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Title: Poly(A) binding proteins in human mitochondria
Author: Wydro, Mateusz
ISNI:       0000 0001 3573 5339
Awarding Body: Newcastle University
Current Institution: University of Newcastle upon Tyne
Date of Award: 2008
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Mitochondria are organelles present in all nucleated eukaryotic cells. They play a central role in the conversion of metabolic fuels to a readily ·utilisable source of energy in the form of ATP. One of the most distinct features of mitochondria is the possession of their own genome (mtDNA). The mammalian mitochondrial genome encodes only 13 peptides, all of which all are involved in oxidative phosphorylation, in addition to all the RNA components necessary for the intra-mitochondrial translation machinery. Since mitochondria comprise greater than 1000 proteins, the majority of mitochondrial proteins are encoded by the nucleus, synthesized in the cytosol and then imported into mitochondria. In mammals, all mRNA species encoded by mtDNA exhibit constitutive oligo- or poly- adenylation. The poly(A) tails are generally 50-60 nt in length and stable, suggesting that they are protected from nucleases, potentially by the existence of a mitochondrial poly(A) binding protein. In the eukaryotic cytosol, a number of forms of poly(A)-binding proteins (PABPs) exist and are central to the regulation of RNA stability and translation. To date, no such protein has been isolated from mammalian mitochondria. Extensive in silico studies have not yielded any candidates, and so a biochemical approach with an extended FPLC purification scheme has been used to isolate the putative candidates for this activity from rat liver mitochondria. A subset of fractions .derived from this purification have been analysed by electrophoretic mobility shift assay and a number have demonstrated specific poly(A) binding activity using an in vitro derived radiolabelled substrate. The Mass spectro~copy analysis of these 'poly(A) binding' fractions indicated that the highest abundance proteins are ACATI and ACAA2. These are mitochondrial proteins with well characterised metabolic activities but no recognised RNA binding motifs. Over- Poly(A) binding proteins in human mitochondria Mateusz Wydro expression of recombinant ACATI and ACAA2 followed by in vitro studies revealed that both candidates exhibit specific poly(A) binding with weak affinity. The physiological role of this interaction, if any, remains unclear. A second approach that I have taken has been to target a known cytosolic poly(A) binding protein, PABPC1, to the human mitochondria in an inducible manner and observe any consequent changes in mt-mRNA metabolism. Here, I show that mitochondrially targeted (mt-PABPCI) is able to interact with the mitochondrial mRNA poly(A) region. Binding of mt-PABPCI resulted in shortening of all investigated mitochondrial mRNAs, but does not induce a concomitant loss of RNA stability. In response to mt-PABPC1 binding, however, mitochondrial translation is compromised, leading to a severe respiratory phenotype, consistent with the loss of mitochondrially-encoded polypeptides. I conclude that the mitochondrial translation inhibition is due to mt-PABPCI competition with endogenous protein(s) that interact with the poly(A) that are necessary to promote mitochondrial protein synthesis.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available