Post-transcriptional control of the MCT-1-associated protein DENR/DRP by RNA-binding protein AUF1.

There is often a poor correlation observed between protein and RNA in eukaryotic systems, supporting the emerging pardigm that many of the abnormalities in a cancer cell's proteome may be achieved by differential recruitment of mRNAs to polysomes referred to as the translational profile. The MCT-1 oncogene product has recently been shown to interact with the cap complex and to modulate the translational profile of cell lines when MCT-1 was highly expressed. The MCT-1 protein modifies mRNA translational profiles through its interaction with DENR/DRP, a protein containing an SUI1 domain involved in recognition of the translation initiation codon. It has been shown previously that the protein levels of DENR/DRP go up in parallel with increasing cell density, however the mechanism(s) underlying this increase is poorly understood at present. The 3'-untranslated region (3'UTR) of DENR/DRP was found to have a high number of uracyl (U)- and adenine (A)-rich sequences (AREs). Many RNA-binding proteins (RBPs) have been shown to recognize and bind to mRNAs that contains AREs generally present in the 3'UTR of mRNAs. RBPs binding to AREs such as AUF1, BRF1, KSRP, and TTP are known to regulate mRNA turnover, while TIAR and TIA-1 influence mRNA translation. We assessed the association of several ARE binding proteins with DENR/DRP mRNA by reverse transcription of the RNA obtained after immunoprecipitation of cell lysates from HEK 293 cells growing at varying levels of cell density. HEK 293 cells were transfected with an AUF1 silencing vector (shRNA), and protein levels of DENR/DRP were analyzed by Western blotting. We demonstrated that both HuR and AUF1 bind to discrete regions of DENR/DRP mRNA and that AUF1 silencing increases DENR/DRP protein levels. Our data established a cell density-dependent interaction of AUF1 protein with DENR/DRP mRNA that modulates DENR/DRP protein levels.