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Title: Investigating human mitochondrial ribonucleases
Author: Bobrowicz, Agnieszka Joanna
ISNI:       0000 0004 2740 9754
Awarding Body: University of Newcastle Upon Tyne
Current Institution: University of Newcastle upon Tyne
Date of Award: 2011
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Mitochondria are present in all nucleated eukaryotic cells and generate virtually all the energy for cellular processes. Mitochondria possess their own genome, and mutations in mtDNA or in nuclear genes encoding mitochondrial proteins have been associated with a number of human disorders, and the decline of the activity and efficiency of the oxidative phosphorylation has been linked to the majority of age- related degenerative diseases. Investigation of the mechanisms that govern mtDNA expression is therefore critical to understand their role in physiological and disease conditions. Human mt-mRNA turnover remains one of the unsolved puzzles. Neither the possible pathways nor the enzymes involved have been described. Mitochondrial mRNAs are polyadenylated, but the role ofpoly(A) tails and the existence of a protective poly(A)-binding protein is uncertain. In the studies presented in this thesis the extended biochemical fractionation of mitochondria from rat liver or human cell line combined with in vitro enzymatic analyses was used to isolate putative ribonucleases involved in the metabolism of mitochondrial mRNA. This resulted in identification ofHRSP12 as a potential candidate. The intracellular localisation ofHRSP12 could not be determined unequivocally, and further study will assess whether any of the several splicing variants ofHRSP12 is indeed a mitochondrial enzyme involved in mRNA degradation. To improve future research into factors involved in mitochondrial mRNA degradation a modified purification strategy was initiated and optimised during the course of this work, with the aim to preserve the native interactions of putative multiprotein machineries controlling and facilitating mitochondrial gene expression. In an alternative approach to studying the mechanism of mt-mRNA degradation the role of polyadenylation in mitochondrial transcript stability was investigated. The exogenous poly(A)-exoribonuclease PARN was targeted to the mitochondria of human cell line. Ensuing deadenylation of mitochondrial mRNAs revealed that while the effect of deadenylation on the steady-state levels of transcripts varies between the mRNA species, polyadenylation is not an absolute requirement for stability of mt-mRNA. Moreover, following transcript deadenylation by exogenous PARN, synthesis of the mitochondrially-encoded polypeptides appeared to be compromised, therefore the results of this study strongly suggest that polyadenylation of mitochondrial mRNA is essential in regulating protein synthesis in human mitochondria.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available