Molecular Basis of UG‐Rich Element Recognition by ESRP2 RRM3

Epithelial splicing regulatory protein 2 (ESRP2) plays a pivotal role in alternative splicing regulation, particularly in maintaining epithelial cell identity and suppressing epithelial-to-mesenchymal transition (EMT). Despite its biological significance, the structural basis for its RNA-binding specificity remains poorly understood. In this study, we report the solution structure and RNA-binding properties of the RNA Recognition Motif (RRM3) of human ESRP2 using an integrative approach combining nuclear magnetic resonance (NMR) spectroscopy, ITC, molecular docking, and MD simulations. Our structural analysis revealed that ESRP2-RRM3 adopts a canonical RRM fold (βαββαβ), featuring a positively charged β-sheet surface conducive to RNA interaction. ITC assays demonstrated that RRM3 binds UG-rich RNA sequences with moderate affinity, and NMR titrations identified key interacting residues within the conserved RNP motifs, particularly F522 and R480. RNA docking and MD simulations further corroborated these interactions, revealing π-π stacking and hydrogen bonding at the protein-RNA interface. These findings represent the first atomic-level characterization of ESRP2's interaction with its RNA targets and provide mechanistic insight into how it may guide alternative splicing events in vivo. This work lays the groundwork for understanding the modular RNA recognition by ESRP2's multiple RRMs and its broader role in splicing regulation, development, and cancer suppression.