Identification of the stress granule transcriptome via RNA-editing in single cells and in vivo

Wessel van Leeuwen; Michael VanInsberghe; Nico Battich; Fredrik Salmén; Alexander van Oudenaarden; Catherine Rabouille

doi:10.1016/j.crmeth.2022.100235

Identification of the stress granule transcriptome via RNA-editing in single cells and in vivo

Wessel van Leeuwen, Michael VanInsberghe, Nico Battich, Fredrik Salmén, Alexander van Oudenaarden, Catherine Rabouille

Hubrecht Institute

Research output: Contribution to journal/periodical › Article › Scientific › peer-review

5 Citations (Scopus)

Abstract

Stress granules are phase-separated assemblies formed around RNAs. So far, the techniques available to identify these RNAs are not suitable for single cells and small tissues displaying cell heterogeneity. Here, we used TRIBE (target of RNA-binding proteins identified by editing) to profile stress granule RNAs. We used an RNA-binding protein (FMR1) fused to the catalytic domain of an RNA-editing enzyme (ADAR), which coalesces into stress granules upon oxidative stress. RNAs colocalized with this fusion are edited, producing mutations that are detectable by VASA sequencing. Using single-molecule FISH, we validated that this purification-free method can reliably identify stress granule RNAs in bulk and single S2 cells and in Drosophila neurons. Similar to mammalian cells, we find that stress granule mRNAs encode ATP binding, cell cycle, and transcription factors. This method opens the possibility to identify stress granule RNAs and other RNA-based assemblies in other single cells and tissues.

Original language	English
Pages (from-to)	100235
Journal	Cell reports methods
Volume	2
Issue number	6
DOIs	https://doi.org/10.1016/j.crmeth.2022.100235
Publication status	Published - 20 Jun 2022

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10.1016/j.crmeth.2022.100235

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title = "Identification of the stress granule transcriptome via RNA-editing in single cells and in vivo",

abstract = "Stress granules are phase-separated assemblies formed around RNAs. So far, the techniques available to identify these RNAs are not suitable for single cells and small tissues displaying cell heterogeneity. Here, we used TRIBE (target of RNA-binding proteins identified by editing) to profile stress granule RNAs. We used an RNA-binding protein (FMR1) fused to the catalytic domain of an RNA-editing enzyme (ADAR), which coalesces into stress granules upon oxidative stress. RNAs colocalized with this fusion are edited, producing mutations that are detectable by VASA sequencing. Using single-molecule FISH, we validated that this purification-free method can reliably identify stress granule RNAs in bulk and single S2 cells and in Drosophila neurons. Similar to mammalian cells, we find that stress granule mRNAs encode ATP binding, cell cycle, and transcription factors. This method opens the possibility to identify stress granule RNAs and other RNA-based assemblies in other single cells and tissues.",

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T1 - Identification of the stress granule transcriptome via RNA-editing in single cells and in vivo

AU - van Leeuwen, Wessel

AU - VanInsberghe, Michael

AU - Battich, Nico

AU - Salmén, Fredrik

AU - van Oudenaarden, Alexander

AU - Rabouille, Catherine

PY - 2022/6/20

Y1 - 2022/6/20

N2 - Stress granules are phase-separated assemblies formed around RNAs. So far, the techniques available to identify these RNAs are not suitable for single cells and small tissues displaying cell heterogeneity. Here, we used TRIBE (target of RNA-binding proteins identified by editing) to profile stress granule RNAs. We used an RNA-binding protein (FMR1) fused to the catalytic domain of an RNA-editing enzyme (ADAR), which coalesces into stress granules upon oxidative stress. RNAs colocalized with this fusion are edited, producing mutations that are detectable by VASA sequencing. Using single-molecule FISH, we validated that this purification-free method can reliably identify stress granule RNAs in bulk and single S2 cells and in Drosophila neurons. Similar to mammalian cells, we find that stress granule mRNAs encode ATP binding, cell cycle, and transcription factors. This method opens the possibility to identify stress granule RNAs and other RNA-based assemblies in other single cells and tissues.

AB - Stress granules are phase-separated assemblies formed around RNAs. So far, the techniques available to identify these RNAs are not suitable for single cells and small tissues displaying cell heterogeneity. Here, we used TRIBE (target of RNA-binding proteins identified by editing) to profile stress granule RNAs. We used an RNA-binding protein (FMR1) fused to the catalytic domain of an RNA-editing enzyme (ADAR), which coalesces into stress granules upon oxidative stress. RNAs colocalized with this fusion are edited, producing mutations that are detectable by VASA sequencing. Using single-molecule FISH, we validated that this purification-free method can reliably identify stress granule RNAs in bulk and single S2 cells and in Drosophila neurons. Similar to mammalian cells, we find that stress granule mRNAs encode ATP binding, cell cycle, and transcription factors. This method opens the possibility to identify stress granule RNAs and other RNA-based assemblies in other single cells and tissues.

U2 - 10.1016/j.crmeth.2022.100235

DO - 10.1016/j.crmeth.2022.100235

M3 - Article

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JO - Cell reports methods

JF - Cell reports methods

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ER -

Identification of the stress granule transcriptome via RNA-editing in single cells and in vivo

Abstract

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