Abstract
Photoactivatable fluorescent proteins enable precise analysis of subcellular protein dynamics, but a comparable approach for photoactivated tagging subcellular RNA remains lacking. Here, we develop PA-Broccoli, a photoactivatable fluorescent RNA tag that mimics photoactivatable green fluorescent proteins (PA-GFP). Similar to PA-GFP, PA-Broccoli is initially nonfluorescent but emits intense green fluorescence after ultraviolet light irradiation. Notably, complete photoactivation of PA-Broccoli results in a 6000-fold fluorescence enhancement with a rapid activation ( t 1/2 = ∼3 s), outperforming PA-GFP in sensitivity and speed by 1–2 orders of magnitude. Using PA-Broccoli, we first uncovered that cytoplasmic RNAs display restricted mobility compared to proteins. In addition, we revealed that circular RNAs undergo Ran-GTP-dependent nuclear export and slower cytoplasmic accumulation than linear mRNAs. Third, real-time imaging of mRNA within stress granules indicates that mRNA exchange throughout these granules is energy-dependent. These findings establish PA-Broccoli as a transformative tool for live-cell RNA tracking, offering unprecedented insights into RNA dynamics and regulatory mechanisms.