Expression of proteins in spatially restricted patterns frequently relies on prior localization of the encoding mRNAs. One conserved RNA transport mechanism, used in multiple cell types, involves a stem-loop transport signal within the mRNA, and proteins (Egalitarian and Bicaudal-D) to bind the signal and link the RNA cargo with the dynein motor for transit along microtubules. This transport system also anchors the mRNAs at their destination, although anchoring is poorly understood. The initial step in localization of Drosophila oskar mRNA - transport from the nurse cells to the oocyte - relies on this conserved transport system. However, oskar mRNA differs from simple examples of this system, in that the mRNA must then be transferred to a different localization machinery for a later step in localization. Notably, the cis-acting localization signals in oskar mRNA that direct the initial transport step are individually weak, even though transport is robust. Replacing the multiple weak signals with a single strong signal disrupts the later step in localization. This suggests that weak association with the machinery for the initial transport step allows the mRNA to be handed off to the other type of machinery. A model is proposed to explain what makes the oskar transport signals weak, and how the conflicting requirements for weak signals yet highly efficient transport can both be met. In part, this model relies on Staufen protein to inhibit association of the oocyte transport machinery with the oskar oocyte transport signals. Staufen is more conventionally thought to act in the later step of oskar mRNA localization, although the staufen mutant phenotype is equally consistent with both models.
One Aim i s to confirm, using an affinity purification approach, that oskar relies on the conserved transport system for its initial step of localization and to reveal candidates for other contributing factors.
The second Aim i s to test two predictions of what makes the individual transport signals weak: modest affinity for the recognition factor, Egalitarian; and displacement of Egalitarian by Staufen after the mRNA arrives in the oocyte.
The third Aim i s to ask if the contribution of Staufen to localization of oskar mRNA is limited to the novel role postulated here, or if Staufen also has the conventional role, or both. The novel role for Staufen could explain how it contributes to a wide variety of forms of post-transcriptional regulation, a possibility that will be tested.
A final Aim i s to exploit unique features of this system in a sensitized genetic assay to evaluate candidate transport/anchoring factors, and to screen for such factors. Further analysis of these factors should provide substantial insights into many aspects of mRNA localization and anchoring.
Localization of mRNAs is a strategy widely used to achieve local protein expression. Often, an mRNA must be transferred between different molecular motors or localization machineries to reach its final destination, but how this occurs is unknown. This proposal aims to exploit unique features of a well-developed experimental system to test a specific model for such a transfer.
