Translational Control Protein 80 Stimulates IRES-Mediated Translation of p53 mRNA in Response to DNA Damage

Figure 1

(a) TCP80 has increased binding to the p53 mRNA following DNA damage. MCF-7 cells were transfected with pcDNA3.1/HisB/TCP80 that encodes for the Xpress-tagged TCP80 protein. Twenty-four hours following transfection, the cells were treated with or without 10 μM etoposide for 2 hours. They were then lysed in polysome lysis buffer and incubated with protein G-plus agarose beads coated with the anti-Xpress antibody. The TCP80 and mRNA complexes (mRNP) were immunoprecipitated and mRNA was extracted from the immunoprecipitate as described in Experimental procedures. RT-PCR was then performed to reverse-transcribe and amplify the p53 IRES sequence (~145 bp). (b) TCP80 positively affects the p53 IRES activity in response to DNA damage. MCF-7 cells were cotransfected with pRF or pR5UTRF along with either pcDNA3.1 or pcDNA3.1/HisB/TCP80. Twenty-four hours following the transfection, the cells were treated with or without etoposide for 2 hours. The cells were then lysed and a dual-luciferase assay was performed to detect firefly (Fluc) and renilla (Rluc) luciferase activities as described in Experimental procedures. The results presented are average ± SEM from three individual experiments.

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