Deciphering structural and mechanistic characteristics of ribosome rescue pathways in mitochondria

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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.