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Title: Studies on the cellular localisation of Poly ADP Ribose Polymerase (PARP)-14
Author: Yu, Sheng-Chun
ISNI:       0000 0005 0732 5091
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2014
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The poly(ADP-ribose) polymerase (PARP) family consists of 17 proteins, some of which are involved in DNA repair, regulation of transcription and mediation of telomere length. PARP-14 is the largest molecule in this family and has already been shown to play a nuclear role in regulating Stat6-activated transcription, TH2 differentiation, and preventing apoptosis in B cells. This thesis describes work that has explored the hypothesis that PARP-14 also has a role in the cytoplasm. Based on observations that GFP-tagged full length PARP-14 and a GFP-tagged fragment of PARP-14 consisting of the region from the third macro domain (M3) to the WWE domain (M3-WWE) both localise to granules in the cytoplasm of Hela cells, we initially used M3-WWE as bait in a yeast two-hybrid screen to search potential binding partners. This failed to identify any definite and relevant hits for follow-up. Using PHYRE (a web-based protein fold recognition programme established at Imperial College), we subsequently discovered three previously unrecognized degenerate KH domains in PARP-14, with one (KH3) situated between the third macro domain and WWE domains. Using a panel of GFP-fusion proteins and confocal microscopy, we found that KH3-GFP localises to cytoplasmic granules in Hela cells in a similar manner to full length PARP-14, which in turn showed a similar distribution to that of antibody-detected endogenous PARP-14 in mouse macrophages. Using a panel of antibodies against protein components of stress granules, exosomes and lysosomes, we found that PARP-14 colocalises with stress granules in cells stressed by sodium arsenite, but with exosome complex proteins in cells activated by LPS. Co-immunoprecipitation experiments confirmed that KH3-GFP associates with eIF3η and TTP and that these associations were RNA-dependent manner. These observations support a role for PARP-14 in regulating RNA processing in stress granules and exosomes, and suggest that the KH3 domain is critical for PARP-14 cytoplasmic granule localisation.
Supervisor: Haskard, Dorian Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral