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

ABSTRACT 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, N 6 ‐methyladenosine (m 6 A) 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 m 6 A erasers. Using RNAs with m 6 A 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 m 6 A was near the G‐tetrad core, such as the HIV‐1 U3‐region G‐quadruplex‐forming sequence. Conversely, m 6 A 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 m 6 A eraser protein activity. These findings highlight the importance of the RNA structural context in regulating m 6 A erasers and suggest that G‐quadruplexes may serve as regulatory elements in epitranscriptomic control.