<i>In vivo</i> discovery of RNA proximal proteins via proximity-dependent biotinylation

RNA molecules function as messenger RNAs (mRNAs) that encode proteins and noncoding transcripts that serve as adaptor molecules, structural components, and regulators of genome organization and gene expression. Their function and regulation are largely mediated by RNA binding proteins (RBPs). Here we present RNA proximity labelling (RPL), an RNA-centric method comprising the endonuclease-deficient Type VI CRISPR-Cas protein dCas13b fused to engineered ascorbate peroxidase APEX2. RPL discovers target RNA proximal proteins <i>in vivo</i> via proximity-based biotinylation. RPL applied to <i>U1</i> identified proteins involved in both <i>U1</i> canonical and noncanonical functions. Profiling of poly(A) tail proximal proteins uncovered expected categories of RBPs and provided additional evidence for 5ʹ-3ʹ proximity and unexplored subcellular localizations of poly(A)⁺ RNA. Our results suggest that RPL allows rapid identification of target RNA binding proteins in native cellular contexts, and is expected to pave the way for discovery of novel RNA–protein interactions important for health and disease.