Long noncoding RNA regulation of neural stem cells

 描述(申请人提供)：长非编码RNA(LncRNAs)--长于200个核苷酸的转录本，几乎没有蛋白质编码潜力的证据--与广泛的人类神经疾病有关，包括癌症、发育迟缓、精神分裂症和阿尔茨海默病。虽然哺乳动物基因组已经被发现转录数以千计的lncRNAs，但很少有lncRNAs在体内的功能和分子机制方面得到表征。在对成人室-室下区(V-SVZ)神经发生中的lncRNA进行全基因组分析时，我们发现了一种新的lncRNA转录本，命名为Pinky(Pnky)。我们最近证明了Pnky调节胚胎和出生后大脑NSCs的神经元产生。Pnky是一种神经特异性的核转录产物。在V-SVZ神经源性谱系中，Pnky在神经干细胞中表达，并在神经元分化过程中下调。在出生后的V-SVZ神经干细胞中，Pnky基因敲除增强了神经元的谱系承诺，扩大了运输放大细胞的数量，使神经元产量增加了数倍。Pnky在进化上是保守的，并在发育中的人脑NSCs中表达。在胚胎小鼠皮质中，Pnky基因敲除增加了神经元分化并耗尽了NSC数量。质谱仪、Western印迹和RNA免疫沉淀分析表明，Pnky在物理上与PTBP1相互作用，PTBP1是一种已知的神经发生、脑肿瘤、直接细胞重编程和RNA剪接的调节因子。在神经干细胞中，Pnky和PTBP1调控与细胞表型相关的一组核心转录本的表达和选择性剪接。自那以后，我们培育了一只Pnky条件性基因敲除(Pnky-CKO)小鼠，这种Pnky缺乏症的遗传模型在体外和体内都发生了Pnky基因敲除。这项拟议工作的总体目标是了解Pnky的体内功能和机制。目的1通过体内Pnky缺陷和转基因表达的研究，探讨Pnky在成人V-SVZ神经发生中的作用。初步数据，我们在V-SVZ生物学方面的专业知识，以及使用多种互补的方法来操纵Pnky的表达，支持目标1的可行性。目标2是确定Pnky调节神经发生的机制(S)。Pnky和PTBP1是否在功能上相互作用将通过分析生物学表型、转录组变化和RNA-蛋白质相互作用来研究。其他与Pnky相互作用的因子的发现将为研究其他潜在的LncRNA机制提供基础。除了初步数据，与Aaron Diaz博士(生物信息学)、Nigan Krogan博士(质谱学)、Seth Blackshaw博士(蛋白质微阵列)和Hiten Madhani博士(RNA剪接、RNA-蛋白质相互作用)的合作支持AIM 2的可行性。这些对lncRNA发育和机制功能的了解将为lncRNAs如何导致神经系统疾病提供关键的见解，并可能为lncRNAs作为治疗靶点的发展提供信息。

 DESCRIPTION (provided by applicant): Long noncoding RNAs (lncRNAs) - transcripts longer than 200 nucleotides with little evidence of protein coding potential - have been implicated in a wide range of human neurological disorders including cancer, developmental delay, schizophrenia and Alzheimer's disease. While the mammalian genome has been discovered to transcribe many thousands of lncRNAs, very few lncRNAs have been characterized in terms of in vivo function and molecular mechanism. In a genome-wide analysis of lncRNAs in adult ventricular- subventricular zone (V-SVZ) neurogenesis, we identified a novel lncRNA transcript named Pinky (Pnky). We have recently demonstrated that Pnky regulates the production of neurons from NSCs of the embryonic and postnatal brain. Pnky is a neural-specific, nuclear lncRNA transcript. In the V-SVZ neurogenic lineage, Pnky is expressed in NSCs and becomes downregulated during neuronal differentiation. In postnatal V-SVZ NSCs, Pnky knockdown potentiates neuronal lineage commitment and expands the transit-amplifying cell population, increasing neuron production several-fold. Pnky is evolutionarily conserved and expressed in NSCs of the developing human brain. In the embryonic mouse cortex, Pnky knockdown increases neuronal differentiation and depletes the NSC population. Mass spectrometry, Western blot, and RNA immunoprecipitation analysis indicates that Pnky physically interacts with PTBP1, a known regulator of neurogenesis, brain tumors, direct cell reprogramming, and RNA splicing. In NSCs, Pnky and PTBP1 regulate the expression and alternative splicing of a core set of transcripts that relates to the cellular phenotype. We have since generated a Pnky conditional knockout (Pnky-cKO) mouse, and this genetic model of Pnky-deficiency phenocopied Pnky knockdown both in vitro and in vivo. The overall goal of the proposed work is to understand the in vivo function and mechanism of Pnky. Aim 1 is to determine the role of Pnky in adult V-SVZ neurogenesis by studying Pnky-deficiency and Pnky transgenic expression in vivo. Preliminary Data, our expertise in V-SVZ biology, and the use of multiple, complementary approaches for manipulating Pnky expression support the feasibility of Aim 1. Aim 2 is to determine the mechanism(s) by which Pnky regulates neurogenesis. Whether Pnky and PTBP1 functionally interact will be investigated with the analysis of biological phenotypes, transcriptome changes, and RNA-protein interactions. The discovery of additional factors that interact with Pnky will provide the basis for investigating other potential lncRNA mechanisms. In addition to Preliminary Data, collaborations with Dr. Aaron Diaz (bioinformatics), Dr. Nevan Krogan (mass spectrometry), Dr. Seth Blackshaw (protein microarrays), and Dr. Hiten Madhani (RNA splicing, RNA-protein interactions) support the feasibility of Aim 2. Such knowledge of lncRNA developmental and mechanistic function will provide critical insight into how lncRNAs can underlie neurological disease and may inform the development of lncRNAs as therapeutic targets.