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

核编码的tRNA基因的初级转录本经过各种处理后成为成熟的tRNA，具有在细胞质中翻译的功能。在这些过程中，剪接是含有内含子的Pre-RNA成熟所必需的。Pre-tRNA的剪接被认为发生在细胞核中。在酵母中，切割前tRNAs外显子-内含子连接的tRNA剪接内切酶由Sen54p、Sen2p、Sen34p和SenlSp四个亚基组成，被认为是内核膜的完整膜蛋白。通过这项研究，我们发现大多数Sen2p、Sen54p和内切酶活性并不定位于细胞核，而是伴随在线粒体的表面。缺乏内切酶活性的酵母突变细胞在胞浆中积累未剪接的前tRNAs。通过线粒体靶向信号和线粒体外膜完整膜蛋白Tom70P的跨膜区(Tom70N)人工固定在线粒体表面的Sen54p衍生物可以在功能上取代真正的Sen54p。另一方面，具有核定位信号的Sen54p衍生物并不完全活跃。Sen54p中线粒体靶向信号的部分缺失影响了其功能，但在该缺失突变体中加入Tom70N可抑制其缺陷。这些结果强烈表明，tRNA剪接的早期步骤不是核事件，前tRNA在成熟过程中通过线粒体表面采取复杂的途径。

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.