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

干细胞具有分裂和产生两个具有不同身份的子细胞的独特能力。这通常涉及到一个再生干细胞和一个分化细胞的产生。在哺乳动物的大脑中，这一过程允许神经干细胞产生正在发育的皮层的神经元和胶质细胞，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尚不清楚。我们可以在其他模式生物如果蝇中找到线索。在果蝇生殖细胞和神经干细胞中，已经证明某些mrna被运输到分裂细胞的一半，因此只有一个子细胞继承它们。通常，这些mrna产生的蛋白质驱使遗传子细胞沿着分化途径前进。这种机制的破坏会导致发育中的苍蝇产生戏剧性的结果，通常会使它们的胚胎无法形成适当的身体计划。在我的博士后研究中，我想知道这种不对称的mRNA定位机制在哺乳动物大脑的发育中是否起着重要作用。我证明了这种机制，至少部分地，在神经干细胞中是保守的。我发现mrna对于赋予神经谱系很重要，而参与其定位的蛋白质在分化细胞分裂过程中是不对称分布的。当我破坏这个定位复合体时，细胞不再能够不对称分裂，相反，产生了太多的神经元，以牺牲干细胞池为代价。然而，仍然存在两个基本问题。首先，mrna在什么时候被确定为不对称分布？其次，当定位发生时，这些mrna是如何维持在翻译抑制状态的？这里提出的研究计划旨在解决这两个问题。我的假设是，注定不对称分布的mrna在输出之前在细胞核中被识别。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的现象越来越明显。我的目标是确定剪接机制的哪些组成部分参与鉴定mRNA的定位，以及它们如何与细胞质内发现的成分相互作用，以将mRNA运送到适当的目的地。在这种转运过程中，人们认为mRNA保持沉默状态是为了防止在不需要的区域产生蛋白质。我之前已经证明，RNA结合蛋白和翻译抑制因子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Pum2及其旁系Pum1，以及类似的翻译抑制因子武藏1和武藏2，是否在神经前体的不对称RNA定位中起沉默作用。这两个基因家族已经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履行这一角色的理想候选者。这两个核心问题将使用完善的分子、细胞和生化技术来解决，这些技术允许在培养和完整的大脑中对神经干细胞进行遗传操作。这一发现将促进我们对神经前体细胞的不对称细胞分裂以及这一事件如何参与调节大脑发育的理解。

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.