Emerging Technologies in RNA–Protein Interaction Analysis

RNA–protein interactions (RPIs), mediated primarily by RNA-binding proteins (RBPs), are central to post-transcriptional gene regulation and govern RNA splicing, transport, localization, translation, and decay. Dysregulation of RBPs and their associated RNA networks contributes to diverse pathologies, including cancer, neurodegenerative disorders, and viral infections. However, profiling RPIs remains a challenge due to their inherent transience, low binding affinity, and shifting spatial dynamics. This review provides a comprehensive and systematic overview of current methodologies for investigating RPIs. We discuss RNA-centric and protein-centric strategies. In addition, imaging-based approaches are evaluated for their capacity to resolve spatial and temporal dynamics of RBP–RNA interactions in situ. We compare these methodologies in terms of resolution, sensitivity, specificity, and biological applicability, emphasizing the importance of integrative strategies for constructing high-resolution, context-dependent RPI maps in physiological and disease settings.