Live-cell mapping of organelle-associated RNAs via proximity biotinylation combined with protein-RNA crosslinking
Author(s)Kaewsapsak, Pornchai; Shechner, David Michael; Mallard, William; Rinn, John L; Ting, Alice Y
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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, 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.
DepartmentMassachusetts Institute of Technology. Department of Chemistry
eLife Sciences Organisation, Ltd.
Kaewsapsak, Pornchai et al. “Live-Cell Mapping of Organelle-Associated RNAs via Proximity Biotinylation Combined with Protein-RNA Crosslinking.” eLife 2017, 6 (December 2017): e29224 © 2017 Kaewsapsak et al
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