In this study, I have shown that XStau 1 and 2 are expressed throughout oogenesis, embryogenesis and in the adult <i>Xenopus</i>. Immunostaining and confocal microscopy has demonstrated that both XStaul 1 and 2 localise to the vegetal cortex during oogenesis and colocalise with the ER but not the MC, at a time consistent with a role in the vegetal localisation of late pathway mRNAs. Biochemical analysis has shown that on meiotic maturation XStau 1 and 2 become phosphorylated and that this phosphorylation coincides with their rearrangement at the vegetal cortex in mature eggs, where they no longer co-localise with ER. Using gel filtration separation, I have shown that XStaufen 1 and 2 are part of large RNP complexes with a minor proportion present in smaller particles. RNA-independent ligands have been identified by co-immunoprecipitation with XStaul antibody, and have been sequenced by mass spectrometry and their identities confirmed by Western blot analysis and in <i>in vitro</i> binding assays. Binding patterns identified by immunoprecipitation did not include proteins with a role in mRNA localisation, but instead suggest a role for XStau1 in translational control and included CPEB, Xp54, p100, ePABP and XUpf1. This was confirmed in MS2 tethering assays where XStau1 but not XStau2 was shown to repress the translation, but not stability, of a firefly luciferase reporter mRNA 1.5 to 2 fold. These studies extend our understanding of the vertebrate Staufen proteins in the control of gene expression and indicate that in <i>Xenopus</i> oocytes, XStau1 mediates translational repression.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:595474 |
| Date | January 2007 |
| Creators | Allison, R. J. |
| Publisher | University of Cambridge |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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