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Mol Cell. 2019 Aug 22;75(4):875-887.e5. doi: 10.1016/j.molcel.2019.07.030.

Proximity RNA Labeling by APEX-Seq Reveals the Organization of Translation Initiation Complexes and Repressive RNA Granules.

Author information

1
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
2
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; RNA Systems Biochemistry Laboratory, RIKEN Cluster for Pioneering Research, Wako, Saitama 351-0198, Japan; Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Tokyo 277-8561, Japan.
3
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA. Electronic address: ingolia@berkeley.edu.

Abstract

Diverse ribonucleoprotein complexes control mRNA processing, translation, and decay. Transcripts in these complexes localize to specific regions of the cell and can condense into non-membrane-bound structures such as stress granules. It has proven challenging to map the RNA composition of these large and dynamic structures, however. We therefore developed an RNA proximity labeling technique, APEX-seq, which uses the ascorbate peroxidase APEX2 to probe the spatial organization of the transcriptome. We show that APEX-seq can resolve the localization of RNAs within the cell and determine their enrichment or depletion near key RNA-binding proteins. Matching the spatial transcriptome, as revealed by APEX-seq, with the spatial proteome determined by APEX-mass spectrometry (APEX-MS), obtained precisely in parallel, provides new insights into the organization of translation initiation complexes on active mRNAs and unanticipated complexity in stress granule composition. Our novel technique allows a powerful and general approach to explore the spatial environment of macromolecules.

KEYWORDS:

APEX-Seq; RNA localization; proximity labeling; subcellular localization

PMID:
31442426
PMCID:
PMC6834362
[Available on 2020-08-22]
DOI:
10.1016/j.molcel.2019.07.030
[Indexed for MEDLINE]

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