Live-Cell RNA Imaging with Metabolically Incorporated Fluorescent Nucleosides.

TitleLive-Cell RNA Imaging with Metabolically Incorporated Fluorescent Nucleosides.
Publication TypeJournal Article
Year of Publication2022
AuthorsWang, D, Shalamberidze, A, A Arguello, E, Purse, BW, Kleiner, RE
JournalJ Am Chem Soc
Volume144
Issue32
Pagination14647-14656
Date Published2022 Aug 17
ISSN1520-5126
KeywordsCytidine, Nucleosides, RNA
Abstract

<p>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. In particular, metabolic feeding with the tricyclic cytidine-derived nucleoside tC combined with 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 NaAsO treatment. Furthermore, 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.</p>

DOI10.1021/jacs.2c04142
Alternate JournalJ Am Chem Soc
PubMed ID35930766
Grant ListR01 GM132189 / GM / NIGMS NIH HHS / United States