Deciphering structural and mechanistic characteristics of ribosome rescue pathways in mitochondria

线粒体是一种半自主的细胞器，对大多数真核细胞的内环境稳定至关重要。它们拥有自己的基因组，在线粒体基质中转录和翻译。由于特定的线粒体基因组编辑技术以及体外翻译系统的不可用，我们缺乏对线粒体中翻译过程的详细了解。然而，最近冷冻EM技术和蛋白质组学方法的改进首次使人们能够深入了解线粒体翻译。但是如果在蛋白质合成过程中出现问题会发生什么呢？到目前为止，线粒体中的核糖体拯救途径的信息很少，我们将在拟议的项目中解决这个问题。我们的工作将主要集中在C12 orf 65和mtRF 1，这两个基因与细菌释放因子具有同源性。尽管最近的cryo-EM结构揭示了涉及C12 orf 65的线粒体拯救机制的第一个快照，但关于这一过程中事件的顺序以及其他因素的参与的信息仍然很少。为了进一步了解C12 orf 65介导的拯救机制，我们将对分离的C12 orf 65-线粒体复合物进行冷冻电镜分析。根据我们的初步数据，我们将能够解决涉及C12 orf 65的几个平移状态，从而为现有知识增加重要的细节。为了解开未知蛋白mtRF 1的功能，我们将首先探索mtRF 1的相互作用组。我们将应用几种策略来捕获mtRF 1-线粒体相互作用，这将连续用于mtRF 1拯救机制的结构研究。此外，我们将探索应用单分子技术结合时间分辨冷冻电镜的线粒体拯救的动力学。最后，为了在机制水平上理解拯救过程，我们将联合收割机结构研究与功能测定相结合，包括下一代RNA测序辅助核糖体分析和细胞培养中的其他生化测定。总之，该项目将为线粒体翻译的质量控制提供详细的机制见解，这可能为线粒体疾病的治疗提供新的靶点。

Mitochondria are semi-autonomous organelles, which are essential for the homeostasis of most eukaryotic cells. They harbor their own genome, which is transcribed and translated within the mitochondrial matrix. Due to the unavailability of specific mitochondrial genome editing techniques, as well as in vitro translation systems, we lack a detailed understanding of the translation process in mitochondria. Yet, recent improvements of cryo-EM techniques and proteomic approaches enabled first insights into mitochondrial translation. But what happens if something goes wrong during protein synthesis? So far, there is little information about ribosome rescue pathways in mitochondria and we will address this topic in the proposed project. Our work will mainly focus on C12orf65 and mtRF1, which show homology to bacterial release factors. Even though a recent cryo-EM structure revealed a first snapshot of the mitochondrial rescue mechanism involving C12orf65, there is still little information on the sequence of events during this process and the involvement of other factors. To gain further insights into the C12orf65-mediated rescue mechanism we will perform cryo-EM analysis on isolated C12orf65-mitoribosome complexes. According to our preliminary data, we will be able to resolve several translational states involving C12orf65 and consequently add significant details to the current knowledge. To unravel the function of the uncharacterized protein mtRF1, we will first explore the interactome of mtRF1. We will apply several strategies to trap mtRF1-mitoribosome interactions, which will consecutively be used for structural studies on the mtRF1 rescue mechanism. Additionally, we will explore the dynamics of mitoribosome rescue applying single-molecule techniques combined with time-resolved cryo-EM. Finally, to understand the rescue processes on a mechanistic level, we will combine structural studies with functional assays, including next-generation RNA sequencing-assisted ribosome profiling and other biochemical assays in cell culture. Together, this project will provide detailed mechanistic insights into quality control of mitochondrial translation, which might offer new targets for the treatment of mitochondrial diseases.