Long noncoding RNA regulation of neural stem cells

神经干细胞的长非编码RNA调控

基本信息

批准号：
9105277
负责人：
DANIEL A LIM
金额：
$ 34.67万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-02-15 至 2021-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9105277
关键词：
Address Adult Alternative Splicing Alzheimer&apos s Disease Bioinformatics Biological Biology Brain Brain Neoplasms Cell Lineage Cells Code Collaborations Complex Data Development Developmental Delay Disorders Embryo Genetic Models Goals Human Human Genome Immunoprecipitation In Vitro Knockout Mice Knowledge Malignant Neoplasms Mass Spectrum Analysis Mental disorders Messenger RNA Molecular Mus Names Neurodegenerative Disorders Neuronal Differentiation Neurons Nuclear Nucleotides Outcome Phenocopy Phenotype Population Process Production Protein Microchips Proteins RNA RNA Processing RNA Splicing RNA-Protein Interaction Regulation Ribonucleoproteins Role Schizophrenia Testing Transcript Transgenic Organisms Untranslated RNA Ventricular Western Blotting Work base daughter cell genetic approach genome-wide analysis human disease in vivo insight knock-down mammalian genome nerve stem cell nervous system disorder neurodevelopment neurogenesis novel postnatal progenitor programs public health relevance relating to nervous system stem cell population subventricular zone therapeutic target therapy development transcriptome

项目摘要

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.

描述（由申请人提供）：长非编码 RNA (lncRNA)——长度超过 200 个核苷酸的转录物，几乎没有证据表明具有蛋白质编码潜力——与多种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。已发现哺乳动物基因组可转录数千个 lncRNA，但很少有 lncRNA 在体内功能和分子机制方面得到表征。通过对成人心室-室下区 (V-SVZ) 神经发生中的 lncRNA 进行全基因组分析，我们发现了一种名为 Pinky (Pnky) 的新型 lncRNA 转录本。我们最近证明，Pnky 调节胚胎和出生后大脑 NSC 的神经元产生。 Pnky 是一种神经特异性核 lncRNA 转录物，在 V-SVZ 神经源性谱系中，Pnky 在 NSC 中表达，并在神经元分化过程中下调。在出生后的 V-SVZ NSC 中，Pnky 敲低可增强神经元谱系定向并扩大转运放大细胞群，使神经元产量增加数倍。质谱、Western blot 和 RNA 免疫沉淀分析表明 Pnky 与 PTBP1 存在物理相互作用。神经发生、脑肿瘤、直接细胞重编程和 RNA 剪接的已知调节因子在 NSC 中，Pnky 和 PTBP1 调节与细胞表型相关的一组核心转录物的表达和选择性剪接。 (Pnky-cKO) 小鼠，以及 Pnky 缺陷表型 Pnky 敲低的遗传模型在体外和体内的总体目标是了解。 Pnky 的体内功能和机制目标 1 是通过研究体内 Pnky 缺陷和 Pnky 转基因表达、我们在 V-SVZ 生物学方面的专业知识和用途来确定 Pnky 在成人 V-SVZ 神经发生中的作用。操纵 Pnky 表达的多种互补方法支持目标 1 的可行性。目标 2 是确定 Pnky 调节神经发生的机制。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 PTBP1 的功能相互作用。除了初步数据外，与 Pnky 相互作用的其他因素的发现将为研究其他潜在的 lncRNA 机制提供基础。 Aaron Diaz 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）支持了目标 2 的可行性。有关 lncRNA 发育和机制功能的知识将为了解 lncRNA 如何成为神经系统疾病的基础提供重要见解，并可能为 lncRNA 作为治疗靶点的开发提供信息。