Novel regulatory networks driving human ribosome biogenesis

项目摘要/摘要 在过去的15年里，公认的人类遗传病的数量出现了爆炸性增长 制造核糖体，核糖体疾病，其中大部分是遗传性疾病。尽管有明显的影响， 人类疾病中核糖体生物发生的异常，人类细胞中的核糖体生物发生才刚刚开始 有待调查和澄清。现在的关键挑战是查明人类核糖体是如何制造的 细胞，以定义这一关键过程在不同组织和不同类型的细胞中是如何调节的 胚胎发育，并探索这一过程中的失败如何导致人类 核糖体。 作为更好地探索人类细胞核糖体形成机制的一种新方法， 我们已经成功地开发出一种独特的、高度定量的、基于图像的细胞检测方法，它可以报告 核仁功能障碍(《细胞报告2018》)。我们已经在近二倍体中进行了全基因组siRNA筛选 MCF10A人乳腺上皮细胞鉴定核仁数量变化的细胞蛋白。此屏幕 是第一次使用核仁数量作为终点的RNAi运动，也是第一次进行此类筛选 在不同于Hela细胞的人类细胞系中。这种无偏见的筛查方法揭示了许多新的细胞 以前与制造核糖体无关的蛋白质(HITS)。 我们已经获得并成功验证了核仁和非核仁的命中结果，从而提出了这一假设 非核仁蛋白是人类细胞核糖体生物发生的新的间接调节因子。使用 定制的技术策略，我们将进一步定义HITS的子集在核糖体生物发生中的作用 人类组织培养细胞，揭示了独特的调控途径。此外，我们还将它们链接到一个 以非洲爪哇为模式系统研究胚胎发育的关键要求。曾经被看做 作为一个“管家”细胞器，我们的工作将突出核仁对人类的重要贡献。 遗传病和先天畸形。

Project Summary/Abstract Over the last 15 years there has been an explosion in the number of recognized human genetic diseases of making ribosomes, the ribosomopathies, most of which are inherited conditions. Despite the clear impact of abnormalities in ribosome biogenesis on human disease, ribosome biogenesis in human cells is just beginning to be investigated and elucidated. Key challenges now are to pinpoint how ribosomes are made in human cells, to define how this critical process is regulated in different tissues and diverse cell types throughout embryonic development, and to probe how failures in this process lead to the human diseases of making ribosomes. As one novel approach to better probe the mechanisms underlying how ribosomes are made in human cells, we have successfully developed a unique, highly-quantitative and image-based cellular assay that reports nucleolar dysfunction (Cell Reports 2018). We have conducted a genome-wide siRNA screen in near-diploid MCF10A human breast epithelial cells to identify cellular proteins that change nucleolar number. This screen was the first RNAi campaign to use nucleolar number as an endpoint, and the first screen of its type carried out in a human cell line other than Hela cells. This unbiased screening approach revealed many new cellular proteins (the “hits”) not previously connected to making ribosomes. We have obtained and successfully validated both nucleolar and non-nucleolar hits, prompting the hypothesis that the non-nucleolar proteins are novel indirect regulators of ribosome biogenesis in human cells. With tailored technological strategies, we will further define the role of a subset of the hits in ribosome biogenesis in human tissue culture cells, shedding light on unique regulatory pathways. In addition, we will link them to a critical requirement in embryonic development using Xenopus tropicalis as a model system. Once looked upon as a “housekeeping” organelle, our work will highlight the nucleolus as a significant contributor to human genetic disease and congenital malformations.