Role of Bruno phosphorylation in translational regulation of oskar. Goheun Kim¹, Keiji Sato², Akira Nakamura², Paul Macdonald¹. 1) Molecular Cell & Developmental Biology, University of Texas at Austin, Austin, TX; 2) Laboratory for Germline Development, RIKEN Center for Developmental Biology, Kobe, Japan.

   Oskar (Osk) is a posterior body patterning determinant in Drosophila and is highly concentrated at the posterior pole of the oocyte. Tight spatial and temporal restriction of the Osk patterning activity is essential for proper development of the embryo. Bruno (Bru) directly binds to the osk mRNA and represses translation during mRNA localization to the posterior pole. After osk mRNA localization, repression must be alleviated to allow accumulation of Osk protein. In one model for repression, Bru bound to osk mRNA recruits Cup, which in turn binds eIF4E and prevents its interaction with eIF4G. In another model, Bru promotes oligomerization of multiple osk mRNAs into large particles that are inaccessible to the translational machinery. The interactions of Bru with RNA and proteins must underlie its repressive activity, and may be disrupted for release from repression. We show that Bru dimerizes, and have investigated interaction of Bru with itself and with Cup. We also show that Bru is phosphorylated, and have investigated how this modification affects the binding activities of Bru. Using GST pull-down assays we show that two domains of Bru, aa1-146 and aa334-416, contribute to both interactions. Deletion of the N-terminal domain dramatically reduces binding, and the isolated N-terminal domain binds both Bru and Cup. A small fraction of Bru in ovaries is phosphorylated. Several predicted sites of phosphorylation by PKA in Bru lie within the N-terminal domain. These could be targets for inhibition of protein interactions, and thereby mediate inactivation of repression. Phospho-mimetic mutations in these sites interfere with Bru binding to both itself and Cup, while corresponding phospho-silent mutations have no effect. The same phospho-silent mutations interfere with in vitro phosphorylation of Bru by purified PKA. We propose that local phosphorylation of Bru by PKA at the posterior of the oocyte alleviates Bru-dependent translational repression of osk.