RBProximity-CLIP Enables Subcellular Mapping of RNA-Binding Protein Interactions at Nucleotide Resolution

Nowak, I.; Polash, A. H.; Huynh, H. T.; Kaur, M.; Lobo, V.; Scutenaire, J.; Fong, M.; Alluhaibi, G.; Anastasakis, D. G.; Hafner, M.; Benhalevy, D.; Sarshad, A.

doi:10.64898/2025.12.12.693770

bioRxiv · 10.64898/2025.12.12.693770

RBProximity-CLIP Enables Subcellular Mapping of RNA-Binding Protein Interactions at Nucleotide Resolution

Nowak, I.·Polash, A. H.·Huynh, H. T.·Kaur, M.·Lobo, V.·Scutenaire, J.·Fong, M.·Alluhaibi, G.·Anastasakis, D. G.·Hafner, M.·Benhalevy, D.·Sarshad, A.

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Abstract

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.

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Nowak, I., Polash, A. H., Huynh, H. T., Kaur, M., Lobo, V., Scutenaire, J., Fong, M., Alluhaibi, G., Anastasakis, D. G., Hafner, M., Benhalevy, D., Sarshad, A.. 2025-12-15. RBProximity-CLIP Enables Subcellular Mapping of RNA-Binding Protein Interactions at Nucleotide Resolution. https://doi.org/10.64898/2025.12.12.693770

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