Novel regulatory networks driving human ribosome biogenesis

项目总结/摘要 在过去的15年里，人类遗传疾病的数量激增， 制造核糖体，即核糖体病，其中大部分是遗传性疾病。尽管有明显的影响， 核糖体生物合成异常对人类疾病的影响，人类细胞中的核糖体生物合成才刚刚开始 有待调查和阐明。现在的关键挑战是确定核糖体是如何在人体内产生的 细胞，以确定这一关键过程是如何在不同的组织和不同的细胞类型的调节， 胚胎发育，并探讨如何在这一过程中失败导致人类疾病， 核糖体 作为一种更好地探索人类细胞中核糖体形成机制的新方法， 我们已经成功地开发了一种独特的，高度定量和基于图像的细胞分析， 核仁功能障碍（Cell Reports 2018）。我们在近二倍体细胞中进行了全基因组siRNA筛选， MCF 10A人乳腺上皮细胞，以鉴定改变核仁数量的细胞蛋白。此屏幕 这是第一个使用核仁数作为终点的RNAi活动，也是第一次进行这种类型的筛选 在人类细胞系而不是Hela细胞中。这种无偏见的筛选方法揭示了许多新的细胞 蛋白质（“命中”）先前没有连接到制造核糖体。 我们已经获得并成功地验证了核仁和非核仁命中，促使假设 非核仁蛋白是人类细胞中核糖体生物合成的新型间接调节剂。与 量身定制的技术策略，我们将进一步确定一个子集的命中在核糖体生物合成中的作用， 人类组织培养细胞，揭示独特的调控途径。此外，我们还将它们与 胚胎发育的关键要求，使用非洲爪蟾作为模型系统。曾经被视为 作为一个“管家”细胞器，我们的工作将突出核仁作为人类的重要贡献者， 遗传疾病和先天畸形。

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.