Mechanism of Mitochondrial Ribosome Assembly

线粒体核糖体组装机制

• 批准号: 8962407
• 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
• 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/8962407
• 关键词: 3' 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; 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 Binding; 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; public health relevance; research study; respiratory; response; stable isotope

 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。这些蛋白质在线粒体核糖体上翻译，线粒体核糖体是通过将三种核糖体 RNA 与导入线粒体的约 80 种核编码蛋白质组合而成的。这些线粒体核糖体蛋白 (MRP) 最近才被鉴定，其中包括 MRPS16、MRPS22、MRPL3 和 MRPL44 在内的几种蛋白与遗传性疾病有关。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，这使其成为一个丰富的研究课题。氨基糖苷类引起的耳聋和神经退行性疾病中线粒体核糖体的组装和功能发生改变。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观察到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，某些蛋白质可能在转录仍在继续的同时，在 mtDNA 核仁处结合新合成的 rRNA；这些是早期核糖体组装蛋白的候选者。其他的则在核糖体不再与核仁紧密相连后才加入核糖体。我们建议使用精细的脉冲追踪方法来增进对线粒体糖体组装机制、动力学和效率的理解。最近的冷冻电子显微镜研究发现 tRNAvaline 是大亚基的新成分。我们将测试一个模型，其中串联转录的 12S rRNA-tRNAval-16S rRNA 裂解成三个独立的 RNA 必须与新合成的 MRP 的结合相协调。我们将确定新合成的 tRNAval 是否通过转录偶联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。我们将研究当系统因单个 MRP 或本身不是核糖体成分的组装因子的消耗而受到干扰时，组装过程如何被扭曲。鉴于我们发现早期组装发生在 mtDNA 核仁上，我们将继续努力研究核仁结构无序的细胞中的线粒体核糖体组装。我们假设，如果改变的核结构影响 rRNA 合成和加工，这可能会导致 MRP 积累，而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。影响：拟议的研究将为线粒体核糖体组装过程提供机制上的见解，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反应。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。