mRNA Alternative Splicing Regulatory Networks in the Specification of Cortical Interneuron Subtypes

皮质中间神经元亚型规范中的 mRNA 选择性剪接调控网络

• 批准号: 10188959
• 负责人: Melissa McKenzie Campbell
• 金额: $ 10.99万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-13 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10188959
• 关键词: Address; Adoption; Adult; Alternative Splicing; Bioinformatics; Biology; Brain; Cell Separation; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Complement; D Cells; Data Set; Development; Developmental Process; Disease; Embryo; Environment; Fluorescence; Foundations; Ganglia; Health; In Vitro; Interneurons; Knock-out; Knowledge; Medial; Messenger RNA; Nervous system structure; Neurodevelopmental Disorder; Neurons; Output; Pattern; Process; Proteins; Publishing; RNA Editing; RNA Splicing; RNA analysis; RNA-Binding Proteins; Regulation; Reporter; Research; Rodent; Role; Signal Transduction; Somatostatin; System; Testing; Training; Transcript; Transcriptional Regulation; Variant; Visual Cortex; WNT Signaling Pathway; Work; base; cell type; comparative; design; experimental study; genetic regulatory protein; in vivo; insight; mRNA Precursor; neurodevelopment; programs; single-cell RNA sequencing; stem cell biology; success; tool; transcriptomics

Project Summary/Abstract Alternative splicing of pre-mRNAs is extensively employed by the nervous system to expand the transcriptomic manifold. Regulated in specific cellular contexts by multiple RNA-binding proteins (RBPs), this process is a major contributor to cellular identity that acts orthogonally to transcriptional regulation and has been implicated in many neurodevelopmental disorders. Understanding and integrating the impact of alternative RNA splicing on the establishment and organizational hierarchy of interneuron subtype specification is therefore of high value, though there has been relatively little progress in this direction. To address this gap, we have designed a high throughput system to determine the role of alternative splicing regulatory networks in cortical interneuron subtype specification. We hypothesize that alternative splicing is an informative component of interneuron cell fate decisions. Using scRNA-seq datasets with in-depth and full transcript read coverage across diverse neuronal cell types, we will identify key RBPs by highly correlated expression with cell-type specific splicing patterns together with integrative analysis of RBP target networks. We will then test the role of these RBPs by CRISPR knockout in mESCs differentiated into interneuron subtypes using a dual interneuron lineage fluorescent reporter line that we have developed. Training in splicing analysis in the expert environment of the Zhang lab is an ideal complement to the trainee’s previous extensive training in interneuron biology. The proposed studies and training plan will provide a fertile basis for a productive independent research program synthesizing traditional neurodevelopment with state of the art bioinformatics. Alternative splicing and cellular diversity are known to be closely intertwined and important in mature neurons; a better understanding of the inception of these relationships will be greatly impactful to our understanding of the nervous system in health and neurodevelopmental disease.

项目概要/摘要 神经系统广泛采用前体 mRNA 的选择性剪接来扩展转录组 歧管。这个过程在特定的细胞环境中受到多种 RNA 结合蛋白 (RBP) 的调节，是一个重要的过程。 细胞身份的贡献者，其作用与转录调控正交，并与许多 神经发育障碍。了解并整合选择性 RNA 剪接对 因此，中间神经元亚型规范的建立和组织层次结构具有很高的价值 这方面的进展相对较小。为了解决这一差距，我们设计了高吞吐量 确定选择性剪接调节网络在皮质中间神经元亚型中的作用的系统 规格。我们假设选择性剪接是中间神经元细胞命运的重要组成部分 决定。使用 scRNA-seq 数据集，对不同神经元进行深入且完整的转录本读取覆盖 细胞类型，我们将通过与细胞类型特异性剪接模式高度相关的表达来识别关键 RBP 以及 RBP 目标网络的综合分析。然后我们将通过 CRISPR 测试这些 RBP 的作用 使用双中间神经元谱系荧光报告基因敲除分化为中间神经元亚型的 mESC 我们开发的生产线。在张实验室的专家环境中进行剪接分析培训是理想的选择 对学员之前在中间神经元生物学方面的广泛培训的补充。拟议的研究和培训 该计划将为综合传统的富有成效的独立研究计划提供肥沃的基础 神经发育与最先进的生物信息学。已知选择性剪接和细胞多样性 在成熟神经元中紧密相连且重要；更好地了解这些的开始 人际关系将极大地影响我们对健康和神经系统的理解 神经发育疾病。