Discovery of New Regulators of Human Ribosome Biogenesis [electronic resource].

9781085777674

Ann Arbor : ProQuest Dissertations & Theses, 2019.

1 online resource (288 p.)

Access is available to the Yale community.

Ribosomes are macromolecular machines that perform the essential function of protein synthesis in human cells. The process of making a ribosome is incredibly intricate and requires all 3 RNA polymerases, a host of assembly factors, and approximately 80 different ribosomal proteins (r-proteins). Ribosome biogenesis begins in the nucleolus, proceeding outward towards the cytoplasm where the ribosomes function in protein synthesis. Despite the importance of this biological process in cellular growth and development, many questions remain regarding the synthesis of ribosomes. Still less is known about this process in human cells, as the foundational knowledge of ribosome biogenesis was developed in the budding yeast Saccharomyces cerevisiae. This work therefore seeks to answer key questions about the factors regulating the synthesis of ribosomes in human cells.To identify new regulators of ribosome biogenesis in human cells, a high-throughput, genome-wide RNAi screen was performed. The screen took advantage of the fact that the structure of the nucleolus is intricately linked with its function in making ribosomes. After 72 hours of protein depletion, reductions in the number of nucleoli per cell nucleus were observed, and this readout of reduced nucleolar number was used as a proxy for changes in nucleolar function. In all, the screen identified 139 proteins required for nucleolar formation and function in human cells. Further testing of 20 high-confidence screen hits revealed functions for 18 in the nucleolar processes of rDNA transcription (7/20), pre-ribosomal RNA processing (16/20), and global protein synthesis (14/20). The protein hits from the screen function in a plethora of cellular processes including cell division, development, proliferation, and more. The screen will therefore continue to serve as a rich resource to increase our understanding of the essential biological process of making ribosomes in humans.Additional studies of one screen hit, Paired Box 9 (PAX9), reveled a new role for this protein in human ribosome biogenesis. In human cells, depletion of PAX9 results in decreased nucleolar number as well as defects in small subunit (SSU) pre-ribosomal RNA processing and global protein synthesis. Two transcriptomics analyses, RNA-sequencing and RNA Polymerase II (RNAPII) Chromatin immunoprecipitation sequencing (ChIP-seq), revealed that PAX9 acts as a RNAPII transcription factor to drive the expression of a number of mRNAs that encode proteins critical for making ribosomes. In addition, the role of PAX9 in human ribosome biogenesis is conserved to a model organism, the African clawed frog, Xenopus tropicalis (X. tropicalis). This function for PAX9 in making ribosomes connects its role as an RNAPII transcription factor to both ribosome biogenesis and craniofacial development.

Dissertations & Theses @ Yale University.

Books / Online / Dissertations & Theses

English

January 17, 2020

Thesis (Ph.D.)--Yale University, 2019.

Yale University. Molecular Biophysics and Biochemistry.