Mechanistic analysis of nanos mRNA translational regulation

Nanos is critical for patterning the posterior half of the embryo during early development. Loss of nanos leads to embryos that fail to develop abdominal segments, while overexpression or mislocalization of nanos leads to embryos that develop additional abdominal fates at the expense of anterior fates. A gradient of Nanos protein is synthesized from maternally deposited nanos mRNA that is localized to the posterior of the embryo, and localization of nanos is necessary to activate its translation and produce this gradient. However, only 4% of the total pool of nanos mRNA is actually localized and it is essential that the unlocalized mRNA be translationally repressed for proper development to occur. Translational repression of nanos is conferred by the translational control element in the nanos 3'UTR. One key component of the embryonic translational repression machinery, the protein Smaug, has been identified, and recently a second protein, Glorund, has been shown to repress nanos translation during oogenesis. Smaug has been proposed to repress nanos by blocking translational initiation. The mechanism by which Glorund acts to repress translation in the ovary is not known. Polysome sedimentation gradients have shown that translationally repressed nanos RNA is associated with polysomes, suggesting that at least some aspects of translational control may occur post-initiation.;To determine whether repression acts at initiation, post-initiation, or both, we have developed a Drosophila ovarian extract that faithfully recapitulates nanos translational repression and have used this extract to further characterize the mode of repression in both the embryo and the ovary. Our results indicate that while translational repression of nanos mRNA in the embryo extract occurs entirely at translational initiation, translational repression in the ovarian extract operates by two modes of repression. One of these acts at initiation and has a strong dependence on the presence of a poly(A) tail, while the other acts post-initiation in a cap-independent manner.;In a second approach to studying translational control of nanos , we have examined the regulation of nanos at the neuromuscular junction. During embryonic development in Drosophila, Nanos acts together with the RNA-binding protein Pumilio to direct posterior patterning. Nanos and Pumilio also function to regulate synaptic growth and plasticity at the neuromuscular junction, but in that context appear to act in opposition to each other. Postsynaptic Pumilio negatively regulates the expression of several proteins, including eIF4E, the Glutamate RIIA receptor subunit, and Nanos itself. In contrast, Nanos positively regulates Glutamate RIIA translation. We have examined whether the observed regulation of Glutamate RIIA and nanos by Pumilio could result from a direct interaction using a series of gel mobility shift assays. Our results demonstrate that Pumilio protein binds selectively and directly to the Glutamate RIIA 3'UTR as well as to the nanos 3'UTR, and identifies a novel binding target for Pumilio.