The role of the mRNA decay factor CNOT3 in cortical development

摘要 大脑皮质发育过程中基因表达的适当空间和时间控制 对维持神经前体细胞和神经分化至关重要。而转录调控 长期以来一直被认为是控制皮质发育过程中基因表达的关键， 人们对RNA衰变的贡献知之甚少。尽管如此，对基因表达的错误调控 转录后水平正在成为神经发育疾病的主要原因，包括智力疾病。 残疾和自闭症。新近发现，作为mRNA稳定性调节因子的CNOT3在一个队列中发生了突变 患有智力残疾和自闭症的患者。更多的研究表明，CNOT3调节 转录本的稳定性涉及细胞周期调节和干细胞命运。尽管有联系 在CNOT3，mRNA降解，神经发育疾病，CNOT3如何对大脑发育做出贡献， 以及它在这一过程中的关键目标，都是未知的。我将使用CRE-LOX条件来解决这一知识差距 去除神经前体细胞中CNOT3表达的基因敲除小鼠模型。初步数据显示，在 胚胎期(E)14.5天(约为神经发生中期)，CKO小鼠表现出小头畸形、细胞凋亡和 活跃增殖的细胞数量减少。在这项建议中，我将在这些观察和 测试总体假设，即CNOT3介导的mRNA稳定性调节是正常皮质所必需的 发展。目的1研究CNOT3中断对皮质生成的影响，并使用实时成像 在祖先命运决策中监控CNOT3要求的方法。《目标2》使用了最先进的 全球监测神经前体细胞mRNA稳定性并评估CNOT3后果的技术 缺失对mRNA稳定性的影响。这些方法将揭示以前未知的CNOT3在皮质中的作用。 同时提供了一个前所未有的关于大脑皮层发育过程中RNA稳定性的观点，以及它是如何 错误的监管会导致疾病。

ABSTRACT Proper spatial and temporal control of gene expression during development of the cerebral cortex is critical for the maintenance of neural progenitors and for neural differentiation. While transcriptional regulation has long been recognized as critical to the control of gene expression during cortical development, the contribution of RNA decay is less well understood. Despite this, mis-regulation regulation of gene expression at the post-transcriptional level is emerging as a major cause of neurodevelopmental diseases, including intellectual disability and autism. CNOT3, a regulator of mRNA stability, has recently been found to be mutated in a cohort of patients suffering from intellectual disability and autism. Additional studies have shown that CNOT3 regulates the stability of transcripts encoding proteins involved in cell cycle regulation and stem cell fate. Despite the links between Cnot3, mRNA degradation, neurodevelopmental disease, how Cnot3 contributes to brain development, and its key targets in this process, are unknown. I will address this knowledge gap using a Cre-lox conditional knockout mouse model to ablate Cnot3 expression in neural progenitors. Preliminary data indicates that at embryonic day (E)14.5 (approximately mid-neurogenesis), cKO mice exhibit microcephaly, apoptosis, and a reduction in the number of actively proliferating cells. In this proposal, I will build upon these observations and test the overall hypothesis that that CNOT3-mediated regulation of mRNA stability is required for proper cortical development. Aim 1 investigates the impact of Cnot3 disruption across corticogenesis, and uses live imaging approaches to monitor the requirements for CNOT3 in progenitor fate decisions. Aim 2 uses state-of-the-art technology to globally monitor mRNA stability in neural progenitors and assess the consequences of Cnot3 deletion on mRNA stability. These approaches will reveal previously uncharacterized role for CNOT3 in cortical development, while providing an unprecedented view of RNA stability during cortical development, and how its mis-regulation contributes to disease.