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Elife. 2017 Dec 14;6. pii: e29224. doi: 10.7554/eLife.29224.

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

Kaewsapsak P1,2,3,4, Shechner DM5,6,7, Mallard W5,6,7, Rinn JL5,6,7, Ting AY1,2,3,4,7.

Author information

1
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, United States.
2
Department of Genetics, Stanford University, Stanford, United States.
3
Department of Biology, Stanford University, Stanford, United States.
4
Department of Chemistry, Stanford University, Stanford, United States.
5
Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, United States.
6
Department of Molecular and Cellular Biology, Harvard University, Cambridge, United States.
7
Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, United States.

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.

KEYWORDS:

APEX2; Horseradish peroxidase; RNA localization; RNA sequencing; biochemistry; cell biology; human; peroxidase; subcellular transcriptome

PMID:
29239719
PMCID:
PMC5730372
DOI:
10.7554/eLife.29224
[Indexed for MEDLINE]
Free PMC Article

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