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Title: Elucidating the RNA-binding mechanisms and functions of E3 ubiquitin ligase TRIM25 in cell biology and innate immunity
Author: Heikel, Gregory
ISNI:       0000 0004 8508 8493
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2019
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Pathogenic viruses have a huge impact on human health and have caused numerous major epidemics both in the past and during the 21st century. The innate immune system is the body's first line of defence against viruses, with pattern recognition receptors recognising molecules unique to viruses and triggering the expression of interferons and other anti-viral cytokines, leading to the formation of an anti-viral state. The Tripartite Motif Containing 25 (TRIM25) is an E3 ubiquitin ligase thought to be a key component in the activation of signalling by the pattern recognition receptor Retinoic Acid-Inducible Gene I Protein (RIG-I), which recognises viral RNAs with a 5'-triphosphate moiety. TRIM25 has recently been identified as an RNA-binding protein, raising the question of whether its RNA-binding activity is important for its role in innate immunity. In this thesis, I demonstrated that TRIM25's RNA-binding activity is mediated by its C-terminal PRY/SPRY domain and is required for its E3 ligase activity. I also generated TRIM25 knockout cells using a CRISPR/Cas9 strategy in HeLa and HEK293 cell lines and showed that deletion of TRIM25 does not generally affect levels of the mRNA binding partners of TRIM25 identified by a genome-wide Cross-Linking Immunoprecipitation (CLIP) screen. Finally, I showed that although deletion of TRIM25 in HEK293 cells reduced their ability to restrict Influenza A virus infection, it did not affect activation of RIG-I signalling pathway in response to 5'- triphosphate RNA. This suggests that TRIM25 is redundant for RIG-I signalling in HEK293 cells and its role in restricting Influenza A virus infection is unrelated to its role in the RIG-I pathway. These findings have opened new lines of investigations into functional and molecular roles of TRIM25 in cell biology and control of pathogenic infections and I have generated tools to aid in these investigations.
Supervisor: Michlewski, Gracjan ; Granneman, Sander Sponsor: Wellcome Trust
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: 5'ppp-RNA ; RIG-I signalling process ; TRIM25 ; innate immunity