Keywords
Demethylation activity, Enzymology, Dimethyladenosine