Identifying Non-Coding and Protein Regulators of Ribosome Biogenesis
Date of Award
Spring 2023
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Genetics
First Advisor
Baserga, Susan
Abstract
Ribosomes are the macromolecular structures necessary for all cells to produce their proteins and ultimately perform functions. The process the cell undertakes to makes ribosomes, termed ribosome biogenesis, is complex and energetically taxing. Ribosome biogenesis begins in the nucleolus and is completed in the cytoplasm allowing the production of proteins during protein synthesis. Our understanding of this essential process has been studied intensely in lower organisms, in particular the yeast Saccharomyces cerevisiae. However, much remains unexamined in human cells. The need for a detailed understanding of ribosome biogenesis in human cells is underscored by the role aberrant ribosome biogenesis plays in the development of rare genetic disease—termed ribosomopathies, i.e. Diamond-Blackfan anemia or Schwachman-Diamond syndrome—and cancer. Technological advances have enabled many discoveries about the regulation of ribosome biogenesis. This includes the use of chemoproteomics to discover alternative proteins (alt-proteins). In this thesis, I discuss the discovery of alt-RBM10 (MINAS-60), which also exemplifies a rare negative regulator of late 60S maturation. These technological advances have also allowed numerous high-throughput screens to identify novel human regulators of ribosome biogenesis. I surveyed and compared the datasets from these screens and the nucleolar proteome to identify areas of overlap and current gaps in knowledge. While each screen uncovered unique regulators of ribosome biogenesis, substantial overlap of all datasets was not seen. These screens, taken together, do show a connection between large ribosomal subunit factors and nucleolar form, which is an interesting avenue to explore in more detail in the future. Finally, I conducted a high-throughput screen to identify novel long non-coding RNA regulators of ribosome biogenesis. Following 72-hour siRNA depletion of lncRNAs, nucleoli were counted for changes in the number of nucleoli per nucleus, as our laboratory has previously performed. Robust statistical analyses and filtering revealed 72 lncRNAs that decrease the number of nucleoli per nucleus to one, and 17 that increase the number of nucleoli per nucleus to 5 or more. Hitpick and siRNA deconvolution validation screens were performed to solidify a list of 21 validated, high-confidence lncRNAs that alter nucleolar number. Of these 21 hits, 19 were found to significantly affect nucleolar rRNA biogenesis as assessed by inhibition of 5-EU incorporation. qRT-PCR analysis revealed 12 of these transcripts were robustly expressed in MCF10A cells, with two shown to be depleted by siRNA. Taken together, I discovered 10 lncRNA hits expressed in MCF10A, 2 of which can be depleted by siRNAs, and remain to be followed up on for mechanistic analysis.
Recommended Citation
Harold, Cecelia Marie, "Identifying Non-Coding and Protein Regulators of Ribosome Biogenesis" (2023). Yale Graduate School of Arts and Sciences Dissertations. 983.
https://elischolar.library.yale.edu/gsas_dissertations/983
