Live-cell mapping of organelle-associated RNAs via proximity biotinylation combined with protein-RNA crosslinking

The spatial organization of RNA within cells is a crucial factor influencing a wide range of biological functions throughout all kingdoms of life. However, a general understanding of RNA localization has been hindered by a lack of simple, high-throughput methods for mapping the transcriptomes of subcellular compartments. Here, we develop such a method, termed APEX-RIP, which combines peroxidase-catalyzed, spatially restricted in situ protein biotinylation with RNA-protein chemical crosslinking. We demonstrate that, using a single protocol, APEX-RIP can isolate RNAs from a variety of subcellular compartments, including the mitochondrial matrix, nucleus, bulk cytosol, and endoplasmic reticulum (ER), with specificity and sensitivity that rival or exceed those of conventional approaches. We further identify candidate RNAs localized to mitochondria-ER junctions and nuclear lamina, two compartments that are recalcitrant to classical biochemical purification. Since APEX-RIP is simple, versatile, and does not require special instrumentation, we envision its broad application in a variety of biological contexts. 27 samples total, from five experimental courses (negative controls, and mitochondrial, nuclear, cytosolic, and endoplasmic reticulum-targeted APEX-RIP). Negative controls: 4 samples (2 replicates, with paired inputs and mock RIPs); Mitochondrial APEX-RIP: 6 samples (3 replicates, with paired inputs and RIPs); Nuclear APEX-RIP: 6 samples (3 replicates, with paired inputs and RIPs); Cytosolic APEX-RIP: 6 samples (6 replicates, with paired inputs and RIPs); ER-targeted APEX-RIP ("KDEL"): 4 samples (2 replicates, with paired inputs and RIPs).