RNA molecules are highly compartmentalized in eukaryotic cells, with their localizations intimately linked to their functions. Despite the importance of RNA targeting, our current knowledge of the spatial organization of the transcriptome has been limited by a lack of analytical tools. In this study, we develop a chemical biology approach to label RNAs in live cells with high spatial specificity. Our method, called CAP-seq, capitalizes on light-activated, proximity-dependent photo-oxidation of RNA nucleobases, which could be subsequently enriched via affinity purification and identified by high-throughput sequencing. Using this technique, we investigate the local transcriptomes that are proximal to various subcellular compartments, including the endoplasmic reticulum and mitochondria. We discover that messenger RNAs encoding for ribosomal proteins and oxidative phosphorylation pathway proteins are highly enriched at the outer mitochondrial membrane. Due to its specificity and ease of use, CAP-seq is a generally applicable technique to investigate the spatial transcriptome in many biological systems.
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We acknowledge funding from the National Key R&D Program of China (Ministry of Science and Technology, grant nos. 2017YFA0503600 and 2018YFA0507600), the National Natural Science Foundation of China (grant nos. 91753131 and 21675098), Beijing Natural Science Foundation (grant no. 5182011), the Interdisciplinary Medicine Seed Fund of Peking University (grant no. BMU2017MC006), Li Ge-Zhao Ning Life Science Junior Research Fellowship and Beijing Advanced Innovation Center for Structural Biology. P.Z. and J.W. are sponsored by the National Thousand Young Talents Award. We thank X. Chen (Peking University, Beijing, China), K. Sarkisyan (Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia) and A.Y. Ting (Stanford University, USA) for providing plasmids, and J. Yang (Peking University, Beijing, China) for providing HEK293T cells. T. Ding assisted with photo-oxidation product characterization. C. Xiao assisted with streptavidin dot blot analysis. We thank Y. Wang, M. Conrad and Y. Sun for advice, and the National Center for Protein Sciences at Peking University in Beijing, China, for assistance with Fragment Analyzer 12.
The authors declare no competing interests.
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Wang, P., Tang, W., Li, Z. et al. Mapping spatial transcriptome with light-activated proximity-dependent RNA labeling. Nat Chem Biol 15, 1110–1119 (2019) doi:10.1038/s41589-019-0368-5