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

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: About 6 x 105 cells were seeded in 60-mm plates and transfected the next day with 50 pmol of siRNA targeting YBX1 (ThermoFisher Scientific, Cat.#4390824, siRNA ID s9731) or a control siRNA (ThermoFisher Scientific, Cat.#4390843) preincubated for 20 min at room temperature with 5 µL of Lipofectamine? RNAiMAX transfection reagent (ThermoFisher Scientific, Cat.#13778150) in OPTI-MEM medium. Cells were collected and split in two 72 h after transfection. Total proteins were extracted with RIPA buffer on one half of the cells and used for western blot to assess the silencing efficiency. Total RNAs were extracted on the second half of the cells with TRIzol™ Reagent (ThermoFisher Scientific, Cat.#15596018) and treated with DNAse I using the Direct-zol RNA Miniprep kit (Zymo research, Cat.#R2052) according to the manufacturer's instructions. Each siRNA transfection was performed in biological triplicate. rRNA-depleted libraries were generated from 1 µg of total RNA using the NEBNext® rRNA Depletion Kit v2 (New England Biolabs, Cat.#E7400X) and NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (New England Biolabs, Cat.#E7760L) according to the manufacturer's instructions. The quality of the initial RNA material and of the final cDNA libraries was assessed using a TapeStation system (Agilent Technologies). Final libraries were sequenced on an Illumina NovaSeq X Plus platform (Illumina) to generate paired-end reads (2 × 50 bp).