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

项目摘要/摘要 神经系统广泛使用前-mRNAs的选择性剪接来扩大转录 多方面的。在特定的细胞环境中，这个过程受到多个RNA结合蛋白(RBPs)的调节，是一个主要的 细胞身份的贡献者，与转录调控相互作用，并与许多 神经发育障碍。理解和整合替代RNA剪接对 因此，建立和组织中间神经元亚型规范具有很高的价值 在这个方向上，进展相对较小。为了弥补这一差距，我们设计了一种高吞吐量 确定替代剪接调控网络在皮质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信息成分 决定。使用具有跨不同神经元的深入和完整的转录阅读的scRNA-seq数据集 细胞类型，我们将通过与细胞类型特定剪接模式高度相关的表达来识别关键限制性商业惯例 并对RBP目标网络进行了综合分析。然后，我们将通过CRISPR测试这些RBP的作用 双重中间神经元系荧光报告基因敲除小鼠胚胎干细胞分化为中间神经元亚型 这是我们开发的产品线。在张实验室的专家环境中进行剪接分析培训是理想的 补充受训人员之前在中间神经元生物学方面的广泛培训。拟议的学习和培训 计划将为一个富有成效的独立研究计划提供肥沃的基础，该计划将综合传统的 神经发展与最先进的生物信息学。另类剪接和细胞多样性是已知的 紧密交织在成熟神经元中很重要；更好地理解这些 人际关系将极大地影响我们对神经系统在健康和 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.