Import of RNA into Mitochondria

将 RNA 导入线粒体

• 批准号: 7081269
• 负责人: MARK E SCHMITT
• 金额: $ 21.82万
• 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 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7081269
• 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。 该提案中概述的研究的主要目标是了解 RNA 分子如何导入线粒体区室并确定参与此过程的机制。 作为模型系统，我们一直使用酿酒酵母 RNase MRP，这是一种核糖核蛋白核糖核酸内切酶，在细胞 RNA 加工中至少具有两个作用，一个在线粒体区室中，在线粒体 DNA 复制的启动中发挥直接作用，另一个在核仁中发挥作用。 该提案的具体目标是了解核糖核蛋白酶 RNase MRP 的 RNA 亚基导入线粒体的机制和途径。 线粒体 DNA 因突变、缺失或耗竭而丢失与心脏病、糖尿病、肌肉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患有这些疾病的患者提供更好的治疗。 了解 RNA 如何导入线粒体将为线粒体 DNA 突变患者提供一种基因治疗模式，而无需改造线粒体。实验基于这样的基本原理：RNase MRP 的 RNA 成分的基因是核编码的；因此，MRP RNA需要从其在细胞核中的转录位点转运至线粒体并与其同源蛋白组装。 酵母中的 RNase MRP 酶提供了一个理想的系统，可以使用生化和遗传方法研究 RNA 导入线粒体。 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) 使用新开发的体外RNA导入系统来阐明RNA导入的生化和分子要求。 2) 分离线粒体输入特异的 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输入线粒体基质所需的序列。 3) 这些突变体线粒体代谢各个方面的表征，包括线粒体呼吸丧失、DNA 维持、翻译、RNA 加工以及突变体 MRP RNA 在我们的体外系统中的导入。 4) 鉴定导入线粒体的其他RNA分子。 这些实验应该提供有关这一基本过程的丰富知识。