Systematic functional dissection of neuronal transcriptome diversity

项目摘要 神经元转录组多样性的系统功能分析 细胞类型特异性选择性剪接（AS）极大地放大了神经元转录组的多样性。适当 这种分子复杂性的调节及其在发育过程中的建立对于成熟至关重要。 和维持它们的体内平衡。多种RNA结合蛋白（RBP）已被发现。 被鉴定为控制神经元特异性剪接。我们率先制定了“以限制性商业惯例为中心”的战略， 使用整合的方法精确重建特定类别神经元RBP的剪接调控网络， 分析框架结合了多种形式的实验和计算数据。这些努力 生成了发育调控外显子的优先列表，这些外显子将被详细研究，以提高我们的 理解AS在神经元分化的各个阶段的功能重要性。但 该领域的主要障碍是我们目前无法有效地询问大量 剪接变体。为了填补这一空白，我们建议开发一种“外显子为中心”的策略，使用外显子- 特异性基因筛选，直接系统地剖析特异性剪接的功能作用 神经发育过程中的变异对于初步研究，我们的重点是确定替代外显子， 在小鼠脊髓运动神经元体外模型系统中调节轴突形态发生 胚胎干细胞（mES）。在目标1中，我们将建立一个大规模的基因组编辑平台， 通过基于慢病毒的CRISPR/Cas9介导的基因组在mES细胞中的单个替代外显子 工程.我们设计了一种克隆策略，使我们能够构建一个CRISPR文库， 靶向单个替代外显子以触发特异性外显子缺失的成对引导RNA（gRNA）。参数 为了优化文库的复杂性，将建立病毒递送和基因组编辑的效率。 在目标2中，我们将对约100个优先的神经元替代外显子进行初步筛选，并识别出这些外显子。 对轴突生长很重要为了进行基于神经元形态学分析的筛选， 由于缺乏可靠的报告基因，我们提出了一种策略，以获得克隆突变mES细胞群， 以高通量的形式转导细胞池。这些携带个体突变的克隆系将被 进行神经元分化、高通量成像和表型分析。这一战略 将允许敏感地检测在轴突生长中显示精细形态缺陷的突变体，这将是 基因分型并进一步验证。因此，我们的方法将结合联合收割机的优点， 灵活.这项研究将建立一种非常有效的方法，将我们对基因功能的认识扩展到 单个剪接变异体。这种策略可以很容易地适应于研究潜在的分子程序 神经分化，迁移和正常和病理情况下的功能。

Project summary Systematic functional dissection of neuronal transcriptome diversity Cell type-specific alternative splicing (AS) enormously amplifies the neuronal transcriptome diversity. Proper regulation of such molecular complexity and its establishment during development is critical for the maturation of nerve cells and maintenance of their homeostasis. Multiple RNA-binding proteins (RBPs) have been identified to control neuron-specific splicing. We pioneered the development of an “RBP-centric” strategy to reconstruct precisely the splicing regulatory networks of specific classes of neuronal RBPs using an integrative analysis framework that combines multiple modalities of experimental and computational data. These efforts generated prioritized lists of developmentally regulated exons that will be studied in details to improve our understanding of the functional importance of AS at various stages of neuronal differentiation. However, a major roadblock for the field is our current inability to efficiently interrogate the function of a vast number of splice variants. To fill in this gap, we propose to develop an “exon-centric” strategy using an exon- specific genetic screen to dissect directly and systematically the functional role of specific splice variants during neural development. For a pilot study, our focus is to identify alternative exons that regulate axon morphogenesis in an in vitro model system of spinal motor neurons derived from mouse embryonic stem (mES) cells. In Aim 1, we will establish a large-scale genome-editing platform to delete individual alternative exons in mES cells through lentivirus-based, CRISPR/Cas9-mediated genome engineering. We designed a cloning strategy that will allow us to build a CRISPR library with a large pool of paired guide RNAs (gRNAs) targeting individual alternative exons to trigger specific exon deletion. Parameters for optimizing the complexity of the library, viral delivery, and efficiency of genome editing will be established. In Aim 2, we will perform a pilot screen of ~100 prioritized neuronal alternative exons and identify those important for axon outgrowth. To perform this screening based on analysis of neuronal morphology in the absence of a reliable reporter, we propose a strategy to derive clonal mutant mES cell populations from transduced cell pools in a high-throughput format. These clonal lines carrying individual mutations will be subject to paralleled neuronal differentiation, high-throughput imaging and phenotypic analysis. This strategy will allow sensitive detection of mutants showing fine morphological defects in axon growth, which will be genotyped and further validated. Our approach will therefore combine advantages of being both scalable and flexible. This study will establish a very effective method to extend our knowledge of gene function to the level of individual splice variants. This strategy can be readily adapted to study the molecular programs underlying neural differentiation, migration, and function in normal and pathological contexts.