Date of Award

5-2017

Document Type

Honors Thesis

Degree Name

Bachelor of Science

Department

Biological Science

First Advisor

Prashanth Rangan

Second Advisor

Pooja Flora

Abstract

Stem cells have the intrinsic ability to divide to form a stem cell daughter, while also retaining the ability to self-renew. These qualities are critical, as improper stem cell maintenance is implicit in aging and provokes diseases such as cancer. We previously revealed that during Drosophila oogenesis, transcriptional silencing mediated by polar granule component (pgc) alters the germ line cell cycle to promote germ line stem cell (GSC) differentiation. Our work with pgc suggested that cell cycle regulation might be an important general mechanism to facilitate division GSCs into daughter stem cells (or cystoblast, CBs), and differentiation of the CB into a mature egg. Consequentially, we aimed to the test the hypothesis that differences in global gene expression occur in each phase of the cell cycle in both GSCs and CBs to promote a stem cell or CB fate, respectively. We marked and quantified the number of GSCs and CBs in each phase of the cell cycle by harnessing the fluorescence ubiquitination cell cycle indicator system (FUCCI) to label cells. Additionally, we successfully isolated FUCCI-labeled single cells for subsequent separation by fluorescence activated cell sorting (FACS). Moreover, we endeavored to isolate both GSCs and CBs in each phase of the cell cycle by FACS. Once separated, we will perform genomic analyses on each cell type by RNA-seq to generate a database of the cell cycle-dependent transcriptional landscape of differentiation. Thus, our work provides an invaluable tool for future investigations that examine the mechanisms that regulate stem cell fate. Maternal deposition of mRNAs to the developing egg is critical to establish the future generation’s developmental program. However, how these RNAs remain under strict translational regulation remains elusive. Therefore, we asked how maternal RNAs are continually repressed, using polar granule component (pgc) as a model system. Preliminary results from our lab reveal that the pgc 5’UTR is required but not sufficient for suppressing Pgc expression in the GSCs. On the other hand, pgc 3’UTR is required and sufficient to suppress its translation throughout oogenesis. To determine cis and trans-acting factors that control pgc translation, we previously carried out a phylogenetic analysis of its 3’UTR and identified conserved binding sites for translational repressor Pumilio (Pum). We found that Pum binds to the 3’UTR of pgc and together with its binding partners Nanos and Twin (CCR4 de-adenylase), represses pgc translation only in GSCs. Here, we show that the NOT complex, recruited by CCR4, also regulates Pgc expression in the GSCs and later stages of differentiation. Interestingly, previous studies have shown that Me31B interacts with the CCR4-NOT complex in the 3’ end and cap proteins in the 5’ end to suppress translation. As a result, we investigated if Me31B is also regulating Pgc expression in the GSCs via this complex. Remarkably, we found that loss of me31b in the germline upregulates Pgc expression in the GSCs and that pgc mRNA associates with Me31b. Altogether our data elucidates that an intricate protein complex in the GSCs bridges pgc’s 5’ and 3’UTR ends, masking it from translational machinery. We are currently identifying if there is a network of germline mRNAs that could be similarly regulated during Drosophila development.

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Biology Commons

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