Analyzing the Composition and Organization of Ribonucleoprotein Complexes by APEX-Seq.

Diverse protein-RNA complexes assemble in cells, and their composition and localization regulate the fate of mRNAs. Here, we detail APEX-Seq, an experimental strategy to capture protein-RNA interactions and profile their sub-cellular organization by in vivo proximity labeling and high-throughput sequencing. APEX-Seq relies on direct proximity labeling of RNAs by the peroxidase enzyme APEX2, which can be targeted to specific sites in the cell or fused to proteins of interest. Direct RNA proximity labeling promises new insights into the dynamic behavior of RNA, addressing length scales beyond direct physical contact but too short for microscopy. APEX-Seq should be widely applicable to diverse biological questions and in many cell types, enabling comprehensive studies of the spatial transcriptome and its dynamics over time.