The subcellular localization of messenger RNAs is a widespread mechanism for localizing cytoplasmic proteins in cells. This proposal addresses the mechanism by which localized mRNAs are anchored in a specific subcellular region, a process that probably involves the attachment of RNA-protein complexes to the cytoskeleton. In the model system under consideration in this proposal, two mRNAs, bicoid and htsN4 are each localized at the anterior margin of Drosophila oocytes. It has been proposed that the swallow protein acts to link the mRNAs to the microtubule cytoskeleton, perhaps through the microtubule motor molecule dynein. The experiments proposed here will investigate several features of this model: Hypothesis 1: The dynein motor complex plays an essential role in anchoring bicoid and htsN4 mRNAs at the anterior oocyte margin. This idea will be tested by determining whether dynein mutants affect bicoid/htsN4 mRNA localization. Hypothesis 2: The swallow protein is associated with the dynein motor complex and mediates RNA localization through this association. This idea will be tested by determining the subcellular localization and co-localization of the swallow protein, determining the effects of dynein mutants on swa protein localization, and by identifying and mapping interacting domains in proteins that associate with swallow. Hypothesis 3: The effects of swallow mutations on cytoskeletal organization are indirect, caused by the failure to localize htsN4 mRNA and the adducin-like protein it encodes. Swallow mutant embryos have defects in the cytoskeleton that have confused past attempts to understand the role of swallow in development. Here we test the idea that htsN4 mRNA and protein mislocalization are sufficient to account for the observed cytoskeletal defects, and that swa only has an indirect role in cytoskeletal organization.
