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

Alejandro Padròn, Shintaro Iwasaki, View ORCID ProfileNicholas T. Ingolia
doi: https://doi.org/10.1101/454066
Alejandro Padròn
1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720 USA
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Shintaro Iwasaki
1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720 USA
2RNA Systems Biochemistry Laboratory, RIKEN Cluster for Pioneering Research, Wako, Saitama, 351-0198 Japan
3Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, 277-8561 Japan
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Nicholas T. Ingolia
1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720 USA
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  • ORCID record for Nicholas T. Ingolia
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Abstract

Diverse ribonucleoprotein complexes control messenger RNA 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) provides new insights into the organization of translation initiation complexes on active mRNAs, as well as exposing unanticipated complexity in stress granule composition, and provides a powerful and general approach to explore the spatial environment of macromolecules.

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Posted October 25, 2018.
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Proximity RNA labeling by APEX-Seq Reveals the Organization of Translation Initiation Complexes and Repressive RNA Granules
Alejandro Padròn, Shintaro Iwasaki, Nicholas T. Ingolia
bioRxiv 454066; doi: https://doi.org/10.1101/454066
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Proximity RNA labeling by APEX-Seq Reveals the Organization of Translation Initiation Complexes and Repressive RNA Granules
Alejandro Padròn, Shintaro Iwasaki, Nicholas T. Ingolia
bioRxiv 454066; doi: https://doi.org/10.1101/454066

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