Functional characterization of the role of Bruno protein in translational regulation and germ line development in Drosophila melanogaster
Both body axes of the Drosophila egg are determined by localization of several mRNAs to specific regions within the oocyte. One of these mRNAs, oskar (osk), is required for posterior body patterning. Localization and translational control are both crucial for the correct deployment of osk. Bruno (Bru) binds specifically to the 3’UTR of the osk mRNA and represses osk translation. In this dissertation, I first describe a genetic screen looking for dominant modifiers of the arrest (aret) mutant phenotype (aret encodes Bru). Two modifiers suggested additional targets for Bru action. One is Star, a gene that contributes to provision of Gurken activity. The second suggested target is a gene acting in the Delta signaling pathway. A final modifier, Lk6, encodes a protein kinase predicted to regulate eIF4E. I also took a biochemical approach trying to understand how Bru regulates osk translation. Bru protein contains three RNA Recognition Motifs, but the remainder of the protein had no known function. I identified a domain, which is required for interaction to Bru itself, Cup and Apontic. Subsequent analysis of mutant forms of Bruno defective in these interactions led us to an unexpected discovery that Bru also acts as an activator of osk translation. Parallel analysis of Bru binding sites in osk 3’UTR fully support the notion that Bru has a dual role. There are two clusters of Bru Recognition Elements in either end of osk 3’UTR. Point mutations in one cluster cause overproduction of Osk protein while point mutations in the other cluster largely prevent translation of the message. To understand the molecular basis of the opposing roles of Bru, I used quantitative methods to better define and compare the binding of Bru to the different regulatory elements: those that either repress or activate osk mRNA translation. Using purified components I found that Bru binds to two clusters of binding sites in the osk 3’UTR differently, in terms of affinity, cooperativity and apparent compaction of the RNA. This work raises the possibility that the details of how Bru binds its substrate may determine whether it acts as a repressor or an activator.