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Title: The role of BRF1 and BRF2 in the immune response
Author: Crawford, Rebecca
ISNI:       0000 0004 2678 291X
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2009
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Tristetraprolin (TTP), butyrate response factor 1 (BRF1) and butyrate response factor 2 (BRF2) are members of a family of zinc finger containing ARE binding proteins known as the Zfp36 family. They all possess a conserved tandem zinc finger domain, which facilitates their binding to mRNAs that contain adenosine/uridine rich elements (ARE) in their 3' untranslated region (3'UTR). Binding to the target mRNA results in its destabilisation. Several mRNAs containing an ARE in their 3'UTR are stabilised by the mitogen activated protein kinase (MAPK) p38 pathway. Both the expression and the mRNA destabilising function of TTP are controlled by the p38 MAPK pathway. Much less is known about BRF1 and BRF2 functions, and it is not clear whether their expression or function is regulated by the p38 MAPK pathway. So far no difference has been seen in the binding specificities of the three proteins in vitro, however the phenotypes of knockout mice suggest that they have distinct functions, and may have different mRNA targets in vivo. Western blotting and quantitative PCR (qPCR) have been used to investigate the expression of members of the Zfp36 family. RNA interference was used to knock down the expression of BRF1 and BRF2 in HeLa cells, and the effects on p38-regulated inflammatory mediator expression were examined. BRF1-/- mouse embryonic fibroblast (MEF) cell lines were used to investigate the function of this family member. No evidence that BRF1 or BRF2 contributes to the post-transcriptional regulation of inflammatory mediators by the p38 MAPK pathway in HeLa cells or MEFs was found. BRF1 null MEFs over-expressed IL-6, protein, IL-6 mRNA and Cox-2 mRNA but did not over-express KC protein. The hypothesis that BRF1 is regulating IL-6 and Cox-2 by controlling mRNA stability was disproved. As a result investigation of the transcriptional regulation of these genes was researched. Primary transcript qPCR showed that both IL-6 and Cox-2 undergo more rapid transcription in BRF1-/- MEFs. This suggests that IL-6 and Cox-2 are indirect targets of BRF1 and that their regulation is through a transcription factor which is itself a target of BRF1.
Supervisor: Clark, Andy Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral