The long term objective of the proposed research is to understand the basic mechanisms of messenger RNA (mRNA) localization, a phenomenon that occurs in polarized cells from a variety of animals, as well as plants and yeast, and contributes to diverse processes ranging from intercellular signaling to synaptic function and plasticity. The example studied here, localization of bicoid (bcd) mRNA in the Drosophila oocyte and embryo, is essential for embryonic body patterning. ? ? The goals of the proposal are to understand how bcd mRNA is recognized by the localization machinery, and how recognition is integrated with the movement and anchoring steps of mRNA localization. Recognition appears to involve a protein complex that binds specifically to a minimal form of the localization signal. Coupling of RNA recognition with RNA movement and/or anchoring is suggested by the presence in the complex of Swallow, a protein that is required for one or both of these steps of localization. We propose to complete the identification of the components of the complex, of which there are about 10 proteins (Aim I). Most or all of the proteins will be tested genetically for a role in mRNA localization, using a sensitive RNA injection assay that has important advantages over the classical in situ hybridization assay. The nature of the defects, as well as the distributions of the proteins from the complex, detected in fixed tissues and live ovaries, will suggest likely roles for individual proteins: in RNA recognition, movement or anchoring (Aim II). To better understand the recognition process, and to test our hypothesis that recognition of the bcd mRNA localization signal relies on the sum of many low affinity protein:RNA interactions, and not a single high affinity interaction (as is typical of the protein: nucleic acid interactions that underlie most forms of genetic regulation), we will determine which proteins from the complex is required for specific binding (Aim III). To gain insights into the integration of RNA recognition with movements and anchoring, we will determine how Swallow is associated with the complex components that mediate RNA binding (Aim IV). Finally, as a complement to the studies on the trans-acting factors, we will use the RNA injection assay to extend our analysis of the cis-acting localization signal, with an emphasis on understanding the contributions of different sequences to the different stages of localization. ? ?
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