Long noncoding RNA regulation of neural stem cells

 描述（由申请人提供）：长非编码RNA（lncRNA）-长于200个核苷酸的转录物，几乎没有蛋白质编码潜力的证据-与广泛的人类神经系统疾病有关，包括癌症、发育迟缓、精神分裂症和阿尔茨海默病。虽然已经发现哺乳动物基因组转录成千上万的lncRNA，但很少有lncRNA在体内功能和分子机制方面被表征。在成人脑室-脑室下区（V-SVZ）神经发生中lncRNA的全基因组分析中，我们鉴定了一种名为Pinky（Pnky）的新型lncRNA转录本。我们最近已经证明，Pnky调节胚胎和出生后大脑的神经干细胞的神经元的产生。Pnky是一种神经特异性的核lncRNA转录物。在V-SVZ神经源性谱系中，Pnky在NSC中表达，并且在神经元分化期间下调。在出生后的V-SVZ NSC中，Pnky敲低增强了神经元谱系定型并扩大了传递放大细胞群，使神经元产生增加了数倍。Pnky在进化上是保守的，并在发育中的人脑的NSC中表达。在胚胎小鼠皮层中，Pnky敲低增加神经元分化并耗尽NSC群体。质谱、蛋白质印迹和RNA免疫沉淀分析表明，Pnky与PTBP 1发生物理相互作用，PTBP 1是已知的神经发生、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PTBP 1调节与细胞表型相关的核心转录物的表达和选择性剪接。此后，我们产生了Pnky条件性敲除（Pnky-cKO）小鼠，并且这种Pnky缺陷的遗传模型在体外和体内表型模仿Pnky敲除。这项工作的总体目标是了解Pnky的体内功能和机制。目的1通过研究Pnky基因缺陷和Pnky转基因鼠V-SVZ神经发生的体内表达，探讨Pnky基因在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，以及使用多种互补方法来操纵Pnky表达支持目标1的可行性。目的2是确定Pnky调节神经发生的机制。Pnky和PTBP 1是否在功能上相互作用将通过分析生物表型、转录组变化和RNA-蛋白质相互作用来研究。与Pnky相互作用的其他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迪亚兹博士（生物信息学）、Nevan Krogan博士（质谱）、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）的合作支持了Aim 2的可行性。这种lncRNA发育和机制功能的知识将为lncRNA如何成为神经系统疾病的基础提供关键的见解，并可能为lncRNA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.