School of Life Sciences, Tsinghua University, Beijing, China.
College of Chemistry and Molecular Engineering, Peking University, Beijing, China.
Nat Chem Biol. 2019 Nov;15(11):1110-1119. doi: 10.1038/s41589-019-0368-5. Epub 2019 Oct 7.
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.
RNA 分子在真核细胞中高度分隔，其定位与其功能密切相关。尽管 RNA 靶向的重要性，但由于缺乏分析工具，我们目前对转录组的空间组织的了解受到限制。在这项研究中，我们开发了一种化学生物学方法，以高空间特异性标记活细胞中的 RNA。我们的方法称为 CAP-seq，利用光激活、接近依赖性的 RNA 核碱基光氧化，随后可以通过亲和纯化进行富集，并通过高通量测序进行鉴定。使用这项技术，我们研究了与各种亚细胞区室（包括内质网和线粒体）接近的局部转录组。我们发现编码核糖体蛋白和氧化磷酸化途径蛋白的信使 RNA 在外膜上高度富集。由于其特异性和易用性，CAP-seq 是一种普遍适用于许多生物系统中研究空间转录组的技术。
