Identification of the stress granule transcriptome via RNA-editing in single cells and in vivo
van Leeuwen, Wessel; VanInsberghe, Michael; Battich, Nico; Salmén, Fredrik; van Oudenaarden, Alexander; Rabouille, Catherine
(2022) Cell reports methods, volume 2, issue 6
(Article)
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
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(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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Keywords: Drosophila, hyperTRIBE, neurons, RNA, RNA-editing, S2 cells, single cell, stress granules, VASA-seq, Biotechnology, Biochemistry, Biochemistry, Genetics and Molecular Biology (miscellaneous), Genetics, Radiology Nuclear Medicine and imaging, Computer Science Applications
ISSN: 2667-2375
Publisher: Cell Press
Note: Funding Information: We thank Geert Kops and Jacques Bothma for critically reading the manuscript. We thank Anko de Graaff of the Hubrecht Imaging Facility for help with the imaging and Buys de Barbanson for help with data analysis. This work was supported by a European Research Council Advanced grant ( ERC-AdG 742225-IntScOmics ) and Nederlandse Organisatie voor Wetenschappelijk Onderzoek ( NWO ) TOP award ( NWO-CW 714.016.001 ). This work is part of the Oncode Institute , which is partly financed by the Dutch Cancer Society . In addition, we thank the Hubrecht Sorting Facility and the Utrecht Sequencing Facility, subsidized by the University Medical Center Utrecht, Hubrecht Institute, Utrecht University, and the Netherlands X-omics Initiative (NWO project 184.034.019). Funding Information: We thank Geert Kops and Jacques Bothma for critically reading the manuscript. We thank Anko de Graaff of the Hubrecht Imaging Facility for help with the imaging and Buys de Barbanson for help with data analysis. This work was supported by a European Research Council Advanced grant (ERC-AdG 742225-IntScOmics) and Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO) TOP award (NWO-CW 714.016.001). This work is part of the Oncode Institute, which is partly financed by the Dutch Cancer Society. In addition, we thank the Hubrecht Sorting Facility and the Utrecht Sequencing Facility, subsidized by the University Medical Center Utrecht, Hubrecht Institute, Utrecht University, and the Netherlands X-omics Initiative (NWO project 184.034.019). Conceptualization, W.v.L. M.V. C.R. and A.v.O.; methodology, W.v.L. M.V. N.B. and F.S.; validation, W.v.L. M.V. and C.R.; formal analysis, W.v.L. M.V. and N.B.; investigation, W.v.L. and M.V.; resources, C.R. and A.v.O.; data curation, W.v.L. M.V. N.B. and C.R.; writing – original draft, W.v.L. and C.R.; writing – review & editing, W.v.L. M.V. C.R. and A.v.O.; visualization, W.v.L. and M.V.; supervision: C.R. and A.v.O. The authors declare no competing interests. Publisher Copyright: © 2022 The Author(s)
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