Demonstration that a mRNA Binding Protein is Responsible for GADD45 mRNA Destabilization

摘要

We are studying the post-transcriptional control of expression of the p53-inducible antiproliferative gene known as Growth Arrest and DNA Damage induced gene 45 (GADD45) Using regions of the GADD45 mRNA 3'-untranslated region (UTR) in KNA gel shift assays, we have observed that glutamine causes distinct changes in RBP activities in cytoplasmic and nuclear protein extracts. KNA- affinity columns were used to enrich extracts for RBPs binding to the distal region of GADD45 3'-UTR. Eluded proteins were subjected to Western blotting, to detect specific RBPs, as well as proteomic analysis. Data suggest that the distal region of the GADD45 3'-UTR is bound by the destabilizing RBP known as Au-binding factor 1 (AUFl) . Western blotting demonstrated that AUFl is present in extracts from both glutamine-fed and glutamine-starved cells. However, when cytoplasmic extracts from glutamine-starved cells are subjected to RNA-affinity purification, the AUFl does not bind to the distal GADD45 3'-UTR region. These results suggest that the effect of glutamine deprivation on GADD45 mRNA stability is orchestrated by AUFl binding, either through inhibition of AUFl RNA binding activity or through competitive inhibition by another RBP. Regions of GADD45 mKNA are now being tested for the ability to confer glutamine-mediated instability to fusion mRNAs.