%0 Generic
%A Kulkarni, Meeta Girish
%D 2012
%F heidok:13310
%K ARE , Butyrate response factor 2 , 14-3-3 , MK2
%R 10.11588/heidok.00013310
%T Characterization of Butyrate response factor 2 and study of its role in AU-rich element-mediated mRNA decay
%U https://archiv.ub.uni-heidelberg.de/volltextserver/13310/
%X AU-rich element-mediated mRNA decay (AMD) is a prominent mode of mRNA degradation in the cell considering 10-15 % of mRNA in the cells include an AU-rich element (ARE) in their 3’ UTR. These mRNAs code for proteins involved in important cellular processes, namely, growth and maintenance, development, transcription, inflammation, apoptosis, etc. AMD is majorly promoted by the TTP family of proteins consisting of TTP, BRF1, and BRF2, which bind to the AREs in the 3’UTR of mRNAs and accelerate their degradation. The TTP family members show diverse characteristics inspite of bearing extensive homology amongst each other. In my study, I attempted to characterize the BFR2 protein and determine its possible role in AMD. Experiments in this study suggested that BRF2 degrades mRNA in an ARE-dependent manner and that BRF2 binds to 14-3-3 in a phosphorylation-dependent manner. I studied to greater detail the binding characteristics of BRF2 with 14-3-3 as a possible means of regulation of the AMD activity of BRF2. I could establish that S123 and S257 are the binding sites for 14-3-3 on BRF2 and that mutating both sites together is necessary for abolishing 14-3-3 binding. MK2 kinase seemed to affect phosphorylation of BRF2 and 14-3-3 binding, since 14-3-3 binding appeared reduced when an inactive form of MK2 was co-expressed. However, unlike observed in TTP and BRF1, MK2 did not appear to reduce AMD efficiency of BRF2 in the given experimental conditions. This could possibly indicate that BRF2 might be resistant to inactivation by stress-induced kinases. The 14-3-3 binding mutants showed a slightly more nuclear presence than the BRF2 wildtype which remained predominantly cytoplasmic. On exposure to arsenite stress, a part of the nuclear fraction of the BRF2 wildtype protein seemed to shift to the cytoplasm. All 14-3-3 binding mutants spontaneously localized to P bodies equally efficiently and to arsenite-induced stress granules with slightly varying efficiencies. Preliminary co-immunoprecipitation experiments suggest of interactions between BRF2 and the mRNA deadenylation machinery. BRF2:aa1-275 (N-terminus + RNA binding region) and BRF2:aa97-275 (RNA binding region) were seen to localize to the nucleus. This could be because of lack of the nuclear export signal (NES) from the C terminus. Interestingly, BRF2:aa1-275 shifted to the cytoplasm on exposure to oxidative stress, which could indicate presence of an additional NES in the N-terminus which is active in oxidative stress. It was seen that the central RBD region consisting of the two 14-3-3 sites and two zinc fingers was sufficient to cause AMD of ß-globin-ARE reporter mRNA. BRF2:aa97-275 despite being predominantly nuclear is still capable of promoting AMD, and AMD has hitherto not been shown to take place in the nucleus. This opens up an interesting idea as to whether AMD could actually be nucleus-associated.