Identification of the Main Translation Release Factor in Human Mitochondria

Mitochondria, the powerhouses of the cell, possess their own translational system seemingly inherited from their proteo-bacterial ancestor. Termination of translation in bacteria is mediated by Release Factors 1, 2 and 3 (RF1, 2 & 3), the first two of which recognise stop codons UAA, UAG and UGA, while RF3 enhances the efficiency of termination. Codon discrimination in RF1 is ascribed to a highly conserved three-peptide motif, namely 'PXT', in its anticodon-like domain. In human mitochondria where translation termination is triggered by four stop codons (UAA, UAG, AGG and AGA), only one candidate gene (MTRFl) was purported in silico to encode a functional mitochondrial release factor. Having searched the human genome database, we have found another candidate (UniprotKB/TrEMBL Q96EX4), potentially capable of acting as mitochondrial release factor based upon its high similarity to bacterial RFI and MTRFI. An alignment analysis of the highly conserved PXT motif revealed that the new candidate harbours the same three-peptide motif in the form of 'PKT', whereas the previously identified mtRF1 carried a hexa-peptide motif of'PEVGLS'. GFP fusion studies demonstrated co-localization of this new candidate with mitochondria. But to confirm whether this new candidate is actually imported into mitochondria, a radiolabelled product was generated in vitro and incubated with isolated rat liver mitochondria. N-terminal cleavage was demonstrated and the matured protein became insensitive to added proteinase, consistent with this protein accessing the mitochondrial matrix. In vitro translation termination assays indicated that this new candidate is capable of codon discrimination, recognising UAA and UAG as stop codons, thus implicating the new protein as the main translational release factor in human mitochondria. Further, this protein was shown to be capable of rescuing the non-respiratory phenotype of the I1mr/] yeast strains (8. cerevisiae and S. pombe) in non-fermentable carbon sources owing to restoration of mitochondrial respiration. RNAi knock-down of mtRF1a in human HeLa cells increased mitochondrial mass and ROS generation, in addition to retarding their growth in galactose medium consistent with the new candidate having a vital role in human mitochondrial respiration. This protein has now been re-designated as 'mtRF1a', mutations of which may prove responsible for mitochondrial disorders in some patients.EThOS - Electronic Theses Online ServiceGBUnited Kingdo