Live-cell mapping of organelle-associated RNAs via proximity biotinylation combined with protein-RNA crosslinking.
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Abstract | 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. |
Year of Publication | 2017
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Journal | Elife
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Volume | 6
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Date Published | 2017 12 14
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ISSN | 2050-084X
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DOI | 10.7554/eLife.29224
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PubMed ID | 29239719
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PubMed Central ID | PMC5730372
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Grant list | R01 CA186568 / CA / NCI NIH HHS / United States
U01 DA040612 / DA / NIDA NIH HHS / United States
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