Posttransscriptional regulation of the nanos mRNA in Drosophila melanogaster

Deadenylation is the first and often rate-limiting step in mRNA decay. Deadenylation is also used as a means of translational repression. Translation of the nanos (nos) mRNA of Drosophila is repressed by the protein Smaug. Smaug also induces deadenylation by the CCR4-NOT complex. We have reproduced both deadenylation and translational repression in extracts of Drosophila embryos. The deadenylation activity, which is associated with rapidly sedimenting particles, depends on the Smaug binding sites in the RNA and on an ATP-regenerating system. Micrococcal nuclease inhibits the reaction. The proposal aims to identify the factors responsible for the reaction by an affinity purification approach with the goal of analyzing the mechanism of accelerated deadenylation. Deadenylation contributes to translational repression of nos, but a second, deadenylation-independent mechanism is also operating. We will test the model that repression depends on Smaug-dependent recruitment of the protein Cup, which then displaces the initiation factor eIF4G from the cap-binding protein eIF4E. We will also examine whether the repressed nos mRNA is in fact on polysomes, as previously reported, or whether it forms non-polysomal repressed RNPs. We will examine which role the factors identified by affinity-purification play in translational repression.