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Background: Dysregulation of tumor necrosis factor (TNF)-α has been implicated in the pathogenesis of a variety of diseases including cancer, sepsis and inflammatory processes like ulcerative colitis and rheumatoid arthritis. The expression of TNF-α is therefore tightly controlled, and post-transcriptional pathways, such as RNA stabilization and translational silencing, play a major role in this regulation. While the AU-rich element (ARE) in the 3’- untranslated region (UTR) is essential in post-transcriptional control, the function of non-ARE cis elements is less understood. In this study, we investigated expression of TNF-α in neuroblastoma and glioma cells and discovered a novel region in the 3’UTR upstream from the AREs that negatively regulates RNA stability and expression.

Methods: Reporter constructs containing portions of the TNF-α 3’-UTR fused downstream of the luciferase coding sequence were cloned and transfected into SHEP and U251 cells. Luciferase activity was measured by a luminescence assay. RNA kinetics were performed using actinomycin D, and real time PCR was used for RNA quantitation. UV cross-linking was performed on cell extracts using radiolabeled probes derived from the 3’UTR.

Results: The intact 485-nucleotide TNF-α 3’-UTR suppressed luciferase activity by ten-fold compared with the antisense 3’-UTR and a control sequence of similar length from Bluescript. Surprisingly, deletion of the two AREs only increased luciferase activity by three-fold. Further mapping of the 3’UTR revealed a 120-nucleotide element immediately upstream from the first ARE that suppressed luciferase expression by more than three-fold. The suppressive effect of this element is comparable to that of the AREs. An antisense version of this region, along with other non-ARE regions of the 3’UTR yielded no suppression. RNA kinetics indicated that this element was destabilizing, similar to the full-length UTR. UV cross-linking revealed the binding of two major proteins (80-kDa and 52-kDa).

Conclusion: Our results indicate a novel, non-ARE element in the TNF-α 3’UTR that confers mRNA destabilization and down-regulation. Further characterization of this element, and the two RBPs binding to it, may reveal an additional control point for maintaining tight expression of this cytokine.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA