Epitranscriptomic Control of Local Gene Expression in Neural Stem Cells

摘要 在大脑发育过程中，神经元由神经干细胞产生，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学，具有长的基底突起，延伸至 Pia以形成端脚。基底突作为神经元迁移的支架起着关键作用， 也影响神经发生。尽管它们对神经发育很重要，但我们对 这些基底放射状神经胶质结构内的分子调节仍然很差。我们组最近 发现RGC末端含有一个可以局部翻译的特定转录组。这 表明局部转录组调控对于控制RGCs中的基因表达是重要的。 然而，我们对调节RGCs内mRNA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的生物学效应。 在基因表达调控中起重要作用的转录组特征。m6A是 在大脑中特别丰富，最近的研究表明，动态甲基化使 细胞来微调转录组子集的表达。此外，m6A甲基转移酶， 对于促进干细胞（包括RGCs）的分化是必需的。我们的初步数据 表明m6A存在于RGC末端局部转录组中，提示了有趣但 mRNA甲基化控制这一重要干细胞亚细胞事件的可能性未经检验 人口这一提议将检验新的假设，即RGC亚细胞区室含有 不同的甲基化mRNA库和mRNA修饰有助于局部基因 在发育中的大脑中的表达调控。我们将首先采用我们开发的新方法， RGC末端隔离组结合全球m6A映射策略， RGC中的甲基化。我们将确定转录本，其定位到端足取决于 m6A，并测试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影响端足定位。发现FMRP如何瞄准 RGC中的RNA是重要的，因为FMRP突变影响皮质发育并导致 脆性X染色体综合征。总的来说，这些研究将首次鉴定出含有m6A的 RGC末端的mRNA，并将揭示RGC中局部表达为m6A的转录本。 依赖。这项工作将为今后的研究提供基础，旨在调查 局部RGC mRNA调节对神经干细胞功能和脑发育的影响。

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.