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  • The Functional Role of NRAP in the Nucleolus

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    Author(s)
    Inder, Kerry
    Primary Supervisor
    Kennedy, Derek
    Other Supervisors
    Gabrielli, Brian
    Year published
    2006
    Metadata
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    Abstract
    The nucleolus is the site for rRNA synthesis, a process requiring the recruitment of many proteins involved in ribosomal biogenesis. Nrap is a novel nucleolar protein found to be present in all eukaryotes. Preliminary characterisation of Nrap suggested it was likely to participate in ribosome biogenesis but as with many other nucleolar proteins, the functional role of Nrap is largely unknown. In this study, the role of mammalian Nrap in the nucleolus and in ribosome biogenesis was explored. Initially, a number of tools were generated to investigate Nrap function. This involved raising and purifying a polyclonal antibody ...
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    The nucleolus is the site for rRNA synthesis, a process requiring the recruitment of many proteins involved in ribosomal biogenesis. Nrap is a novel nucleolar protein found to be present in all eukaryotes. Preliminary characterisation of Nrap suggested it was likely to participate in ribosome biogenesis but as with many other nucleolar proteins, the functional role of Nrap is largely unknown. In this study, the role of mammalian Nrap in the nucleolus and in ribosome biogenesis was explored. Initially, a number of tools were generated to investigate Nrap function. This involved raising and purifying a polyclonal antibody against the N-terminal region of Nrap. The anti-Nrap antibody was found to detect two Nrap bands in mouse fibroblast cells, possibly corresponding to the two mouse Nrap isoforms, and . In addition, mammalian expression vectors containing the full Nrap sequence as well as deletion constructs were created. The subcellular localisation of each construct was observed by fluorescent microscopy. It was revealed that recombinant Nrap did not localise to the nucleolus, possibly because it was exported to undergo degradation by the 26S proteasome. Two putative NLSs were found to be responsible for directing Nrap to the nucleus but a region accountable for nucleolar localisation was not identified. The data indicated that multiple domains working together are likely to direct Nrap to the nucleolus. Nrap was also observed to co-localise with nucleolar proteins B23 and p19ARF. Moreover, it was shown by reciprocal immunoprecipitation that these three nucleolar proteins existed in a complex in unsynchronised mouse fibroblast cells. Recent reports demonstrated a complex relationship between B23 and p19ARF although the functional significance remained unclear. Nrap's in vivo association with B23 and p19ARF indicated a specific functional role in the nucleolus. Nrap knockdown using siRNA significantly increased B23 protein levels in a dose-dependent manner and down-regulated p19ARF protein levels at higher siRNA concentration. Preliminary studies also implicated Nrap in cell proliferation through these novel interactions. Both endogenous and recombinant Nrap were found to be highly unstable suggesting that Nrap might regulate B23 and p19ARF through its own tightly regulated stability. Finally, the role of Nrap in rRNA processing was investigated by northern blot analysis. Nrap knockdown was found to affect the levels of 45S, 32S and 28S rRNAs. The changes found may be a consequence of the concurrent perturbation in the levels of B23 and p19ARF caused by Nrap knockdown. As the results were not consistent with previous reports, it was likely that changes to rRNA processing could be contributed to Nrap loss of function. This study demonstrated for the first time a functional role of Nrap in rRNA processing possibly through its association with B23 and p19ARF.
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    Thesis Type
    Thesis (PhD Doctorate)
    Degree Program
    Doctor of Philosophy (PhD)
    School
    School of Biomolecular and Biomedical Sciences
    DOI
    https://doi.org/10.25904/1912/3452
    Copyright Statement
    The author owns the copyright in this thesis, unless stated otherwise.
    Item Access Status
    Public
    Subject
    NRAP
    novel nucleolar protein
    nucleolus
    eukaryotes
    ribosomal biogenesis
    Publication URI
    http://hdl.handle.net/10072/367738
    Collection
    • Theses - Higher Degree by Research

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