Regulation of mRNA translation and stability plays a key role in early development of many organisms and is an essential aspect of gene regulation in germ cells of animals from nematodes to humans. This proposal focuses on understanding the regulation of mRNAs in Drosophila, a leading model system for the study of translational control. Genetic studies have implicated Drosophila Nanos protein in many different aspects of germ cell biology, neural function, and establishment of the embryonic body plan. Its mechanism of action is best understood during body patterning, where it is recruited by Pumilio protein to specific elements in the 3'-UTR of hunchback mRNA;the resulting repressor complex recruits an additional factor and then regulates translation and stability of hunchback mRNA. A similar Nanos Response Element (NRE) is found in the 3'-UTR of bicoid mRNA, but the reason for its conservation is unclear, since bicoid mRNA is not exposed to meaningful levels of Nanos in wild type embryos. Our experiments suggest that the NRE may mediate regulation of bcd mRNA during post- embryonic development, a hypothesis that will be tested in this proposal. Although it is essential for body patterning in flies, the evolutionarily conserved roles of Nanos are in the germ line, where it regulates a number of biological processes. A major focus of this proposal is to understand how Nanos controls two essential events in Drosophila germ cells. Shortly after their birth, Nanos blocks the proliferation of primordial germ cells. We have shown that quiescence is imposed (in part) via Nanos-dependent recruitment of the CCR4 deadenylase to the 3'-UTR of CyclinB, thereby inhibiting translation of the mRNA. In addition to Nanos, we found that other factors present only in the germ cells are required for repression. We will identify these co-repressors to understand the mechanism by which Nanos targets specific mRNAs for regulation in the germ line. Genetic studies have shown that Nanos is required for maintenance of the self-renewing fate of germ line stem cells in flies and mice (and likely in humans too, since Nanos orthologs are expressed specifically in testes). Very few Nanos regulatory targets have been identified, and none that are implicated in germ line stem cell biology. To identify Nanos targets, we have developed a tool that specifically degrades Nanos-bound mRNAs when expressed in flies-- a fusion of the NMD factor Upf1 to Nanos. To define Nanos regulatory targets, we will measure mRNA abundance using microarrays and identify mRNAs depleted by Upf1-Nanos.
The process of turning mRNA into proteins is critical for human germ cells, which give rise to sperm and eggs, and for the normal function of neurons. Because closely related molecules control these events in humans and other organisms, we study the easily manipulated fruit fly to learn how mRNAs are regulated. Our work will have an impact on understanding events that contribute to infertility and neural diseases, as well as shedding light on the properties of germ line stem cells.
