Functional long noncoding RNAs in neural development

摘要 长非编码RNAs(LncRNAs)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。虽然我们知道， 大脑富含特定的lncRNAs，从功能和分子方面研究的相对较少 机制。我们的长期目标是了解lncRNAs在体内的功能和分子机制。 神经发育。这些基础知识对于理解人类社会的这一大方面是至关重要的 非编码基因组调控大脑发育和疾病。我们采取了两种方法来研究 LncRNA功能。第一种方法是一种“传统的”分子遗传学研究，对特定的大脑进行进化研究。 保守的lncRNA，是神经干细胞(NSCs)的有效调节因子。在之前的研究中，我们发现了一种 新的lncRNA转录本，我们命名为Pnky(POU3F2基因间非编码)。在培养的神经干细胞中，要么是Pnky 转录敲除或Pnky条件性敲除(Pnky-CKO)使神经元的产量增加约4倍。 Pnky是体内正常的皮质神经发生所必需的，并通过BAC转基因表达Pnky (BAC-Pnky)完全挽救了Pnky的缺失--包括在转录组水平--表明这一点 LncRNA在反式转录中起作用。Pnky与剪接调节因子PTBP1(多嘧啶束结合)相互作用 蛋白质1)-神经干细胞神经发生的关键调节因子-和Pnky似乎在相同的 分子途径为PTBP1。初步研究表明，Pnky折叠成致密的单分散 包含复杂结构的状态，包括伪结，伪结是已知具有的结构模块 在非编码RNA中的重要作用。根据这些数据，我们假设Pnky包含函数 结构模块，并调节PTBP1的功能。我们的第二种方法是使用系统泛函 筛选以发现lncRNA基因组功能的关键原理。在初步研究中，我们使用CRISPRi 平行筛选10,671个lncRNA和18,905个mRNA基因在神经干细胞神经诱导中的作用 人诱导多能干细胞(IPSCs)。我们还进行了CRISPRi微扰与液滴的耦合。 基于单细胞RNA-Seq(扰动序列)，用于数百次屏幕点击。基于这些系统化研究的结果 研究表明，我们的工作假设是，与编码基因相比，功能性lncRNAs对 在“聚焦”分化到特定神经细胞类型中的作用。为了进一步检验这一假设，我们将研究 LncRNA在人脑有机体中的功能，并扩展我们的屏幕来分析神经发生。确定 LncRNAs和编码基因在基因组水平上的独特功能作用将对 神经发育的转录组和表观组学研究解读。一起，通过研究lncRNA 在个体转录水平和基因组水平上的作用，我们期望获得基本的见解 非编码基因组的这一大方面的功能。

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.