RNA TRANSPORT AND LOCALIZATION IN YEAST IN SITU

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EXCEED THE SPACE PROVIDED. Numerous mRNA species localize within cells. The synthesis ofbeta-actin at the leading edge facilitates cell polarity and motility. In yeast, ASH1 mRNA localizes to the bud tip and is necessary for asymmetric gene expression between the mother and the bud, allowing mating type switching. A major question has been exactly how mRNA localization occurs and what factors are involved. In order to address this, a method for visualizing the movement ofmRNAs within living cells was developed. ASH1 mRNA movements are observed in living yeast cells using a GFP reporter system fused to the RNA-binding protein MS2 and introduction of MS2 sequences into the reporter RNA (Bertrand et al, Mol. Cell 2:437, 1998). This approach showed unequivocally that the mRNA formed a particle that moved on actin cables and that two proteins; She2p, a protein that binds each of the four zipcodes in the ASH1 mRNA, and the myosin, Shelp, are bridged by a third protein, She3p (Long, et al., EMBO J. 19:6592, 2000). We propose to isolate the "locasome", the particle formed by the ASH1 zipcodes. Because of the GFP tagging, this particle will be visually identifiable by light microscopy. By isolating this structure, we will be able to identify the various proteins associated with its (hypothesized) nuclear formation, export, association with the myosin motor, anchoring at the bud tip and translational regulation. These genes will be tested by mutation or deletion in the GFP particle localization assay, after induction by galactose. The visualization of the GFP particle using high-speed microscopy will capture the rapid movements during transcriptional release and movement to the nuclear pores, export and coupling to the cytoplasmic filaments. Increasing the number of MS2 binding sites on the RNA will allow us to detect single RNA molecules. Using these approaches, we will have the opportunity to identify where in the pathway the components of localization and regulation of protein expression operate, with high spatial and temporal resolution. PERFORMANCE SITE ========================================Section End===========================================