Import of RNA into Mitochondria

将 RNA 导入线粒体

• 批准号: 6756516
• 负责人: MARK E SCHMITT
• 金额: $ 22.34万
• 依托单位: UPSTATE MEDICAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6756516
• 关键词: RNA; RNA biosynthesis; Saccharomyces cerevisiae; binding proteins; endoribonucleases; gene mutation; intracellular transport; membrane potentials; mitochondria; mitochondrial DNA; nucleic acid quantitation /detection; nucleic acid sequence; ribonuclease P; ribonucleoproteins

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.

标题：将RNA导入线粒体。这项提案中概述的研究的主要目标是了解RNA分子是如何进入线粒体的，并确定这一过程中涉及的机制。作为一个模型系统，我们一直使用酿酒酵母RNase MRP，这是一种核糖核蛋白内切核酸酶，在细胞RNA加工中至少有两个角色，一个在线粒体中，它在线粒体DNA复制的启动中起直接作用，第二个在核仁中。这一建议的具体目标是了解核糖核蛋白酶RNase MRP的RNA亚单位导入线粒体的机制和途径。线粒体DNA因突变、缺失或耗尽而丢失，与心脏病、糖尿病、肌肉疾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会为这些疾病的患者提供更好的治疗。了解RNA是如何进入线粒体的，将为患有线粒体DNA突变的人提供一种基因治疗模式，而不需要转化线粒体。实验基于这样的理论基础，即RNase MRP的RNA成分的基因是核编码的；因此，MRP RNA需要从其在核中的转录位置运输到线粒体，并与其同源蛋白组装。酵母中的核糖核酸酶MRP酶为利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。下面列举的特定目的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。具体目标包括。1)利用新开发的体外RNA导入系统，阐明了RNA导入的生化和分子要求。2)分离线粒体导入所特有的MRP RNA突变，以确定线粒体底物结合、线粒体特异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，包括线粒体呼吸的丧失、DNA的维持、翻译、RNA的加工以及突变的MRP RNA在我们的体外系统中的输入。4)线粒体导入的其他RNA分子的鉴定。这些实验应该会提供关于这一基本过程的丰富知识。