tRNA dynamics between the nucleus and the cytoplasm

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The aim of this project is to clarify the intracellular localization of tRNA ligase, Rlg1p, and describe nuclear-cytoplasmic dynamics of tRNAs during their maturation. In the term of the research, I focused on the two major points listed above using yeast Saccharomyces cerevisiae, and also started development of the in vitro system to analyze intracellular dynamics of tRNAs.1) I found that Rlg1p is mainly localized in the cytosol not in the nucleus, using immuno-cytochemical techniques and cell fractionation techniques with specific antibodies against Rlg1p and with tag-fused Rlg1p. I could not find any nuclear localization signal in Rlg1p through the analysis of Rlg1p partial deletions. On the other hand Rlg1p with a mitochondrial anchor signal could substitute the authentic Rlg1p, indicating that Rlg1p stays and functions in the cytoplasm.2) I developed a system to trace a particular tRNA molecule with ^3H-uracil pulse-labeling and hybrid-precipitation techniques. Using the system, I found that pre-tRNAs accumulated in the cytosol of the temperature-sensitive tRNA splicing mutant are not dead-end products but can be chased into mature tRNAs. The results indicate that tRNA splicing occurs not in the nucleus but in the cytosol.3) Using art expression system of S.pombe tRNA in S.cerevisiae cells and heterokaryon assay utilizing yeast mating procedures, I demonstrated that mature tRNA is re-imported into the nucleus for the first time.These results force us to change our image of uni-directional movement, from the nucleus to the cytoplasm, of tRNAs during their maturation.