Mechanism of tRNA Import Into Trypanosome Mitochondria

描述（由申请人提供）：线粒体是大多数真核细胞中发现的基本细胞器，提供多种代谢功能。完全活跃的线粒体需要由线粒体和核基因组编码的蛋白质和rna。在人类中，至少有600种核编码蛋白质被输入线粒体，另外13种蛋白质由线粒体基因编码。这些有限数量的线粒体编码蛋白的翻译需要线粒体核糖体、翻译因子和转移rna。最近对进化多样性生物体的研究表明，部分或全部线粒体trna可以被核编码，并且必须输入线粒体。在锥虫中，所有线粒体trna都是核编码的，并且大多数都定位于线粒体和细胞质。虽然在阐明真菌和哺乳动物线粒体中蛋白质输入的机制方面取得了显著进展，但对线粒体RNA输入的机制知之甚少。本提案的总体目标是剖析tRNA在非洲锥虫中的生物合成和贩运途径。为实现这一目标，提出了以下具体目标。在Specific Aim I中，我们将通过体外转录和完整细胞中tRNA运输的动力学分析来研究锥虫tRNA的生物合成途径。在Specific Aim II中，将确定特定序列和/或结构基序在tRNA从细胞核输出和输入线粒体中的作用。最后，在Specific Aim III中，将确定与前体和成熟tRNA相互作用以影响tRNA运输并最终影响线粒体进口的锥虫蛋白。锥虫提供了一个有吸引力的模型来研究tRNA运输途径，因为最近已经开发出体外和体内系统来研究tRNA合成和线粒体输入。此外，线粒体tRNA输入可能具有治疗潜力，因为与锥虫不同，所有哺乳动物线粒体tRNA都是由线粒体基因编码的。

DESCRIPTION (provided by the applicant): Mitochondria are essential organelles found in most eukaryotic cells and provide for diverse metabolic functions. Fully active mitochondria require proteins and RNAs encoded both by the mitochondrial and nuclear genomes. In humans, at least 600 nuclear encoded proteins are imported into mitochondria and another 13 proteins are encoded by mitochondrial genes. The translation of this limited number of mitochondria-encoded proteins requires mitochondrial ribosomes, translation factors and transfer RNAs. Recent studies, of evolutionary diverse organisms, have shown that some or all of the mitochondrial tRNAs can be nuclear-encoded and must be imported into the mitochondria. In trypanosomes, all mitochondrial tRNAs are nuclear encoded and most are localized to both the mitochondrion and cytosol. While remarkable progress has been made in the elucidation of the mechanism of protein import into fungal and mammalian mitochondria, little is known about the mechanism of mitochondrial RNA import. The overall goal of this proposal is to dissect the pathway of tRNA biosynthesis and trafficking in African trypanosomes. To accomplish this goal the following specific aims are proposed. In Specific Aim I, the biosynthetic pathway for trypanosome tRNAs will be investigated by in vitro transcription and kinetic analysis of tRNA trafficking in intact cells. In Specific Aim II, the role of specific sequence and/or structural motifs in tRNA export from the nucleus and import into the mitochondria will be determined. Finally, in Specific Aim III, trypanosome proteins that interact with precursor and mature tRNAs to influence tRNA trafficking and ultimately mitochondrial import will be identified. Trypanosomes offer an attractive model to study tRNA trafficking pathways since both in vitro and in vivo systems have recently been developed to study both tRNA synthesis and mitochondrial import. In addition, mitochondrial tRNA import may have therapeutic potential since, in contrast to trypanosomes, all mammalian mitochondrial tRNAs are encoded by mitochondrial genes.