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Analysis of physiological meanings of mitochondrial localization of yeast tRNA splicing endonuclease

酵母tRNA剪接核酸内切酶线粒体定位的生理意义分析

基本信息

批准号：
12680609
负责人：
YOSHIHI Tbhn
金额：
$ 0.7万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12680609/
关键词：
tRNA splicing nuclear-cytoplasmic transpo endonuclease mitochondria

项目摘要

Primary transcripts of nuclear-encoded tRNA genes receive various processing to become mature tRNA functional for translation in cytoplasm. Among such processing, splicing is essential for maturation of intron-containing pre-RNAs.The splicing of pre-tRNAs has been believed to occur in the nuclei. In yeast, Saccharomyces cerevisiae, tRNA splicing endonuclease that cleaves exon-intron junctions of pre tRNAs consists offour subunits, Sen54p, Sen2p, Sen34p and SenlSp, and has been supposed as an integral membrane protein of inner uclear envelope. Through this research project, we show that the majority of Sen2p, Sen54p and the endonuclease activity are not localized in the nuclei but associated on the surface of mitochondria. Yeast mutant cells deficient in the endonuclease activity accumulate unspliced pre-tRNAs in cytosol. A Sen54p derivative artificially fixed on the mitochondrial surface by fusing a itochondrial targeting signal and a transmembrane domain ofTom70p (Tom70N), an integral membrane protein of outer mitochondrial membrane, can functionally replace authentic Sen54p. On the other hand, Sen54p derivatives with nuclear localization signals are not fully active. Partial deletion of the mitochondrial targeting signal in Sen54p compromises its function, but addition of Tom70N to this deletion mutant suppresses its defect. These results strongly suggest that an early step of tRNA splicing is not a nuclear event and that pre-tRNAs take a complex route via the mitochondrial surface during their maturation.

核编码的tRNA基因的初级转录物接受各种加工以成为成熟的tRNA，其在细胞质中具有翻译功能。在这些加工过程中，剪接对于含内含子的前体RNA的成熟是必不可少的，前体tRNA的剪接被认为发生在细胞核中。在酿酒酵母（Saccharomycescerevisiae）中，tRNA剪接内切酶切割前tRNA的外显子-内含子连接，由四个亚基组成，Sen 54 p、Sen 2 p、Sen 34 p和SenlSp，被认为是核内被膜的一个完整的膜蛋白。通过本研究项目，我们发现大部分的Sen 2 p，Sen 54 p和核酸内切酶活性并不位于细胞核中，而是与线粒体表面相关。缺乏内切核酸酶活性的酵母突变体细胞在胞质溶胶中积累未剪接的前tRNA。通过融合线粒体靶向信号和线粒体外膜的整合膜蛋白Tom 70 p（Tom 70 N）的跨膜结构域，将Sen 54 p衍生物人工固定在线粒体表面，可以在功能上替代真实的Sen 54 p。另一方面，具有核定位信号的Sen 54 p衍生物不是完全活性的。Sen 54 p中线粒体靶向信号的部分缺失损害了其功能，但向该缺失突变体中添加Tom 70 N抑制了其缺陷。这些结果有力地表明，tRNA剪接的早期步骤不是核事件，并且前tRNA在其成熟期间通过线粒体表面采取复杂的路线。