RBProximity-CLIP enables subcellular mapping of RNA-binding protein interactions at nucleotide resolution

RNA-binding proteins (RBPs) regulate gene expression through interactions with RNA molecules. Many RBPs reside in multiple compartments within cells, partitioning their accessible RNA targets and regulome. To characterize RBP-RNA interactions at subcellular resolution, we develop RBProximity-CLIP, which enables compartment-specific capture and profiling of individual RBP-RNA interactions by combining APEX2-based proximity labeling and 4-thiouridine–enhanced RBP-RNA crosslinking, with sequential RBP- and biotin-affinity purifications. Using RBProximity-CLIP, we profile subcellular RNA interactions of AGO2, YBX1, and ELAVL1. We reveal distinct, compartment-specific RNA-binding patterns by these RBPs through canonical motif recognition.