eprintid: 24701
rev_number: 14
eprint_status: archive
userid: 3811
dir: disk0/00/02/47/01
datestamp: 2018-06-20 09:57:54
lastmod: 2018-06-20 13:17:56
status_changed: 2018-06-20 09:57:54
type: doctoralThesis
metadata_visibility: show
creators_name: Bordi, Matteo
title: Investigating the RNA-binding activity of the Drosophila melanogaster germline inducer Oskar
subjects: ddc-500
subjects: ddc-570
divisions: i-140001
divisions: i-850800
adv_faculty: af-14
abstract: In many animals, a specialized cytoplasm forms within the oocyte that harbors all the molecular factors required for germ cell fate specification and is defined as the germ plasm. In Drosophila melanogaster, the germ plasm (or pole plasm), is assembled at the posterior pole of the oocyte in a stepwise process triggered by Oskar protein. oskar mRNA transcribed in the nuclei of nurse cells is actively transported into, and to the posterior pole of, the oocyte. At the posterior pole, oskar mRNA is translated into Oskar protein, which recruits the other pole plasm components required for germline specification and posterior patterning in the embryo.
Recently, it was shown that Oskar binds polyadenylated mRNAs in vivo and that the C-terminal domain of the protein binds RNA in vitro. In that study, by using UV crosslinking and immunoprecipitation experiments, I showed that Oskar associates in vivo with three mRNAs involved in posterior patterning and germ cell fate specification: nanos, polar granule component and germ cell-less. In order to identify Oskar’s binding site(s) on its target transcripts I applied the iCLIP method to early Drosophila embryos. To this end, I analyzed the efficiency of three key steps of the iCLIP protocol: RNAse digestion, 3’ end dephosphorylation, and adapter ligation. I found that, while the 3’ end dephosphorylation was generally efficient, the ligation of an adapter to RNA was a limiting step in the protocol. By performing a series of optimization experiments, I established new reaction conditions for adapter ligation that increased the efficiency of the reaction significantly.
Even after optimization of the iCLIP protocol, the data produced in the Oskar iCLIP were inconclusive, due at least in part to the low affinity of Oskar for RNA. Hence, positional information regarding Oskar’s interaction with specific RNAs in vivo is still lacking. Such information would enable validation and further characterization of the RNA-binding activity of Oskar, providing important new insight into the molecular mechanisms underlying Oskar’s unique pole plasm inducing activity.
The optimized iCLIP protocol I developed should be applicable to other RNA-binding proteins in Drosophila and in other model organisms.
date: 2018
id_scheme: DOI
id_number: 10.11588/heidok.00024701
ppn_swb: 1657590380
own_urn: urn:nbn:de:bsz:16-heidok-247010
date_accepted: 2017-11-21
advisor: HASH(0x55a9a64f5a88)
language: eng
bibsort: BORDIMATTEINVESTIGAT2018
full_text_status: public
citation:   Bordi, Matteo  (2018) Investigating the RNA-binding activity of the Drosophila melanogaster germline inducer Oskar.  [Dissertation]     
document_url: https://archiv.ub.uni-heidelberg.de/volltextserver/24701/1/Matteo%20Bordi%20PhD%20Thesis.pdf