PT - JOURNAL ARTICLE AU - Pornchai Kaewsapsak AU - David M. Shechner AU - William Mallard AU - John L. Rinn AU - Alice Y. Ting TI - Live-cell mapping of organelle-associated RNAs via proximity biotinylation combined with protein-RNA crosslinking AID - 10.1101/153098 DP - 2017 Jan 01 TA - bioRxiv PG - 153098 4099 - http://biorxiv.org/content/early/2017/06/21/153098.short 4100 - http://biorxiv.org/content/early/2017/06/21/153098.full AB - The spatial organization of RNA within cells is a crucial factor in a wide range of biological functions, spanning 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 higher specificity and coverage than do conventional approaches. We furthermore 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.