Dynamics and regulation of the mitochondrial translation machinery

by Thomas Schöndorf
Doctoral thesis
Date of Examination:2023-12-14
Date of issue:2024-11-15
Advisor:Prof. Dr. Peter Rehling
Referee:Prof. Dr. Marina Rodnina
Referee:Prof. Dr. Markus Bohnsack
Referee:Dr. Johannes Söding
Referee:Prof. Dr. Stefan Jakobs
Referee:Prof. Dr. Ralph Kehlenbach
Persistent Address: http://dx.doi.org/10.53846/goediss-10866

 

 

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Abstract

English

Throughout this project, we have successfully established and implemented a ribosome profiling protocol tailored for examination of translating mitochondrial ribosomes, thereby affording a deeper understanding of mitochondrial translation dynamics. Our adapted Ribosome profiling approach has uncovered instances of ribosomal stalling and alterations in translation velocity, potentially implicating a direct connection with co-translational membrane insertion. We were able to shed further light into the co-translationally regulation of translation speed for mRNAs encoding proteins within the ND2-module of complex I, particularly in response to the perturbation introduced by the loss of MITRAC15. Furthermore, our investigation into the consequences of C12ORF62 depletion, which has been demonstrated to induce a high reduction in COX1 protein levels, while the impact at the mRNA level remains comparatively reduced. Additionally, we validated the specificity and efficacy of a morpholino-based approach for translational inhibition within isolated mitochondria. The deployment of morpholinos designed to target specific segments of COX1, COX2, and ND2 mRNAs has afforded an insightful perspective on their respective effects, manifesting as alterations in ribosomal footprints. In summary, our refined ribosome profiling methodology has yielded an in-depth comprehension of the nuanced dynamics governing mitochondrial translation. This includes an elucidation of early co-translational events and the elucidation of key regulatory roles played by factors such as MITRAC15 and C12ORF62. This investigative framework can be further supplemented with complementary biochemical assays to enhance our knowledge of mitochondrial translation, including the precise mapping of binding sites for assembly factors and translational activators. The integration of RNAseq analysis holds the potential to enhance the congruence between ribosomal footprints and de novo protein synthesis. Furthermore, the refinement of nuclease-based techniques may pave the way for investigations of individual codons, as well as the exact cofactor interaction sites.
Keywords: translation; mitochondria; ribosome