Single-cell in situ analysis of RNA modifications in intact tissues

项目摘要 细胞RNA具有100多种不同的化学修饰，它们共同构成了 epitranscriptome。先前的研究表明，RNA修饰途径对高等真核生物至关重要， 这些途径的改变与许多人类疾病有关。但目前尚不清楚 为什么我们需要如此复杂的RNA化学库以及RNA修饰如何影响基因表达 在具有不同细胞类型的生物组织中。研究RNA修饰的主要瓶颈是 缺乏具有亚细胞和单细胞分辨率的测量工具。批量表位组测序 这些方法只能测量来自数百万个细胞的修饰位点的平均分布， 不同细胞类型中表转录组状态的内在异质性。此外，空间信息在 从组织中提取RNA，防止在组织背景下进一步分析RNA修饰模式 形态学和亚细胞区室。为了解决这些限制，我们建议创建尖端的 用于RNA修饰的三维（3D）原位测序的平台。然后我们将利用新的工具 在完整的细胞中以单细胞分辨率分析由RNA修饰介导的基因调控机制， 生物组织使用小鼠脑组织中的N6-甲基腺苷（m6 A）通路作为我们的模型系统，我们 将（a）开发创新的和广泛适用的RNA修饰3D原位测序方法;（B） 在完整的小鼠脑组织中以单细胞分辨率分析m6 A编码;（c）研究m6 A修饰如何在小鼠脑组织中表达。 各种类型的细胞共同和相互作用地调节大脑功能。总的来说，这项建议将导致 (1)一个变革性的工具箱，用于单细胞表观转录组学分析，在各种生物学领域具有广泛的应用。 组织，（2）小鼠大脑中空间分辨的单细胞RNA修饰图谱，以及（3）新的科学 了解RNA修饰如何影响组织功能。从长远来看，我们的目标是揭示新的原则， 在复杂的生物系统中以亚细胞和单细胞分辨率进行转录后基因调控， 以及发现健康和疾病中RNA的新化学指纹。

PROJECT SUMMARY Cellular RNAs have more than 100 different kinds of chemical modifications, which together comprise the epitranscriptome. Previous studies have revealed that RNA-modifying pathways are vital to higher eukaryotes, and the alteration of these pathways is associated with a number of human diseases. However, it is still unclear why we need such a complex chemical repertoire for RNA and how RNA modifications impact gene expression in biological tissues with diverse cell types. The major bottleneck for investigating RNA modifications has been the lack of measurement tools with subcellular and single-cell resolutions. Bulk epitranscriptomic sequencing methods can only measure the averaged distribution of modification sites from millions of cells, obscuring the intrinsic heterogeneity of epitranscriptomic states in distinct cell types. Moreover, spatial information is lost during RNA extraction from tissues, preventing further analysis of RNA modification patterns in the context of tissue morphology and subcellular compartments. To address such limitations, we propose to create cutting-edge platforms for three-dimensional (3D) in situ sequencing of RNA modifications. Then we will utilize the new tools to analyze gene regulation mechanisms mediated by RNA modifications at single-cell resolution in intact biological tissues. Using the N6-methyladenosine (m6A) pathway in mouse brain tissue as our model system, we will (a) develop innovative and broadly applicable 3D in situ sequencing methods for RNA modifications; (b) profile the m6A code at single-cell resolution within intact mouse brain tissue; (c) study how m6A modifications in various cell types work collectively and interactively to regulate brain function. In total, this proposal will lead to (1) a transformative toolbox for single-cell epitranscriptomic profiling with broad applications in various biological tissues, (2) a spatially resolved single-cell RNA modification atlas in the mouse brain, and (3) new scientific understanding on how RNA modifications impact tissue function. In the long term, we aim to reveal new principles of post-transcriptional gene regulation at subcellular and single-cell resolutions in complex biological systems, as well as discover new chemical fingerprints of RNAs in health and disease.