FMR1 KH0-KH1 domains coordinate m6A binding and phase separation in Fragile X syndrome.

Fragile X messenger ribonucleoprotein 1 (FMR1) regulates neurodevelopment through m6A RNA interactions, yet the domain-specific roles of KH0 and KH1 in RNA binding and disease pathogenesis remain poorly understood. Using mutagenesis and AlphaFold3 structural modeling, we identify KH1 as the primary m6A-binding interface, while the KH0 domain (particularly Arg138) modulates liquid-liquid phase separation (LLPS). Pathogenic mutations in KH0 impair RNA binding and promote aberrant LLPS aggregation, whereas m6A-modified RNA suppresses LLPS formation at KH0. Structural simulations uncover synergistic interactions between KH0 and KH1 mediated by hydrophobic and electrostatic networks. These domain-specific cooperations establish a mechanistic link between m6A dysregulation, pathological phase separation, and Fragile X syndrome pathogenesis. Our findings nominate KH0 as a potential therapeutic target for RNA-driven neurodevelopmental disorders.