Mechanism of tRNA Import Into Trypanosome Mitochondria

描述(由申请人提供)：线粒体是在大多数真核细胞中发现的基本细胞器，并提供不同的代谢功能。充分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种核编码蛋白被输入线粒体，另外13种蛋白质由线粒体基因编码。这种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的研究表明，部分或全部线粒体tRNA可以是核编码的，必须输入到线粒体中。在锥虫体内，所有线粒体tRNAs都是核编码的，大多数定位于线粒体和胞浆。虽然在阐明蛋白质进入真菌和哺乳动物线粒体的机制方面已经取得了显著的进展，但对线粒体RNA的导入机制却知之甚少。这项提案的总体目标是剖析tRNA生物合成和贩运非洲锥虫的途径。为实现这一目标，提出了以下具体目标。在具体目标I中，将通过tRNA在完整细胞中的体外转录和动力学分析来研究锥虫tRNAs的生物合成途径。在特定目标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到线粒体中的作用。最后，在特定目标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输入的锥虫蛋白。锥体为研究tRNA转运途径提供了一个很有吸引力的模型，因为最近开发了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导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.