2025/12/15 by Iwona Nowak, Ahsan Habib Polash, Hang T Huynh +9 · 1 voice
Biochemistry, Genetics and Molecular Biology · #RNA Research and Splicing #Nuclear Structure and Function #Biotin and Related Studies
paper · doi:10.64898/2025.12.12.693770
openalex publication_date 2025/12/15 · openalex created_date 2025/12/16 · openalex updated_date 2026/07/14
RNA-binding proteins (RBPs) enable post-transcriptional gene regulation (PTGR) through specific interactions with RNA molecules, influencing processes ranging from nuclear processing and export to cytoplasmic localization, translation, storage and degradation. A key determinant of PTGR processes is the subcellular compartmentalization of RBPs, which dictates RNA targets they can access and the regulation performed in that environment. To characterize RBP-RNA interactions at subcellular resolution, we developed RBProximity-CLIP. RBProximity-CLIP enables compartment-specific isolation and profiling of individual RBP-RNA interactions by combining APEX2-based proximity labeling and 4-thiouridine-enhanced RNA-protein crosslinking, with sequential RBP- and biotin-affinity purifications. Using this approach, we profiled the RNA targets of three RBPs, AGO2, YBX1, and ELAVL1, across the cytoplasmic, nuclear, and nucleolar compartments, revealing nucleus-specific miRNA-mediated AGO2 targets, as well as subsets of YBX1 and ELAVL1 targets that differ by compartment, yet share identical binding motifs. RBProximity-CLIP enables specific and sensitive detection of compartment-specific RBP-RNA interactomes, thereby providing new insight into spatial gene regulation by RBPs.