Structural Control of RNA Demethylation: G‐Quadruplex Proximity Suppresses ALKBH5 Activity

RNA modifications and higher-order structures function as regulatory layers of RNA metabolism beyond sequence context. However, their interplay remains largely unexplored. Among RNA modifications, N⁶-methyladenosine (m⁶A) is the most abundant internal modification in mRNAs, regulated by writer, reader, and eraser proteins; the AlkB family dioxygenases, AlkB homolog 5 (ALKBH5), and fat mass- and obesity-associated protein (FTO). In addition, G-quadruplex RNA is a representative structure involved in key biological processes. This study investigated how G-quadruplex structures influence the demethylation efficiency of m⁶A erasers. Using RNAs with m⁶A positioned adjacent to the G-tetrad core or within a flexible loop of G-quadruplex structures, we showed that G-quadruplex formation markedly suppressed ALKBH5 activity compared with FTO, particularly when m⁶A was near the G-tetrad core, such as the HIV-1 U3-region G-quadruplex-forming sequence. Conversely, m⁶A within a long, flexible loop, remained accessible to ALKBH5. The G-quadruplex-specific ligand BRACO19, which stabilizes G-quadruplex structures, further confirmed the structural sensitivity of ALKBH5. To our knowledge, this is the first study to demonstrate a direct link between G-quadruplex structures and m⁶A eraser protein activity. These findings highlight the importance of the RNA structural context in regulating m⁶A erasers and suggest that G-quadruplexes may serve as regulatory elements in epitranscriptomic control.