PROJECT SUMMARY Our long-term research goal is to understand post-transcriptional mechanisms that control gene activity during early animal development. We focus on intracellular mRNA localization and translational control, which play crucial roles in regulating the production of proteins from maternally supplied transcripts. Because these transcripts, which control the initial developmental program of nearly all animals, are pre-loaded in the egg, the spatial and temporal expression of the proteins they encode must be exerted post-transcriptionally. In animals as diverse as flies and frogs, mRNA localization and local control of translation produce asymmetric protein distributions required for axis formation, patterning, and germline development. Often many different transcripts must be localized concurrently to various subcellular locations. Additionally, translational control must be superimposed to repress unlocalized transcripts and activate properly localized transcripts. How specificity is conferred on these processes, so that each transcript is targeted to its correct destination and translated appropriately, is poorly understood. Our research has capitalized on the Drosophila egg, which relies heavily on maternal transcripts, to investigate mechanisms of mRNA localization and its coupling to translational control. Our early studies focusing on nanos mRNA led to the discovery of a diffusion-and-entrapment mechanism used by numerous transcripts for localization to the specialized germ plasm at the posterior of the oocyte. Produced by the ovarian nurse cells and then transferred to the oocyte, these transcripts are co- packaged at the posterior end into ribonucleoprotein complexes (RNPs) called germ granules. Later during embryogenesis, germ granule mRNAs are segregated as a cohort to the primordial germ cells, where they are required for germline development. Despite their shared dependence on germ granule localization tor translational activation, different transcripts have distinct temporal demands. Our recent studies have led to a stepwise model for germ granule assembly that provides a framework for understanding the composition, structure, and translational properties of RNPs and their functions. Determining the specific roles of shared and RNA-specific proteins in controlling RNP assembly and translation will, in turn, be fundamental to a deeper understanding of mRNA localization as a mechanism for generating protein ? and consequently cellular ? asymmetries. To elucidate how localized assembly and function of complex RNA granules is controlled, we will take advantage of quantitative high resolution imaging, in vivo fluorescent RNA labeling, and new biochemical strategies to identify cis-acting regulatory elements and interacting proteins that mediate both individualistic and coordinate RNA behaviors. Ribosome footprinting, a genome-level approach for monitoring translation, will be employed to investigate mechanisms that impose translational arrest on unlocalized transcripts. Finally, we will use high resolution imaging of protein synthesis in vivo to decipher the relationship between germ granule association and translational activity.