ORIGINAL ARTICLE Effect of a natural mutation in the 5 0 untranslated region on the translational control of p53 mRNA D Khan, A Sharathchandra 1 , A Ponnuswamy 1 , R Grover and S Das Tumor-suppressor protein p53, the ‘guardian of the genome’, is critical in maintaining cellular homeostasis and genomic stability. Earlier, we have reported the discovery of internal ribosome entry sites (IRESs) within the p53 mRNA that regulate the translation of the full length and its N-terminal-truncated isoform, DN-p53. Polypyrimidine tract-binding protein (PTB) is an IRES trans-acting factor that positively regulates the IRES activities of both p53 isoforms by relocating from nucleus to the cytoplasm during stress conditions. Here we have demonstrated the putative contact points of PTB on the p53 IRES RNA. Studies on mutations that occur naturally in the 5 0 untranslated region (5 0 UTR) in p53 mRNA were lacking. We have investigated a naturally occurring C-to-T single-nucleotide polymorphism (SNP) first reported in human melanoma tumors. This SNP is at position 119 in the 5 0 UTR of p53 mRNA and we demonstrate that it has consequences on the translational control of p53. Introduction of this SNP has led to decrease in cap-independent translation from p53 5 0 UTR in bicistronic reporter assay. Further, the effects of this SNP on cap-independent translation have been studied in the context of p53 cDNA as well. Interestingly, the 5 0 UTR with this SNP has shown reduced binding to PTB that can be corroborated to its weaker IRES activity. Previously, it has been shown that G2–M checkpoint, DNA-damaging stress and oncogenic insult favor IRES-mediated translation. Under similar conditions, we demonstrate that this SNP interferes with the enhancement of the IRES activity of the 5 0 UTR. Taken together, the results demonstrate for the first time that SNP in the 5 0 UTR of the p53 mRNA might have a role in translational control of this critical tumor-suppressor gene. Oncogene (2013) 32, 4148–4159; doi:10.1038/onc.2012.422; published online 1 October 2012 Keywords: p53 mRNA; translational control; SNP; IRES; PTB; internal initiation INTRODUCTION Human TP53 gene encodes the tumor-suppressor p53 protein that has a major role in maintaining genomic integrity and cellular homeostasis and inhibiting cell transformation. It is triggered by cellular stresses, such as DNA damage, oncogene activation, mitotic impairment and oxidative stress, that in turn lead to DNA repair, cell-cycle arrest, senescence or apoptosis. 1,2 It is one of the most commonly mutated genes in majority of cancers. 3 The p53 pathway is often activated during conditions such as ER stress or apoptosis that lead to global attenuation of cap- dependent translation. An alternative mode of cap-independent translation initiation mediated by internal entry of ribosomes on p53 mRNA maintains the expression of p53 and its N-terminal- truncated isoform DN-p53 (alternatively, p53/47 or D40p53). Thus, p53 mRNA has additional layers of regulation that decide the production of relative amounts of isoforms. Two internal ribosome entry site (IRES) elements residing within p53 mRNA mediate its translation under conditions where cap-dependent translation is reduced. The first IRES, located in the 5 0 untranslated region (UTR), was shown to regulate p53 translation in response to etoposide- induced DNA damage, where cap-dependent translation is inhibited by increased association of translation initiation factor eIF4E with its binding protein 4E-BP1. 4 A number of proteins, such as ribosomal protein L26 and nucleolin, were identified to interact with p53 5 0 UTR and regulate its translation both at basal levels and in response to different stress conditions. 5 A second IRES, located in the coding sequence between the translation initiation codons of p53 and its translational isoform DN-p53, was shown to regulate DN-p53 translation. 6,7 The IRES responsible for the translation of p53 was observed to be most active at the G2–M transition. As p53 activity is required during this phase of the cell cycle, IRES-mediated translation might be required for maintaining basal levels of p53 protein at the M phase when there is a decrease in canonical cap-dependent translation. On the other hand, the activity of IRES responsible for the translation of DN-p53 predominates at the G1–S transition. 7 DN-p53 translation is upregulated in response to ER stress, under conditions in which cap-dependent translation is inhibited by eIF2a phosphorylation. 6,8 IRES-mediated translation of p53 has been linked to disease. A recent study has shown that during oncogene-induced senes- cence, a switch between cap- and IRES-dependent translations occurs. During this switch, the IRES element positioned in the 5 0 UTR of p53 is engaged and facilitates p53 translation. 9 This study concludes that in forms of X-linked dyskeratosis congenita where dyskerin is mutated, IRES-dependent translation is compro- mised in the face of oncogenic insult. Another independent study has come up with similar results. 10 Several studies have demonstrated the requirement of cellular RNA-binding proteins, termed as IRES trans-acting factors (ITAFs), as critical for the function of cellular and viral IRESs, that might act as RNA chaperones either to attain or maintain the correct three-dimensional IRES structure required for the efficient assembly of initiation complex and/or the recruitment of ribosomes. 11 Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India. Correspondence: Professor S Das, Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India. E-mail: sdas@mcbl.iisc.ernet.in 1 These authors contributed equally to this work. Received 8 September 2011; revised 5 June 2012; accepted 2 August 2012; published online 1 October 2012 Oncogene (2013) 32, 4148–4159 & 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13 www.nature.com/onc