Specialized Translational Control of Stem Cell Differentiation and Embryonic Development

控制基因在空间和时间上的表达对于使细胞“知道”它们在细胞中的位置起着重要作用。 胚胎的发育和将来的发展，这一过程通常被称为细胞特化。几十年的研究已经证明 证实了转录和转录后基因表达调控的多个层次 级别，它协调这一过程。相反，基因表达的翻译控制收到的却更少 实验注意。最值得注意的是，流行的教条是，在蛋白质生产水平上，核糖体- 尽管一个极其复杂的分子机器-在翻译过程中具有结构性而不是调节性功能 MRNAs。我们的发现已经建立了一个新的研究领域，证明了核糖体在控制中是高度调控的。 哺乳动物组织构型和形成的发育基因调控网络的表达 身体计划。在我们最新的研究中，我们已经确定了代表的胚胎干细胞中的整个生物学途径。 由于特定核糖体的翻译偏好，这些核糖体的核糖体蛋白(RP)的组成或 我们最近发现的与新的核糖体相关蛋白(RAP)直接相关的相互作用 哺乳动物的核糖体。我们进一步显示了核糖体在关键细胞器附近的异质性 在亚细胞空间内控制局部蛋白质生产的机制。这些发现改变了我们对 基因调控和打开研究对细胞控制至关重要的另一层基因表达的新门户 规格、组织图案化和胚胎发育。在这项提案中，我们将进行高度多学科的 描述这一新的调控代码的方法，用于关键发育网络的电路的翻译控制。 在Aim1中，我们将扩展我们关于核糖体异质性的新路线图，这一路线图由不同的核糖体在 原代人类胚胎干细胞分化到生物体水平。特别是，我们将利用新的遗传工具来研究 体内核糖体生物学。使用这种方法，我们将描述单个RP可以控制 胚胎发育中最重要的一步，即持续的近轴中胚层形成及其在翻译中的作用 控制WNT信号通路，这反映了在调节主要信号通路方面的一个新步骤 发展。在AIM2中，我们将采用系统级别的方法来描述核糖体作为关键调节因子的作用 细胞命运的转变。我们将利用新技术来强制和诱导地选择性地从 首次发现了细胞质核糖体，并评估了它们在干细胞分化中的个体功能 中胚层和内胚层谱系。在Aim3中，我们将从功能上描述乳腺-GREAD中的替代RP类似物 我们令人信服的初步数据表明，它们在 换成哺乳期。我们假设，翻译控制是合成大量对乳制品至关重要的蛋白质所必需的。 新生儿的食物。在更基本的水平上定义核糖体介导的控制基因的特异性和动力学 对于我们理解核糖体中的去调控如何改变基因的精确控制，调控将是无价的。 人类先天出生缺陷的潜在表现。

Control of gene expression in space and time plays an important role in enabling cells to “know” where they are in the developing embryo and what to become, a process often referred to as cellular specification. Decades of research have demonstrated numerous layers of regulation in control of gene expression, at both the transcriptional and post-transcriptional level, which coordinate this process. Translational control of gene expression has, on the contrary, received less experimental attention. Most notably, the prevailing dogma has been that at the level of protein production, the ribosome - although an immensely complex molecular machine- possesses a constitutive rather than regulatory function in translating mRNAs. Our findings have established a new field of study by demonstrating that ribosomes are highly regulatory in control of the expression of developmental gene regulatory networks underlying tissue patterning and formation of the mammalian body plan. In our most recent studies, we have identified entire biological pathways in embryonic stem cells represented by the translational preferences of specific ribosomes, that differ in the composition of their ribosomal proteins (RPs) or the interaction of novel ribosome-associated proteins (RAPs) that we have recently identified that directly associate with mammalian ribosomes. We have further shown ribosome heterogeneity in proximity to key cellular organelles as a mechanism to control localized protein production within subcellular space. These findings change our understanding of gene regulation and open a new portal of study into an additional layer of gene expression vital to control of cell specification, tissue patterning, and embryonic development. In this proposal we will undertake a highly multidisciplinary approach to characterize this novel regulatory code for translational control of the circuitry of key developmental networks. In Aim1 we will extend our new roadmap of ribosome heterogeneity indicated by the presence of distinct ribosomes during primary human ES cellular differentiation to an organismal level. In particular, we will leverage novel genetic tools to study ribosome biology in-vivo. Using this approach, we will delineate the mechanisms by which a single RP can control a paramount step in embryonic development, namely sustained paraxial mesoderm formation, and its role in translational control of the WNT signaling pathway, which reflects a novel step in the regulation of a major signaling pathway in development. In Aim2 we will undertake a systems level approach to characterize the role of ribosomes as key regulators of cell fate transitions. We will utilize novel technologies to forcibly and inducibly remove specific RPs selectively from cytoplasmic ribosomes for the first time and assess their individual functions on stem cells differentiation down the mesoderm and endoderm lineages. In Aim3 we will functionally characterize alternative RP paralogs in mammary-glad development for which our compelling preliminary data indicate that they translate distinct subsets of mRNAs during the switch to lactation. We hypothesize that translation control is required to synthesize copious milk proteins critical for neonate sustenance. Defining at a more basic level the specificity and dynamics of ribosome-mediated control gene regulation will be invaluable for our understanding of how deregulations in the ribosome alter accurate control of gene expression underlying human congenital birth defects.