Functional proximity mapping of RNA binding proteins uncovers a mitochondrial mRNA anchor that promotes stress recovery

Proximity labeling (PL) with genetically-targeted promiscuous enzymes has emerged as a powerful tool for unbiased proteome discovery. By combining the spatiotemporal specificity of PL with methods for functional protein enrichment, it should be possible to map specific protein subclasses within distinct compartments of living cells. Here we demonstrate this capability for RNA binding proteins (RBPs), by combining peroxidase-based PL with organic-aqueous phase separation of crosslinked protein-RNA complexes (“APEX-PS”). We validated APEX-PS by mapping nuclear RBPs, then applied it to uncover the RBPomes of two unpurifiable subcompartments - the nucleolus and the outer mitochondrial membrane (OMM). At the OMM, we discovered the RBP SYNJ2BP, which retains specific nuclear-encoded mitochondrial mRNAs during translation stress, to promote their local translation and import of protein products into the mitochondrion during stress recovery. APEX-PS is a versatile tool for compartment-specific RBP discovery and expands the scope of PL to functional protein mapping. Graphic Abstract