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Mapping spatial transcriptome with light-activated proximity-dependent RNA labeling

Nature Chemical Biology volume 15, pages 1110–1119 (2019)Cite this article

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Abstract

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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Fig. 1: Development and characterization of CAP-seq method.
Fig. 2: CAP-seq reveals subcellular transcriptome in the mitochondrial matrix.
Fig. 3: Mapping of the ER-proximal transcriptome.
Fig. 4: Mapping local transcriptome at the OMM.
Fig. 5: Validation of RNAs identified in OMM CAP-seq.
Fig. 6: OMM CAP-seq with drug perturbation.

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Data availability

All data presented in this study are available in the main text and Supplementary Information. Sequencing data are included in Source Data Figs. 2–4 and 6a,d.

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Acknowledgements

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.

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Author notes

  1. These authors contributed equally: Pengchong Wang, Wei Tang, Zeyao Li.

Authors and Affiliations

  1. School of Life Sciences, Tsinghua University, Beijing, China

    Pengchong Wang, Zeyao Li & Jianbin Wang

  2. College of Chemistry and Molecular Engineering, Peking University, Beijing, China

    Pengchong Wang, Zhongyu Zou, Ying Zhou & Peng Zou

  3. Synthetic and Functional Biomolecules Center, Peking University, Beijing, China

    Pengchong Wang, Zhongyu Zou, Ying Zhou & Peng Zou

  4. Beijing National Laboratory for Molecular Sciences, Peking University, Beijing, China

    Pengchong Wang, Zhongyu Zou, Ying Zhou & Peng Zou

  5. Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Peking University, Beijing, China

    Pengchong Wang, Zhongyu Zou, Ying Zhou & Peng Zou

  6. Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China

    Wei Tang & Ran Li

  7. Peking-Tsinghua Center for Life Sciences, Beijing, China

    Wei Tang, Ran Li, Jianbin Wang & Peng Zou

  8. Peking-Tsinghua-NIBS Joint Graduate Program, Tsinghua University, Beijing, China

    Zeyao Li

  9. Department of Chemical Biology, College of Chemistry, Nankai University, Tianjin, China

    Tianyu Xiong

  10. Chinese Institute for Brain Research, Beijing, China

    Jianbin Wang

  11. PKU-IDG/McGovern Institute for Brain Research, Beijing, China

    Peng Zou

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Contributions

P.W., W.T. and P.Z. conceived the project. P.W., W.T., Z.L., J.W. and P.Z. designed the experiments. P.W. and W.T. performed all experiments, unless otherwise noted. Z.L. and J.W. designed sequencing data analysis. Z.Z. and R.L. carried out mass-spec experiments. Y.Z. carried out probe synthesis. P.W. and T.X. carried out FISH experiments. P.W., W.T., Z.L., J.W. and P.Z. analyzed data. P.W., W.T. and P.Z. wrote the paper with input from all authors.

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Correspondence to Jianbin Wang or Peng Zou.

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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). https://doi.org/10.1038/s41589-019-0368-5

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