Date of Award

Spring 2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular Biophysics and Biochemistry

First Advisor

Baserga, Susan

Abstract

Nucleoli are dynamic nuclear condensates in eukaryotic cells that originate through ribosome biogenesis at loci that harbor the ribosomal DNA. These loci are known as nucleolar organizer regions and there are 10 in a human diploid genome. While there are 10 nucleolar organizer regions however, the number of nucleoli observed in cells is variable. Furthermore, changes in number are associated with disease, with increased numbers and size common in aggressive cancers. In the near-diploid human breast epithelial cell line, MCF10A, the most frequently observed number of nucleoli is 2-3 per cell. While ribosome biogenesis is an essential biological process that is common among all life forms, studies that elaborate on the complexities of ribosome biogenesis in higher eukaryotes, like humans, are few. In this dissertation, to identify novel regulators of ribosome biogenesis in higher eukaryotes, I used quantitative imaging of MCF10A cells to perform a high-throughput siRNA screen for proteins that, when depleted, increase the percentage of nuclei with ≥5 nucleoli. Unexpectedly, this screening approach led to the identification of proteins associated with the cell cycle. Functional analysis on a subset of hits further revealed not only proteins required for progression through S and G2/M phase, but also proteins required explicitly for the regulation of RNA polymerase I transcription and protein synthesis. Thus, results from this screen for increased nucleolar number highlight the significance of the nucleolus in human cell cycle regulation, linking RNA polymerase I transcription to cell cycle progression. In this dissertation, I also applied this high-throughput screening approach to cancer drug discovery. Because ribosome biogenesis is essential for cell growth and is linked to the pathogenesis of cancer, targeting the nucleolus has become an attractive target for the development of novel therapies. Screening a library of ~4,000 FDA-approved drugs revealed over 100 compounds that regulate nucleolar number, with antineoplastic agents being the most common identified. Expanding the search to a library of ~25,000 novel, synthetic compounds also revealed additional regulators of nucleolar number that are structurally distinct from the FDA-approved drugs and harbor promise as putative new cancer therapies. The discoveries described herein broaden our understanding of nucleolar biology in higher eukaryotes and will provide a foundation for the development of novel and more effective therapeutics for the treatment of cancer. `

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