RNA regulatory networks in neuronal cell type diversity and function

项目摘要 神经元细胞类型多样性和功能中的RNA调控网络 哺乳动物的大脑可能是身体中最复杂的器官，其正常功能需要 许多不同类型的兴奋性和抑制性神经元的协调，形成不同的功能回路。 神经元细胞类型的表征不仅是了解大脑如何工作的基础， 特定的细胞类型在多种神经元疾病中受到选择性影响，以及如何逆转这一过程。 最近根据大量基因表达谱定义了大量神经元细胞类型， 单个细胞，并且这些细胞类型被组织成分层细胞分类。选择性剪接是一种 产生具有不同功能的多种转录物和蛋白质变体的机制，从而提供了一种主要的 哺乳动物分子多样性的驱动力。我的研究小组的首要目标是了解 选择性剪接的贡献和潜在的RNA调控网络在大脑和大脑相关的 紊乱包括我们在内的多个研究小组先前的工作明确地表明， 与非神经元组织相比，它具有独特的剪接调节程序。我们的研究还显示， 建立由多种RNA结合蛋白（RBP）调控的泛神经元剪接程序， 神经发育，并证明了Rbfox蛋白家族在调节轴突生长中的重要作用。 成熟和神经元兴奋性。然而，选择性剪接如何有助于不同的分子 目前对皮层中不同神经元细胞类型的分布了解甚少。我们假设 由高度调节的替代外显子产生的转录组多样性是一个主要组成部分， 神经元细胞类型的身份和功能。在本申请中，我们描述了我们对神经元细胞的初步分析， 小鼠皮层中的类型特异性可变剪接调节，这为以下内容提供了强有力的支持 我们想追求的具体目标：1）进行神经元细胞类型特异性替代的系统分析 使用深度sc-RNA-seq数据进行剪接以鉴定新的神经元亚类和调节子。2)验证我们 计算预测和表征神经元细胞类型特异性剪接调节的机制， 使用两个互补的模型系统的功能：GABA能的两个主要亚类的区别 中间神经元起源于尾侧（CGE）和内侧（MGE）神经节隆起，和GABA能神经元- Ank 3/Ankyrin G特异性微外显子。为了实现我们的目标，我们将采用多学科方法， 整合了尖端的统计机器学习方法和多种体外和体内实验 模型这项研究将产生一个全球性的和机制的看法，精确的选择性剪接调控， 不同的神经元细胞类型，并阐明其对神经元结构和功能特性的影响。

PROJECT SUMMARY RNA regulatory networks in neuronal cell type diversity and function The mammalian brain is probably the most complex organ in the body and its proper function requires coordination of many diverse types of excitatory and inhibitory neurons that form distinct functional circuitries. Characterization of neuronal cell types is fundamental to understand not only how the brain works, but also how specific cell types are selectively affected in multiple neuronal disorders and how this process can be reversed. A large number of neuronal cell types were recently defined based on their gene expression profiles in bulk and single cells, and these cell types are organized into a hierarchical cell taxonomy. Alternative splicing is a mechanism to generate multiple transcript and protein variants with distinct functions, thus providing a major driving force of the molecular diversity in mammals. The overarching goal of my research group is to understand the contribution of alternative splicing and the underlying RNA-regulatory networks in the brain and brain-related disorders. Previous work from multiple groups, including ours, unambiguously demonstrated that the brain has unique splicing-regulatory programs compared to non-neuronal tissues. Our studies also revealed the establishment of a pan-neuronal splicing program regulated by multiple RNA-binding proteins (RBPs) during neural development, and demonstrated the important role of the Rbfox protein family in regulating axonal maturation and neuronal excitability. However, how alternative splicing contributes to the distinct molecular profiles of diverse neuronal cell types in the cortex is currently poorly understood. We hypothesize that the transcriptome diversity generated by highly regulated alternative exons is a major component that specifies neuronal cell type identity and function. In this application, we describe our preliminary analysis of neuronal cell type-specific alternative splicing regulation in mouse cortex, which provides strong support for the following specific aims we would like to pursue: 1) Perform systematic analysis of neuronal cell type-specific alternative splicing to identify novel neuronal subclasses and regulators using deep sc-RNA-seq data. 2) Validate our computational predictions and characterize mechanisms of neuronal cell type-specific splicing regulation and function using two complementary model systems: the distinction of two major subclasses of GABAergic interneurons originating from caudal (CGE) and medial (MGE) ganglionic eminences, and a GABAergic neuron- specific microexon in Ank3/Ankyrin G. To achieve our goal, we will use a multidisciplinary approach that integrates cutting-edge statistical an machine learning methods and multiple in vitro and in vivo experimental models. This study will generate a global and mechanistic view of precise alternative splicing regulation across diverse neuronal cell types and illuminate its impact on neuronal structural and functional properties.