Mechanism of Mitochondrial Ribosome Assembly

 描述(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，只允许表达13种蛋白质，这些蛋白质都是呼吸所需的复合体的基本亚基。这些蛋白质被翻译到线粒体核糖体上，线粒体核糖体是由三个核糖体RNA与大约80个输入线粒体的核编码蛋白组合而成的。这些线粒体核糖体蛋白(MRPs)直到最近才被发现，其中包括MRPS16、MRPS22、MRPL3和MRPL44等几个蛋白与遗传性遗传疾病有关。尽管它至关重要，但有丝分裂体组装的途径实际上是未知的，这确立了这是一个肥沃的研究课题。在氨基糖苷类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，导致有丝分裂蛋白失衡，也可能是随着年龄的增长而观察到的进行性线粒体功能障碍的原因。我们建议使用我们开发的一种新的方法来研究线粒体核糖体组装，该方法使用细胞培养中的稳定同位素脉冲追赶标记(脉冲追赶SILAC)和质谱学。我们广泛的初步结果表明，某些蛋白质与新合成的rRNA结合在mtDNA类核上，可能是在转录继续的同时；这些蛋白质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核糖体紧密相连后才加入。我们建议使用改进的脉冲追逐方法来提高对有丝分裂核糖体组装的机制、动力学和效率的理解。最近的冷冻电子显微镜研究发现，tRNAvaline是大亚基的一种新成分。我们将测试一个模型，在该模型中，转录的12S rRNA-tRNAval-16S rRNA必须与新合成的MRP的结合相协调，才能被切割成三个单独的RNA。我们将确定新合成的tRNAval是否通过转录耦合组装过程与转录的16S rRNA一起被整合到核糖体中，或者是否先前存在的tRNAval拷贝被招募到核糖体中。我们将研究当系统受到单个MRP的耗尽或本身不是核糖体成分的组装因子的干扰时，组装过程是如何扭曲的。鉴于我们发现早期组装发生在线粒体DNA类核，我们将继续努力研究具有无序类核结构的细胞中的有丝分裂体组装。我们假设，如果改变类核结构影响rRNA的合成和处理，这可能导致MRP的积累，而这些MRP不能有效地参与核糖体组装，从而导致线粒体未折叠蛋白反应。影响：拟议的研究将提供对有丝分裂体组装过程的机械性洞察，阐明其对RNA加工的依赖，并调查改变组装的病理变化的后果，包括触发未折叠的蛋白质反应。从长远来看，这将为大大提高对有丝分裂体生物发生的了解铺平道路，这对于了解线粒体翻译缺陷引起的线粒体疾病的发病机制是必不可少的。

 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.