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.
This proposal is designed to answer important questions about the connections between making ribosomes and human disease. Ribosomes are the cellular factories that synthesize proteins in all human cells. As both cancer and genetic diseases of newborn babies are linked to abnormalities in the making of ribosomes, our work will be crucial for elucidating the underlying mechanisms of these diseases.
