tRNA dynamics between the nucleus and the cytoplasm

本项目的目的是阐明tRNA连接酶Rlg1p在细胞内的定位，并描述tRNA在成熟过程中的核质动态。在本研究中，我以酿酒酵母为研究对象，重点研究了上述两个主要方面，并开始了tRNAs细胞内动力学分析系统的开发。1)利用免疫细胞化学技术和细胞分离技术，发现Rlg1p主要定位于胞浆，而不是胞核。通过对Rlg1p部分缺失的分析，在Rlg1p中没有发现任何核定位信号。另一方面，带有线粒体锚定信号的Rlg1p可以替代真正的Rlg1p，表明Rlg1p在细胞质中保留并发挥作用。2)我建立了一个用~3H-尿嘧啶脉冲标记和杂交沉淀技术追踪特定tRNA分子的系统。使用该系统，我发现在温度敏感的tRNA剪接突变体的胞浆中积累的前tRNA不是死端产物，但可以被追逐到成熟的tRNA中。3)利用酿酒酵母细胞中pombe tRNA的ART表达系统和酵母交配过程中的异核体分析，首次证明了成熟的tRNA可以再次进入细胞核，这些结果迫使我们改变了我们对tRNA在成熟过程中从细胞核到细胞质的单向运动的看法。

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.