Department of Biology, Indiana University, Bloomington, IN 47405, USA.
Department of Biology, Indiana University, Bloomington, IN 47405, USA
Development. 2021 Jan 7;148(1):dev196568. doi: 10.1242/dev.196568.
The adult intestinal epithelium is a model system for stem cell biology, but its utility is limited by current biochemical methods that lack cell type resolution. Here, we describe a new proximity-based profiling method that relies upon a GAL4 driver, termed (), that is exclusively expressed in intestinal progenitor cells. This method uses UV crosslinked whole animal frozen powder as its starting material to immunoprecipitate the RNA cargoes of transgenic epitope-tagged RNA binding proteins driven by When applied to the general mRNA-binder, poly(A)-binding protein, the RNA profile obtained by this method identifies 98.8% of transcripts found after progenitor cell sorting, and has low background noise despite being derived from whole animal lysate. We also mapped the targets of the more selective RNA binder, Fragile X mental retardation protein (FMRP), using enhanced crosslinking and immunoprecipitation (eCLIP), and report for the first time its binding motif in cells. This method will therefore enable the RNA profiling of wild-type and mutant intestinal progenitor cells from intact flies exposed to normal and altered environments, as well as the identification of RNA-protein interactions crucial for stem cell function.
成年肠道上皮是干细胞生物学的模型系统，但由于缺乏具有细胞类型分辨率的当前生化方法，其用途受到限制。在这里，我们描述了一种新的基于邻近性的分析方法，该方法依赖于一种称为 GAL4 的驱动子（），该驱动子仅在肠祖细胞中表达。该方法使用 UV 交联的整个动物冷冻粉末作为起始材料，通过 来免疫沉淀由转基因表位标记 RNA 结合蛋白驱动的 RNA 货物。当应用于一般的 mRNA 结合蛋白聚（A）结合蛋白时，该方法获得的 RNA 图谱可识别出经过祖细胞分选后发现的 98.8％的转录本，并且尽管源自整个动物裂解物，但背景噪声较低。我们还使用增强交联和免疫沉淀（eCLIP）对更具选择性的 RNA 结合蛋白 Fragile X 智力迟钝蛋白（FMRP）的靶标进行了作图，并首次在 细胞中报告了其结合基序。因此，该方法将能够对暴露于正常和改变的环境的完整苍蝇中的野生型和突变体肠祖细胞进行 RNA 分析，以及鉴定对干细胞功能至关重要的 RNA-蛋白质相互作用。
