Photoactivatable RNA Tags for Subcellular Photolabeling of RNA
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 (<i>t</i><sub>1/2</sub> = ∼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.
Authors
- Zhenyin Chen (ORCID: https://orcid.org/0000-0002-1118-1690)
- Caihong Zeng (ORCID: https://orcid.org/0000-0002-3816-5164)
- Xiaoxiao Rong (ORCID: https://orcid.org/0009-0001-8979-7196)
- Pingyong Xu (ORCID: https://orcid.org/0000-0002-8779-6931)
- Samie R. Jaffrey (ORCID: https://orcid.org/0000-0003-3615-6958)
- Haodong Jiang (ORCID: https://orcid.org/0000-0001-6760-5336)
- Yuan Lin (ORCID: https://orcid.org/0000-0001-8961-8971)
- Liuqin He (ORCID: https://orcid.org/0000-0003-1184-6944)
- Fangqing Zhao (ORCID: https://orcid.org/0000-0002-6216-1235)
- Xing Li (ORCID: https://orcid.org/0000-0003-3149-9783)
- Yulong Yin (ORCID: https://orcid.org/0000-0002-0248-6900)
- Jinyang Zhang (ORCID: https://orcid.org/0000-0002-5163-894X)
- Tao Yao (ORCID: https://orcid.org/0000-0001-8699-8294)
- Wankai Gao
Institutions
- National Academy of Medicine (US)
- Hunan Normal University (CN)
- Chinese Academy of Sciences (CN)
- Cornell University (US)
- Affiliated Hospital of Southwest Medical University (CN)
- Institute of Subtropical Agriculture (CN)
- Institute of Biophysics (CN)
- Laboratoire des Biomolécules (FR)
- University of Chinese Academy of Sciences (CN)
- Institute of Zoology (CN)
- Hebei University (CN)
- Weill Cornell Medicine (US)
Publication Details
- Journal
- Journal of the American Chemical Society
- Published
- 2025-08-25
- DOI
- https://doi.org/10.1021/jacs.5c07380
- Citations
- 1
- Primary Topic
- RNA Interference and Gene Delivery
- Type
- article
- Field-Weighted Citation Impact
- 0.81
Funders
- National Natural Science Foundation of China
- Natural Science Foundation of Hunan Province
- Beijing Municipal Natural Science Foundation
- Institute of Zoology, Chinese Academy of Sciences
- National Key Research and Development Program of China
- National Institute of Neurological Disorders and Stroke