Characterization of Butyrate response factor-2 (BRF2) in AU-rich element-mediated mRNA decay

AU-rich elements (AREs) are found in many cytokine and proto-oncogene mRNAs, and by mediating rapid mRNA degradation they potently inhibit gene expression at the post-transcriptional level. Out of more than 20 different proteins that specifically bind to AREs, the Tis11 family of tandem zinc finger proteins is of particular importance. The three mammalian members of this family, TTP (tristetraprolin), BRF (butyrate response factor)-1 and BRF2, are all potent inducers of ARE-mediated decay (AMD). These proteins function as adaptors that connect ARE-containing mRNAs to the deadenylation, exosome, decapping, and 5'-3' decay complexes, thereby delivering the bound mRNAs to the general RNA-decay machinery.

In this study, we investigated a possible role for BRF2 in AMD. To achieve this, we tested the mRNA-degrading capacity of BRF2 in a mutant cell line termed SlowC that is defective in AMD due to mutations in the BRF1 gene. By using a GFP-based reporter system, we measured AMD using FACS analysis and Northern blotting. Our analyses indicate that over-expression of BRF2 accelerates reporter-mRNA decay thus restoring AMD in SlowC cells. Drawing analogy with the BRF1 protein, we further characterized the regulation of BRF2. BRF1 is phosphorylated at specific serine residues by Protein kinase B and MAPK-activated protein kinase 2 (MK2). 14-3-3 binds the thus-phosphorylated BRF1 and inhibits its mRNA-decay activity. Here, we studied BRF2 with regard to phosphorylation, subsequent binding to 14-3-3, and the kinase involved in this phosphorylation process. Site-directed mutagenesis was performed to determine putative 14-3-3 binding sites in BRF2. Thereby, we found that serines at positions 123 and 257 in BRF2 are crucial for 14-3-3 binding. Disrupting both sites together abrogated binding of 14-3-3 to BRF2, as determined by co-immunoprecipitation analyses. Moreover, studies subsequent to over-expressing wild-type and dominant negative forms of the MK2 kinase hint at a role of MK2 in phosphorylating BRF2.