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: 10542419
• 负责人: Adam Norris
• 金额: $ 31.45万
• 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 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10542419
• 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; Neurons; Outcome; Pattern; Phenotype; Phosphotransferases; Positioning Attribute; Property; RNA; RNA Splicing; RNA-Binding Proteins; Regulation; Regulator Genes; Reporter; Research; Sensory; Shapes; Sorting; Specific qualifier value; Testing; Touch sensation; Transcript; Transcriptional Regulation; United States National Institutes of Health; Work; cell type; combinatorial; egg; experimental study; gene network; 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.

单个神经元的开发和功能由其独特的转录组特性定义，但 尽管最近的努力分类了单个神经元转录组，但我们对 基因调节因子指定个体神经元转录组的因果机制。在 特别是，关于如何调节基因表达各个层的因素，例如转录 因子（TFS）和RNA结合蛋白（RBP）协调控制单神经元的转录组。这 提案旨在通过利用线虫秀丽隐杆线虫的独特特性来填补空白 机械学研究了体内特定模型神经元的协调转录组调节。秀丽隐杆线虫神经系统的良好描述和不变的谱系，结合强大的遗传技术， 将使TF-RBP控制神经元发育的详细解剖。最近的其他工具 在实验室中开发和改编，包括组合CRISPR/CAS9，单个神经元在体内替代 剪接记者和神经元特异性FACS分类，然后是RNA SEQ，将揭示机制和 体内单个神经元协调调节的后果。该提议的目的是定义 TF-RBP对遗传相互作用并结合塑造神经元特异性转录组。这 假设是TFS和RBP的细胞特异性组合在特定目标网络上收敛以定义 神经元转录组。秀丽隐杆线虫中的初步体内数据证明了这一假设 （a）某些TFS和RBPS组合定义剪接选择，包括保守神经元的剪接 激酶SAD-1中的单个神经元（例如接触式神经元）以及（b）神经元TFS和RBPS中的激酶1 遗传相互作用以影响神经元功能和行为。该假设将通过 在以下目的中提出的实验：1）确定神经元TFS的分子机制 和RBP，我们已经确定在触摸神经元中协调控制SAD-1替代剪接，2）定义 当这些调节因素或其它们的功能障碍神经元转录组失调的功能后果 目标成绩单丢失，3）系统地识别神经元TF和RBPS协调控制 神经元的命运和功能在特定的可牵引神经元细胞类型中。拟议工作的预期结果 确定坐标TF-RBP控制对单个神经元的机制和功能后果 转录组。提出的方法是创新的，因为它通过检查因果而偏离现状 跨基因跨基因的机制和后果 体内调节。这很重要，因为有望推进单神经元转录组学领域 在体内单个神经元中的因果机制，功能后果和协调调节。 最终，这些发现将使我们对神经系统的发展方式和指定。