Thermodynamics and Binding Mechanism of Insulin-like Growth Factor Binding Protein 1 (IGF2BP1) and Y3 RNA Interaction

Y3 RNA is a conserved noncoding RNA involved in RNA-protein interactions, stress responses, and post-transcriptional regulation. It interacts with various RNA-binding proteins, including Insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), which regulates mRNA stability, localization, and translation. However, the molecular basis of Y3 RNA recognition by IGF2BP1 remains unclear. In this study, we analyzed the structural conservation of Y3 RNA and characterized its interaction with IGF2BP1. Phylogenetic analysis showed that Y3 RNA is highly conserved across mammals, with over 90% sequence similarity and a preserved secondary structure featuring an apical stem-loop, lower stem, and pyrimidine-rich internal loop. UV melting experiments confirmed the predicted structural elements, revealing two distinct melting transitions corresponding to the upper and lower stems. Electrophoretic mobility shift assays (EMSA) and isothermal titration calorimetry (ITC) demonstrated that the IGF2BP1 binding to Y3 RNA is multimeric. It occurs via a biphasic mechanism as demonstrated by thermodynamic studies, primarily mediated by the KH3-4 di-domain. Mutational analysis identified the pyrimidine-rich internal loop as the key binding site, with minimal contributions from the apical stem-loop and lower stem. Thermodynamic studies revealed that one binding event is entropically driven, followed by the binding interactions which are enthalpically favorable. Our findings provide new insights into the conserved structural features of Y3 RNA and its recognition by IGF2BP1. Identifying key RNA elements involved in binding enhances our understanding of Y3 RNA's functional roles and its potential impact on RNA-protein networks.