Localization-dependent translation requires a functional interaction between the 5' and 3' ends of oskar mRNA.

The precise restriction of proteins to specific domains within a cell plays an important role in early development and differentiation. An efficient way to localize and concentrate proteins is by localization of mRNA in a translationally repressed state, followed by activation of translation when the mRNA reaches its destination. A central issue is how localized mRNAs are derepressed. In this study we demonstrate that, when oskar mRNA reaches the posterior pole of the Drosophila oocyte, its translation is derepressed by an active process that requires a specific element in the 5' region of the mRNA. We demonstrate that this novel type of element is a translational derepressor element, whose functional interaction with the previously identified repressor region in the oskar 3' UTR is required for activation of oskar mRNA translation at the posterior pole. The derepressor element only functions at the posterior pole, suggesting that a locally restricted interaction between trans-acting factors and the derepressor element may be the link between mRNA localization and translational activation. We also show specific interaction of two proteins with the oskar mRNA 5' region; one of these also recognizes the 3' repressor element. We discuss the possible involvement of these factors as well as known genes in the process of localization-dependent translation.

Oskar protein interaction with Vasa represents an essential step in polar granule assembly.

The posterior pole plasm of the Drosophila egg contains the determinants of abdominal and germ-cell fates of the embryo. Pole plasm assembly is induced by oskar RNA localized to the posterior pole of the oocyte. Genetics has revealed three additional genes, staufen, vasa, and tudor, that are also essential for pole plasm formation. Staufen protein is required for both oskar RNA localization and translation. Vasa and Tudor are localized dependent on Oskar protein and are required to accumulate Oskar protein stably at the posterior pole. We have explored interactions between these gene products at the molecular level and find that Oskar interacts directly with Vasa and Staufen, in a yeast two-hybrid assay. These interactions also occur in vitro and are affected by mutations in Oskar that abolish pole plasm formation in vivo. Finally, we show that in the pole plasm, Oskar protein, like Vasa and Tudor, is a component of polar granules, the germ-line-specific RNP structures. These results suggest that the Oskar-Vasa interaction constitutes an initial step in polar granule assembly. In addition, we discuss the possible biological role of the Oskar-Staufen interaction.

Translational control of oskar generates short OSK, the isoform that induces pole plasma assembly.

At the posterior pole of the Drosophila oocyte, oskar induces a tightly localized assembly of pole plasm. This spatial restriction of oskar activity has been thought to be achieved by the localization of oskar mRNA, since mislocalization of the RNA to the anterior induces anterior pole plasm. However, ectopic pole plasm does not form in mutant ovaries where oskar mRNA is not localized, suggesting that the unlocalized mRNA is inactive. As a first step towards understanding how oskar activity is restricted to the posterior pole, we analyzed oskar translation in wild type and mutants. We show that the targeting of oskar activity to the posterior pole involves two steps of spatial restriction, cytoskeleton-dependent localization of the mRNA and localization-dependent translation. Furthermore, our experiments demonstrate that two isoforms of Oskar protein are produced by alternative start codon usage. The short isoform, which is translated from the second in-frame AUG of the mRNA, has full oskar activity. Finally, we show that when oskar RNA is localized, accumulation of Oskar protein requires the functions of vasa and tudor, as well as oskar itself, suggesting a positive feedback mechanism in the induction of pole plasm by oskar.

Nuclear and mitochondrial forms of human uracil-DNA glycosylase are encoded by the same gene.

Recent cloning of a cDNA (UNG15) encoding human uracil-DNA glycosylase (UDG), indicated that the gene product of M(r) = 33,800 contains an N-terminal sequence of 77 amino acids not present in the presumed mature form of M(r) = 25,800. This led to the hypothesis that the N-terminal sequence might be involved in intracellular targeting. To examine this hypothesis, we analysed UDG from nuclei, mitochondria and cytosol by western blotting and high resolution gel filtration. An antibody that recognises a sequence in the mature form of the UNG protein detected all three forms, indicating that they are products of the same gene. The nuclear and mitochondrial form had an apparent M(r) = 27,500 and the cytosolic form an apparent M(r) = 38,000 by western blotting. Gel filtration gave essentially similar estimates. An antibody with specificity towards the presequence recognised the cytosolic form of M(r) = 38,000 only, indicating that the difference in size is due to the presequence. Immunofluorescence studies of HeLa cells clearly demonstrated that the major part of the UDG activity was localised in the nuclei. Transfection experiments with plasmids carrying full-length UNG15 cDNA or a truncated form of UNG15 encoding the presumed mature UNG protein demonstrated that the UNG presequence mediated sorting to the mitochondria, whereas UNG lacking the presequence was translocated to the nuclei. We conclude that the same gene encodes nuclear and mitochondrial uracil-DNA glycosylase and that the signals for mitochondrial translocation resides in the presequence, whereas signals for nuclear import are within the mature protein.