Mechanism of grk mRNA anchoring during Drosophila oogenesis

Abstract

Messenger RNA localization is a widespread mechanism of posttranscriptional regulation of gene expression in multicellular organisms ranging from yeast to mammals. In Drosophila oocytes, gurken (grk) mRNA is transported by Dynein to produce a local secreted signal to the overlying follicle cells. This signal is responsible for setting up the primary axes in the oocyte. grk mRNA is transcribed in nurse cells and transported into the oocyte where it localizes at two distinct stages of oogenesis, thus targeting the translation of Grk/TGFalpha protein, first to the posterior and later to the dorso-anterior (DA) corner where it is translated. Gurken protein signals to the overlying follicle cells to establish the dorsal fate of the oocyte. grk transcripts are transported by Dynein in EM-dense particles on microtubules. These particles are not associated with vesicles nor membrane-bound and contain many copies of grk mRNA, Dynein and hnRNP Squid. At the DA corner transport particles assemble into large EM-dense cytoplasmic anchoring complexes called Sponge Bodies. In this thesis I present evidence that at their dorso-anterior destination, grk transcripts are statically anchored by Dynein, independently of functional Egalitarian and Bicaudal D, which are required for Dynein transport. I show by the disrupting the protein’s function after it has fulfilled its role in transport that hnRNP Squid is involved in the formation and maintenance of these Sponge Bodies. I provide evidence by EM and fluorescent microscopy that Sponge Bodies share many of components of translational regulation pathways found in Processing Bodies. I show by small RNA interference experiments and by genetic analysis that the structural role of Dynein heavy chain is a unique feature of the Sponge Bodies and that such a function does not occur in Processing Bodies in Drosophila. I show that the localization and anchoring of RNA in Sponge Bodies is not a unique feature of grk mRNA but that I factor RNA is also localized to Sponge bodies. The work presented tries to elucidate the function of Sponge Bodies in translational control of grk mRNA and illustrates by EM the dynamic nature of the Sponge Body structure during oogenesis. My results suggest that Sponge Bodies are RNA granules that are similar to Processing Bodies in a way that they are involved in translational regulation but unlike Processing Bodies depend on Dynein for their structural integrity. I propose that Sponge Bodies are RNA dependent granules that form by the recruitment of proteins involved in the anchoring and translational regulation.