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. While we obtained and successfully validated both nucleolar and non- nucleolar hits, we hypothesize that the non-nucleolar proteins are novel indirect regulators of ribosome biogenesis in human cells. One intriguing non-nucleolar protein with a human disease connection is the PAX9 protein, a protein that has not been implicated in any aspect of ribosome biogenesis previously. We selected the PAX9 protein because mutation in PAX9 leads to human genetic diseases of craniofacial development in many different populations worldwide (www.ncbi.nlm.nih.gov/gene/5083). Patients are afflicted with varying degrees of missing teeth (oligodontia), cleft palate and skeletal abnormalities. PAX9 is also a transcription factor for RNA polymerase II (RNAPII). In support of the hypothesis that PAX9 plays a role in making ribosomes in the nucleolus, we present extensive evidence in Preliminary Results demonstrating a role for PAX9 in nucleolar pre-18S rRNA processing with implications for overall cellular protein synthesis. Prior to this work, it was not known that PAX9 functions in ribosome biogenesis. This intriguing finding adds to the growing body of knowledge that links craniofacial abnormalities of development to the making of ribosomes. Building on our new findings, we propose the following Specific Aims: 1) To probe the mechanism of how siRNA depletion of the validated hit, PAX9, results in defective ribosome biogenesis in MCF10A cells and 2) To test the extent to which Pax9 depletion or mutation links craniofacial abnormalities to defective ribosome biogenesis in vivo in the model organism, Xenopus tropicalis. This work is important because it will explore the cellular mechanisms that underlie the function of PAX9 in ribosome biogenesis and provide an increased mechanistic understanding of the craniofacial genetic landscape that connects the basic science of ribosome biogenesis to craniofacial development.
This proposal is designed to answer important questions about the function of the PAX9 protein, a protein that when mutated causes the absence of most or all of the adult teeth as well as other facial malformations in families worldwide. We propose that part of the disease may be due to abnormalities in an unexpected cellular process, the process of making ribosomes, which are the factories that make proteins. A more thorough understanding of the disease mechanism will help us design better preventive therapies.
|Griffin, John N; Sondalle, Samuel B; Robson, Andrew et al. (2018) RPSA, a candidate gene for isolated congenital asplenia, is required for pre-rRNA processing and spleen formation in Xenopus. Development 145:|
|Farley-Barnes, Katherine I; McCann, Kathleen L; Ogawa, Lisa M et al. (2018) Diverse Regulators of Human Ribosome Biogenesis Discovered by Changes in Nucleolar Number. Cell Rep 22:1923-1934|