The Role of HAR1 Non-coding RNA's in Cortical Development

描述（由申请人提供）： 这项研究的长期目标是了解人类基因组中的遗传变化， 人类特有的皮质发育特征的进化基础，并确定其功能 后果大脑的大脑皮层，特别是前额叶皮层，在人类身上已经大大扩展，这种扩展有助于人类独特的认知能力。最终，对人类皮层发育和功能的分子基础的理解将为导致前额叶皮层病变和认知功能受损的药物成瘾等疾病提供治疗靶点。我们最近发现了两个非编码RNA基因（HAR1F和HAR1R），它们是由基因组区域编码的，自我们与黑猩猩的共同祖先以来，这两个基因组区域在人类谱系中变化最大。这些人类特异性变化似乎改变了HAR1 ncRNA的结构。人HAR1F的表达模式表明其在皮质神经元发育中的作用。本研究将通过以下几个方面探索人和小鼠HAR1 ncRNA的功能：目的1：研究神经元分化过程中HAR1的表达模式，并确定HAR1转录本的生物起源和亚细胞定位。为了研究HAR1的生物化学功能，我们需要有表达HAR1的细胞的来源。我们将确定在神经诱导过程中，小鼠和人胚胎干细胞培养中HAR1转录本的表达模式。这些培养物将用于鉴定HAR1表达细胞培养物中HAR1 RNA的生物发生途径、亚细胞位置和成熟形式，这将帮助我们确定HAR1是否起调节转录、RNA加工、翻译或蛋白质活性的作用。目的2：确定HAR1表达的获得和丧失是否会导致体外和体内神经发育的变化。为了探索HAR1的功能作用，我们将使用表达HAR1的细胞培养物和小鼠来确定通过调节HAR1表达而改变的途径和过程。我们将通过在培养细胞中过表达HAR1 ncRNA和通过RNAi抑制内源性HAR1表达来研究调节HAR1表达对神经分化测定和基因表达的下游影响。然后，我们将确定这些扰动是否会影响体外神经分化，以及它们是否会影响基于微阵列的方法的基因表达。我们还将测试HAR1F和HAR1R转录本之间的交叉调节。基于人Har1的表达模式，我们预测它参与皮质神经发生或皮质迁移。我们计划使用子宫内电穿孔技术检查异位表达HAR1在体内分化皮质祖细胞中的作用，并测试皮质大小、图案和结构是否发生变化，以更全面地表征HAR1 ncRNA在皮质发育中的作用。 公共卫生相关性： 该项目旨在表征我们最近发现的两个重叠的非蛋白质编码RNA基因，它们与调节人类大脑皮层的发育有关。这些基因是从人类基因组中自我们与黑猩猩的共同祖先以来变化最显著的区域转录的，因此可能有助于人类特定的皮质发育方面。更好地了解人类大脑发育可能会导致新的方法来治疗与皮质病变和认知功能受损（如药物滥用）相关的神经系统疾病。

DESCRIPTION (provided by applicant): The long-term goal of this research is to understand the genetic changes in the human genome that underlie the evolution of human-specific features of cortical development and to identify their functional consequences. The cerebral cortex of the brain, particularly the prefrontal cortex, has greatly expanded in humans, and this expansion contributes to uniquely human cognitive abilities. Ultimately, an understanding of the molecular basis of human cortical development and function will provide therapeutic targets for diseases such as drug addiction that lead to pathologies in the prefrontal cortex and impaired cognitive function. We recently identified two non-coding RNA genes (HAR1F and HAR1R) encoded by the region of the genome that changed most dramatically in the human lineage since our common ancestor with chimpanzees. These human-specific changes appear to have altered the structure of the HAR1 ncRNAs. Human HAR1F is expressed in a pattern suggesting a role in cortical neuron development. This proposal will explore the function of human and mouse HAR1 ncRNAs through the following specific aims: Aim 1: Characterize the HAR1 expression pattern during neuronal differentiation and determine the biogenesis and subcellular localization of HAR1 transcripts. In order to study the biochemical functions of HAR1 we need to have a source of HAR1 expressing cells. We will determine the expression pattern of HAR1 transcripts in mouse and human embryonic stem cell cultures during neural induction protocols. These cultures will be used to identify the biogenesis pathway, subcellular location and mature form of HAR1 RNAs in the HAR1 expressing cell cultures, which will help us determine whether HAR1 acts to regulate transcription, RNA processing, translation or protein activity. Aim 2: Determine if gain and loss of HAR1 expression leads to changes in neural development in vitro and in vivo. To explore the functional role of HAR1, we will identify pathways and processes altered by modulating HAR1 expression using HAR1-expressing cell cultures and mice. We will examine the downstream effects of modulating HAR1 expression on neural differentiation assays and gene expression by overexpressing HAR1 ncRNAs and by inhibiting endogenous HAR1 expression by RNAi in cultured cells. We will then determine if these perturbations affect neural differentiation in vitro and if they affect gene expression using a microarray based approach. We will also test for cross-regulation between the HAR1F and HAR1R transcripts. Based on the expression pattern of human Har1, we predict that it is involved in cortical neurogenesis or cortical migration. We plan to examine the effect of ectopically expressing HAR1 in differentiating cortical progenitors in vivo using in utero electroporation techniques and test if there are changes in cortical size, patterning, and structure to more fully characterize the role of HAR1 ncRNAs in cortical development. PUBLIC HEALTH RELEVANCE: This project aims to characterize two overlapping non-protein-coding RNA genes we recently identified that are implicated in regulating the development of the human cerebral cortex. These genes are transcribed from the region of the genome most significantly changed in humans since our common ancestor with chimpanzees and may therefore contribute to human-specific aspects of cortical development. A better understanding of human brain development may lead to new approaches for treating neurological disorders associated with cortical pathologies and impaired cognitive functions such as drug abuse.