Deciphering structural and mechanistic characteristics of ribosome rescue pathways in mitochondria
Biochemistry
Final Report Abstract
The basic aspects of protein synthesis are shared between all kingdoms of life. It is a multistep process, which is catalyzed by ribosomes. Yet, organisms have evolved distinct structural components and regulatory mechanisms, which help to adapt to the respective requirements. Interestingly, eukaryotic cells harbor two separate protein synthesis pathways. While most proteins are encoded in the nucleus and translated in the cytoplasm, 13 proteins are encoded on mitochondrial DNA and translated in the mitochondrial matrix. Those proteins are all part of the OXPHOS system. Consequently, a correct mitochondrial translation is essential for a functional energy metabolism of cells. The final step of protein synthesis is translation termination. In this step, the ribosome reaches a stop codon, which is specifically recognized by a release factor mediating release of the fully translated protein. In human mitochondria, the canonical stop codons UAG and UAA are recognized by the canonical release factor mtRF1a. However, two out of the 13 mitochondrial transcripts do not possess these stop codons at the end of their open reading frames (ORFs). Instead, their ORFs terminate with AGG and AGA codons, respectively. Besides several attempts to characterize the release mechanism at these non-canonical stop codons, it has remained an unsolved research topic. In the first part of this research project, we characterized the mitochondrial release factor mtRF1 and demonstrate its involvement in the release at AGG/AGA codons. By performing next-generation RNA sequencing-assisted ribosome profiling in HEK293 mtRF1 KO cells, we reveal specific mitoribosome stalling events at AGG/AGA codons in the absence of mtRF1. In addition, using an in vitro reconstituted mitochondrial translation system, we demonstrate the specific peptide release activity of mtRF1 at these two codons. Finally, we investigate the cellular consequences, caused by the loss of mtRF1, and demonstrate transcript-specific downstream effects. Together, this part of the study not only unravels the function of mtRF1, but also provides a more comprehensive picture on the last steps of mitochondrial translation. In the second part of this project, we characterized the mitochondrial release factor mtRFR. Due to the lack of a codon recognition domain, mtRFR was suggested to act as a ribosome rescue factor recognizing stalled mitoribosomes. Furthermore, a recent cryo-EM study demonstrated its binding together with MTRES1 to the split large subunit of the mitoribosome, which might represent an intermediate in the rescue process. To investigate further the rescue mechanism of mtRFR, we performed immunoprecipitation of a Flag-tagged version of mtRFR and analyzed the eluate via mass-spectrometry and cryo-EM. Besides confirming the binding of mtRFR together with MTRES1 to the large subunit of the mitoribosome, our data suggests affinity of mtRFR to the small subunit/monosome. Yet, the binding mode remains elusive. Furthermore, we characterized the cellular consequences of loss of MTRFR. We demonstrate that the absence of mtRFR causes a strong defect on mitochondrial translation and OXPHOS complex activity, which, however, is not associated with specific stalling events of mitoribosomes on mitochondrial transcripts. In conclusion, our data support a fundamental role of mtRFR in mitochondrial translation, which needs to be elucidated in further studies.
Publications
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Human mitochondria require mtRF1 for translation termination at non-canonical stop codons. Nature Communications, 14(1).
Krüger, Annika; Remes, Cristina; Shiriaev, Dmitrii Igorevich; Liu, Yong; Spåhr, Henrik; Wibom, Rolf; Atanassov, Ilian; Nguyen, Minh Duc; Cooperman, Barry S. & Rorbach, Joanna
