RNA trafficking in mitochondria

线粒体是大多数真核细胞必需的细胞器。它们提供多种代谢功能 包括产生能量和用于生物合成过程的特殊代谢物。组装 由线粒体和核基因组编码的功能性线粒体、蛋白质和RNA， 必需的.在人类中，至少有1,000种核编码蛋白被输入线粒体，而只有13种蛋白被输入线粒体。 电子传递链的蛋白质由线粒体基因编码。这几个翻译， 必需的线粒体编码的蛋白质需要线粒体核糖体、翻译因子和转运， RNA，其中的一个子集必须从细胞质输入到线粒体基质中。的近期研究 进化上多样化的生物体已经表明，多种RNA在细胞核内编码并输入 从细胞质进入线粒体。我们对线粒体输入途径的总体理解， 编码的蛋白质非常详细。相比之下，对线粒体输入机制知之甚少 对于核编码RNA或如何使用这些新知识将特定RNA递送到线粒体中。 我们的竞争R 01更新申请的修订后的总体目标是剖析主要和 也许只有RNA进入线粒体的途径，并开发靶向核编码的策略， 在报告构建体中将RNA导入线粒体和用于治疗由线粒体RNA突变引起的疾病 线粒体DNA（mtDNA）。在单独的研究中，我们将识别导入到细胞中的RNA队列。 使用下一代RNA-Seq和耦合的生物信息学方法对线粒体进行分析。此前，我们曾报道 研究表明，RNA加工酶多核苷酸磷酸化酶（PNIPs）出乎意料地 定位于线粒体膜间隙（IMS），在那里它起着细胞核转录的看门人的作用。 从细胞质中输入的编码RNA。有趣的是，PNTR只存在于 生物，如苍蝇，蠕虫和哺乳动物，表明其功能仅限于高等真核生物。 为了实现我们修改后的提案目标，保留项目的原始本质，我们 确定了两个具体的研究目标。在目标1中，除《禁止化学武器公约》外， 核编码的RNA将被鉴定。在目标2中，报告基因和校正核编码的RNA构建体将 被工程化以确定RNA序列和RNA输入机制的结构要求， 开发治疗由线粒体DNA突变引起的疾病的策略。在这些研究中，我们将 我们已经建立了不同的模型系统来操纵PNTs水平和活动的优势。 除了增加我们对RNA输入机制的基本知识外， 线粒体和学习规则操纵这种RNA输入途径，我们的更新应用程序可能 对公众健康产生广泛的影响，因为我们的方法可以提供一种方法， 治疗几十种由线粒体DNA突变引起的人类疾病。

Mitochondria are essential organelles for most eukaryotic cells. They provide diverse metabolic functions including the production of energy and specialized metabolites for biosynthetic processes. To assemble functional mitochondria, proteins and RNAs encoded by both the mitochondrial and nuclear genomes are required. In humans, at least 1,000 nuclear encoded proteins are imported into mitochondria, whereas only 13 proteins of the electron transport chain are encoded by mitochondrial genes. The translation of these few but essential mitochondrial encoded proteins requires mitochondrial ribosomes, translation factors, and transfer RNAs, a subset of which must be imported from the cytosol into the mitochondrial matrix. Recent studies in evolutionarily diverse organisms have shown that multiple RNAs are encoded within the nucleus and imported from the cytosol into mitochondria. Our overall understanding of mitochondrial import pathways for nuclear encoded proteins is quite detailed. By contrast, very little is known about the mitochondrial import mechanism for nuclear encoded RNAs or how this new knowledge can be used to deliver specific RNAs into mitochondria. The revised overall goals of our competitive R01 renewal application are to dissect the main and perhaps only pathway of RNA import into mitochondria and to develop strategies that target nuclear encoded RNAs to mitochondria in reporter constructs and for the treatment of diseases caused by mutations of mitochondrial DNA (mtDNA). In separate studies, we will identify the cohort of RNAs that are imported into mitochondria using next generation RNA-Seq and coupled bioinformatics approaches. Previously, we reported studies that showed that the RNA processing enzyme polynucleotide phosphorylase (PNPase) is unexpectedly localized to the mitochondrial intermembrane space (IMS), where it functions as a gatekeeper for nuclear encoded RNAs that are imported from the cytosol. Interestingly, PNPase is only present in the genomes of organisms such as flies, worms, and mammals, suggesting that its functions are limited to higher eukaryotes. To accomplish our revised proposal goals that retain the original essence of the project, we have identified two specific study aims. In Aim 1, additional regulatory components beyond PNPase for the import of nuclear encoded RNAs will be identified. In Aim 2, reporter and corrective nuclear encoded RNA constructs will be engineered to determine the RNA sequence and structural requirements of the RNA import machinery and to develop strategies to treat diseases caused by mutations in mtDNA. For these studies we will take advantage of different model systems that we have established to manipulate PNPase levels and activities. In addition to increasing our fundamental knowledge about the mechanisms of RNA import into mitochondria and learning the rules for manipulating this RNA import pathway, our renewal application may have a broad impact on public health because our approach could provide a method to ameliorate or possibly cure dozens of human diseases caused by mtDNA mutations.