Date of Award

1-1-2023

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Biological Sciences

Content Description

1 online resource (x, 184 pages) : illustrations (some color)

Dissertation/Thesis Chair

Prashanth Rangan

Committee Members

Andy Berglund, Gaby Fuchs, Florence Marlow

Keywords

Drosophila, Germ cells, Gametogenesis, Biological control systems

Subject Categories

Biology

Abstract

To launch the next generation, all sexually reproducing animals must undergo the process of producing mature eggs and sperm. This process is known as gametogenesis. During gametogenesis key developmental checkpoints are met to ensure proper egg and sperm production. Model organisms, such as Drosophila melanogaster, have been used to study gametogenesis to gain insight into the conserved mechanisms that can be attributed to human infertility and disease due to the availability of genetic tools, quick life cycle and the conservation of regulatory mechanisms. During Drosophila oogenesis there are many key developmental transitions that must be completed to produce a fully mature and functioning egg. These transitions include but are not limited to Germline Stem Cell (GSC) differentiation and maintenance, completion of meiosis and deposition of the maternal contribution. One key transition is the after the GSC differentiates into a GSC daughter. In the GSC daughter, transcriptional silencing occurs which promotes differentiation. During this silencing, mitotic genes are silenced while meiotic gene transcription is simultaneously promoted. In addition, the GSC daughter must inhibit transposon activity which could be detrimental to the developing egg due to random insertion of the transposon into the genome. Another key transition is after the GSC daughter differentiates and begins to transition from a mitotic to meiotic program. During this period the transcriptional instruction is limited due to transcriptional silencing and thus post-transcriptional regulation must occur to ensure mRNAs that are required for this transition are translated in a timely manner. Some known mechanisms of post- transcriptional regulation include alternative splicing, RNA modifications, ribosome production, RNA degradation and translational regulation. Many proteins that are involved in these mechanisms are conserved in humans making Drosophila an excellent model to study the post-transcriptional mechanisms during GSC differentiation. In part of my studies, I found that GSC daughter the histone modifier Little Imaginal Discs (lid), or Kdm5, which is an H3K4me3 is required to establish the proper chromatin landscape and for proper egg production as kdm5 mutants accumulate GSC daughter cells. Furthermore, I found that kdm5 ensures that the proper chromatin landscape is established which mediates silencing of transposons, burdock. The accumulating GSC daughter cells in kdm5 mutants activate a P53 mediated checkpoint which blocks further differentiation, causing an accumulation of single cells. Furthermore, loss of P53 in kdm5 mutants partially rescues the differentiation defect. Thus, in this work it was discovered that there is a key P53 mediated checkpoint in the GSC daughter to ensure the proper chromatin landscape prior to differentiation. In another study, a mass spectrometry screen was coupled to an unbiased RNA Interference (RNAi) screen and analyses of the identified genes which identified critical RNA modifications that are required for Drosophila oogenesis. This revealed that pseudouridine was abundant and required for proper oogenesis. Specifically, the H/ ACA box, a conserved pseudouridine synthase, is required for depositing pseudouridine on rRNA during the meiotic stages of oogenesis. Proper pseudouridylation of rRNA promotes the translation of meiotic RNAs such as RNA-binding Fox protein 1 (Rbfox1) and Bruno1. These RNAs contain a highly conserved and repeating CAG motif that codes for the amino acid glutamine (Q). Furthermore, the H/ ACA box increases ribosome biogenesis and this increase is required to translate the meiotic CAG containing mRNAs. Moreover, modulating the Target of Rapamycin (TOR) pathway, a critical pathway that modulates ribosome biogenesis, either through genetics or by the TOR pathway inhibitor, Rapamycin, can alter translation of CAG containing mRNAs and reporter. Thus, meiotic CAG containing mRNAs are sensitive to ribosome levels. The repeating CAG motif codes for the amino acid glutamine (Q) which are commonly associated with a group of diseased proteins known as polyQ proteins. Expansions of the CAG repeat results in large Q expansions that cause proteins to stick to one another resulting in and diseases such as Huntington’s disease. Our data suggests that presence of the repeating CAG motif may act as a ribosomal sink preventing translation of the mRNAs containing the motif. Thus, the dulate the TOR pathway may alleviate the neuronal degeneration associated with polyQ diseases by preventing translation of mRNAs that contain the CAG motif. Although further studies are needed, our data suggests the repurposing of drugs that affect TOR pathway. Combined, this work illustrates key regulatory mechanisms at both a transcriptional and post-transcriptional level that are required for GSC differentiation and to successfully launch the next generation.

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