During the elongation of the polypeptide chain in eukaryotic protein synthesis, GTP-bound eukaryotic translation elongation factor 1A recruits the aminoacyl tRNA to the A-site of the ribosome. The GDP-GTP recycling is catalysed by the elongation factor 1B complex (eEF1B) which in higher eukaryotes consists of three different subunits: alpha, delta and gamma. Previous studies on eEF1B focused mainly on biochemical analysis and reports of overexpression in tumours and correlation to decreased survival rate but not a lot is known about is biology. The aim of this PhD is to characterise the eEF1B subunits at the molecular level in view of their potential involvement in tumourigenesis using a variety of bioinformatic and laboratory techniques. All three subunits were found to be ubiquitously expressed at mRNA and protein levels in all mouse tissues analysed. In addition, eEF1Bβ has several transcript variants in mice derived from alternative splicing and multiple isoforms, including a brain and testis specific heavier isoform and a muscle-specific form in addition to other forms. The characteristics of each eEF1B subunit were catalogued by further bioinformatic analysis. eEF1Bα was not detectable at early mouse developmental stages, eEF1Bβ showed stronger expression at pre-natal and early post-natal stages than adult stage whereas eEF1Bγ is ubiquitously expressed at similar levels throughout mouse development. In adult mice and human tissues, eEF1B subunits appeared to be expressed in different cell types and cell sub-populations. Surprisingly, cytoplasmic and some nuclear expression was observed in vivo. This nuclear expression pattern could not be observed in cell lines and it was not related to the cell cycle stage in vitro. The expression of eEF1B subunits did not change during the cell cycle except eEF1Bγ which was highly expressed in S-phase arrested cells. Knockdown by siRNAs of eEF1B subunits leads to decreased proliferation, increased number of cells in G0/G1 phase and increase in apoptosis in HeLa, HCT116, DLD1 and HepG2 cells. In contrast, overexpression in HeLa cells with a V5-tagged constructs lead to increased proliferation, increased number of cells in the G2/M phase and increased viability. Knockdown of eEF1Bα and eEF1Bβ leads to a reduction in eEF1Bγ levels; it is therefore possible that the phenotype shown by the knockdown of each subunit individually might be due to the reduced levels of eEF1Bγ. However, overexpression of each subunit did not affect the protein levels of the other subunits. The presence of multiple forms, the complex expression pattern and distribution of each eEF1B subunit in mouse and human tissues, and the knockdown and overexpression effect on cells suggests that the eEF1B complex might have different quaternary forms throughout development and in different cell types, possibly a more intricate role in translation, potential non-canonical functions any of which may be implicated in the potential role of eEF1B subunits in tumourgenesis.

Eukaryotic protein translation elongation is tightly controlled by several regulation factors. Eukaryotic translation elongation factor 1B (eEF1B) is the GTP exchange factor for eukaryotic translation elongation factor 1A (eEF1A), which is a G-protein transporting aminoacyl-tRNA to the A site of the ribosome in a GTP dependent manner. The structure of the heavy complex composed of eEF1B and eEF1A (eEF1H) has been widely studied and several models have been proposed, but it is yet not clear how the subunits of the two proteins interact with each other. eEF1B is made up of three subunits, eEF1Bα, eEF1Bδ and eEF1Bγ, and each subunit has been found to be over expressed in different types of cancer. A copy number variant near the eEF1Bδ gene is associated with amyotrophic lateral sclerosis. The two isoforms of eEF1A, eEF1A1 and eEF1A2, are 92% identical, but only eEF1A1 was found to interact with eEF1B subunits in yeast two hybrid (Y2H) experiments. The aims of this PhD project are to investigate the potential involvement of eEF1B in disease, as well as the relationship between eEF1B and eEF1A2. All three eEF1B subunits were present in almost all the cell types and mouse tissues tested. eEF1Bδ showed different variants, the heaviest of which is tissue specific and expressed only in brain and spinal cord. eEF1Bα and eEF1Bδ showed certain abnormalities in transformed cell lines, although in the breast cancer tissues tested no apparent change in eEF1B expression was found. Knockdown of eEF1B did not significantly affect NSC34 cell viability over short periods. In spinal cord sections from motor neurone disease (MND) patients, half of the cases showed a change of eEF1B protein expression compared to normal spinal cord, with either a higher level in glial cells, or a lower level in motor neurones. eEF1B and eEF1A2 were found to be co-expressed in mouse motor neurones, and proximity ligation assay also detected physical interactions between both eEF1A isoforms and eEF1B subunits in mammalian cells, contrary to the previous Y2H study. Experiments in a mouse model with no eEF1A2 expression also support this finding. In heart and skeletal muscle from wasted mice where eEF1A is absent the expression of eEF1Bα and eEF1Bδ was down regulated at both protein and mRNA level, suggesting that eEF1A2 and eEF1B not only physically interact, but also show an interdependence in expression. Overall the results from cultured cells, mouse and human tissues in this study demonstrate the potential involvement of eEF1B in MND, and its interaction with eEF1A, which contributes to the understanding of the non-canonical functions of eEF1B and the structure of eEF1H.