SNAMA is a 142 kDa Drosophila melanogaster protein, which consists of the uncharacterized conserved domain with no name (DWNN), zinc and RING finger-like motifs. The primary structure of SNAMA suggests that it might play an important role in cell cycle regulation and apoptosis. Previous studies revealed that homozygous SNAMA mutants underwent ectopic apoptosis which resulted in recessive lethality. SNAMA orthologues such P2P-R, PACT and RBBP6 are involved in cell cycle regulation, whereas Mpe1 is involved in mRNA processing. The aim of this study was to map out the role of SNAMA by isolating proteins which interact with it. DWNN was inserted into pGEX6P-2, phylexzeo plasmid (bait) and the Drosophila 0-12 hours cDNA library inserted in pJG4-5 (prey). The bait and the prey plasmid were used to transform appropriate yeast cells to probe for interacting proteins in yeast two hybrid assays, whereas the pGEX6P-2 was used for heterologous overexpression of DWNN in E. coli. Immunoprecipitation assays were also carried out with the crude protein extract from embryos, adult wild type, SNAMA mutant flies and the overexpressed protein using antibodies against SNAMA, Drosophila p53 Human DWNN and GST. The hybrid assay did not produce any interactors. Some of the proteins obtained from the immunoprecipitations were isolated and sequenced. The proteins identified were hsp82, Hsp70 and CG2985-PA. Data obtained from the immunoprecipitations suggest that SNAMA like Dmp53 might be involved in cell cycle regulation.

Master of Science - Science / DWNN-deficient Chinese Hamster Ovary cells have been found to be resistant to staurosporine-induced apoptosis. The human DWNN gene is located on chromosome 16p21, with 18 exons and is 36 kb long. It is alternatively spliced at exon 16 and makes two major mRNA transcripts, 1.1 and 6.1 kb, encoding 13 kDa and 200 kDa proteins respectively. The purpose of the study was to elucidate the possible role of DWNN in cervical cancer and apoptosis, to establish tissue distribution and expression levels of DWNN at protein and mRNA levels in cervical cancer. In situ hybridization studies showed elevated levels of the three mRNA transcripts in cervical cancer as compared to the normal tissues. The transcripts were localized in the nuclei of invaded stroma, moderately differentiated islands of tumours, dysplastic epithelium and some infiltrating lymphocytes. Immunocytochemistry showed that DWNN proteins were highly expressed in the dysplastic epithelium, dysplastic endocervical glands, moderately and well differentiated islands of tumours and the invaded stroma. Image analysis indicated elevated expression levels in the islands of tumours. Apoptosis detection by TUNEL revealed high apoptotic levels in the invaded stroma and moderately differentiated islands of tumours and this significantly correlated with DWNN localization. Proliferation assay using Ki67 antibody was found to be indirectly directly proportional to DWNN expression. Antiapoptotic Bcl-2 expression levels were found to be inversely proportional to the expression levels of DWNN. The up-regulated levels of DWNN in cervical cancers in contrast to normal tissues suggest DWNN to be proapoptotic, as there were elevated levels of apoptosis in the same sites where there were high levels of DWNN expression and Bcl-2 was down-regulated in the same sites. DWNN expression significantly correlated with apoptotic levels and was indirectly proportional to ki67 in human cervical cancers. Real Time PCR also confirmed the up-regulation in levels of DWNN in cervical cancer. This study suggests that the DWNN gene may be involved in apoptosis. Further characterization of this gene could lead to its manipulation as a diagnostic marker and a potential therapeutic target for cancer treatment.

Student Number : 9105022E - PhD thesis - School of Molecular and Cell Biology - Faculty of Science / SNAMA is the Drosophila melanogaster homologue of a group of proteins that are known to bind p53 and the retinoblastoma protein (Rb). This multi domain protein consists of a conserved N-terminal domain called Domain With No Name (DWNN), a zinc finger, a cysteine rich RING finger-like domain, a probable p53 binding region, and a glutamic acid-rich and lysine-rich region. These associated domains indicate that SNAMA plays an important regulatory role in the cell and may function in RNA processing and in apoptosis. The DWNN domain was first identified in Cytotoxic T-cell resistant Chinese hamster ovary (CHO) cells using promoter trap mutagenesis to screen for genes involved in apoptosis. Subsequently, this domain was identified in other eukaryotic organisms including animals and plants. The SNAMA transcriptional unit consists of 9 exons and 8 introns that code for a 1231 amino acid protein with the 76 residue N-terminal DWNN domain. The DWNN domain has a 23.5% sequence identity to the ubiquitin protein and a predicted folded structure similar to ubiquitin. Western blots identified multiple bands indicative of ubiquitin tagged proteins. Taken together this suggests a role in the ubiquitin pathway either as an ubiquitin domain protein or the DWNN domain of SNAMA tagging other proteins. The cysteine rich RING finger-like domain has a histidine to serine substitution at the fourth position of the putative RING finger and represents a distinct class of RING finger-like proteins that could have ubiquitin ligase activity. Northern blot analysis identified a single 4.6 kbp transcript expressed abundantly throughout development early in embryogenesis but reduced in older embryos and in adult male and females. SNAMA probably interacts with Dmp53 as a suppressor of apoptosis or a negative regulator of an activator of apoptosis. It is a vital gene required for development, as the mutant P-element insertion line in which the Pelement is inserted in the first intron of SNAMA is lethal when homozygous. Acridine orange staining of these mutant flies showed a direct correlation between the presence of SNAMA and apoptosis. An increase in the levels of apoptosis occurred in embryos with relatively low levels of SNAMA expression. The mode of this action is either direct, or via other proteins that are involved in the apoptotic pathway.

Retinoblastoma Binding Protein 6 (RBBP6) is a 200 KDa protein shown to play a role in 3'- polyadenylation of mRNA transcripts, as well as to function as an E3 ligase catalysing ubiquitination of cancer-associated proteins. RBBP6 has been previously reported to localise to nuclear speckles, which are thought to play a role in mRNA splicing, presumably as a result of its RS domain, which is known to target mRNA splicing factors to nuclear speckles. However recent studies in our laboratory have shown that isoform 3 of RBBP6, consisting mainly of the DWNN domain, also localises to speckles in resting cells, but more strongly in cells subjected to various stresses, suggesting that the DWNN domain may be the speckle-targeting domain. / Magister Scientiae - MSc (Biotechnology)

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.

>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.

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.