Beyond Transcription - microRNA Regulation of Neuronal Development

超越转录 - microRNA 对神经元发育的调节

• 批准号: 10598035
• 负责人: GIORDANO LIPPI
• 金额: $ 72.39万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10598035
• 关键词: Ablation; Affect; Binding; Biology; Brain; CRISPR-mediated transcriptional activation; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Complex; DNA; Data; Development; Disease; Engineering; Ensure; Evolution; Excision; Failure; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Goals; In Vitro; Individual; Knowledge; Light; Link; Maps; Mediating; Messenger RNA; MicroRNAs; Modernization; Molecular; Morphology; Mutate; Neurobiology; Neurodevelopmental Disorder; Neurons; Neurosciences; Phenotype; Play; Positioning Attribute; Post-Transcriptional Regulation; Proteins; Regulation; Reporter; Repression; Research; Resolution; Role; Schizophrenia; Structure; Testing; Time; Transcription Repressor; Transcriptional Activation; Transcriptional Regulation; Translational Repression; Untranslated RNA; Work; autism spectrum disorder; cell type; derepression; design; dosage; excitatory neuron; gamma-Aminobutyric Acid; gene discovery; gene repression; in vivo; innovation; loss of function; neuron development; neuronal survival; novel; novel strategies; postnatal development; posttranscriptional; prevent; programs; tool; transcriptomics

ABSTRACT As the brain matures, each neuronal cell type must execute a specialized developmental program to build a cell with the appropriate features. These programs are composed of waves of gene expression that turn on and off with exquisite precision as the cell goes through sequential developmental stages. In fact, failure of these pro- grams to unfold correctly is linked to several neurodevelopmental disorders. Modern transcriptomics has allowed us to map developmental trajectories of different neuronal subtypes at increasingly high resolution. However, this information alone is not sufficient. In fact, layers of sophisticated post-transcriptional regulation of gene ex- pression by microRNAs (miRNA) help execute these incredibly complex developmental programs. MiRNAs act as post-transcriptional repressors, ensuring that their mRNA targets are not expressed at an inappropriate time or place. We and others demonstrated that removal of a single brain-enriched miRNA can have profound con- sequences for brain development, but because each miRNA represses hundreds of different targets, it is hard to pinpoint precise molecular mechanisms. In light of this information, it is necessary to radically change the way we study miRNAs. Here, we propose two novel strategies that do not focus on single miRNAs and are designed to immediately identify molecular mechanisms of post-transcriptional regulation of gene expression. The first approach is based on the fact that, at times, de-repression of a single miRNA-target interaction (MTI) can be sufficient to induce a phenotype. Hence, in Aim 1 we establish a pipeline to perform a large-scale test of all MTIs in excitatory principal neurons (PN), independently of which miRNA is binding to them, to identify which ones are critical for their development. To do so, we engineered tools to map and manipulate cell type-specific MTIs, and fluorescent reporters that function as fast readouts of the developmental stage of PNs. The second approach takes advantage of the fact that, often, multiple miRNAs converge on the same target to ensure tight regulation. If evolution imposed multiple layers of repression on the same target, then controlling its protein levels must be essential to maintain the proper developmental trajectory. Thus, in Aim 2 we establish a pipeline to identify the targets most heavily repressed by miRNAs and study the functional consequences of their complete de-repres- sion on the developmental trajectory of PNs. For both aims, we propose to investigate how de-repression of a single MTI or of a single miRNA target affects the structure, function, and connectivity of developing cortical PNs both in vitro and in vivo. With this proposal we expect to greatly expand our understanding of broad post-tran- scriptional mechanisms of PN development, both at single MTI resolution and at the level of single target re- pressed by many miRNAs. Such knowledge will be key not only for basic neurobiology, but also to identify how failure in miRNA repression could lead to neurodevelopmental disorders.

抽象的 随着大脑的成熟，每种神经元细胞类型都必须执行一个专门的发展程序以构建单元 具有适当的功能。这些程序由打开和关闭的基因表达波组成 随着单元格经历顺序的发育阶段，具有精确的精度。实际上，这些亲 克正确展开的克与几种神经发育障碍有关。现代转录组学允许 美国以越来越高的分辨率绘制不同神经元亚型的发育轨迹。然而， 仅此信息就不够。实际上，基因的复杂后转录调节层 MicroRNA（miRNA）的压力有助于执行这些令人难以置信的复杂发展计划。 mirnas法 作为转录后的阻遏物，确保其mRNA靶标在不适当的时间不表达 或位置。我们和其他人表明，去除单一富含脑部的miRNA可以具有深刻的影响 大脑发育的序列，但由于每个miRNA都压抑了数百个不同的目标，所以很难 查明精确的分子机制。鉴于这些信息，有必要从根本上改变方式 我们研究miRNA。在这里，我们提出了两种新的策略，这些策略不专注于单个miRNA，并且是设计的 立即确定基因表达转录后调节的分子机制。第一个 方法是基于以下事实：有时，单个miRNA靶向相互作用（MTI）的抑制可以是 足以诱导表型。因此，在AIM 1中，我们建立了一条管道，以对所有MTI进行大规模测试 在兴奋性主神经元（PN）中，与miRNA与它们的结合无关，以确定哪些 对于他们的发展至关重要。为此，我们设计了映射和操纵细胞类型特异性MTI的工具， 和荧光记者的功能作为PNS发育阶段的快速读数。第二种方法 利用这一事实，通常多个miRNA会在同一目标上汇聚以确保严格的调节。 如果进化在同一目标上施加了多层抑制层，则控制其蛋白质水平必须为 维持适当的发展轨迹至关重要。因此，在目标2中，我们建立了一个管道来确定 目标最严重地被miRNA抑制，并研究其完全取消代表的功能后果 对PNS的发育轨迹的影响。对于这两种目标，我们建议研究如何消除A的抑制 单个MTI或单个miRNA靶标影响发展皮质PN的结构，功能和连通性 体外和体内。通过这项建议，我们希望大大扩展我们对广泛的后传播的理解 单个MTI分辨率和单个目标的级别，PN开发的拼写机制 受许多mirnas的压力。这些知识不仅对基本神经生物学是关键，而且要确定如何 miRNA抑制失败可能导致神经发育障碍。