Specialized Translational Control of Stem Cell Differentiation and Embryonic Development

控制基因在空间和时间上的表达在使细胞“知道”它们在细胞中的位置方面起着重要作用。 发育胚胎和成为什么，这一过程通常被称为细胞特化。几十年的研究 证明了在转录和转录后水平上控制基因表达的许多调节层 这一过程的协调。相反，基因表达的翻译控制受到的关注较少。 实验注意力最值得注意的是，流行的教条是，在蛋白质生产的水平上，核糖体- 尽管这是一个极其复杂的分子机器，但它在翻译过程中具有组成性而非调节性功能， mRNA。我们的发现建立了一个新的研究领域，证明核糖体在控制中具有高度的调节作用。 哺乳动物的组织模式和形成的基础发育基因调控网络的表达 身体计划。在我们最近的研究中，我们已经确定了胚胎干细胞中的整个生物学途径， 通过特定核糖体的翻译偏好，其核糖体蛋白质（RP）的组成不同，或者 新的核糖体相关蛋白（RAP）的相互作用，我们最近发现，直接相关的 哺乳动物核糖体。我们进一步表明，核糖体的异质性接近关键细胞器，作为一个重要的细胞， 在亚细胞空间内控制局部蛋白质产生的机制。这些发现改变了我们对 基因调控，并打开了一个新的门户网站的研究到一个额外的基因表达层至关重要的控制细胞 规格，组织图案和胚胎发育。在这份提案中，我们将进行一项高度多学科的 方法来表征这种新的调控代码的关键发育网络的电路的翻译控制。 在Aim 1中，我们将扩展我们新的核糖体异质性路线图，该路线图由不同核糖体的存在指示， 原代人ES细胞分化为生物体水平。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，我们将描绘一个单一的RP可以控制一个机制， 胚胎发育的重要步骤，即持续的近轴中胚层形成，及其在翻译中的作用 WNT信号通路的控制，这反映了一个新的步骤，在调节一个主要的信号通路， 发展在Aim 2中，我们将采用系统水平的方法来表征核糖体作为关键调节器的作用 细胞命运的转变。我们将利用新的技术，强制和诱导地选择性地去除特定的RP， 细胞质核糖体，并评估其对干细胞分化的个体功能， 中胚层和内胚层谱系。在Aim 3中，我们将在功能上表征乳腺癌中的替代RP旁系同源物。 我们令人信服的初步数据表明，它们在发育过程中翻译不同的mRNA子集。 转为哺乳期。我们假设，翻译控制是合成丰富的乳蛋白所必需的， 新生儿营养在更基础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的失调如何改变基因的精确控制， 表达潜在的人类先天性出生缺陷。

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.