tRNA dynamics between the nucleus and the cytoplasm

本项目的目的是阐明tRNA连接酶Rlg1p的细胞内定位，并描述其成熟过程中tRNA的核质动力学。在研究过程中，我以酿酒酵母（Saccharomycescerevisiae）为研究对象，围绕上述两个主要方面展开研究，并建立了tRNA的细胞内动力学体外分析系统。1）利用免疫细胞化学技术和细胞分级分离技术，结合Rlg1p特异性抗体和融合标签的Rlg1p，发现Rlg1p主要定位于胞浆，而非细胞核。通过对Rlg1p部分缺失的分析，未发现Rlg1p的核定位信号。另一方面，带有线粒体锚信号的Rlg1 p可以替代真正的Rlg1 p，这表明Rlg1 p在细胞质中存在并发挥作用。2）我开发了一个系统，用^3 H-尿嘧啶脉冲标记和杂交沉淀技术追踪特定的tRNA分子。使用该系统，我发现在温度敏感的tRNA剪接突变体的胞质溶胶中积累的前tRNA不是死端产物，而是可以被追赶成成熟的tRNA。结果表明，tRNA的剪接不是发生在细胞核中，而是发生在细胞质中。3）利用粟酒裂殖酵母tRNA在酿酒酵母细胞中的表达系统和利用酵母交配程序进行的异核体实验，首次证明了成熟的tRNA被重新输入细胞核，这一结果改变了人们对tRNA在成熟过程中从细胞核到细胞质单向运动的认识。

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.