Live-Cell RNA Imaging with Metabolically Incorporated Fluorescent Nucleosides

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• Current cellular RNA imaging methods are limited, often restricting studies to fixed samples or individual RNA molecules.
• Investigating dynamic changes in bulk RNA within living cells is crucial for understanding cellular processes.

Purpose of the Study:

• To develop a novel platform for fluorescence imaging of bulk RNA dynamics in living cells.
• To investigate RNA metabolism and localization during cellular responses to oxidative stress.

Main Methods:

• Metabolic incorporation of fluorescent bicyclic and tricyclic cytidine analogues into cellular RNA via uridine-cytidine kinase 2 overexpression.
• Confocal imaging of metabolically labeled RNA in living cells.
• Analysis of RNA synthesis, degradation, and trafficking dynamics.

Main Results:

• Demonstrated successful metabolic labeling and live-cell imaging of bulk RNA using fluorescent cytidine analogues (tC).
• Observed significantly accelerated bulk RNA turnover in cells treated with sodium arsenite (NaAsO2) to induce oxidative stress.
• Identified novel cytoplasmic RNA granules containing oxidative stress-induced transcripts, distinct from stress granules and P-bodies, which co-localize with DDX6.

Conclusions:

• The developed platform enables high-resolution, live-cell imaging of bulk RNA dynamics.
• Oxidative stress profoundly impacts cellular RNA metabolism, leading to accelerated turnover and the formation of specific RNA granules.
• This approach provides new insights into cellular transcriptome dynamics and stress responses.