Date of Award

1-1-2021

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Biological Sciences

Content Description

1 online resource (xv, 186 pages) : color illustrations.

Dissertation/Thesis Chair

Prashanth Rangan

Committee Members

Miler Lee, Marlene Belfort, Cara Pager, Gabriele Fuchs

Keywords

Drosophila, Germline, Helicase, mRNA, Oogenesis, Germ cells, RNA splicing, Catalytic RNA, Messenger RNA

Subject Categories

Molecular Biology

Abstract

Gametogenesis, the process of creating egg or sperm, is required for launching successive generations of sexually reproducing organisms. The developmental milestones that occur during gamete production have been studied for decades and are of critical interest to gain insight to conserved features of human fertility. Drosophila has been used for over a century as an efficient research model and remains pivotal in uncovering fundamental biological paradigms. During Drosophila egg production, or oogenesis, several developmental transitions must be traversed to ensure completion of oogenesis including: Germline Stem Cell (GSC) maintenance and differentiation, mitotic and meiotic cell divisions, and production of maternally contributed mRNAs for the oocyte. The diverse biological processes occurring during oogenesis makes the female germ line a salient model for understanding how cellular fate transitions are mediated. In Drosophila, the molecular processes that govern germline development are diverse and largely dictated by RNA regulation. Due to silencing of the genome during meiotic divisions, there is little instructive transcription during oogenesis, barring a few examples, to mediate these critical transitions. Thus, several layers of post-transcriptional regulation ensure that the mRNAs required for these processes are translated and expressed in a timely manner and as needed during germline differentiation. Broadly, these layers of regulation include alternative splicing, RNA modifications, ribosome production, RNA degradation, and translational regulation. Many of the proteins and pathways involved in these regulatory activities are conserved in humans, making the Drosophila germline an efficient and elegant model for studying the role of post-transcriptional regulation during stem cell differentiation and meiosis. Specifically, a class of well-conserved, secondary structure remodeling proteins, called RNA helicases, are pivotal during these molecular processes, and required for the successful production of gametes. The wide diversity and differential utility of RNA helicases allow for their participation in many RNA processes. In this work, a germline specific RNA interference (RNAi) knockdown screen of helicases revealed that many RNA helicases play important and unique roles during Drosophila oogenesis. The helicases identified are required for oogenesis and facilitate processes such as RNA splicing, translational regulation, ribosome biogenesis and RNA turnover, and are highlighted in this work. Specific helicases were characterized during egg production and the unique phenotypes observed upon their depletion shed light on their molecular roles, providing information to a deeper biological function. RNA helicases involved in the ubiquitous processes of splicing and translation initiation resulted in interesting morphological phenotypes when depleted. Specifically, Me31b, a well conserved RNA helicase, was found to negatively regulate the translation of a consequential germline gene called polar granule component (pgc). Me31b binds pgc mRNA and inhibits its translation, presumably through interactions with the 5’ cap and partnering proteins such as dGe-1. Unsurprisingly, many helicases that mediate the crucial process of ribosome biogenesis were found to be required for maintenance of the germ line. However, three of these previously uncharacterized helicases which we named Aramis, Athos and Porthos, were found to have a unique requirement in GSC differentiation and cell cycle progression. We found that the production of ribosomes gates the translation of important ribosomal proteins, and that this regulation occurs through LA-related protein (Larp) and terminal oligopyrimidine (TOP) motifs in the 5’ UTRs of target mRNAs. This work elucidates the tissue-specific requirement of a seemingly ubiquitous process such as ribosome biogenesis in the germline. Lastly, an RNA helicase known through homology to facilitate cytoplasmic mRNA degradation, named Twister (tst) in Drosophila, is responsible for curating mRNAs expressed in early stages of oogenesis, so that subsequent stages can properly progress, and maternal mRNAs can be abundantly produced for the egg. Tst-regulated RNAs are targeted during translation as ribosome association increases during GSC differentiation. Interestingly, what was thought to be a housekeeping protein involved in mRNA turnover has a distinct role in the germline, sculpting the maternal mRNA contribution during the transition from a GSC to an oocyte. This work illustrates that mRNA degradation pathways sculpt the transcriptome during oogenesis to successfully launch the next generation. Thus, RNA helicases have specific, diverse and noteworthy roles in regulating a variety of molecular processes required for oogenesis.

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