The formation of the embryonic axes is in Drosophila controlled by the localization of determinants in form of mRNAs within the oocyte. The formation of anterior-posterior polarity is secondarily regulated by translational control of these mRNAs. Dr. Macdonald and his group work on localization and translation. The anterior morphogen of the Drosophila embryos is bicoid (bcd) and the posterior patterning is controlled by oskar (osk) and nanos (nos). All three mRNAs are localized during oogenesis and are only translated after this localization occurs. The translational control has at least two separate components. Translation is repressed in the oocyte, and only when the mRNA is localized is it actively translated, while the repression of unlocalized RNA is maintained. This proposal's aim is to study the translational control of osk. Dr. Macdonald identified elements in the 3'UTR of osk RNA that are essential for translational repression several years ago. He then identified a protein, Bruno (Bru), binding to these elements. In mutants in arrest (aret), encoding Bru, oogenesis stops before the translational control of osk occurs. However, Macdonald has developed an in vitro translation system using ovary extracts, and has studied the function of Bruno in this system. After demonstrating that the in vitro system reproduces in vivo results, he has shown that the length of the polyA tail does not influence Bru regulated translation. He has also established that the 5'cap probably does not regulate Bru function either. In a two-hybrid screen looking for Bru interactors several proteins were identified. One of these is Apontic (Apt) that may function redundantly with another Bru interactor p50. Another interactor is a translation initiation factor elF-5. A third interactor is squid, which functions in the localization of the gurken RNA. These results as well as the lab's biochemical results are consistent with the existence of a protein complex containing RNA localization and translational control proteins. Aubergine Aub has been identified as a translational activator of osk. Macdonald has cloned the gene and found that it encodes an elf-2C translation initiation factor conserved in all eukaryotes. Dr. Macdonald has also identified sequences (SRE) in the 3' UTR of nos essential in the regulation of nos translation. Using a biochemical approach he has identified a protein, Smaug (Smg), binding to the SREs, and has studied its function in the in vitro translation system. The proposal is to continue with this general approach.
The first aim i s a structure function analysis in vitro. Deleted Bru and then point mutants will be used to map RNA binding, translational repression, and specific protein- interaction domains. One hypothesis of how Bru functions in translational repression is that it competes for binding elF-5 with the translation machinery. This possibility will be tested.
Aim two is to follow up the in vitro experiments by confirming them through in vivo experiments in the fly.
Aim three is to isolate mutants in the elF-5 gene.
Aim four is the biochemical isolation of a complex containing Bru and functions in translational repression. These experiments are already underway.
Aim five is to identify known proteins in the complex by western blots. Peptides from unknown protein bands will be sequenced and the corresponding genes cloned. The co-localization of the proteins identified as part of the complex will be analyzed in various ways, in live ovaries as well as fixed material. The sixth and last aim is to characterize Aub activity, in particular, to determine if it functions like elF2C.
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