>Magister Scientiae - MSc / Retinoblastoma binding protein 6 (RBBP6) is a 250 kDa RING finger-containing protein whose function is known to be mediated through interaction with other proteins. RBBP6 plays a role in the regulation of the tumour suppressor protein p53 and is also thought to be involved in mRNA splicing although its role has yet to be characterised. A recent study utilising a yeast 2-hybrid screen identified the cancer-associated protein known as YB-l as an interacting partner of RBBP6, and showed that RBBP6 ubiquitinates YB-I, leading to its degradation in the proteasome.Human Y-box binding protein 1 (YB-I) is member of the cold-shock domain family of proteins, which regulates a number of growth related genes through both transcriptional and translational mechanisms. YB-l is a cell-survival factor whose expression is increased in proliferating normal and cancer cells. It also protects cells against p53-mediated apoptosis by repressing the p53- promoter and down-regulating endogenous p53. The interaction between RBBP6 and YB-l involves the RING finger-like domain ofRBBP6 and the C-terminal62 amino acids ofYB-l. As a means of further localising the interaction, truncated fragments derived from the C-terminal region of YB-I, were tested for their interaction with the RING finger domain of RBBP6 using three different assays: a directed yeast 2-hybrid assay, co-immunoprecipitation and NMR chemical shift perturbation analysis. Our results suggest that the entire 62 amino acid region at the C-terminal domain ofYB-l may be involved in the interaction with RBBP6. Using chemical shift perturbation analysis, this study provides an indication of where YB-l binds to the RING fmger. This represents the first step towards the design of therapeutics aimed at modulating the interaction between RBBP6 and YB-l as a means of targeting the oncogenic effects ofYB-l. In order to identify E2 enzymes involved in the ubiquitination of YB-I, we examined the efficiencies of selected E2s in an in vitro ubiquitination assay. UbcH5c and UbcH7 were both found to catalyse the ubiquitination of YB-l in conjuction with RBBP6, whereas Ubc 13 was not. Finally, we show using NMR that two single-point mutations of the RING finger-like domain are sufficient to abolish homodimerisation of the domain. These will be used in future studies to investigate the requirement for homodimerisation on the ubiquitination activity of RBBP6. www.etd.

>Magister Scientiae - MSc / This thesis describes the cloning and recombinant expression of domains from the human RBBP6 protein for future in vitro binding studies with pRb and p53. RBBP6 is a splicing-associated protein that is known to interact with both p53 and the Retinoblastoma gene product (pRb), and has recently been shown to be highly upregulated in oesophageal cancer. The pRb binding domain (RbBD) and the p53 binding domain (p53BD) were each expressed using the glutathione-S-transferase (GST) tag affinity system, and affinity purified using a glutathione-linked agarose column. Purified fusion proteins were cleaved to separate the target protein from GST using PreScission ™ Protease, for which there is a recognition sequence located immediately upstream of the multiple cloning site on the pGEX-6P series of plasmids. The pRb binding and p53 binding domains were further purified using cation exchange chromatography. Mass spectrometry confirmed that the RbBD was expressed as a single species of the expected molecular weight. However preliminary NMR analysis suggested that the domain was not fully folded. A total yield of 8 mg of protein was achieved from 11 of culture, which make it feasible to express 15Nand 12Clabelled samples for NMR. The p53BD was found to be expressed at lower levels and subject to C-terminal degradation, which suggest that the C-terminus is unstructured most likely due to the presence of polylysine tail. Human pRb protein was also successfully expressed and purified using the GST affinity system. Human p53 protein was expressed but was found to be insoluble and attempts to purify it were not pursued. Attempts to confirm the interactions between human RBBP6 and p53 and pRb proteins are on-going but fall outside the scope of this thesis. Expression constructs for the RING and zinc finger domains from human RBBP6 were also cloned into the pGEX system for future structural studies using NMR. Both domains were found to be expressed as soluble fusion proteins in preliminary expression studies.

<p>The aim of this thesis was to investigate the cellular role of the mouse RBBP6 gene using the interference RNA (RNAi) gene targeting technology and also to understand the relevance of two promoters for the RBBP6 gene.</p>

<p>The aim of this thesis was to investigate the cellular role of the mouse RBBP6 gene using the interference RNA (RNAi) gene targeting technology and also to understand the relevance of two promoters for the RBBP6 gene.</p>

Philosophiae Doctor - PhD / The aim of this thesis was to investigate the cellular role of the mouse RBBP6 gene using the interference RNA (RNAi) gene targeting technology and also to understand the relevance of two promoters for the RBBP6 gene. / South Africa