COORDINATE CONTROL OF INDIVIDUAL NEURONAL TRANSCRIPTOMES BY TRANSCRIPTION FACTORS AND RNA BINDING PROTEINS

单个神经元的发育和功能是由它们独特的转录特性定义的，但 尽管最近努力对单个神经元转录本进行了编目，但我们对 基因调控因子指定单个神经元转录本的因果机制。在……里面 特别是，对于调节基因表达的各个层面的因素，例如转录，人们知之甚少 因子(TF)和RNA结合蛋白(RBPs)协同控制单个神经元的转录。这 该提案旨在通过利用线虫秀丽线虫的独特特性来填补这一空白 从机制上研究体内特定模型神经元的协同转录调控。线虫神经系统的描述良好且不变的谱系，结合强大的基因技术， 将使我们能够详细剖析Tf-RBP对神经元发育的控制。最近添加了其他工具 在实验室开发和适应，包括组合CRISPR/Cas9，体内单神经元替代 剪接记者，以及神经元特异性FAC排序，然后RNA序列，将揭示机制和 体内单个神经元协调调节的后果。这项提案的目标是界定 Tf-RBP对，它们在基因上相互作用，并组合形成神经元特异性转录本。这个 假设是TF和RBPs的细胞特定组合在特定的目标网络上聚合，以定义 神经转录本。这一假设得到了线虫体内初步数据的支持，这些数据表明 (A)某些转录因子和限制性商业惯例组合定义了剪接选择，包括保守神经元的剪接 单个神经元，如触摸感觉神经元中的激酶SAD-1，以及(B)神经元转录因子和限制性商业惯例 基因的相互作用会影响神经元的功能和行为。这一假说将由 提出的实验目的如下：1)确定神经元转录因子的分子机制 和我们已经确定的限制性商业惯例协调控制触摸神经元中的SAD-1选择性剪接，2)定义 当这些调节因子或它们的基因转录异常时，触觉神经元转录本的功能后果 目标转录本丢失，3)系统地识别协调控制的神经元转录因子和限制性商业惯例 神经元在特定易处理神经细胞类型中的命运和功能。拟议工作的预期结果 是为了确定协调Tf-RBP控制单个神经元的机制和功能后果 抄本。建议的方法是创新的，因为它通过检查因果关系来脱离现状。 跨多层基因的单神经元转录调控的机制和后果 体内调节。这项研究意义重大，因为它有望推动单神经元转录学领域的发展 体内单个神经元的因果机制、功能后果和协调调节。 最终，这些发现将使我们了解神经系统是如何发展和具体化的。

The development and function of individual neurons are defined by their unique transcriptomic properties, but despite recent efforts cataloguing single neuron transcriptomes, there remains a gap in our understanding of the causal mechanisms by which gene regulatory factors specify individual neuronal transcriptomes. In particular, little is known about how factors regulating various layers of gene expression, e.g. transcription factors (TFs) and RNA binding proteins (RBPs), coordinately control the transcriptomes of single neurons. This proposal aims to fill the gap by leveraging unique properties of the nematode Caenorhabditis elegans to mechanistically investigate coordinated transcriptomic regulation of specific model neurons in vivo. The well-described and invariant lineage of the C. elegans nervous system, combined with powerful genetic techniques, will enable detailed dissection of TF-RBP control over neuronal development. Additional tools recently developed and adapted in the lab, including combinatorial CRISPR/Cas9, single-neuron in vivo alternative splicing reporters, and neuron-specific FACS sorting followed by RNA Seq, will reveal mechanisms and consequences of coordinated regulation of single neurons in vivo. The objective of this proposal is to define TF-RBP pairs that genetically interact and combinatorially shape neuron-specific transcriptomes. The hypothesis is that cell-specific combinations of TFs and RBPs converge on specific target networks to define neuronal transcriptomes. This hypothesis is supported by preliminary in vivo data in C. elegans showing that (a) certain TFs and RBPs combinatorially define splicing choices including splicing of the conserved neuronal kinase sad-1 in individual neurons such as the touch-sensing neurons, and (b) neuronal TFs and RBPs genetically interact to affect neuronal function and behavior. The hypothesis will be further tested by the experiments proposed in the following aims: 1) Determine molecular mechanisms by which the neuronal TFs and RBPs we have identified coordinately control sad-1 alternative splicing in touch neurons, 2) Define functional consequences of dysregulated touch neuron transcriptomes when these regulatory factors or their target transcripts are lost, and 3) Systematically identify neuronal TFs and RBPs coordinately controlling neuron fate and function in specific tractable neuronal cell types. The expected outcomes of the proposed work are to determine mechanisms and functional consequences of coordinate TF-RBP control over single neuron transcriptomes. The proposed approach is innovative as it departs from the status quo by examining causal mechanisms and consequences of single-neuron transcriptomic regulation across multiple layers of gene regulation in vivo. It is significant because it is expected to advance the field of single-neuron transcriptomics into causal mechanisms, functional consequences, and coordinated regulation in single neurons in vivo. Ultimately, these findings will inform our understanding of how nervous systems develop and are specified.