RBProximity-CLIP: A Method for Profiling the Interactome of an RNA-Binding Protein at Subcellular Resolution

RNA-binding proteins (RBPs) are central players in post-transcriptional gene regulation (PTGR), and their mode of action often depends on subcellular localization. Conventional crosslinking and immunoprecipitation (CLIP) methods map the RNA targets of RBPs with nucleotide resolution but lack spatial specificity, making it impossible to distinguish how one RBP can contribute to biological processes across distinct compartments simultaneously. Here, we present RBProximity-CLIP, an extension of Proximity-CLIP that integrates APEX2-mediated proximity labeling with UV crosslinking of 4-thiouridine-labeled RNAs, followed by sequential biotin- and RBP-specific immunoprecipitations, to capture RBP-RNA complexes with both spatial and protein specificity. RBProximity-CLIP generates nucleotide-resolution maps of RBP-RNA interactions and enables proteomic characterization of complete ribonucleoprotein (RNP) compositions. Using Y-box binding protein 1 (YBX1) as a model RBP, we provide a detailed protocol for RBProximity-CLIP.