Hubrecht Institute of the KNAW & UMC Utrecht, Utrecht, the Netherlands.
Section Cell Biology, Center for Molecular Medicine, University Medical Center Utrecht, Utrecht, the Netherlands.
Cell Rep Methods. 2022 Jun 20;2(6):100235. doi: 10.1016/j.crmeth.2022.100235.
Stress granules are phase-separated assemblies formed around RNAs. So far, the techniques available to identify these RNAs are not suitable for single cells and small tissues displaying cell heterogeneity. Here, we used TRIBE (target of RNA-binding proteins identified by editing) to profile stress granule RNAs. We used an RNA-binding protein (FMR1) fused to the catalytic domain of an RNA-editing enzyme (ADAR), which coalesces into stress granules upon oxidative stress. RNAs colocalized with this fusion are edited, producing mutations that are detectable by VASA sequencing. Using single-molecule FISH, we validated that this purification-free method can reliably identify stress granule RNAs in bulk and single S2 cells and in neurons. Similar to mammalian cells, we find that stress granule mRNAs encode ATP binding, cell cycle, and transcription factors. This method opens the possibility to identify stress granule RNAs and other RNA-based assemblies in other single cells and tissues.
应激颗粒是围绕 RNA 形成的相分离组装体。到目前为止，可用于鉴定这些 RNA 的技术不适用于显示细胞异质性的单细胞和小组织。在这里，我们使用 TRIBE（通过编辑鉴定的 RNA 结合蛋白的靶标）来分析应激颗粒 RNA。我们使用 RNA 结合蛋白（FMR1）融合到 RNA 编辑酶（ADAR）的催化结构域，该结构域在氧化应激下凝聚成应激颗粒。与该融合蛋白共定位的 RNA 经过编辑，产生可通过 VASA 测序检测到的突变。使用单分子 FISH，我们验证了这种无需纯化的方法可以可靠地鉴定批量和单个 S2 细胞以及神经元中的应激颗粒 RNA。与哺乳动物细胞类似，我们发现应激颗粒 mRNA 编码 ATP 结合、细胞周期和转录因子。该方法为鉴定应激颗粒 RNA 和其他基于 RNA 的组装体提供了在其他单细胞和组织中的可能性。
