Investigation of the dual role of mL62 during the mitoribosome assembly and rescue
by Venkatapathi Challa
Date of Examination:2024-06-11
Date of issue:2024-12-19
Advisor:Dr. Ricarda Richter-Dennerlein
Referee:Prof. Dr. Henning Urlaub
Referee:Prof. Dr. Ralph Kehlenbach
Persistent Address:
http://dx.doi.org/10.53846/goediss-10971
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Abstract
English
Mitochondria are eukaryotic organelles, which have their own DNA (mtDNA) that encode for 13 proteins that are essential for the oxidative phosphorylation. The mitochondrial ribosome (mitoribosome) is responsible for the synthesis of these 13 proteins. The 55S human mitoribosome is formed by a 39S large mitoribosomal subunit (mtLSU), consisting of 52 proteins, the 16S mt-rRNA and the mt-tRNAVal, and the 28S small mitoribosomal subunit (mtSSU), containing 30 proteins and 12S mt-rRNA. Mitoribosome biogenesis is a highly complex and hierarchical process. This involves a wide range of components essential for importing mitoribosomal proteins (MRPs), maturing the rRNAs and coordinating their stepwise assembly. The translation cycle follows the four steps of initiation, elongation, termination and ribosome recycling. Certain events such as mRNA truncation or aminoacyl tRNA starvation can stall the ribosome during translation. Such complexes need to be rescued. Two distinct ribosome rescue pathways have been suggested for the two release factors ICT1/mL62 and C12orf65/mtRF-R based on in vitro and structural studies. Besides of being a member of the mitochondrial release factor family, mL62 is also an integral constituent of the central protuberance (CP) of the mtLSU. This suggests a dual role of mL62 in mitoribosome rescue and assembly. However, all these data are based on in vitro studies, and it remain to be addressed whether mL62 is required as a rescue factor in native conditions and what are the consequences if mL62 as a rescue factor is missing. Similarly, based on structural analysis the association of C12orf65 with split mtLSU with a peptidyl-tRNA has been demonstrated suggesting that C12ORF65 is part of the mitochondrial ribosome-associated quality control (mtRQC), however, it is unclear how the stalled ribosome becomes dissociated in the first place and what are the consequences of C12ORF65 ablation. Therefore, in this current study I mainly focused on the dual role of mL62/ICT1 during mitoribosome assembly and rescue. Furthermore, the ribosome rescue function of mL62 was compared with C12ORF65 to ascertain the importance of both proteins in mitochondrial functions. In the absence of mL62 the ribosome assembly was severely impacted, mainly, the CP of the mtLSU is not able to form and join the mtLSU efficiently. The proposed late binding proteins like uL23m and the cluster of proteins that it belongs to are joining the mtLSU independent of the CP cluster. Recent structural studies demonstrated the order of GTPBP10 and GTPBP5 activity on the assembly of mtLSU and in this current study the same order of activity was observed. The loss of efficient mtLSU formation led to defective monosome levels and it further impacted the mitochondrial translation. Additionally, in the absence of mL62 catalytic activity the mitochondrial translation was also impacted but when it compared to C12ORF65 loss it demonstrated that C12ORF65 has more impact on the translation meaning that the C12ORF65 mediated mtRQC pathway is more important than the mL62 mediated ribosome rescue.
Keywords: Mitochondria; Mitoribosome; Mitoribosome assembly; Mitoribosome rescue; mL62; C12ORF65; Leigh syndrome; Ophthalmoplegia
