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

RNA-binding proteins (RBPs) orchestrate post-transcriptional gene regulatory (PTGR) processes through specific interactions with RNA molecules, which are tightly regulated in both space and time, including through the subcellular compartmentalization of RBPs and their target RNAs. To characterize these interactions in specific subcellular compartments, we developed RBProximity-CLIP, an approach that integrates targeted APEX2-based proximity labeling with 4-thiouridine–enhanced RNA–protein crosslinking to enable the isolation and profiling of target sites of individual RBPs in a compartment-specific manner . Using this approach, we profiled three RBPs—AGO2, YBX1, and ELAVL1—within the cytoplasmic, nuclear, and nucleolar compartments. RBProximity-CLIP enabled robust isolation of compartment specific RBP-RNA interactome and new insights of compartmental specific gene regulation of distinct RBPs. Overall design: We developed RBProximity-CLIP methodology for isolating and profiling RNA binding proteins (herein: AGO2, YBX1 and ELAVL1) in cytoplasm, nucleus and nucleolar compartments.