Eukaryotic cells possess organelles allowing biochemical pathways components to be in close physical proximity and in environments optimal for activity. However, such organization generates cell biological traffic problems to effectively deliver only appropriate macromolecules to particular subcellular destinations and to maintain cellular organization throughout the cell cycle. Moreover, there must be effective communication between the various compartments so that each is coupled to the cellular metabolic state and to the cell cycle and there must be surveillance mechanisms able to recognize and correct or prevent mistakes. Using yeast as a model system, our primary focus is to understand the paths of tRNA export from the nucleus to the cytosol, the coupling of tRNA processing and nuclear export to cellular metabolism and the cell cycle, and the nature of the surveillance systems that assure fidelity of these important processes. (1) We will rigorously test the hypothesis that aminoacyl tRNA synthetases enter the nucleus to proof tRNAs prior to their export. (2) As the only known exportin for tRNA is unessential, it is likely that tRNA exits the nucleus by more than a single route. We will test the hypothesis that Ccalp provides an alternative path and we will undertake genetic studies to identify other paths. (3) We will study the mechanisms involved in communication of metabolism in the cytosol with tRNA processing and nuclear export. We will test the hypothesis that amino acid availability and pre-tRNA splicing signal each other. In searches for tRNA export mutants we identified ctf13, a known centromere-binding protein. We will test the hypothesis that Ctfl3p has dual functions in mitosis and tRNA nuclear export, coupling macromolecular biosynthesis to cell cycle progression. (4) A secondary focus concerns the mechanisms of action and epigenetic inheritance of dsRNAi, likely to involve RNA metabolism and nucleus/cytosol exchange. Preliminary studies indicate that dsRNAi-mediated gene silencing occurs in budding yeast. We will identify gene products functioning in dsRNAi-mediated silencing and its epigenetic inheritance.
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