Import of RNA into Mitochondria

题目：RNA导入线粒体。这项研究的主要目标是了解RNA分子是如何进入线粒体腔室的，并确定这一过程中涉及的机制。作为一个模型系统，我们一直在使用酿酒酵母RNase MRP，这是一种核糖核蛋白核糖核酸内切酶，在细胞RNA加工中至少有两个作用，一个在线粒体室中，它在线粒体DNA复制的起始起直接作用，另一个在核核中起作用。本提案的具体目标是了解核糖核蛋白酶RNase MRP的RNA亚基输入线粒体的机制和途径。线粒体DNA的突变、缺失或缺失与心脏病、糖尿病、肌病以及最近的衰老有关。了解RNase MRP在线粒体DNA复制和功能中的作用可能为这些疾病的患者提供更好的治疗方法。了解rna是如何导入线粒体的，将为线粒体DNA突变的人提供一种无需改造线粒体的基因治疗模式。实验是基于RNase MRP的RNA成分基因是核编码的这一基本原理；因此，MRP RNA需要从其在细胞核中的转录位点运输到线粒体，并与其同源蛋白组装。酵母中的RNase MRP酶为使用生化和遗传学方法研究RNA进入线粒体提供了一个理想的系统。下面列举的具体目标旨在利用生物化学和遗传学来确定RNA输入线粒体的机制和机制。具体目标将包括：1)利用新开发的体外RNA导入系统阐明RNA导入的生化和分子需求。2)分离线粒体导入特异性的MRP RNA突变，以确定线粒体底物结合、线粒体特异性蛋白结合和导入线粒体基质所需的序列。3)这些突变体在线粒体代谢的各个方面的特征，包括线粒体呼吸丧失、DNA维持、翻译、RNA加工和突变体MRP RNA在体外系统中的输入。4)鉴定其他导入线粒体的RNA分子。这些实验应该提供关于这一基本过程的丰富知识。

Title: Import of RNA into Mitochondria. The main goal of the research outlined in this proposal is to understand how an RNA molecule is imported into the mitochondrial compartment and to identify the machinery involved in this process. As a model system we have been using the Saccharomyces cerevisiae RNase MRP, a ribonucleoprotein endoribonuclease that has at least two roles in cellular RNA processing, one in the mitochondrial compartment where it plays a direct role in the initiation of mitochondrial DNA replication and a second in the nucleolus. The specific objective for this proposal is to understand the mechanism and pathway by which the RNA subunit of the ribonucleoprotein enzyme RNase MRP is imported into mitochondria. Loss of mitochondrial DNA through mutation, deletion or depletion has been implicated in heart disease, diabetes, muscle myopathies, and more recently aging. Understanding the role of RNase MRP in mitochondrial DNA replication and function may provide better treatments for patients with these diseases. Understanding how RNAs are imported into mitochondria would provide a mode of gene therapy for people with mitochondrial DNA mutations without the need to transform mitochondria. Experimentation is based on the rationale that the genes for the RNA component of RNase MRP is nuclear encoded; hence, the MRP RNA needs to be transported from its site of transcription in the nucleus to mitochondria and be assembled with its cognate proteins. The RNase MRP enzyme in the yeast offers an ideal system to study RNA import into the mitochondrion using both biochemical and genetic methodologies. The specific aims enumerated below are designed to use both biochemistry and genetics to identify the mechanism and machinery of RNA import into mitochondria. Specific aims will include. 1) Use of a newly developed in vitro RNA import system to elucidate the biochemical and molecular requirements for RNA import. 2) Isolation of mutations in the MRP RNA that are specific for mitochondrial import to determine the sequences required for mitochondrial substrate binding, mitochondrial specific protein binding and import into the mitochondrial matrix. 3) Characterization of these mutants for various aspects of mitochondrial metabolism including loss of mitochondrial respiration, DNA maintenance, translation, processing of RNAs and import of the mutant MRP RNA in our in vitro system. 4) Identification of other RNA molecules imported into mitochondria. These experiments should provide a wealth of knowledge about this fundamental process.