Live-Cell RNA Imaging with Metabolically Incorporated Fluorescent Nucleosides

Danyang Wang, Ana Shalamberidze, A. Emilia Arguello, Byron W. Purse, Ralph E. Kleiner

Research output: Contribution to journalArticlepeer-review

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. 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 NaAsO2 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.

Original languageEnglish (US)
Pages (from-to)14647-14656
Number of pages10
JournalJournal of the American Chemical Society
Volume144
Issue number32
DOIs
StatePublished - Aug 17 2022

All Science Journal Classification (ASJC) codes

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

Fingerprint

Dive into the research topics of 'Live-Cell RNA Imaging with Metabolically Incorporated Fluorescent Nucleosides'. Together they form a unique fingerprint.

Cite this