High-resolution Profiling of RNA-Protein Interaction in Living Cells

RNA-protein interactions play essential roles in regulating a wide range of cellular processes. Profiling the protein interactome of specific RNA loci enables understanding the underlying molecular mechanisms of RNA regulatory processes. Here we describe an RNA-centric proximity labeling approach for profiling the protein interactome of particular RNA sequences in living cells. In this method, CIRTS3-miniTurbo fusion proteins assemble with specific gRNAs to bind to target RNA and subsequently covalently tag the interacting proteins of target RNA in situ. We first profiled the protein interactome of circRNA mCherry and found that HNRNPK modulates the expression level of circmCherry and some endogenous circRNAs. We then employed our method to in-situ capture the interacting proteins of the rG4 structure in the BCL-2 transcript. We validated the interaction between RBM12 and BCL-2 rG4 structure and showed that RBM12 modulates the translation of the BCL-2 transcript by recruiting ribosomal proteins. Our studies provide a generally applicable strategy for profiling the protein interactome of specific RNA sequences in living cells. To investigate the effect of HNRNPK on the production of endogenous circRNAs, we overexpressed the HNRNPK protein, and the circRNA expression levels were quantified by circRNA-seq.