Functional long noncoding RNAs in neural development

神经发育中的功能性长非编码RNA

基本信息

批准号：
10632048
负责人：
DANIEL A LIM
金额：
$ 61.01万
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：
2022
资助国家：
美国
起止时间：
2022-07-01 至 2027-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10632048
关键词：
Alzheimer&apos s Disease Bacterial Artificial Chromosomes Biological Process Brain CRISPR interference Cell Proliferation Code Communities Coupled Data Development Developmental Delay Disorders Disease Drug Targeting Genes Genetic study Genome Goals Human Human Cell Line Human Genome In Vitro Individual Knowledge Malignant Neoplasms Maps Mental disorders Messenger RNA Molecular Molecular Genetics Names Nature Neurodegenerative Disorders Neurons Organoids Outcome Pathway interactions Polypyrimidine Tract-Binding Protein Production RNA Splicing Research Role Schizophrenia Structure Testing Transcript Transgenes Transplantation Untranslated RNA Work cell type conditional knockout data resource epigenomics genome-wide human disease human stem cells in vivo induced pluripotent stem cell insight knock-down nerve stem cell nervous system disorder neural neurodevelopment neurogenesis novel single-cell RNA sequencing therapeutic target therapy development transcriptome transcriptomics transdifferentiation

项目摘要

ABSTRACT Long noncoding RNAs (lncRNAs) have been implicated in a wide range of human neurological disorders including cancer, developmental delay, psychiatric and neurodegenerative disease. While it is known that the brain is enriched in specific lncRNAs, relatively few have been characterized in terms of function and molecular mechanism. Our long-term goal is to understand the function and molecular mechanisms of lncRNAs in neurodevelopment. Such fundamental knowledge is critical to understanding how this large aspect of the noncoding genome regulates brain development and disease. We have taken two approaches for the study of lncRNA function. The first approach is a “traditional” molecular-genetic study of a brain-specific, evolutionarily conserved lncRNA that is a potent regulator of neural stem cells (NSCs). In previous studies, we identified a novel lncRNA transcript that we named Pnky (Pou3f2 intergenic non-koding). In cultured NSCs, either Pnky transcript knockdown or Pnky conditional knockout (Pnky-cKO) increases neuronal production by ~4-fold. Pnky is required for proper cortical neurogenesis in vivo, and the expression of Pnky from a BAC transgene (BAC-Pnky) fully rescues Pnky-deletion – including at the level of the transcriptome – indicating that this lncRNA functions in trans. Pnky interacts with the splicing regulator PTBP1 (Polypyrimidine tract binding protein 1) – a critical regulator of neurogenesis from NSCs – and Pnky appears to function in the same molecular pathway as PTBP1. Preliminary Studies demonstrate that Pnky folds into a compact, monodisperse state that contains intricate structures including a pseudoknot, which is a structural module known to have important function in noncoding RNAs. Given these data, we hypothesize that Pnky contains functional structural modules and regulates the function of PTBP1. Our second approach is to use systematic functional screens to discover key principles of lncRNA genome function. In Preliminary Studies, we used CRISPRi to screen in parallel 10,671 lncRNA and 18,905 mRNA genes for roles in the neural induction of NSCs from human induced pluripotent stem cells (iPSCs). We also performed CRISPRi perturbation coupled with droplet- based single-cell RNA-Seq (Perturb-Seq) for hundreds of screen hits. Based on results from these systematic studies, our working hypothesis is that functional lncRNAs – in comparison to coding genes – are enriched for roles in “focusing” differentiation to specific neural cell types. To further test this hypothesis, we will study lncRNA function in human brain organoids and extend our screens to analyze neurogenesis. Determining the unique functional roles of lncRNAs and coding genes at genome-scale will have important, broad impact on the interpretation of transcriptomic and epigenomic studies of neurodevelopment. Together, by studying lncRNA function at the level of individual transcripts and also at genome scale, we expect to gain fundamental insights into the function of this large aspect of the noncoding genome.

抽象的长链非编码 RNA (lncRNA) 与多种人类神经系统疾病有关包括癌症、发育迟缓、精神疾病和神经退行性疾病。大脑富含特定的lncRNA，但在功能和分子方面被表征的相对较少我们的长期目标是了解 lncRNA 的功能和分子机制。这些基础知识对于理解神经发育的这一重要方面至关重要。非编码基因组调节大脑发育和疾病我们采取了两种方法来研究。第一种方法是对大脑特定的、进化的“传统”分子遗传学研究。在之前的研究中，我们发现了一种保守的 lncRNA，它是神经干细胞 (NSC) 的有效调节因子。在培养的 NSC 中，我们将其命名为 Pnky（Pou3f2 基因间非编码）的新 lncRNA 转录物，或者是 Pnky。转录下调或 Pnky 条件敲除 (Pnky-cKO) 敲除可将神经元产量增加约 4 倍。 Pnky 是体内正常皮质神经发生所必需的，并且 BAC 转基因表达 Pnky (BAC-Pnky) 拯救完全拯救 Pnky 删除 – 包括在转录组水平 – 表明这 Pnky 与剪接调节因子 PTBP1（多嘧啶束结合）相互作用。蛋白质 1) – NSC 神经发生的关键调节因子 – 和 Pnky 似乎以相同的方式发挥作用初步研究表明 Pnky 折叠成紧凑的单分散分子途径。包含复杂结构（包括伪结）的状态，这是已知具有的结构模块考虑到这些数据，我们认为 Pnky 包含功能性的 RNA。我们的第二种方法是使用系统功能。在初步研究中，我们使用 CRISPRi 进行筛选以发现 lncRNA 基因组功能的关键原理。平行筛选 10,671 个 lncRNA 和 18,905 个 mRNA 基因，了解其在 NSC 神经诱导中的作用我们还对人类诱导多能干细胞 (iPSC) 进行了 CRISPRi 扰动与液滴-结合。基于单细胞 RNA 测序 (Perturb-Seq) 的数百个筛选结果基于这些系统的结果。研究中，我们的工作假设是，与编码基因相比，功能性 lncRNA 富集了为了进一步检验这一假设，我们将研究“聚焦”分化为特定神经细胞类型的作用。 lncRNA 在人脑类器官中发挥作用，并扩展我们的筛选来分析神经发生。 lncRNA和编码基因在基因组规模上的独特功能作用将对通过研究 lncRNA 共同解释神经发育的转录组学和表观基因组学研究。在个体转录本水平和基因组规模上发挥作用，我们希望获得基本的见解深入了解非编码基因组这一重要方面的功能。