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mRNA组织在不同的细胞过程和疾病中起着重要作用。总的来说， 区室化细胞（例如，神经元和胚胎），亚细胞mRNA定位提供了核心机制 蛋白质合成的时空调控。自从最初发现亚细胞mRNA分布以来， 1983年，高通量成像和测序方法已经揭示，在许多细胞类型中， RNA定位于不同的区室。例如，神经元中的许多轴突相关mRNA将转运 沿着沿着长（>100μm）的轴突到达“需要的部位”，这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， 异常的空间RNA组织和越来越多的疾病，包括肌萎缩性侧索硬化症 (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， 其允许追踪和操纵内源性mRNA在原代细胞中的空间定位， 在体内，空间组织的机制和功能相关性只被探索了一小部分， mRNA的数量。在这个建议中，我们寻求建立一套技术作为研究的新基础 空间RNA生物学，通过开发一个允许复杂计算分析的集成框架， 实时RNA跟踪，以及原位和体内任何内源性mRNA的可编程空间操作， 在高通量（> 1，000个mRNA平行）规模上。为了实现这一目标，我们将从建立一个深入的 学习框架，可以分析不同细胞类型中的空间定位RNA，并预测其相关的 调节因素（例如，RNA基序、RNA结合蛋白）。这将提供一个空间RNA组织图谱 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- 介导的转录本组织（CRISPR-TO），用于任何靶mRNA定位的可编程操作。 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mRNA定位的相关性与前所未有的轻松和时空分辨率。三是 试图应用这一框架，通过高通量研究mRNA在原代神经元中的定位功能 操纵> 1，000个mRNA，以揭示轴突导向、生长锥发育和突触形成的功能。 活动将在体内验证选定的功能性mRNA（>100）。最后，我们将把这个框架应用于 探讨神经系统疾病脊髓RNA定位异常的病理机制 肌肉萎缩症（SMA）的体外和体内研究。我们不仅要剖析mRNA组织与 和SMA病理学，而且还探索用于潜在治疗的调节RNA定位的策略。我们 设想该提案将导致新的突破性见解的机制和功能作用， 全细胞mRNA的空间组织的细胞和生理功能，还没有被揭示之前。