Vorschau

PDF, Englisch (Matteo Bordi PhD Thesis) - Hauptdokument Download (4MB) | Nutzungsbedingungen

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.