In vivo discovery of RNA proximal proteins in human cells via proximity-dependent biotinylation

Abstract

RNA molecules function as messengers or noncoding adaptor molecules, structural components, and regulators of genome organization and gene expression. Their roles and regulation are mediated by other molecules they interact with, especially RNA binding proteins (RBPs). Here we report RNA proximity labeling (RPL), an RNA-centric method based on fusion of an endonuclease-deficient Type VI CRISPR-Cas protein (dCas13b) and engineered ascorbate peroxidase (APEX2) to discover in vivo target RNA proximal proteins (RPPs) through proximity-based biotinylation. U1 RPPs enriched by proximity-based biotinylation included both U1 snRNA canonical and noncanonical functions-related proteins. In addition, profiling of poly(A) tail proximal proteins uncovered expected categories of RBPs for poly(A) tails and also provided novel evidence for poly(A)+ RNA 5-3 proximity and expanded subcellular localizations. Our results suggest that RPL is a rapid approach for identifying both interacting and neighboring proteins associated with target RNA molecules in their native cellular contexts.