Colocalization CLIP (coCLIP) for subcellular resolution of RBP–RNA interactions. (A) An APEX2 enzyme is localized to a specific compartment, depicted here as either nuclear, cytoplasmic, or within stress granules as a G3BP1 fusion. The proximity labeling reaction using biotin phenol and hydrogen peroxide is performed, followed by UV cross-linking. (B) After cell lysis, the sample undergoes antibody-based RBP–CLIP. A second streptavidin pulldown following RBP IP enables the capture of local RBP targets. (C) Immunofluorescence for streptavidin reactive signal with and without labeling reagents for arsenite-stressed Huh7.5 cells with nuclear, cytoplasmic, or G3BP1 localized APEX2. Images were captured at 20× magnification. Scale bar, 10 µm. (D) Example autoradiograms for input HuR CLIP (left) or cytoplasmic HuR coCLIP (right) from two replicates. The size of HuR is indicated with an arrow and excised HuR:RNA complexes are indicated with boxes. (OD) RNAse overdigestion, (-Lab) no label. (E) HuR peak counts (left) and proportions (right) for mRNAs within input CLIP or from nuclear, cytoplasmic, or G3BP1 coCLIP in Huh7.5 cells. (F) HuR peak counts (left) and proportions (right) as in E for ncRNA peaks. See also Supplemental Figures S1–S3.
