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 under-go 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.