This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. mRNA localization plays a pivotal role in the establishment of polarity in the Drosophila egg and embryo. It typically begins with the transcription and packaging of the localized mRNAs in RNP complexes in nurse cell nuclei. These complexes 'mask'the mRNAs, preventing them from being translated while in transit, and 'mark'them for localization. The masked mRNAs are transported through the nurse cells and deposited in the oocyte. Depending upon their cis-acting localization elements, the mRNAs are then targeted to specific locations in the oocyte where they are anchored to the cytosketal network. Translational activation of the localized message provides a spatially restricted source of the protein product. Alternatively the mRNAs can be stored in a masked form until an appropriate signal, such as egg deposition, activates translation. This provides a mechanism for coordinating the localized synthesis of the protein with other developmental events. While the importance of mRNA localization (coupled with 'on site'translational regulation) was first documented in fly ovaries and early embryos, it is now clear that this regulatory mechanism is employed in many different contexts and occurs in virtually every eukaryote. For example, in yeast, the asymmetric segregation of ash-1 mRNA to the daughter cell provides a mechanism for ensuring that mating type switching does not occur in that cell. mRNA localization in somatic cells also occurs during the processes of movement, differentiation or growth. In neuronal cells, mRNAs encoding MAP2, a dendrite-specific microtubule-associated protein, and aCaMKII accumulate in the dendrites, but not in the axons. Localized mRNA translation has also been implicated in synaptic plasticity and learning and memory. In all of these cases, it is likely that the steps involved in mRNA localization and translational regulation are similar in broad outline to those described above in fly ovaries The proposed studies focus on the Drosophila orb gene which is one of the key components of the fly ovarian mRNA localization machinery. Orb is a founding member of the highly conserved CPEB family of RRM RNA binding proteins. In previous studies, we have shown that orb functions at the last step in the mRNA localization process, regulating the translation of the mRNA once it is on site. Orb binds to the 3? UTR of many localized mRNAs in vivo oskar (osk) Bicudal-D (Bic-D), and K(10) and is required to promote the translation of these mRNAs. In addition, Orb protein autoregulates its own expression by binding to the 3? UTR of localized orb mRNA and activating translation. This positive feedback loop is critical for the proper expression of Orb in the developing oocyte. The goal of our studies is to better understand how Orb autoregulates its own expression and how it controls the on site translation of mRNAs encoding factors critical for establishing oocyte/nurse cell identity and defining the polarity of the egg chamber and embryo. Part of the answer to these questions will come from identifying and characterizing other genes that are important for Orb autoregulation and/or function. For this purpose we propose to isolate proteins that are associated with Orb in vivo and identify them by Mass Spec analysis. To validate these biochemical studies we have devised a sensitized orb mutant background. The sensitized background will permit us to test mutations in candidate genes which encode proteins that are associated Orb in vivo for genetic interactions with Orb.
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