A translation repressor element resides in the 3' untranslated region of human p53 mRNA Loning Fu 1 , WeiLi Ma 1 and Samuel Benchimol* ,1,2 1 Ontario Cancer Institute/Princess Margaret Hospital, 610 University Avenue, Toronto, Ontario, M5G 2M9, Canada; 2 Department of Medical Biophysics, University of Toronto, 610 University Avenue, Toronto, Ontario, M5G 2M9, Canada The 3' untranslated region of human p53 mRNA represses translation both in vitro and in vivo. Here, we identify a cis-acting 66-nucleotide U-rich sequence in the human p53 mRNA 3' untranslated region that mediates translational repression. Using UV cross- linking, we detect a 40 kDa protein that interacts speci®cally with the p53 3'UTR containing the repressor element. Enhanced translation of p53 mRNA contributes to the accumulation of p53 protein in cells exposed to g- radiation and could be a consequence of relieving the inhibition mediated by the repressor element. Keywords: p53; translational control; 3' UTR Introduction The p53 tumor suppressor protein plays a critical role in the cellular response to DNA damage leading to cell cycle arrest in G1 or apoptosis depending on cell type or culture conditions (Clarke et al., 1993; Kastan et al., 1991; Kuerbitz et al., 1992; Lowe et al., 1993a,b). Although p53 mRNA is ubiquitously expressed in mammalian cells, p53 protein levels are usually low or undetectable in normal cells owing to the protein's short half-life (Oren et al., 1981; Rogel et al., 1985) and slow rate of synthesis (Fu et al., 1996; Mosner et al., 1995). DNA strand breaks produced by g-irradiation or by DNA repair intermediates following treatment with UV-radiation or chemotherapeutic agents result in the accumulation of p53 protein and in the activation of its transcriptional activity (Fritsche et al., 1993; Kastan et al., 1991; Lu and Lane, 1993). The rapid and transient up-regulation of p53 protein is mediated primarily through a post-transcriptional mechanism involving increased p53 mRNA translation (Fu and Benchimol, 1997; Mosner et al., 1995) and increased p53 protein stability (Maltzman and Czyzyk, 1984). The rate of p53 protein synthesis was shown to increase in UV-treated cells as assessed by the incorporation of [ 35 S]methionine during a 30-min metabolic labelling assay (Maltzman and Czyzyk, 1984). Moreover, the ability of protein synthesis inhibitors to block the increase in p53 protein after DNA damage is consistent with translation playing a role in this process (Fritsche et al., 1993; Kastan et al., 1991; Tishler et al., 1993). While elevated levels of p53 protein are believed to be important to initiate the events that lead to G1 arrest or apoptosis after DNA damage, there is compelling evidence that post- translational modi®cation of p53 protein is also required to activate the latent sequence-speci®c DNA binding and transactivation functions of p53 (reviewed in Giaccia and Kastan, 1998). The complexity of the mechanism(s) governing p53 protein accumulation in response to DNA damage is underscored by the ®ndings that the kinetics and duration of p53 protein accumulation dier in dierent cells and in response to dierent DNA damaging agents (Lu and Lane, 1993). Moreover, distinct pathways appear to be activated in UV- and g-irradiated cells to elevate p53 protein levels (Maki and Howley, 1997). The relative importance of increased p53 mRNA translation and increased p53 protein stability is likely to vary in dierent cell types and in response to dierent DNA damaging agents. We reported previously that human p53 mRNA was more actively translated in g-irradiated cells and that a region in the distal end of the 3'UTR of human p53 mRNA was capable of repressing translation of a chimeric reporter RNA both in vitro (Fu et al., 1996) and in vivo (Fu and Benchimol, 1997). Moreover, chimeric transcripts bearing the distal end of the p53 3'UTR were translationally activated in cells exposed to g-radiation (Fu and Benchimol, 1997). In this study, the translational repressor element in the p53 3'UTR was mapped and found to reside within a 66 nt U-rich sequence at the 5' boundary of an Alu-like element. Using UV cross-linking assays, we have detected a 40 kDa protein that interacts speci®cally with the p53 mRNA 3'UTR containing the repressor element. Results A 66 nt U-rich sequence within the human p53 3'UTR mediates translational repression To identify the nucleotide sequence element present on the distal end of the p53 3'UTR that mediates translational repression, a series of chimeric luciferase reporter constructs bearing dierent regions of the human p53 3'UTR was constructed, and the transla- tional eciency of the resulting transcripts was determined in stably transfected MCF-7 clones. The constructs are depicted in Figure 1a. Brie¯y, DA constructs contain a deletion of the entire Alu-like element (nt 715 ± 1068), DU constructs contain a deletion of the U-rich sequence present at the 5' end of the Alu-like element (nt 716 ± 782), DXH constructs contain a deletion of the distal end of the Alu-like element but retain the U-rich region (nt 862 ± 1068); constructs with a 5' pre®x contain the entire p53 *Correspondence: S Benchimol Received 19 April 1999; revised 29 June 1999; accepted 29 June 1999 Oncogene (1999) 18, 6419 ± 6424 ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $15.00 http://www.stockton-press.co.uk/onc