>Magister Scientiae - MSc / Retinoblastoma Binding Protein 6 (RBBP6) is a 200 kDa RING finger-containing protein that plays a role in 3'-end poly-adenylation of mRNA transcripts as well as acting as an E3 ubiquitin ligase against a number of proteins involved in tumourigenesis, including p53. Since the human protein is too large and poorly structured for heterologous expression in bacteria, it would be advantageous to identify smaller fragments suitable for expression in bacteria. Many E3 ubiquitin ligases form homo-dimers and dimerisation is important for their activity; structural studies of the isolated RING finger of RBBP6 showed that it forms a weak homo-dimer. This poses the question of whether the complete RBBP6 protein forms homo-dimers in vivo, and, if so, whether a fragment of RBBP6 containing the RING finger could be identified which would be suitable for structural as well as functional studies. Such a construct would allow detailed investigation of the homo-dimeric state of the fragment, the relationship between dimerisation and ubiquitination activity, and the role of domains such as the DWNN domain and zinc finger in ubiquitination. A fragment consisting of the first 335 residues of RBBP6, dubbed R3 because it contained the first three domains of the protein, was expressed, along with three variants expressing mutations known to disrupt the dimerisation of the isolated RING finger. Size exclusion chromatography showed that R3 forms a strong homo-dimer that was not disrupted by the mutations, suggesting that additional parts of R3 outside of the isolated RING finger form part of the interface. To identify whether this included the DWNN domain or the zinc finger, a shorter fragment dubbed R2, excluding the N-terminal DWNN domain, was cloned and expressed. This was also found to form a strong homo-dimer, suggesting that the DWNN domain may not form an essential part of the dimer interface. Availability of the RING finger samples and monomerising mutations allowed investigation of whether the RING finger from RBBP6 was able to auto-ubiquitinate itself. Using a fully in vitro ubiquitination assay supplemented with intact proteasomes purified from human cell lysates, we found that wild type RING auto-ubiquitinates itself very efficiently, catalysing its own destruction in the proteasome. This provides an answer to the question of why RBBP6 is so difficult to detect in mammalian cells. Surprisingly, monomeric mutant RING fingers were also able to auto-ubiquitinate and catalyse their own destruction, although perhaps not as efficiently as wild type. This result would appear to rule out the hypothesis that dimerisation of RBBP6 is required for ubiquitination activity. Finally, samples of the RING finger from human MDM2 were expressed in bacteria and used to investigate whether the RING fingers of RBBP6 and MDM2 interact directly with each other. If so, this may provide a mechanism whereby RBBP6 and MDM2 cooperate in ubiquitination of p53. The results of a GST pull down assay using GST-MDM2-RING as ''bait'' and RBBP6-RING as ''prey'' provides evidence that such an interaction between the RING does exist. This work lays the foundation for future structural studies of the RING-RING hetero-dimer using protein Nuclear Magnetic Resonance Spectroscopy.

Philosophiae Doctor - PhD (Biochemistry) / Human Retinoblastoma Binding Protein 6 (RBBP6) is a 200 kDa protein that has been implicated in a number of crucial cellular processes. It forms part of the mRNA 3'-end processing complex in both humans and yeast, and it contains an RS-like domain and interacts with core splicing proteins, suggesting multiple roles in mRNA processing. Through its RING finger domain it has been implicated in catalysing ubiquitination of the tumour suppressor p53, the oncogene Y-Box Binding Protein 1 (YB-1) and the DNA replication-associated protein zBTB38. It is one of only a few proteins known to bind to both p53 and pRb. At the N-terminus of the protein is the DWNN domain, an ubiquitin-like domain which is found only in this protein family. Four protein isoforms of RBBP6 have been identified in humans, all of which contain the DWNN domain: isoform 1 contains 1972 residues, isoform 2 contains 1758 residues and isoform 4 contains 952 residues. Isoform 3, which contains the first 101 residues of the full length protein (isoform 1), including the DWNN domain, followed by an unique 17-amino acid tail, is reported to be expressed independently of the other isoforms and to be down-regulated in a number of cancers.

Masters of Science / Retinoblastoma Binding Protein 6 (RBBP6) is a RING finger-containing protein which plays a critical role in the 3'-end processing of mRNA transcripts. It is a constituent of the human pre-mRNA processing complex but also interacts directly with core splicing-associated proteins. RBBP6 also interacts with both major tumour suppressor proteins p53 and pRb and is known to play a critical role in suppression of p53 during development, in cooperation with MDM2. Through its RING finger it interacts with the C-terminus of the oncogenic protein Y-Box Binding Protein 1 (YB-1) both in vitro and in vivo, catalysing its ubiquitination and degradation in the proteasome. YB-1 is closely associated with tumour progression, poor patient prognosis and chemotherapeutic resistance, making it a promising target for therapeutic intervention. Unpublished data from our laboratory suggests that RBBP6 is able to poly-ubiquitinate YB-1 in vitro, using UbcH1 as the ubiquitin- conjugating enzyme (E2). This study aims to identify RBBP6 RING protein-protein interactions involved in the down regulation of YB-1 by RBBP6. These interactions include the C-terminal fragment of YB-1 (substrate), MDM2 (E3) and UbcH1 (E2). The C-terminal fragment of YB-1, denoted YB-1₂₂₀₋₃₂₄, was successfully cloned and expressed in bacteria and demonstrated to interact directly with the RBBP6 RING finger domain in in vitro affinity pull down assays. This is in good agreement with our unpublished data that RBBP6 is able to ubiquitinate full length YB-1 as well as the YB-1₂₂₀₋₃₂₄ fragment. UbcH1 was successfully expressed and shown to interact directly with RBBP6 RING in in vitro affinity pull down assays. This is also in agreement with our data showing that RBBP6 is able to ubiquitinate YB-1 using UbcH1 as E2. ¹⁵N-labelled samples of RBBP6 RING was successfully expressed in bacteria and used to investigate the putative interaction with UbcH1 in NMR-based chemical shift perturbation assays. However no interaction was observed, possibly because the sample of UbcH1 was subsequently found using mass spectrometery to be partially degraded. GST-tagged RBBP6 RING was able to precipitate MDM2 from HeLa lysate. This extends previous reports that full length RBBP6 and MDM2 interact directly and play a role in the suppression of p53 during development. The result was validated by showing that GST-MDM2 was able to precipitate RBBP6 RING in in vitro. This study includes a side project which involved the cloning and expression of DWNN-GG. GST-HADWNN-GG was successfully cloned and expressed in bacteria. An HA tag was included immediately upstream of DWNN-GG for immunodetection using anti-HA antibodies; the construct was designed in such as way that it could be re-used to generate HA-tagged versions of existing constructs cloned into pGEX-6P-2. The above findings lay the foundation for future structural and functional studies of the involvement of RBBP6 in regulation of the cancer-related proteins p53 and YB-1, which may have far-reaching consequences in the fight against cancer. / National Research Foundation (NRF)

>Magister Scientiae - MSc / Retinoblastoma Binding Protein 6 (RBBP6) is a 200 KDa multi-domain protein that has been shown to play a role in mRNA processing, cell cycle arrest and apoptosis. RBBP6 interacts with tumour suppressor proteins such as p53 and pRb and has been shown cooperate with Murine Double Minute 2 (MDM2) protein in catalyzing ubiquitination and suppression of p53. Unpublished data from our laboratory has suggested that RBBP6 and MDM2 interact with each other through their RING finger domains. RBBP6 has also been shown to have its own E3 ubiquitin ligase activity, catalyzing ubiquitination of Y-Box Binding Protein 1 (YB-1) in vitro and in vivo. YB- 1 is a multifunctional oncogenic protein that is generally associated with poor prognosis in cancer, tumourigenesis, metastasis and chemotherapeutic resistance. Unpublished data from our laboratory shows that RBBP6 catalyzes poly-ubiquitination of YB-1, using Ubiquitin-conjugating enzyme H1 (UbcH1) as E2 ubiquitin conjugating enzyme. / 2022-02-25

Philosophiae Doctor - PhD / Retinoblastoma binding protein 6 (RBBP6) is a 250 kDa protein that is implicated in mRNA processing and ubiquitination functions and has been shown to be highly up-regulated in a number of cancers. In humans and mice,RBBP6 interacts with both tumour suppressors p53 and pRb, suggesting that it is involved in regulation of transcription, induction of apoptosis and cell cycle control. Knock-out of an RBBP6 homologue PACT resulted in p53 dependent cell cycle arrest and apoptosis. Although the biological functions of RBBP6 remain largely unclear, it is possible that its functions are mediated through interaction with other cellular proteins. Since it is possible to unveil novel functions of a target protein through identifying its interacting protein partners,this study aims to further characterize the functions of RBBP6 through identifying novel protein interacting partners using a yeast 2-hybrid screen.In order to identify interaction partners of RBBP6, two well characterized domains of RBBP6, the N-terminal ubiquitin-like DWNN domain and RING finger domain, were used as baits in a yeast 2-hybrid screen of a human testis cDNA library. Putative interactors were verified using in vitro and in vivo immunoprecipitation assays. The RING finger domain was shown to interact with transcriptional factors Y-Box binding protein 1 (YB-1) and zinc finger and BTB containing protein 38 (zBTB38), resulting in their ubiquitination. In the case of YB-1 ubiquitination was correlated with a decrease in the intra-cellular levels of YB-1, suggesting that ubiquitination leads to degradation in the proteosome. The DWNN domain was shown to interact with a splicing associated small nuclear ribonucleoprotein polypeptide G (snRPG) and heat shock protein 70 (Hsp70).The results of this work suggest that, at least in the case of YB-1 and zBTB38,RBBP6 plays a role in the regulation of gene expression by ubiquitination of transcription factors, causing them to be degraded in the proteosome. The study provides further evidence of RBBP6’s involvement in mRNA splicing through its interaction with snRPG. The interaction with Hsp70 suggests a possible role in protein quality control similar to that played by other E3 ligases such as Parkin and CHIP.

Philosophiae Doctor - PhD / Retinoblastoma binding protein 6 (RBBP6) is a 250 kDa protein that is implicated in mRNA processing and ubiquitination functions and has been shown to be highly up-regulated in a number of cancers. In humans and mice, RBBP6 interacts with both tumour suppressors p53 and pRb, suggesting that it is involved in regulation of transcription, induction of apoptosis and cell cycle control. Knock-out of an RBBP6 homologue PACT resulted in p53 dependent cell cycle arrest and apoptosis. Although the biological functions of RBBP6 remain largely unclear, it is possible that its functions are mediated through interaction with other cellular proteins. Since it is possible to unveil novel functions of a target protein through identifying its interacting protein partners, this study aims to further characterize the functions of RBBP6 through identifying novel protein interacting partners using a yeast 2-hybrid screen. In order to identify interaction partners of RBBP6, two well characterized domains of RBBP6, the N-terminal ubiquitin-like DWNN domain and RING finger domain, were used as baits in a yeast 2-hybrid screen of a human testis cDNA library. Putative interactors were verified using in vitro and in vivo immunoprecipitation assays. The RING finger domain was shown to interact with transcriptional factors V-Box binding protein 1 (YB-1) and zinc finger and BTB containing protein 38 (zBTB38), resulting in their ubiquitination. In the case of YB-1 ubiquitination was correlated with a decrease in the intra-cellular levels of YB-1, suggesting that ubiquitination leads to degradation in the proteosome. The DWNN domain was shown to interact with a splicing associated small nuclear ribonucleoprotein polypeptide G (snRPG) and heat shock protein 70 (Hsp70). The results of this work suggest that, at least in the case of YB-1 and zBTB38, RBBP6 plays a role in the regulation of gene expression by ubiquitination of transcription factors, causing them to be degraded in the proteosome. The study provides further evidence of RBBP6's involvement in mRNA splicing through its interaction with snRPG. The interaction with Hsp70 suggests a possible role in protein quality control similar to that played by other E3 ligases such as Parkin and CHIP.