| dc.description.abstracteng | Translation termination is the last step of ribosomal protein synthesis. Dedicated translation factors are imported into mitochondria to convert the information stored in the retained mitochondrial DNA (mtDNA) and to facilitate protein synthesis on mitochondrial ribosomes (mitoribosomes), which. An aberrant expression of mtDNA and a defective mitochondrial translation cause OXPHOS deficiencies associated with severe human diseases.
The genetic code used by mitochondria differs from the universal one: only two conventional stop codons, namely UAG and UAA, are present. Additionally, two mRNAs, encoding for COX1 and ND6, are terminated by AGA and AGG, respectively, which have been re-assigned as non-standard stop codons. It remains to be addressed whether a dedicated release factor or ribosomal frameshifting, resulting in standard stop codons, is responsible for translation termination of these two stop codons. Despite sharing a common ancestor, the bacterial and mitochondrial translation machinery reveal significant differences. This is not only true regarding the structure and composition of the ribosome, but also for the involved translation factors such as termination factors. While in bacteria RF1 and RF2 are required to terminate translation, mtRF1a seems to be the main release factor in human mitochondria by terminating all mitochondrial open reading frames with UAG or UAA stop codons. Interestingly, human mitochondria harbor another release factor, mtRF1, whose function still remains elusive. Although in vitro measurements and high-resolution structural analysis provided mechanistic insights into mtRF1a-mediated peptide release, the consequences of loss of translation termination in human mitochondria remains to be addressed.
In this doctoral thesis, the role of mtRF1 and mtRF1a are analyzed biochemically in respective human knockout cell lines. The results demonstrate that both release factors are required for proper mitochondrial function and confirmed in vivo that mtRF1a is the major mitochondrial release factor responsible for terminating all mtDNA-encoded transcripts, except COX1. Also, MT-ND6, which also harbors a non-standard stop codon, is terminated by mtRF1a. In contrast, loss of mtRF1 leads to an isolated complex IV deficiency as it specifically terminates synthesis of its core component COX1. Furthermore, this work shows that cells developed several mechanisms to rescue this isolated COX deficiency. On the one hand, mitoribosome-associated quality control (mtRQC) pathway is activated, which rescues mitoribosomes stalled upon absence of mtRF1 to maintain certain levels of COX1. This pathway triggers the degradation of respective mRNAs to not overload the system. On the other hand, this thesis shows that remaining COX1 and thus complex IV is immediately incorporated into macromolecular respiratory supercomplexes to stabilize it and protect it from turnover.
Taken together, this thesis unravels the mystery regarding the function of mtRF1, verifies the role of mtRF1a in vivo and therefore advance our understanding regarding translation termination in human mitochondria. | de |