The roles of long non-coding RNAs in neuronal function

目的：我的研究项目的主要目的是了解基因表达是如何被控制的，探索动态调节基因的生物学功能。我的短期目标集中在控制“活动依赖”基因表达程序的机制，该程序调节神经元之间突触连接的结构可塑性。长链非编码rna （Long non-coding RNAs, lncRNAs）是一类新发现的基因调控因子，能够协调蛋白质与核酸之间的相互作用。许多lncrna主要在大脑中表达，但对它们的功能知之甚少。本研究旨在探讨lncrna在神经元基因表达调控和活性依赖性结构可塑性中的作用。*******方法：人类神经元将通过遗传因素的细胞重编程从成纤维细胞中产生。我将使用分子/生化方法来检查lncRNA剪接变体和亚细胞定位。lncRNA将使用病毒载体介导的功能遗传学方法进行操作：CRISPR/Cas9系统将用于基因组编辑，并直接激活（CRISPRa）或抑制（CRISPRi） lncRNA的表达。神经元将通过去极化或兴奋性神经递质受体的激动剂刺激，然后分析活动依赖的信号和基因表达。结构可塑性将使用成像方法来评估树突形态和突触数量。*******具体目的：作为功能实验的靶点，我选择了两个在调节突触功能的信号通路中具有潜在作用的lncrna:(I) PTCHD1-AS，它通过一种未知的机制调节nmda型兴奋性神经递质受体；（ii）灵长类特异性的LNC473，它是一个被认为是由神经元活动强烈诱导的环AMP信号的调节剂。目的1：检测PTCHD1-AS和LNC473的神经元剪接变异和亚细胞定位。目的2：评估PTCHD1-AS及其染色体邻居DDX53在NDMA受体信号传导中的作用。目的3：确定LNC473在调控环amp依赖性基因表达和神经元结构可塑性中的作用。*******影响：许多lncrna主要在大脑中表达，进化迅速，并且不广泛保守。创新的细胞重编程方法为人类lncrna的功能分析提供了独特的机会。通过测试lncrna在调节活动依赖的结构可塑性中的作用，本研究将揭示控制大脑中信息处理和存储的动态过程。此外，对人类神经元lncrna的研究可能为人类大脑的调控和功能复杂性提供见解。******

Objective: The primary objective of my research program is to understand how gene expression is controlled, and to explore the biological functions of dynamically-regulated genes. My short-term aims are focused on the mechanisms governing the “activity-dependent” gene expression program, which regulates the structural plasticity of synaptic connections between neurons. Long non-coding RNAs (lncRNAs) are a newly appreciated class of gene regulatory factor that can coordinate interactions between proteins and nucleic acids. Many lncRNAs are expressed primarily in the brain, but little is known about their functions. The aim of the proposed research is to explore the roles of lncRNAs in the regulation of neuronal gene expression and activity-dependent structural plasticity.*******Approach: Human neurons will be generated from fibroblasts by cellular reprogramming with genetic factors. I will used molecular/biochemical approaches will to examine lncRNA splice variants and subcellular localization. lncRNAs will be manipulated using viral vector-mediated functional genetics approaches: the CRISPR/Cas9 system will be used for genome editing and to directly activate (CRISPRa) or inhibit (CRISPRi) lncRNA expression. Neurons will be stimulated by depolarization or with agonists of excitatory neurotransmitter receptors, followed by analyses of activity-dependent signaling and gene expression. Structural plasticity will be assayed using imaging approaches to assess dendrite morphology and synapse numbers.*******Specific Aims: As targets for functional experiments I selected two lncRNAs with potential roles in the signaling pathways that regulate synapse function: (i) PTCHD1-AS, which regulates NMDA-type excitatory neurotransmitter receptors via an unknown mechanism and (ii) the primate-specific LNC473, which is a putative regulator of cyclic AMP signaling that is robustly induced by neuronal activity. Aim 1: Examine neuronal splice variants and subcellular localization of PTCHD1-AS and LNC473. Aim 2: Evaluate the roles of PTCHD1-AS and its chromosomal neighbor DDX53 in NDMA receptor signaling. Aim 3: Determine the roles of LNC473 in regulating cyclic AMP-dependent gene expression and structural plasticity of neurons.*******Impact: Many lncRNAs are primarily expressed in the brain, evolve swiftly, and are not widely conserved. Innovative cellular reprogramming approaches provide a unique opportunity for functional analyses of human lncRNAs. By testing the roles of lncRNAs in regulating activity-dependent structural plasticity the proposed research will shed light on the dynamic processes that control information processing and storage in the brain. Furthermore, studies of human neuronal lncRNAs may provide insights into the regulatory and functional complexity of the human brain.******