Investigating the mechanism of Staufen-mediated RNA-localization in mammalian neural stem cells

研究诗道芬介导的哺乳动物神经干细胞中 RNA 定位的机制

• 批准号: RGPIN-2014-05890
• 负责人: Vessey, John
• 金额: $ 3.35万
• 依托单位: University of Guelph
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=611908
• 关键词: Investigating; mechanism; Staufen; mediated; RNA

Stem cells have the unique ability to divide and produce two daughter cells with different identities. Typically this involves the production of one recurring stem cell and one cell destined for differentiation. In the mammalian brain, this process allows neural stem cells to produce both the neurons and glia of the developing cortex while at the same time ensuring that their own population is replenished. The mechanisms that facilitate these asymmetric cell divisions are poorly understood. Clues can be found in other model organisms such as Drosophila. In fly germ and neural stem cells, it’s been demonstrated that certain mRNAs are transported to one half of the dividing cell whereby only one daughter inherits them. Typically, these mRNAs produce proteins that drive the inheriting daughter down the differentiation pathway. Disruption of this mechanism leads to dramatic outcomes in the developing fly, typically rendering they embryo unable to form a proper body plan. During my postdoctoral studies, I asked if this mechanism of asymmetric mRNA localization plays a significant role in the development of the mammalian brain. I demonstrated that the mechanism is, at least in part, conserved in neural stem cells. I found that mRNAs important for conferring a neural lineage, and the proteins involved in their localization, are asymmetrically distributed during differentiating cell divisions. When I disrupted this localizing complex, the cells were no longer able to divide asymmetrically and instead, produced too many neurons at the expense of the stem cell pool. However, two fundamental questions remain. First, at what point are mRNAs destined for asymmetric distribution identified? Second, how are these mRNAs maintained in a translationally repressed state as localization occurs? The research program proposed here aims to address these two questions. It is my hypothesis that mRNAs destined for asymmetric distribution are identified in the nucleus prior to export. Based on evidence from other model organisms and on observations in differentiated cells in mammals, it is becoming apparent that the proteins involved in RNA splicing are linked with localization. I aim to determine what components of the splicing machinery are involved in identifying mRNAs destined for localization and how they interact with components found within the cytoplasm to carry mRNA to its proper destination. During this transport, it is thought that the mRNA is kept in a silenced state in order to prevent protein production from occurring in unwanted areas. I have previously demonstrated that the RNA-binding protein and translational repressor, Pumilio 2 (Pum2) is part of the asymmetric RNA granule in neural precursor cells. I aim to determine if Pum2 and its paralogue Pum1, as well as the similar translational repressors Musashi 1 and Musashi 2, act as silencers during asymmetric RNA localization in neural precursors. Both gene families have shown evidence of acting as regulators of translation in other stem cell populations, making them ideal candidates to fulfill this role in neural precursor cells. These two central questions will be addressed using well established molecular, cellular and biochemical techniques that allow for the genetic manipulation of neural stem cells both in culture and in the intact brain. The findings will advance our understanding of asymmetric cell divisions in neural precursor cells and how this event participates in regulating the development of the brain.

干细胞具有独特的分裂能力，可以产生两个不同身份的子细胞。通常，这涉及一个再生干细胞和一个注定要分化的细胞的产生。在哺乳动物的大脑中，这个过程允许神经干细胞产生发育中的皮层的神经元和神经胶质，同时确保它们自己的种群得到补充。促进这些不对称细胞分裂的机制知之甚少。线索可以在其他模式生物如果蝇中找到。在果蝇生殖细胞和神经干细胞中，已经证明某些mRNA被运送到分裂细胞的一半，只有一个子细胞继承它们。通常，这些mRNA产生蛋白质，驱动继承的子体沿着分化途径前进。这种机制的破坏会导致发育中的果蝇出现戏剧性的结果，通常会使胚胎无法形成正确的身体计划。在我的博士后研究期间，我问这种不对称mRNA定位机制是否在哺乳动物大脑的发育中起着重要作用。我证明了这一机制在神经干细胞中至少部分是保守的。我发现，在分化的细胞分裂过程中，对赋予神经谱系重要的mRNA和参与其定位的蛋白质是不对称分布的。当我破坏这种定位复合体时，细胞不再能够不对称地分裂，而是以干细胞库为代价产生了太多的神经元。然而，仍然存在两个基本问题。首先，在什么时候确定的mRNA注定不对称分布？第二，当定位发生时，这些mRNA是如何保持在一个被抑制的状态的？本文提出的研究计划旨在解决这两个问题。这是我的假设，注定不对称分布的mRNA在细胞核中确定出口之前。根据其他模式生物的证据和对哺乳动物分化细胞的观察，参与RNA剪接的蛋白质与定位有关，这一点变得越来越明显。我的目标是确定哪些组件的剪接机制参与识别mRNA的定位，以及它们如何与细胞质内发现的组件进行mRNA到其适当的目的地。在这种运输过程中，mRNA被认为保持在沉默状态，以防止蛋白质产生发生在不需要的区域。我以前已经证明，RNA结合蛋白和翻译抑制蛋白，Pumilio 2（Pum2）是神经前体细胞中不对称RNA颗粒的一部分。我的目的是确定是否Pum2和它的parasitum Pum1，以及类似的翻译阻遏物Musashi 1和Musashi 2，作为沉默剂在不对称RNA定位在神经前体。这两个基因家族都显示出在其他干细胞群体中作为翻译调节因子的证据，使它们成为在神经前体细胞中发挥这一作用的理想候选者。这两个核心问题将使用成熟的分子，细胞和生物化学技术，允许在文化和完整的大脑神经干细胞的遗传操作来解决。这些发现将促进我们对神经前体细胞中不对称细胞分裂的理解，以及这一事件如何参与调节大脑的发育。