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Live-cell RNA imaging with metabolically incorporated fluorescent nucleosides

Danyang Wang, Ana Shalamberidze, A. Emilia Arguello, Byron Purse, Ralph E. Kleiner
doi: https://doi.org/10.1101/2022.03.22.485351
Danyang Wang
1Department of Chemistry, Princeton University, Princeton, NJ 08544, USA
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Ana Shalamberidze
2Department of Chemistry and Biochemistry and the Viral Information Institute, San Diego State University, San Diego, CA 92182, USA
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A. Emilia Arguello
1Department of Chemistry, Princeton University, Princeton, NJ 08544, USA
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Byron Purse
2Department of Chemistry and Biochemistry and the Viral Information Institute, San Diego State University, San Diego, CA 92182, USA
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Ralph E. Kleiner
1Department of Chemistry, Princeton University, Princeton, NJ 08544, USA
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  • For correspondence: rkleiner@princeton.edu
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ABSTRACT

Fluorescence imaging is a powerful method for probing macromolecular dynamics in biological systems, however approaches for cellular RNA imaging are limited to the investigation of individual RNA constructs or bulk RNA labeling methods compatible primarily with fixed samples. Here, we develop a platform for fluorescence imaging of bulk RNA dynamics in living cells. We show that fluorescent bicyclic and tricyclic cytidine analogues can be metabolically incorporated into cellular RNA by overexpression of uridine-cytidine kinase 2 (UCK2). In particular, metabolic feeding with the tricyclic cytidine-derived nucleoside tC combined with quantitative confocal imaging enables the investigation of RNA synthesis, degradation, and trafficking at single-cell resolution. We apply our imaging modality to study RNA metabolism and localization during the oxidative stress response and find that bulk RNA turnover is greatly accelerated upon NaAsO2 treatment. Further, we identify cytoplasmic RNA granules containing RNA transcripts generated during oxidative stress that are distinct from canonical stress granules and P-bodies and co-localize with the RNA helicase DDX6. Taken together, our work provides a powerful approach for live-cell RNA imaging and reveals how cells reshape RNA transcriptome dynamics in response to oxidative stress.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • ↵* email: rkleiner{at}princeton.edu, *email: bpurse{at}sdsu.edu

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted March 22, 2022.
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Live-cell RNA imaging with metabolically incorporated fluorescent nucleosides
Danyang Wang, Ana Shalamberidze, A. Emilia Arguello, Byron Purse, Ralph E. Kleiner
bioRxiv 2022.03.22.485351; doi: https://doi.org/10.1101/2022.03.22.485351
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Live-cell RNA imaging with metabolically incorporated fluorescent nucleosides
Danyang Wang, Ana Shalamberidze, A. Emilia Arguello, Byron Purse, Ralph E. Kleiner
bioRxiv 2022.03.22.485351; doi: https://doi.org/10.1101/2022.03.22.485351

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