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

项目总结/摘要 前体mRNA的选择性剪接被神经系统广泛采用，以扩大转录组， 歧管在特定的细胞环境中，这一过程受到多种RNA结合蛋白（RBP）的调控，是一个重要的细胞周期。 细胞身份的贡献者，正交的转录调控，并已牵连在许多 神经发育障碍理解和整合RNA选择性剪接对 因此，尽管中间神经元亚型规范建立和组织层次具有很高的价值， 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 规范.我们假设选择性剪接是中间神经元细胞命运的信息成分 决策使用scRNA-seq数据集，在不同的神经元中进行深入和完整的转录读段覆盖 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的RBP 结合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 我们开发的线路。在Zhang实验室的专家环境中进行剪接分析的培训是一个理想的选择。 这是对受训者之前在中间神经元生物学方面广泛培训的补充。拟议的研究和培训 该计划将为一个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。可变剪接和细胞多样性是已知的 在成熟神经元中紧密交织和重要;更好地了解这些神经元的起源， 关系将极大地影响我们对健康神经系统的理解， 神经发育疾病。

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.