Epitranscriptomic Control of Local Gene Expression in Neural Stem Cells

神经干细胞局部基因表达的表观转录组控制

• 批准号: 9765015
• 负责人: Kathryn D Meyer
• 金额: $ 23.74万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-02-01 至 2020-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9765015
• 关键词: Adenosine; Binding; Binding Proteins; Brain; Cell Cycle Progression; Cell Separation; Cell physiology; Cells; Cellular Morphology; Code; Coupled; Data; Data Set; Detection; Development; Diagnostic; Event; Exhibits; FMR1; FMRP; Foundations; Fragile X Syndrome; Functional disorder; Future; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Glial Cell Proliferation; Goals; In Vitro; Individual; Intellectual functioning disability; Laboratories; Location; Mediating; Messenger RNA; Methods; Methylation; Methyltransferase; Modification; Molecular; Morphology; Mutation; Neurodevelopmental Disability; Neurodevelopmental Disorder; Neuroglia; Neurons; Nucleotides; Play; Preparation; Process; Proteins; Publishing; RNA; RNA immunoprecipitation sequencing; RNA methylation; RNA-Binding Proteins; Radial; Reader; Regulation; Reporter; Research Design; Resolution; Role; Site; Site-Directed Mutagenesis; Specificity; Structure; Testing; Therapeutic; Transcript; Translating; Translations; Work; base; epitranscriptomics; experimental study; insight; methylome; migration; nerve stem cell; nervous system disorder; neurodevelopment; neurogenesis; neuroregulation; next generation sequencing; novel; recruit; scaffold; stem cell differentiation; stem cell population; tool; transcriptome; transcriptome sequencing; transcriptomics

Abstract During brain development, neurons are generated from neural stem cells, also called radial glial cells (RGCs). These cells exhibit a unique morphology with a long basal process that extends to the pia to form endfeet. Basal processes serve critical roles as scaffolds for neuronal migration and can also influence neurogenesis. Despite their importance for neurodevelopment, our understanding of molecular regulation within these basal radial glial structures remains poor. Our group recently discovered that RGC endfeet contain a specific transcriptome which can be locally translated. This suggests that local transcriptomic regulation is important for controlling gene expression in RGCs. However, our understanding of post-transcriptional mechanisms that regulate mRNAs within RGCs is very limited. Recently, methylation of adenosine residues in RNA (m6A) has emerged as a pervasive feature of the transcriptome which plays important roles in the regulation of gene expression. m6A is particularly abundant within the brain, and recent studies have shown that dynamic methylation enables cells to fine-tune the expression of subsets of the transcriptome. Moreover, the m6A methyltransferase, METTL3, is essential for promoting differentiation of stem cells, including RGCs. Our preliminary data indicate that m6A is present in the local transcriptome of RGC endfeet, suggesting the intriguing but untested possibility that mRNA methylation controls sub-cellular events in this important stem cell population. This proposal will test the novel hypothesis that RGC subcellular compartments contain distinct repertoires of methylated mRNAs and that mRNA modifications contribute to local gene expression regulation in the developing brain. We will first employ novel methods developed by our group for RGC endfeet isolation coupled with global m6A mapping strategies to identify the local methylome in RGCs. We will determine the transcripts whose localization to endfeet is dependent upon m6A and test the impact of RNA methylation upon local translation. We will additionally test the hypothesis that FMRP influences endfeet localization by binding m6A. Discoverying how FMRP targets RNAs in RGCs is important given that FMRP mutation influences cortical development and causes Fragile X syndrome. Collectively, these studies will provide the first identification of m6A-containing mRNAs in RGC endfeet and will uncover the transcripts for which local expression in RGCs is m6A- dependent. This work will provide a foundation for future studies designed to investigate the consequences of local RGC mRNA regulation on neural stem cell function and brain development.

摘要 在大脑发育过程中，神经元由神经干细胞产生，神经干细胞也称为放射状胶质细胞 细胞(RGC)。这些细胞表现出独特的形态，具有延伸到 Pia形成尾足。基本突起作为神经元迁移的支架发挥关键作用，并可以 也会影响神经发生。尽管它们对神经发育很重要，但我们对 这些基底径向神经胶质结构内的分子调节仍然很差。我们的小组最近 发现RGC末端含有一个特定的转录组，可以在当地翻译。这 提示局部转录调控对调控视网膜神经节细胞的基因表达具有重要意义。 然而，我们对调控RGC内mRNAs的转录后机制的理解是 非常有限。最近，RNA(M6A)中腺苷残基的甲基化已经成为一种普遍存在的 在基因表达调控中起重要作用的转录组的特征。M6A是 在大脑中尤其丰富，最近的研究表明，动态甲基化能够 细胞来微调转录组亚群的表达。此外，m6A甲基转移酶， METTL3是促进包括视网膜节细胞在内的干细胞分化所必需的。我们的初步数据 表明M6A存在于RGC末端的局部转录组中，这表明有趣的是 未经测试的可能性，即信使核糖核酸甲基化控制这一重要干细胞的亚细胞事件 人口。这一提议将检验RGC亚细胞隔室含有 不同的mRNAs甲基化谱系及其对局部基因的影响 发育中大脑的表达调控。我们将首先使用由我们的 RGC端脚隔离组结合全局M6A映射策略来识别局部 视网膜节细胞中的甲基组。我们将确定其在末端脚的定位取决于哪些转录本 M6A，并检测RNA甲基化对局部翻译的影响。我们还将测试 假设FMRP通过结合M6A影响终足定位。发现FMRP如何瞄准目标 RGCs中的RNA很重要，因为FMRP突变影响皮质发育和原因 脆性X综合征。总的来说，这些研究将提供第一次鉴定含有m6A的 RGC末端的mRNAs，并将发现RGC中局部表达m6A的转录本- 依附的。这项工作将为未来的研究提供基础，旨在调查 局部RGC基因调控对神经干细胞功能和脑发育的影响。