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Abstract

Asymmetric localization of specific RNAs is essential in early embryo development. During Drosophila melanogaster oogenesis, oskar mRNA is transcribed in nurse cells, transported to the transcriptional silent oocyte and subsequently localized at the posterior pole. The first transport step from the nurse cells into the oocyte is mediated by dynein transport, whereas the second step to the posterior pole is dependent on kinesin. At the posterior pole, oskar mRNA is required for the formation of germ plasm and abdominal segments.

The transport of oskar mRNA by kinesin requires the exon junction complex (EJC) core components eIF4AIII, Mago nashi, Y14 and Barentsz as well as the spliced oskar localization element (SOLE). The exact mechanism by which the EJC and SOLE are involved in kinesin transport has remained unclear. I utilized RNA-protein interaction footprinting to analyze how the protein occupancy on the RNA changes upon disrupting the SOLE secondary structure. Through RNA immunoprecipitation against Mago nashi, I discovered that the presence of the SOLE led to a more stable binding of the EJC to oskar mRNA. This demonstrates that the SOLE has a stabilizing effect on EJC binding to oskar mRNA. Although the EJC and SOLE were found to be dispensable for kinesin recruitment to oskar mRNA, they were essential for oskar mRNA transport, suggesting that the EJC-SOLE interactome plays a role in activating kinesin transport to the posterior pole. I optimized a previously described transcript-specific RNA binding protein (RBP) capture protocol and utilized it to analyze the EJC-SOLE interactome. Flies expressing transgenic lacZ RNA containing the oskar 3’UTR and either wild-type or mutant SOLE were created and used to determine the difference in RBP composition upon disrupting the SOLE secondary structure. I identified RBPs that were specifically bound to the transgenic lacZ RNA with the wild-type SOLE, which localizes to the posterior pole and RBPs bound to the lacZ RNA with a disrupted SOLE, which does not localize. Further investigations will be carried out to explore which of the RBPs are involved in the kinesin-mediated transport of oskar mRNA.