Exploring RNA transcription and turnover <i>in vivo</i> by using click chemistry

TLDR

The study introduces a chemical method that incorporates the uridine analog 5‑ethynyluridine into newly transcribed RNA to enable detection of RNA synthesis in cells. The method uses copper(I)-catalyzed click chemistry to attach fluorescent azides to EU‑labeled RNA, allowing rapid, highly sensitive imaging of RNA turnover in cultured cells and transcription rates in tissues of whole animals. Transcription rates differ markedly across tissues and within organ cell types.

Abstract

We describe a chemical method to detect RNA synthesis in cells, based on the biosynthetic incorporation of the uridine analog 5-ethynyluridine (EU) into newly transcribed RNA, on average once every 35 uridine residues in total RNA. EU-labeled cellular RNA is detected quickly and with high sensitivity by using a copper (I)-catalyzed cycloaddition reaction (often referred to as "click" chemistry) with fluorescent azides, followed by microscopic imaging. We demonstrate the use of this method in cultured cells, in which we examine the turnover of bulk RNA after EU pulses of varying lengths. We also use EU to assay transcription rates of various tissues in whole animals, both on sections and by whole-mount staining. We find that total transcription rates vary greatly among different tissues and among different cell types within organs.