Human ras-GAP120 SH3 domain-binding protein (G3BP) was first identified by its co-immunoprecipitation with rasGAP120, a key regulatory molecule in the ras signalling pathway. Around the same time, a second gene with high homology to G3BP was identified during a general screen for RNA-binding proteins. The two genes were later termed G3BPl and G3BP2 in order of their discovery. G3BP2 has three spliced isoforms (G3BP2a, b, c,), two of which are known to produce proteins (G3BP2a and b). Both G3BPl and G3BP2 (G3BP2a and G3BP2b) have an N-terminal NTF2-like domain, an acid rich domain, multiple PXXP motifs (the minimal consensus for SH3 domain binding) and two domains commonly found in RNA-binding proteins, an RNA recognition motif and an RGG domain. Since their discovery, the G3BP family of proteins have been implicated in ras signalling,
c-myc RNA turnover, NF-kappaB signalling and the ubiquitin proteosome pathway.
The aim of this PhD project was to characterise the G3BP family of proteins. Little was known about G3BP family of proteins when the project was initiated. At the time, G3BPs were implicated in ras signalling and c-myc RNA turnover, two pathways involved in cancer progression. This project was therefore initiated by examining the expression of G3BPs in adult mouse tissues and human cancers. The results presented in Chapter 3 show that G3BPl and G3BP2 proteins are expressed in a tissue and cell specific manner compared to their RNA expression which has previously been shown to be ubiquitous. Some tissues expressed all isoforms of G3BP whereas others only expressed one isoform. The expression of G3BP2 was also examined in human cancers. G3BP2 was inappropriately expressed in a high percentage of human breast tumours. No
significant relationship was found between the expression ofG3BP2 and the stage of the tumour suggesting that the inappropriate expression of G3BP2 is an early event in tumour progression. G3BP2 was also shown localised to the nucleus in several cases of breast cancer suggesting that G3BP2 can shuttle between the nucleus and cytoplasm. The localisation of G3BP2 was further examined during cell cycle progression. G3BP2 was shown to shuttle between the nucleus and cytoplasm during cell cycle progression.
The aberrant expression of G3BP2 in human breast tumours prompted exploration into the regulation of G3BP expression. The experiments, presented in chapter 4, were aimed at determining whether the cis-elements in the 3'UTRs of G3BPs were regulating the expression of these proteins. Luciferase reporter assays were used to determine whether deletion of potential regulatory elements found in the 3'UTRs of G3BPs would alter the level of protein expression. The
results showed no significant difference between the wild type 3'UTRs and mutant 3'UTRs. Chapter 4 also reports the identification of a novel splice variant in the 3'UTR ofMmuG3BP2.
In chapter 5, several web-based bioinformatic resources were utilised to search for additional members of the G3BP family across different species. Three novel homologues of G3BP were identified in zebrafish and were named zfG3BPα, zfG3BPβ and zfG3BPγ. Chapter 5 then explored the in vivo function of zfG3BPs using zebrafish as a genetic model organism. Morpholino antisense oligonucleotides (morpholinos) were used to study the effect of zfG3BP knock-down in zebrafish embryos. Overexpression studies were also performed by injecting in vitro transcribed mRNA into zebrafish embryos. The experiments showed that G3BPs are important for zebrafish development. All three zebrafish homologues were shown to be important for
the development of blood circulation in the yolk sack of embryos, most likely through vasculogenesis. In addition to the defects in blood circulation, the knock-down of zfG3BPβ in zebrafish produced embryos with bent notochords and knock-down of zfG3BPγ severely retarded the development of the zebrafish embryos, possible due to abnormal blood circulation. zfG3BPs are therefore important for normal embryo development in zebrafish. Since early embryonic development is highly conserved throughout the vertebrates, the study of G3BPs in zebrafish will provide direct implications for human embryonic development.