Mechanism of Mitochondrial Ribosome Assembly

线粒体核糖体组装机制

• 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
• 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' 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

 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，通过将三个核糖体RNA与输入线粒体的约80个核编码蛋白质组合而组装。这些线粒体核糖体蛋白（MRPs）最近才被鉴定出来，其中几种，包括MRPS 16，MRPS 22，MRPL 3和MRPL 44，与遗传性遗传疾病有关。尽管其至关重要，线粒体组装的途径几乎是未知的，建立这作为一个肥沃的主题进行研究。线粒体核糖体组装和功能在氨基糖苷类药物诱导的耳聋和神经退行性疾病中发生改变。线粒体核糖体组装受损导致线粒体蛋白失衡也可能导致随着衰老观察到的进行性线粒体功能障碍。我们建议使用一种新的方法，我们开发的研究线粒体核糖体组装使用稳定的同位素脉冲追踪标记细胞培养（脉冲追踪SILAC）和质谱。我们广泛的初步结果表明，某些蛋白质结合新合成的rRNA在mtDNA类核，可能同时转录正在继续，这些是早期核糖体组装蛋白的候选者。其他的只是在核糖体不再与类核紧密连接之后才加入核糖体。我们建议使用改进的脉冲追踪方法，以提高理解的机制，动力学和效率的线粒体组装。最近的冷冻电子显微镜研究发现tRNA缬氨酸作为大亚基的一个新的组成部分。我们将测试一个模型，其中串联转录的12 S rRNA-tRNAval-16 S rRNA切割成三个独立的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16 S rRNA一起沿着掺入核糖体中，或者是否预先存在的tRNAval拷贝被募集到核糖体中。我们将研究如何装配过程是扭曲的，当系统被个别MRP或装配因子本身不是核糖体成分的耗尽所干扰时。鉴于我们的发现，早期组装发生在线粒体DNA的类核，我们将扩大我们的努力，研究细胞中的线粒体组装与无序的类核结构。我们假设，如果改变的类核结构影响rRNA的合成和加工，这可能导致不能有效参与核糖体组装的MRP的积累，导致线粒体未折叠蛋白反应。影响：拟议的研究将提供对线粒体组装过程的机制性见解，阐明其对RNA加工的依赖性，并研究改变组装的病理学改变的后果，包括触发未折叠蛋白质反应。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导致的线粒体疾病的发病机制至关重要。