Mechanisms of Smaug-mediated Post-transcriptional Regulation in the Early Drosophila Embryo

Post-transcriptional regulation of gene expression can be mediated by cis acting elements within mRNAs that are recognized by RNA binding proteins that control mRNA expression. Smaug is a highly conserved sequence specific RNA-binding protein that is essential for early Drosophila development. Smaug binds to well defined cis elements in target mRNAs and uses protein-protein interactions to recruit additional factors that regulate the bound mRNA. In this thesis I will describe my efforts to identify novel Smaug binding proteins to better understand the mechanisms of Smaug-mediated control. My efforts identified several Smaug binding partners including RNA helicases CG7878 and CG10077; Not1, a component of the CCR4/NOT deadenylase; and the miRISC component Argonaute 1. My work and the work of others suggests that CG7878 along with other RNA helicases could function with Smaug to regulate target transcripts. My identification of the Smaug/Not1 interaction was the basis for the discovery that Smaug induces transcript decay through recruitment of the CCR4/Not deadenylase complex. Finally, I employed both in vitro and in vivo experiments to show that Smaug can directly recruit Argonaute 1 to a target mRNA to repress its translation and that this recruitment does not require a miRNA. Argonaute 1’s interaction with the target mRNA requires the Dop kinase suggesting that phosphorylation by Dop of one or more proteins is required for Smaug-mediated recruitment of Argonaute 1.Ph