Date of Award

5-1-2022

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Biological Sciences

Content Description

1 online resource (xiii, 223 pages) : color illustrations.

Dissertation/Thesis Chair

Prashanth Rangan

Committee Members

Thomas Begley, Paolo Forni, Joesph Wade

Keywords

Dashboard, Differentiation, Germline Stem Cell, Oogenesis, Ribosome Biogenesis, Translation Control, Drosophila, Genetic translation, Genetic transcription, Germ cells, Messenger RNA

Subject Categories

Bioinformatics | Biology | Developmental Biology

Abstract

The decision of a stem cell to either self-renew or differentiate is controlled by specific cellularpathways that can act at the level of transcription, translation, or post-translation. To study the regulation of these pathways in-vivo, I have used the female Drosophila germline as a model system. Each of the steps from germline stem cell (GSC) to egg require changes in cellular pathways. These changes can occur at the level of transcription, post-transciption, translation, or post-translation . Decades of research has elucidated many of the changes to gene that occur during oogenesis, however, many players in this process still remain mysterious. My work has helped to identify and characterize novel developmental mechanisms that are required for the successive developmental transitions that take place during oogenesis. I have leveraged RNAseq and polysome-seq to probe the global transcription and translation landscape over development and used the power of Drosophila genetics in concert with these sequencing techniques to identify and characterize misregulated pathways. We have discovered a link between the efficient biogenesis of the translation apparatus, the ribosome, and the translation of the constituent proteins of the ribosome. We found that proper ribosome biogenesis ensures that ribosomal proteins are translated at normal levels by preventing a translation inhibitor called La-related protein (Larp) from binding its ii targets, which primarily consists of ribosomal proteins. We found that one of the mRNAs repressed by Larp is Novel nucleolar protein 1 (Non1 ), which prevents cell cycle arrest in a p53 dependent manner. Therefore, we discovered a novel connection between ribosome biogenesis and cell cycle. Our work demonstrates a novel mechanism by which ribosome biogenesis can be balanced. Broadly, this connection has important implications in how stem cells regulate ribosome production, which is known to play a crucial role in stem cell differentiation. Additionally, I have developed a tool called Oo-site to allow researchers to investigate changes in gene expression at the mRNA level and post-transcriptionally over the course of GSC differentiation. Oo-site can aid the research and hypothesis generation of other researchers in the field by democratizing access to stage specific mRNAseq and polysome-seq data, as well as integrates publicly available single-cell seq data. This tool allows nonbioinformaticians to quickly and easily view expression data across Drosophila GSC differentiation and development. This work has revealed that Orientation Disrupter (Ord), a key meiotic gene is controlled post-transcriptionally, at the level of translation and suggests that other key genes involved in the transition of a GSC from a mitotic to a meiotic fate may be controlled through modulating their translation. Finally, I attempted to characterize the regulatory role the ribosome plays by performing a screen of post-translational modifying enzymes, with the hypothesis that some of these enzymes might act on ribosomal proteins. This screen made use of a dual-luciferase reporter to attempt to monitor changes in translation status. Some limitations of this work have thus far prevented any firm conclusions, however, future work in this area could help in understanding what role the ribosome might play in directly regulating translation, which is iii an emerging area of interest. Overall, my work has emphasized the role the ribosome plays regulating stem cell differentiation. This regulation occurs both directly and indirectly. The ribosome regulates stem cell differentiation directly in that sufficient ribosome levels are required to overcome cell cycle blocks that ensure differentiation occurs properly. Indirectly, the ribosome carries out translation, which my work has demonstrated is a key point of regulation during stem cell differentiation. Moving forward, discovering the factors that enact translation regulation during differentiation is of critical importance to fully understanding stem cell differentiation and therefore differentiation related disease states. Future work should focus on understanding what role ribosomes play in guiding translation through alternative ribosomal protein usages, post-transcriptional modification of rRNA, and post-translational modification of ribosomal proteins.

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