RNA Labeling of Nucleolar Transcripts by 5-Ethynyl Uridine Click Chemistry.

The nucleolus is a dynamic membraneless organelle where ribosomal RNA (rRNA) transcription, processing, and ribonucleoprotein assembly occur. Visualizing nascent RNA within nucleolar condensates reveals how RNA metabolism and phase separation reorganize under stress. This chapter provides a practical protocol to label nascent RNA using 5-ethynyl uridine (5-EU), followed by copper-catalyzed azide-alkyne cycloaddition for fluorescent detection. The method is straightforward, works across mammalian cells, and robustly reports nucleolar transcription under basal and stress conditions (heat shock; low-dose actinomycin D). Step-by-step instructions, reagent setup, image analysis metrics, troubleshooting, safety, and expected results are included so that researchers can reproducibly implement the assay.