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

转录因子和 RNA 结合蛋白对个体神经元转录组的协调控制

• 批准号: 10091530
• 负责人: Adam Norris
• 金额: $ 31.39万
• 依托单位: SOUTHERN METHODIST UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10091530
• 关键词: Affect; Alternative Splicing; Behavior; CRISPR/Cas technology; Caenorhabditis elegans; Cataloging; Cells; Data; Defect; Depressed mood; Development; Dissection; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Techniques; Goals; Human; Individual; Lead; Link; Mediating; Mission; Modeling; Molecular; Molecular Analysis; Nematoda; Nervous system structure; Neurons; Outcome; Pattern; Phenotype; Phosphotransferases; Positioning Attribute; Property; RNA; RNA Splicing; RNA-Binding Proteins; Regulation; Regulator Genes; Reporter; Research; Shapes; Sorting - Cell Movement; Specific qualifier value; Testing; Touch sensation; Transcript; Transcriptional Regulation; United States National Institutes of Health; Work; cell type; combinatorial; egg; experimental study; in vivo; innovation; insight; mutant; neuron development; programs; tool; transcription factor; transcriptome; transcriptome sequencing; transcriptomics

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.

单个神经元的发育和功能由其独特的转录组学特性定义，但 尽管最近的努力编目单神经元转录组，仍然有一个差距，我们的理解， 基因调控因子指定单个神经元转录组的因果机制。在 特别是，很少有人知道如何调节基因表达的各个层次的因素，例如转录 转录因子（TF）和RNA结合蛋白（RBP）协调控制单个神经元的转录组。这 一项提案旨在通过利用秀丽隐杆线虫的独特特性来填补差距， 机制研究体内特定模型神经元的协调转录组调控。C. elegans神经系统，结合强大的遗传技术， 将能够详细剖析TF-RBP对神经元发育的控制。最近的工具 在实验室中开发和适应，包括组合CRISPR/Cas9，单神经元体内替代 剪接报告基因和神经元特异性FACS分选，然后是RNA Seq，将揭示机制， 在体内单个神经元的协调调节的结果。本提案的目的是确定 TF-RBP对基因相互作用并组合形成神经元特异性转录组。的 假设是TF和RBP的细胞特异性组合会聚在特定的目标网络上以定义 神经元转录组。这一假设得到了C.优雅的人表明， (a)某些TF和RBP组合限定剪接选择，包括保守神经元的剪接， 单个神经元如触觉神经元中的激酶sad-1，和（B）神经元TF和RBP 基因相互作用影响神经功能和行为。这一假设将由 实验提出了以下目的：1）确定神经元TF的分子机制， 我们已经确定的RBP协调控制触觉神经元中的sad-1选择性剪接，2）定义 当这些调节因子或它们的受体被激活时， 靶转录物丢失，以及3）系统地鉴定协调控制的神经元TF和RBP 在特定易处理的神经元细胞类型中的神经元命运和功能。拟议工作的预期成果 目的是确定TF-RBP协调控制单个神经元的机制和功能后果 转录组所提出的方法是创新的，因为它从现状出发，通过研究因果关系， 单神经元转录组调控跨基因多层的机制和后果 体内调节。这是重要的，因为它有望推进单神经元转录组学领域 因果机制，功能后果，以及体内单个神经元的协调调节。 最终，这些发现将为我们了解神经系统如何发育和指定提供信息。