The localization of specific mRNAs plays a critical role in cell-fate determination during development and synaptic signaling in neurons. Many localized mRNAs encode proteins that trigger the development of specific body plans or regulate the strength of synaptic connections between neurons involved in memory and higher cognitive function. The intracellular transport of mRNAs by cytoskeleton-based motor proteins is an effective mechanism to localize mRNAs within cells. Although several motor proteins have been implicated in mRNA transport, it is currently unknown how motor proteins bind to specific mRNAs and then rapidly transport those mRNAs over significant distances. To address these questions, this proposal will analyze how Myo4p transports ASH1 mRNA to the bud tip in S. cerevisiae. The interaction between Myo4p and ASH1 mRNA requires 2 proteins: She2p and She3p. She2p and She3p directly associate with specific sequences in ASH1 mRNA and provide a molecular link between Myo4p and ASH1 mRNA. The specific aims of the proposal are: (1) To purify an RNA-Myo4p complex that moves along actin filaments and identify proteins that are required to bind Myo4p to ASH1 mRNA and facilitate transport of ASH1 mRNA. The methods developed to purify specific RNAs in S. cerevisiae will serve as footholds for purifying localized mRNAs from higher eukaryotic cells. (2) Perform an extensive mutational analysis of She2p and She3p to identify the molecular interactions that allow She2p and She3p to differentiate between localized and non-localized mRNAs. a combined genetic and biochemical approach will allow us to identify biologically relevant mechanisms that link motor proteins to specific mRNAs. (3) Reconstitute the interaction Myo4p and RNA purified components and analyze the transport of single Myo4p-RNA complexes in vitro. The use of purified proteins and RNAs to study transport will allow us to test several models of how motor proteins generate rapid and long range transport of cargo.