Opposing roles for Egalitarian and Staufen in transport, anchoring and localization of oskar mRNA in the Drosophila oocyte

Localization of oskar mRNA includes two distinct phases: transport from nurse cells to the oocyte, a process typically accompanied by cortical anchoring in the oocyte, followed by posterior localization within the oocyte. Signals within the oskar 3' UTR directing transport are individually weak, a feature previously hypothesized to facilitate exchange between the different localization machineries. We show that alteration of the SL2a stem-loop structure containing the oskar transport and anchoring signal (TAS) removes an inhibitory effect such that in vitro binding by the RNA transport factor, Egalitarian, is elevated as is in vivo transport from the nurse cells into the oocyte. Cortical anchoring within the oocyte is also enhanced, interfering with posterior localization. We also show that mutation of Staufen recognized structures (SRSs), predicted binding sites for Staufen, disrupts posterior localization of oskar mRNA just as in staufen mutants. Two SRSs in SL2a, one overlapping the Egalitarian binding site, are inferred to mediate Staufen-dependent inhibition of TAS anchoring activity, thereby promoting posterior localization. The other three SRSs in the oskar 3' UTR are also required for posterior localization, including two located distant from any known transport signal. Staufen, thus, plays multiple roles in localization of oskar mRNA. Author summary Within cells, many components must be enriched at specific subcellular locations. One mechanism to achieve the nonrandom distribution of a protein is localization of its cognate mRNA. When localization of an mRNA involves more than one step, the mRNA must be released from the machinery directing the first step to allow the machinery for the next step to perform its function. For the Drosophila oskar mRNA the first step is transport of the mRNA from its site of synthesis to the oocyte, where the next step is localization of the mRNA to the posterior pole of the oocyte. Prior studies suggested that the multiple signals in the oskar mRNA that mediate transport were individually weak by necessity, to facilitate action by the posterior localization machinery. Here, we describe a mutant version of the oskar mRNA whose behavior suggests an overly tenacious association with the transport machinery, leading to failure in posterior localization. Biochemical studies show that the mutant signal has enhanced binding to the Egalitarian RNA binding protein, which mediates transport. Genetic studies show that the Staufen RNA binding protein has an opposing role, facilitating dissociation from the transport machinery to enable posterior localization.