Eukaryotic translation elongation factor eEF1A exists in two closely related variant forms, eEF1A1 and eEF1A2, that are encoded by separate loci. The former is the second most abundant protein in the cell and is almost ubiquitously expressed but eEF1A2 expression is more limited and its presence was defined predominantly in neurons and muscle cells. Both perform equally well in translation elongation and are responsible for delivering aminoacylated tRNA to the A site of the ribosome in a GTP-dependent manner. Translation factor eEF1A2 was identified as an oncogene due to inappropriate expression being observed in the high proportion of ovarian, breast, lung, colon and pancreatic tumours. Additionally, its forced expression in rodent fibroblasts resulted in soft agar colony formation along with tumours when overexpressing cells were injected into nude mice. The mechanism by which eEF1A2 contributes to oncogenesis remains unclear. Gene amplification is not solely responsible for eEF1A2 upregulation and neither activating mutations nor methylation status changes are seen in tumours. Interestingly, no connection of eEF1A1 with any malignancy has been made. It is proposed that the oncogenic properties of eEF1A2 might be associated with its conventional role in translation or perhaps with non-canonical functions that differ from those of the eEF1A1 variant. The main objectives of this PhD project were to elucidate the differential functions of both variants of eEF1A in cancer and to investigate other possible mechanism of eEF1A2 upregulation. In order to compare the contribution of overexpressed eEF1A variants to cellular transformation, stable cell lines were generated in NIH-3T3 mouse fibroblasts and tested in a panel of in vitro transformation assays. Mammalian expression plasmids used for transfection contained each eEF1A variant coding sequence with or without its own 5‟UTR and each variant with the 5‟UTR from the other eEF1A form. Transient transfections with the same mammalian expression plasmids were performed to observe that incorporation of exogenous eEF1A1 and eEF1A2 resulted in a decrease of the endogenous eEF1A1 expression at the mRNA and protein level. The dynamic interplay between exogenous and endogenous variants occurred within the first 48 hours post transfection but Eef1a1 returned to the levels seen in controls as soon as the expression of any of the exogenous eEF1A forms started to decline. In contrast, in almost all tested stable cell lines, the levels of endogenous eEF1A1 remained unchanged, at both the mRNA and protein level. NIH-3T3 lines constitutively expressing eEF1A forms were subsequently subjected to various in vitro transformation assays. Stable cell lines of eEF1A1 coding sequence origin formed colonies and foci but with lower efficiency when compared to the eEF1A2 coding sequence variant. It was also shown that anchorage independent growth and foci formation were affected by incorporating either the eEF1A1 or eEF1A2 5‟UTR in front of either eEF1A1 or eEF1A2 coding sequence. There was no apparent increase in migration and invasion of the cell lines stably expressing eEF1A. No significant association between protein synthesis rate or increased overall eEF1A level and transformed phenotype in all tested stable cell lines was observed. Expression of eEF1A1 or eEF1A2 was also determined immunohistologically in panels of different tumour arrays. Moderate to high expression of eEF1A2 protein was observed in 43% of colorectal cancers analysed. The level of eEF1A2 expression appeared to be inversely correlated (P = 0.024) with metastasis in lymph nodes in one of the tested colorectal tumour arrays. Moreover, no substantial upregulation of eEF1A2 at the protein level was confirmed in hepatocellular carcinoma and malignant melanoma arrays. In contrast, eEF1A1 protein expression was mostly weak or absent in these malignancies.

Integration and expression of exogenous genetic material – in particular, transgenes – into the genomes of model organisms, cell lines or patients is widely used for the creation of genetically modified experimental systems and gene therapy. However, loss of transgene expression due to silencing is still a major hurdle which remains to be overcome. Judicious selection of integration loci can help alleviate the risk of silencing; in recent years the ability to efficiently and specifically target transgene integration has been improved by the advent of site-specific nucleases (SSNs). SSNs can be used to generate double strand breaks (DSBs) in a targeted manner, which increases the efficiency of homologous recombination (HR) mediated transgene integration into predetermined loci. In this work I investigate four human genomic loci for their potential to act as transgene integration sites which will support robust long term expression: the adeno-associated virus (AAV) integration site 1 (AAVS1); the human homologue of the mouse Rosa26 locus (hROSA26); the inosine monophosphate dehydrogenase 2 (IMPDH2) gene and the eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) gene. I also investigate the potential of creating a novel drug-selectable transgene integration system at the IMPDH2 locus to allow for rapid and specific selection of correctly inserted transgenes. In addition to their ability to drive targeted transgene integration, SSNs can be harnessed to specifically disrupt gene function through indel formation following erroneous repair of the induced DSB. Using this strategy, I aimed to answer some important biological questions about eukaryotic translation elongation factor 1 alpha (eEF1A); eEF1A is responsible for providing aminoacylated tRNAs to the ribosome during the elongation phase of protein synthesis. Humans and other vertebrates express two isoforms, eEF1A1 and eEF1A2 (encoded by EEF1A1 and EEF1A2 respectively). During development eEF1A1 is replaced by eEF1A2 in some tissues. The reasons for this remain elusive, but one explanation may lie in the moonlighting functions of eEF1A1, which may not be shared by eEF1A2. Additionally, eEF1A2 can act as an oncogene, while there is no evidence that eEF1A1 is overexpressed in tumours. To begin to untangle these issues I targeted EEF1A1 using SSNs with the aim of making a cell line expressing only the eEF1A2 isoform. This work suggests that eEF1A1 may be essential even in the presence of eEF1A2, though further studies will be required to confirm this.

Hepatitis delta virus (HDV) is the smallest known human RNA pathogen. It requires the human hepatitis B virus (HBV) for virion production and transmission, and is hence closely associated with HBV in natural infections. HDV RNA encodes only two viral proteins - the small and the large delta antigens. Due to its limited coding capacity, HDV needs to exploit host factors to ensure its propagation. However, few human proteins are known to interact with the HDV RNA genome. The current study has identified several host proteins interacting with an HDV-derived RNA promoter by multiple approaches: mass spectrometry of a UV-crosslinked ribonucleoprotein complex, RNA affinity chromatography, and screening of a library of purified RNA-binding proteins. Co-immunoprecipitation, both in vitro and ex vivo, confirmed the interactions of eEF1A1, p54nrb, PSF, hnRNP-L, GAPDH and ASF/SF2 with both polarities of the HDV RNA genome. In vitro transcription assays suggested a possible involvement of eEF1A1, GAPDH and PSF in HDV replication. At least three of these proteins, eEF1A1, GAPDH and ASF/SF2, have also been shown to associate with potato spindle tuber viroid (PSTVd) RNA. Because HDV’s structure and mechanism of replication share many similarities with viroids, subviral helper-independent plant pathogens, I transfected human hepatocytes with RNA derived from PSTVd. Here, I show that PSTVd RNA can replicate in human hepatocytes. I further demonstrate that a mutant of HDV, lacking the delta antigen coding region (miniHDV), can also replicate in human cells. However, both PSTVd and miniHDV require the function of the small delta antigen for successful replication. Our discovery that HDV and PSTVd RNAs associate with similar RNA-processing pathways and translation machineries during their replication provides new insight into HDV biology and its evolution.

Hepatitis delta virus (HDV) is the smallest known human RNA pathogen. It requires the human hepatitis B virus (HBV) for virion production and transmission, and is hence closely associated with HBV in natural infections. HDV RNA encodes only two viral proteins - the small and the large delta antigens. Due to its limited coding capacity, HDV needs to exploit host factors to ensure its propagation. However, few human proteins are known to interact with the HDV RNA genome. The current study has identified several host proteins interacting with an HDV-derived RNA promoter by multiple approaches: mass spectrometry of a UV-crosslinked ribonucleoprotein complex, RNA affinity chromatography, and screening of a library of purified RNA-binding proteins. Co-immunoprecipitation, both in vitro and ex vivo, confirmed the interactions of eEF1A1, p54nrb, PSF, hnRNP-L, GAPDH and ASF/SF2 with both polarities of the HDV RNA genome. In vitro transcription assays suggested a possible involvement of eEF1A1, GAPDH and PSF in HDV replication. At least three of these proteins, eEF1A1, GAPDH and ASF/SF2, have also been shown to associate with potato spindle tuber viroid (PSTVd) RNA. Because HDV’s structure and mechanism of replication share many similarities with viroids, subviral helper-independent plant pathogens, I transfected human hepatocytes with RNA derived from PSTVd. Here, I show that PSTVd RNA can replicate in human hepatocytes. I further demonstrate that a mutant of HDV, lacking the delta antigen coding region (miniHDV), can also replicate in human cells. However, both PSTVd and miniHDV require the function of the small delta antigen for successful replication. Our discovery that HDV and PSTVd RNAs associate with similar RNA-processing pathways and translation machineries during their replication provides new insight into HDV biology and its evolution.

Hepatitis delta virus (HDV) is the smallest known human RNA pathogen. It requires the human hepatitis B virus (HBV) for virion production and transmission, and is hence closely associated with HBV in natural infections. HDV RNA encodes only two viral proteins - the small and the large delta antigens. Due to its limited coding capacity, HDV needs to exploit host factors to ensure its propagation. However, few human proteins are known to interact with the HDV RNA genome. The current study has identified several host proteins interacting with an HDV-derived RNA promoter by multiple approaches: mass spectrometry of a UV-crosslinked ribonucleoprotein complex, RNA affinity chromatography, and screening of a library of purified RNA-binding proteins. Co-immunoprecipitation, both in vitro and ex vivo, confirmed the interactions of eEF1A1, p54nrb, PSF, hnRNP-L, GAPDH and ASF/SF2 with both polarities of the HDV RNA genome. In vitro transcription assays suggested a possible involvement of eEF1A1, GAPDH and PSF in HDV replication. At least three of these proteins, eEF1A1, GAPDH and ASF/SF2, have also been shown to associate with potato spindle tuber viroid (PSTVd) RNA. Because HDV’s structure and mechanism of replication share many similarities with viroids, subviral helper-independent plant pathogens, I transfected human hepatocytes with RNA derived from PSTVd. Here, I show that PSTVd RNA can replicate in human hepatocytes. I further demonstrate that a mutant of HDV, lacking the delta antigen coding region (miniHDV), can also replicate in human cells. However, both PSTVd and miniHDV require the function of the small delta antigen for successful replication. Our discovery that HDV and PSTVd RNAs associate with similar RNA-processing pathways and translation machineries during their replication provides new insight into HDV biology and its evolution.