Systematic functional dissection of neuronal transcriptome diversity

项目总结 神经元转录组多样性的系统功能解剖 细胞类型特异的选择性剪接(AS)极大地放大了神经元转录组的多样性。恰如其分 这种分子复杂性的调节及其在发育过程中的建立是成熟的关键 神经细胞及其动态平衡的维持。多个RNA结合蛋白(RBPs)已经被 被确定为控制神经元特异性剪接。我们率先开发了以RBP为中心的战略，以 用一种整合的方法精确重建特定类型神经元限制性商业惯例的剪接调控网络 综合多种形式的实验和计算数据的分析框架。这些努力 生成了发育调控外显子的优先列表，将对其进行详细研究，以改善我们的 了解AS在神经元分化不同阶段的功能重要性。然而，a 这一领域的主要障碍是我们目前无法有效地审问大量 剪接变体。为了填补这一空白，我们建议开发一种以外显子为中心的策略，使用一个外显子- 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。对于一项试点研究，我们的重点是找出可供选择的外显子 小鼠脊髓运动神经元体外模型系统对轴突形态发生的调控 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来删除 慢病毒为基础的CRISPR/Cas9介导的基因组在MES细胞中的个体可选外显子 工程学。我们设计了一种克隆策略，使我们能够构建一个包含大量 针对单个可选外显子以触发特定外显子缺失的配对引导RNA(GRNA)。参数 为了优化文库的复杂性，将建立病毒传递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试点筛选，并确定这些外显子 对轴突生长很重要。根据对脑内神经元形态的分析进行这项筛查 在缺乏可靠报告的情况下，我们提出了一种策略，从 高通量格式的换能式电池池。这些携带个别突变的克隆系将是 接受平行神经元分化、高通量成像和表型分析。这一战略 将允许敏感地检测突变体，这些突变体在轴突生长中表现出细微的形态缺陷，这将是 进行了基因分型和进一步验证。因此，我们的方法将结合可伸缩和 灵活多变。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 不同的剪接变体。这一策略可以很容易地适应于研究潜在的分子程序 神经在正常和病理环境中的分化、迁移和功能。

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.