Use this URL to cite or link to this record in EThOS:
Title: Control of expression and oncogenic potential of eEF1A2
Author: Wang, Yan
ISNI:       0000 0004 5991 3250
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2014
Availability of Full Text:
Access from EThOS:
Full text unavailable from EThOS. Thesis embargoed until 31 Dec 2100
Access from Institution:
In mammals, there are two isoforms of eukaryotic translation elongation factor 1A (eEF1A) called eEF1A1 and eEF1A2. They share 98% similarity at the amino acid level, and the main function of both is to facilitate the elongation process in protein translation. However, they have very different expression patterns. While eEF1A1 is universally expressed, eEF1A2 is strictly expressed in muscle, brain and heart. The over-expression of eEF1A2 has been found in cancers, such as ovarian and breast cancer. The factors influencing the different expression patterns of the two isoforms and the mechanisms by which eEF1A2 can act as an oncogene are not clear, therefore, the main aim of this study was to further investigate these two areas. The first aim was to find out whether the resveratrol induced down-regulation of eEF1A2 was mediated by miR-663. Western blotting in MCF7 cells showed that the level of endogenous eEF1A2 was decreased after resveratrol treatment while eEF1A1 remained stable. In contrast, NIH-3T3 stable cell lines which stably express the eEF1A2 coding sequence (CDS) only did not show this down-regulation, suggesting that the untranslated regions (UTRs) might play a role in this regulation. I then showed that miR-663 has ability to down-regulate a reporter linked to the UTRs of eEF1A2. The same reporter gene harbouring UTRs in which the binding sites of miR-663 had been deleted also showed down-regulation after resveratrol treatment, suggesting that the UTRs of EEF1A2 are key to the down-regulation of eEF1A2 by resveratrol but that miR-663 does not mediate this decrease. The second project aimed to address why eEF1A2 is an oncogene but eEF1A1 is not. The 3D structure of human eEF1A1 and eEF1A2 shows that the most of the highly conserved amino acids differences between the two isoforms are Ser and Thr residues, which are potential sites for phosphorylation. I mutated these three sites in eEF1A2 expression constructs to the equivalent amino acid from eEF1A1. Firstly, by transient transfection, all the mutant eEF1A2 were shown expressed and the sub-cellular locations of eEF1A2 remain unchanged after site-directed mutagenesis. Then, stable cell lines were generated. Anchorage independent growth (soft agar) and focus formation assays showed that the stable cell lines harbouring wild type eEF1A2 were significantly more transformed that those expressing the eEF1A2 mutants. However, there was no apparent difference in global protein synthesis between these cell lines. The results suggest that the potential phosphorylated sites in eEF1A2 play an important role in its oncogenicity and that this oncogenicity is not related to the canonical function of eEF1A2.
Supervisor: Abbott, Catherine ; Evans, Kathy Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: eukaryotic translation elongation factor ; eEF1A2 ; cancer ; resveratrol ; UTRs ; phosphorylation