Brennan, Ruth (2013) Investigating the cellular localisation of DEAD box helicase DDX3. PhD thesis, National University of Ireland Maynooth.

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Abstract

DDX3 is an RNA helicase that has been shown to have a range of nuclear and cytoplasmic functions, including transcription, translation, splicing and mRNA export. DDX3 has also been shown to play a role in innate immune signalling and tumorigenesis. DDX3 is targeted by multiple viruses: HIV, HCV , HBV and Vaccinia Virus have all been shown to interact with DDX3; either using DDX3 to replicate or inhibiting DDX3’s function in antiviral signalling. Human DDX3 was reported to be exported from the nucleus independently of its N-terminal NES and to interact with CRM-1 through DDX3's C-terminal region. It was suggested that DDX3 acts as a CRM-1 cofactor, rather than cargo, and that it mediates Rev-dependent export of HIV RNAs. I confirmed that DDX3 exports in a CRM-1 dependent manner, and DDX3's N-terminus is required for nuclear export. We have also investigated the nuclear import of DDX3. Putative NLS were found using bioinformatic software, and tested for functionality by mutating key basic residues. We also used a range of nuclear import inhibitors to examine how DDX3 is imported into the nucleus. We found that the DDX3's two Rec-A like domains could be imported independently, and also that DDX3 imported independently of Importin- and Calmodulin. We were also interested as to whether DDX3's cellular localisation was regulated during viral infection, cellular stress and during the cell cycle. We investigated the relationship of DDX3 with HIV and HCV viral proteins, and found that both HIV and HCV target the cellular localisation of DDX3. We found that DDX3 directly interacts with HIV-Rev protein, and also that HIV-Rev recruits DDX3 to the nucleolus. We also found that HCV Core protein recruited DDX3 to cytoplasmic speckles, and prevented the nucleocytoplasmic shuttling of DDX3. We found DDX3 to be recruited to Stress Granules after cellular stress. DDX3's cellular localisation and expression levels were also found to change throughout the cell cycle.

Item Type:	Thesis (PhD)
Keywords:	cellular localisation; DEAD box helicase; DDX3;
Academic Unit:	Faculty of Science and Engineering > Biology
Item ID:	6742
Depositing User:	IR eTheses
Date Deposited:	07 Jan 2016 16:43
URI:
Use Licence:	This item is available under a Creative Commons Attribution Non Commercial Share Alike Licence (CC BY-NC-SA). Details of this licence are available here

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