Mechanism of Mitochondrial Ribosome Assembly

线粒体核糖体组装机制

基本信息

批准号：
9125870
负责人：
DANIEL F. BOGENHAGEN
金额：
$ 32.77万
依托单位：
STATE UNIVERSITY NEW YORK STONY BROOK
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-08-15 至 2019-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9125870
关键词：
3&apos Flanking Region A549 Active Sites Address Affect Aging Amino Acids Aminoglycosides Base Pairing Binding Binding Proteins Biogenesis Cell Culture Techniques Cell Nucleus Cells Complex Coupled Cryoelectron Microscopy Data Defect Degenerative Disorder Dependence Disease Excision Exhibits Gene Expression Genetic Transcription Health Hereditary Disease Human Human Cell Line Human Genetics Immune Sera Immunoblotting Individual Inherited Kinetics Label Lead Light Link Mass Spectrum Analysis Membrane Messenger RNA Metabolic Diseases Methods Methyltransferase Mitochondria Mitochondrial DNA Mitochondrial Diseases Modeling Modification Molecular Chaperones Molecular Weight Monitor Neurodegenerative Disorders Nuclear Participant Pathogenesis Pathway interactions Peptide Hydrolases Physiologic pulse Play Precursor RNA Process Protein Import Proteins Proteomics RNA RNA Processing RNA, ribosomal, 12S Recombinants Recruitment Activity Reporting Research Respiration Respiratory Chain Ribosomal Proteins Ribosomal RNA Ribosomes Role Sedimentation process Series Structure Study Subject Surgical incisions Swelling System Testing Transcript Transfer RNA Translating Translations base deafness human disease improved insight interest membrane assembly mitochondrial dysfunction mitochondrial genome normal aging novel novel strategies protein structure research study respiratory response stable isotope

项目摘要

DESCRIPTION (provided by applicant): Human cells contain many copies of a 16,569 base pair mitochondrial genome to permit expression of only 13 proteins that are all essential subunits of complexes required for respiration. These proteins are translated on mitochondrial ribosomes assembled by combining three ribosomal RNAs with about 80 nucleus-encoded proteins imported into mitochondria. These mitochondrial ribosomal proteins (MRPs) have only recently been identified and several of them, including MRPS16, MRPS22, MRPL3 and MRPL44 have been implicated in inherited genetic disorders. Despite its critical importance, the pathway for mitoribosome assembly is virtually unknown, establishing this as a fertile subject for study. Mitochondrial ribosome assembly and function are altered in aminoglycoside-induced deafness and neurodegenerative diseases. Impaired mitochondrial ribosome assembly resulting in mitonuclear protein imbalance may also contribute to the progressive mitochondrial dysfunction observed with aging. We propose to use a novel approach we developed to study mitochondrial ribosome assembly using stable isotope pulse-chase labeling in cell culture (pulse-chase SILAC) and mass spectrometry. Our extensive preliminary results show that certain proteins bind newly- synthesized rRNA at mtDNA nucleoids, possibly while transcription is continuing; these are candidates for early ribosome assembly proteins. Others only join the ribosome later, after it is no longer tightly linked to the nucleoid. We propose to use refined pulse-chase methods to improve understanding of the mechanism, kinetics and efficiency of mitoribosome assembly. Recent cryo-electron microscopy studies have discovered the tRNAvaline as a novel component of the large subunit. We will test a model in which cleavage of the tandemly transcribed 12S rRNA-tRNAval-16S rRNA into three separate RNAs must be coordinated with binding of newly synthesized MRPs. We will determine whether newly-synthesized tRNAval is incorporated into the ribosome along with the tandemly-transcribed 16S rRNA by a transcription-coupled assembly process, or whether a preexisting copy of tRNAval is recruited into the ribosome. We will study how the assembly process is distorted when the system is perturbed by depletion of an individual MRP or of assembly factors that are not themselves ribosomal components. In light of our finding that early assembly takes place at the mtDNA nucleoid, we will extend our efforts to study mitoribosome assembly in cells with disordered nucleoid structure. We hypothesize that if altered nucleoid structure affects rRNA synthesis and processing, this could lead to accumulation of MRPs that cannot participate efficiently in ribosome assembly, leading to a mitochondrial unfolded protein response. Impact: The proposed research will provide mechanistic insight into the process of mitoribosome assembly, clarify its dependence on RNA processing, and investigate the consequences of pathological alterations that alter assembly, including triggering of the unfolded protein response. Long term, this will pave the way for a vastly improved understanding of mitoribosome biogenesis, which is essential in order to understand the pathogenesis of mitochondrial disorders resulting from mitochondrial translation defects.

描述（由申请人提供）：人类细胞含有 16,569 个碱基对的线粒体基因组的许多拷贝，仅允许表达 13 种蛋白质，这些蛋白质都是呼吸所需复合物的必需亚基，这些蛋白质在通过组合三个核糖体 RNA 组装的线粒体核糖体上进行翻译。大约 80 种核编码蛋白被导入线粒体，这些线粒体核糖体蛋白 (MRP) 最近才被鉴定出来，其中包括一些。 MRPS16、MRPS22、MRPL3 和 MRPL44 与遗传性遗传疾病有关，尽管其至关重要，但线粒体核糖体组装的途径实际上是未知的，这使得线粒体核糖体组装和功能在氨基糖甙类诱发的耳聋和耳聋中成为研究对象。神经退行性疾病。线粒体核糖体组装受损，导致线粒体核蛋白失衡，也可能导致随衰老而出现的进行性线粒体功能障碍。我们建议使用我们开发的一种新方法来研究线粒体核糖体组装，在细胞培养物中使用稳定同位素脉冲追踪标记（脉冲追踪 SILAC）和质谱法。我们广泛的初步结果表明，某些蛋白质在 mtDNA 核仁处结合新合成的 rRNA。，可能在转录继续时；这些是早期核糖体组装蛋白的候选者，在它不再与核糖体紧密连接之后才加入核糖体。最近的冷冻电子显微镜研究发现 tRNA 缬氨酸是大亚基的一种新成分，我们将测试串联转录的 12S 裂解的模型。 rRNA-tRNAval-16S rRNA 分成三个单独的 RNA，必须与新合成的 MRP 的结合相协调，我们将确定是否是新合成的。 tRNAval 通过转录耦合组装过程与串联转录的 16S rRNA 一起整合到核糖体中，或者是否将预先存在的 tRNAval 副本招募到核糖体中。我们将研究当系统受到干扰时组装过程如何被扭曲。单个 MRP 或本身不是核糖体成分的组装因子的耗尽鉴于我们发现早期组装发生在 mtDNA 核仁上，我们将扩展。我们在核糖体结构紊乱的细胞中研究线粒体核糖体组装，结果发现，如果核糖体结构改变影响 rRNA 合成和加工，则可能导致 MRP 积累，无法有效参与核糖体组装，从而导致线粒体未折叠蛋白反应。：拟议的研究将为线粒体核糖体组装过程提供机制见解，阐明其对 RNA 加工的依赖性，并研究改变组装的病理改变的后果，包括触发未折叠的蛋白质从长远来看，这将为极大地改善对线粒体生物发生的理解铺平道路，这对于理解线粒体翻译缺陷引起的线粒体疾病的发病机制至关重要。