Subcellular refinement of RNA binding protein target interactions with colocalization CLIP [CLIP-seq]

RNA binding proteins (RBPs) are essential for RNA metabolism and have profound impacts in both health and disease. The subcellular organization of RBP interaction networks with target RNAs remains largely unexplored. Here, we develop colocalization CLIP, a method that combines CrossLinking and ImmunoPrecipitation (CLIP) with proximity labeling, to explore in-depth the subcellular RNA interactions of the well-studied RNA-binding protein HuR. Using this method, we uncover HuR's dynamic and location-specific interactions with RNA, revealing alterations in sequence preferences and interactions in the nucleus, cytosol, or stress granule compartments. We uncover HuR's unique binding preferences within stress granules during arsenite stress, illuminating intricate interactions that conventional methodologies cannot capture. Overall, coCLIP provides a powerful method for revealing RBP:RNA interactions based on localization, and lays the foundation for an advanced understanding of RBP models that incorporate subcellular location as a critical determinant of their functions. Overall design: To investigate nuclear/cytoplasm/stress granule-specific binding behaviors of HuR, three different cell lines expressing ascorbate peroxidase protein in the respective compartments were generated. Then, proximity labeling of the local proteome was performed by treating the cells with hydrogen peroxide and biotin phenol. Following proximity labeling, UV crosslinking and immunoprecipitation for HuR followed by high-throughput sequencing (CLIP-Seq) were performed in the three cell lines. HuR CLIP-Seq was performed on cytoplasm and nuclear fraction as comparison groups.