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Characterisation of RNA binding proteins and their roles in the Drosophila germline

Vaishali, Vaishali

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Abstract

The important role of RNA binding proteins (RBPs) in regulating the fate and functions of RNAs has led to the development of transcript-specific as well as transcriptome-wide techniques allowing an unbiased and comprehensive identification of RBPs. These methods have extended our knowledge of the extent of RBPs in a cell, and studying the roles of these newly identified RBPs in cellular processes has provided us with novel insights into the RNA binding mechanisms, functions and regulation of RNA binding proteins.

For my PhD work, I assessed the RNA binding functions of two proteins identified in high-throughput screens. The first protein is the Fragile X Mental Retardation protein (FMR1), identified in a transcript-specific pulldown targeted at the Drosophila maternal mRNA oskar. I show that FMR1 is a bona fide component of the oskar RNA-protein complexes that interacts with the oskar 3’UTR in vivo. FMR1 positively regulates Oskar protein levels in the oocyte, without any effect on oskar RNA levels. Oskar protein nucleates germ plasm assembly and germ cell formation in the embryo, and the reduction in Oskar protein levels leads to a reduction in the number of pole cells formed in embryos knocked down for FMR1. Finally, I tried to determine how FMR1 regulates translation, with roles identified as both a repressor and activator of translation. FMR1 contains two types of RNA binding domains: two KH domains and a C-terminal RGG box. I show that, in vitro, FMR1 activates translation through the KH domains and requires the C-terminal RGG box for repression of translation. I have thus identified a new role of FMR1 in germline development in Drosophila melanogaster, and also a putative mechanism of how FMR1 performs antagonistic functions in translation regulation. The second protein I studied is the microtubule binding protein EB1, identified as a putative RNA binding protein in a transcriptome-wide RNA interactome capture study performed in Drosophila embryos. Preliminary data showed that EB1 binds to polyU25 RNA in vitro, and uses the same binding surface for interacting with microtubules and RNA. I show that EB1 binds to microtubules and RNA in a mutually exclusive manner in vitro. Furthermore, I performed a RIP-seq experiment to identify the in vivo targets of EB1, but failed to validate the interaction of any of the top candidates with EB1 in vivo. This does not, however, negate a role of EB1 as an RNA binding protein altogether, as RNA might be regulating the functions of the protein, and this would require further investigation.

Document type: Dissertation
Supervisor: Ephrussi, Dr. Anne
Place of Publication: Heidelberg
Date of thesis defense: 11 July 2022
Date Deposited: 15 Jul 2022 12:20
Date: 2023
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
DDC-classification: 500 Natural sciences and mathematics
Controlled Keywords: RNA, Translation, Drosophila melanogaster
Uncontrolled Keywords: RNA binding Proteins, oskar, germline, EB1, FMR1, iCLIP
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