Rescue of mt DNA-derived defects by mitochondria-tareted mRNA import and translation

项目摘要 已知线粒体功能障碍与许多人类疾病有关。许多线粒体 蛋白质是由位于核DNA(NDNA)中的基因编码的，然而线粒体拥有自己的基因 人类编码37个基因的基因组(MtDNA)。其中，13个编码关键多肽 电子传递链(ETC)复合全酶活性的亚基，使它们成为必不可少的组成部分 线粒体的功能。对核编码线粒体蛋白的研究由于 有大量的方法可以用来操纵核基因组。然而，研究的方法 在mt-DNA中发现的突变的影响是有限的和间接的。具体来说，我们产生线粒体DNA的能力 “转基因”细胞系和动物仍然相当有限。这在我们解决以下问题的能力上留下了很大差距 关于线粒体对疾病的贡献的基本问题。 为了弥补这一差距，我们的实验室最近使用了以前的RNA定位序列 由Koehler和Teitell小组发现，可同时递送本地(mtCO2和mtCO3)和非 线粒体天然的、工程化的(EGFP和DNase1)mRNA。我们的结果表明成功地进口和 这些mRNA在SH-SY5Y人神经母细胞瘤细胞线粒体基质中的翻译。致我们的 知识这是第一次成功地证明了线粒体中非天然蛋白质的翻译。 这项研究的目的是进一步发展这种方法作为研究线粒体的一种手段。 靶向线粒体核转录的线粒体mRNA(mt-mrna)导致的功能障碍 翻译并整合到等全酶中。我们的中心假设是核转录， 线粒体翻译的mRNA可以产生有功能的呼吸链蛋白，并将有助于建模 以及挽救线粒体DNA衍生的线粒体功能障碍。我们的长期目标是研究泛函 疾病相关单核苷酸多态对线粒体功能和脑的影响 小鼠的衰老。(目标1)我们将确定线粒体靶向传递的效率和功能效应 以及翻译外源转录物以并入神经细胞中的ETC全酶。(目标2)我们 将调查线粒体靶向传递和翻译外源转录本是否拯救了 线粒体疾病模型小鼠神经元中线粒体DNA衍生的线粒体功能障碍。 我们提出的研究有望产生一种新的方法，无论是在体外还是在体内，分析 线粒体功能障碍在脑老化中的作用，可进一步用于基础和翻译 研究。我们相信，我们的方法将提供对mtdna突变病理的洞察，并提供 潜在干预的特定途径方法。

PROJECT ABSTRACT Mitochondrial dysfunction is known to be involved in a number of human diseases. Many mitochondrial proteins are encoded by genes located in nuclear DNA (nDNA), however mitochondria possess their own genome (mtDNA) that encodes 37 genes in humans. Among them, 13 encode polypeptides that are critical subunits for electron transport chain (ETC) complex holoenzyme activity, making them essential components for mitochondrial function. Investigation into nuclear encoded mitochondrial proteins has been simplified due to an abundance of methods available for manipulating the nuclear genome. However, methods for studying the effects of mutations found in Mt-DNA are limited and indirect. Specifically, our ability to generate mtDNA “transgenic” cell lines and animals remains quite limited. This leaves a significant gap in our ability to address fundamental questions about mitochondrial contribution to disease. In an attempt to bridge this gap, our lab recently used an RNA localization sequence previously discovered by the Koehler and Teitell group to deliver mRNA of both native (mtCO2 and mtCO3) and non- native, engineered (eGFP and DNase1) mRNA to mitochondria. Our results indicate successful import and translation of these mRNA's within the mitochondrial matrix of SH-SY5Y human neuroblastoma cells. To our knowledge this is the first successful demonstration of translation of a non-native protein within mitochondria. The objective of this study is to further develop this method as a means of studying mitochondrial dysfunction by targeting nucleus-transcribed mitochondrial mRNA (mt-mRNA) transcripts to mitochondria for translation and incorporation into ETC holoenzymes. Our central hypothesis is that nucleus-transcribed, mitochondria-translated mRNA can produce functional respiratory chain proteins and will facilitate modeling and rescue of mtDNA-derived mitochondrial dysfunction. Our long-term goals are to study the functional consequences of disease-associated single nucleotide polymorphisms on mitochondrial function and brain aging in mice. (Aim 1) We will determine the efficiency and functional effects of mitochondrial-targeted delivery and translation of exogenous transcripts for incorporation into ETC holoenzymes in neuronal cells. (Aim 2) We will investigate whether mitochondrial-targeted delivery and translation of exogenous transcripts rescues mtDNA-derived mitochondrial dysfunction in mouse neurons of a mitochondrial disease model. Our proposed studies are expected to generate a novel approach, both in vitro and in vivo, to analyzing the role of mitochondrial dysfunction in brain aging that can be further utilized for basic and translational research. We believe that our approach will provide insight into mtDNA mutation pathology and offer a pathway-specific method for potential intervention.