Manipulating and Interrogating Spatial Transcriptomics

操纵和询问空间转录组学

• 批准号: 10702050
• 负责人: Lei Stanley Qi
• 金额: $ 108.08万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2028-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10702050
• 关键词: Amyotrophic Lateral Sclerosis; Atlases; Axon; Biology; Cell physiology; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Computer Analysis; Development; Disease; Embryo; Fluorescent in Situ Hybridization; Foundations; Fragile X Syndrome; Goals; Image; In Situ; In Vitro; Mediating; Messenger RNA; Methods; Monitor; Neurons; Pathologic; Pathology; Physiological; Play; Protein Biosynthesis; RNA; RNA-Binding Proteins; Regulation; Resolution; Role; Site; Spinal Muscular Atrophy; Synapses; Technology; Therapeutic; Time; Transcript; Untranslated RNA; axon guidance; cell type; deep learning; in vivo; insight; machine learning algorithm; nervous system disorder; novel strategies; single molecule; spatiotemporal; transcriptomics

ABSTRACT Spatial mRNA organization plays a fundamental role in diverse cellular processes and disease. In large, compartmentalized cells (e.g., neurons and embryos), subcellular mRNA localization offers a core mechanism for the spatiotemporal regulation of protein synthesis. Since the initial discovery of subcellular mRNA distribution in 1983, high-throughput imaging and sequencing methods have revealed that, in many cell types, thousands of RNAs are localized to distinct compartments. For example, many axonal-related mRNAs in neurons will transport to the “site of needed” along the very long (>100μm) axon, which likely play an important role in axon development and local synaptic activities. Furthermore, mounting evidence shows a correlation between aberrant spatial RNA organization and an increasing number of diseases, including amyotrophic lateral sclerosis (ALS), fragile X syndrome (FXS), and spinal muscular atrophy (SMA). However, due to a lack of technologies that allow for the tracking and manipulation of the spatial localization of endogenous mRNAs in primary cells and in vivo, the mechanism and functional relevance of spatial organization has only been explored for a small number of mRNAs. In this proposal, we seek to establish a set of technologies as a new foundation to study spatial RNA biology, by developing an integrated framework that allows for sophisticated computational analysis, real-time RNA tracking, and programmable spatial manipulation of any endogenous mRNA(s) in situ and in vivo, on a high-throughput (>1,000 mRNAs in parallel) scale. To achieve this goal, we will start by building a deep learning framework that can analyze spatially localized RNAs in different cell types and predict their associated regulatory factors (e.g., RNA motifs, RNA binding proteins). This will provide an atlas of spatial RNA organization as well as candidate RNAs for functional studies. Next, we will develop two novel approaches, RNA live-cell fluorescent in situ hybridization (RNA-LiveFISH) for single-molecule, real-time dynamic tracking, and CRISPR- mediated transcript organization (CRISPR-TO) for programmable manipulation of any target mRNA localization. The two approaches form a new framework that enables us to study the regulatory mechanism and functional relevance of subcellular mRNA localization with unprecedented ease and spatiotemporal resolution. Third, we seek to apply this framework to study the function of mRNA localization in primary neurons, via high-throughput manipulation of >1,000 mRNAs to uncover functions for axon guidance, growth cone development, and synaptic activities. Selected functional mRNAs (>100) will be verified in vivo. Finally, we will apply the framework to investigate the pathological mechanisms of aberrant RNA localization underlying the neurological disease spinal muscular atrophy (SMA) in vitro and in vivo. We will not only dissect the relationship between mRNA organization and SMA pathology, but also explore the strategy of modulating RNA localization for potential therapeutics. We envision that the proposal will lead to new groundbreaking insights into the mechanism and functional roles of whole-cell mRNA spatial organization for cellular and physiological functions that has not been revealed before.

摘要 空间信使核糖核酸在多种细胞过程和疾病中起着基础性作用。总的来说， 分割的细胞(如神经元和胚胎)，亚细胞信使核糖核酸定位提供核心机制 对蛋白质合成的时空调节。自首次发现亚细胞内的mRNA分布以来 1983年，高通量成像和测序方法揭示，在许多类型的细胞中，数千 RNA定位于不同的隔间。例如，神经元中许多与轴突相关的mRNAs将 沿着很长的(&gt；100μm)轴突的“所需位置”，它可能在轴突中起重要作用 发育和局部突触活动。此外，越来越多的证据表明， 空间RNA结构异常和越来越多的疾病，包括肌萎缩侧索硬化症 骨质疏松症(ALS)、脆性X综合征(FXS)和脊髓性肌萎缩症(SMA)。然而，由于缺乏技术， 允许跟踪和操纵原代细胞中内源性mRNA的空间定位 在活体中，空间组织的机制和功能相关性只被探索了很小的 MRNA的数量。在这个方案中，我们试图建立一套技术作为研究的新基础 空间RNA生物学，通过开发一个允许进行复杂计算分析的集成框架， 实时核糖核酸跟踪，可编程的任何内源基因的空间操纵(S)在原位和体内， 在高吞吐量(并行1,000 mRNAs)规模上。为了实现这一目标，我们将从建设一个深层次的 可以分析不同细胞类型中空间定位的RNA并预测其关联的学习框架 调节因子(例如，RNA基序、RNA结合蛋白)。这将提供空间RNA组织的地图集 以及用于功能研究的候选RNA。接下来，我们将开发两种新的方法，RNA活细胞 荧光原位杂交(RNA-LiveFISH)用于单分子、实时动态跟踪和CRISPR- 介导的转录组织(CRISPR-TO)，用于可编程操作任何靶基因的mRNA定位。 这两种方法形成了一个新的框架，使我们能够研究调控机制和功能 亚细胞信使核糖核酸定位与前所未有的简便性和时空分辨率的相关性。第三，我们 试图应用这一框架，通过高通量，研究初级神经元中mRNA定位的功能 操纵&gt；1,000 mRNA以揭示轴突引导、生长锥发育和突触的功能 活动。选定的功能性mRNAs(&gt；100)将在体内得到验证。最后，我们将该框架应用于 探讨神经性脊髓疾病中RNA异常定位的病理机制 肌萎缩症(SMA)的体外和体内实验。我们不仅将剖析信使核糖核酸组织之间的关系 和SMA病理，但也探索调节RNA定位的策略，以用于潜在的治疗方法。我们 设想该提案将导致对 细胞和生理功能的全细胞信使核糖核酸的空间组织，这是以前没有发现的。