Live-Cell RNA Imaging with Metabolically Incorporated Fluorescent Nucleosides

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.

Figure 1.

Metabolic incorporation of fluorescent nucleosides into cellular RNA. (A) Scheme for incorporation of fluorescent nucleosides through the nucleotide salvage pathway. (B) Structure of fluorescent nucleosides used in this work. (C) Absorption (dashed) and corrected emission (solid) spectra of fluorescent ribonucleoside analogues used in this study. Spectra were recorded at 295 K in 1× PBS buffer, pH 7.4.

Figure 2.

Detection of fluorescent nucleosides incorporation into cellular RNA using imaging and LC-QQQ-MS. (A) Incorporation of fluorescent nucleosides into HeLa Flp-In WT UCK2 cells. UCK2 expression was induced with tetracycline and cells were treated with 500 μM nucleoside for 12 h and imaged. Tetracycline induction was omitted to serve as a control for UCK2 expression. (B) Incorporation of fluorescent nucleosides into HeLa Flp-In Y65G UCK2 cells. Cells were treated and imaged as in (A). (C) Workflow for quantitation of fluorescent nucleosides in cellular RNA using LC-QQQ-MS. (D) RNA incorporation of tC or pyrroloC into HeLa cells transfected with UCK2 constructs. (E) RNA incorporation of tC or pyrroloC in HeLa cell lines stably expressing inducible UCK2 WT or Y65G mutant.

Figure 3.

Phosphorylation of fluorescent nucleosides and cytidine analogues by recombinant UCK2. (A-D) HPLC chromatogram time course of pyrroloC (A), tC (B), ^MeOtC (C) and ^DEAtC(D) phosphorylation by recombinant UCK2 WT. (E) Quantification and kinetic analysis of fluorescent nucleoside phosphorylation reactions. Data represent mean ± SD (n = 3). (F). Overlay of simulated pyrroloC (purple) and cytidine (green) bound to UCK2 (PDB:1UEJ).

Figure 4.

Live cell imaging of cellular RNA dynamics using tC RNA labeling. (A) Representative images of tC incorporation in cells grown in 5% or 10% FBS or in the presence of ActD. (B) Fluorescence intensity quantification of cells treated in (A). Error bars represent mean ± s.d (n= 50 cells from three independent biological replicates). (C) Time-course analysis of tC incorporation into cellular RNA. (D) Fluorescence intensity quantification of cells treated in (C). Error bars represent mean ± s.d. (n= 50 cells from three independent biological replicates). (E) Time-course analysis of tC incorporation into cellular RNA during co-treatment with 200 μM NaAsO₂. (F) Fluorescence intensity quantification of cells treated in (E). Error bars represent mean ± s.d. (n= 50 cells from three independent biological replicates). (G) Representative images of cells pulsed with tC for 4 h and chased with complete medium or with 200 μM NaAsO₂ in medium. (H) Fluorescence quantification of cells treated in (G). Error bars represent mean ± s.d. (n= 25 cells from three independent biological replicates). (I) Colocalization of RNA foci formed under stress conditions with G3BP1, DCP1A or DDX6. (J) Quantification of cellular RNA foci number under stress and RNA synthesis inhibitors treatments. Error bars represent mean ± s.d. (n= 25 cells from three independent biological replicates).**P < 0.01, ****P < 0.0001.