![]() ![]() The optimized iCLIP protocol I developed should be applicable to other RNA-binding proteins in Drosophila and in other model organisms. 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. Hence, positional information regarding Oskar’s interaction with specific RNAs in vivo is still lacking. ![]() By performing a series of optimization experiments, I established new reaction conditions for adapter ligation that increased the efficiency of the reaction significantly.Įven 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. 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. To this end, I analyzed the efficiency of three key steps of the iCLIP protocol: RNAse digestion, 3’ end dephosphorylation, and adapter ligation. In order to identify Oskar’s binding site(s) on its target transcripts I applied the iCLIP method to early Drosophila embryos. 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. Instead of utilizing circular ligation, the eCLIP protocol uses ligation of two separate adapters (an indexed 3 RNA adapter that is ligated to the crosslinked RNA fragment while on immunoprecipitation beads and and 3 ssDNA adapter that is ligated after reverse transcription) added in two separate steps. Recently, it was shown that Oskar binds polyadenylated mRNAs in vivo and that the C-terminal domain of the protein binds RNA in vitro. 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. oskar mRNA transcribed in the nuclei of nurse cells is actively transported into, and to the posterior pole of, the oocyte. 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. 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. Citation of documents: Please do not cite the URL that is displayed in your browser location input, instead use the DOI, URN or the persistent URL below, as we can guarantee their long-time accessibility. ![]()
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