Polypyrimidine tract binding protein regulates IRES-mediated translation of p53 isoforms

Abstract

The p53 tumor suppressor protein plays a key role in maintaining genomic integrity. Enhanced expression of p53 during genotoxic stress is due to both increased protein stability and translational upregulation. Previous reports have shown that p53 mRNA is translated from an alternative initiation codon to produce N-terminal truncated isoform (ΔN-p53) besides fulllength p53. We have demonstrated that two internal ribosome entry sites (IRESs) regulate the translation of p53 and ΔN-p53 in a distinct cell cycle phase-dependent manner. Here, we report that polypyrimidine tract-binding protein (PTB) is a p53 IRES interacting trans-acting factor. PTB protein binds specifically to both the p53 IRESs but with differential affinity. siRNA-mediated knockdown of PTB protein results in reduction of activity of both IRESs and also the levels of both the isoforms. It is well known that DNA-damaging agents such as doxorubicin enhance the expression of p53. Our results indicate that during doxorubicin treatment, PTB protein translocates from nucleus to the cytoplasm, probably to facilitate IRES mediated p53 translation. These observations suggest that the relative cytoplasmic abundance of PTB protein, under DNA-damaging conditions, might contribute to regulating the coordinated expression of the p53 isoforms, owing to the differential affinity of PTB binding to the two p53 IRESs.

Reversible induction of translational isoforms of p53 in glucose deprivation

“…In A549 cells, changes in Δ40p53 levels reflect capindependent-IRES-mediated translation of this isoform. 3,14 Owing to glucose deprivation, there was an increase in Δ40p53 levels at 30 h and this was confirmed with two different antibodies (Figure 5a). p53 mRNA levels do not change significantly over this time period (Supplementary Figure S17A).…”

“…From A549 cells, RNA-protein complexes were immunoprecipitated with anti-SMAR1 antibody. The ITAF PTB, which was shown to bind p53 IRESs, 3 served as a positive control (Figure 5f, anti-PTB bars). RT-qPCR analysis of the SMAR1-immunoprecipitate-associated RNA showed the presence of the RNA corresponding to 1-251 IRES, over and above than that from RNA-protein complexes immunoprecipitated by the IgG-isotype antibody, in non-starved cells (Figure 5f).…”

“…Dual-luciferase constructs of the p53 (1-251) IRES, first (1-134) IRES, second (135-251) IRESs, 3,8 VEGF and HIF1α IRESs (kind gifts from Professor Greg Goodall, Centre of Cancer Biology, Australia and Professor Annapoorni Rangarajan, MRDG, IISc) and HCV IRES 62 were used in quantitative bicistronic assays. WWP luciferase (a kind gift from Professor Kumaravel Somasundaram, IISc) was used for measuring p53-dependent transactivation, CMV-Renilla-luciferase (Promega, Madison, WI, USA) constructs were used to normalize transfection efficiency.…”

“…Polypyrimidine-tract-binding protein (PTB) was shown to have differential affinity for the two IRESs of p53 and regulated p53 IRES functions by translocating from nucleus to cytoplasm on doxorubicin-induced DNA damage. 3 This PTB-mediated control of p53 IRESs has been recently found to play a role in the p53-fibrillarin-rRNA methylation network. 9,10 Single-nucleotide polymorphisms in p53 5'UTR 11 or in the coding region of p53 IRES 7,11,12 decreased p53 IRES activity by altering PTB, MDM2 or hnRNPC1/C2 interactions with this regulatory region in p53 mRNA.…”

“…1 p53 and its N-terminally truncated isoform Δ40p53 (also known as ΔN-p53 or p53/47) are translated by internal ribosome entry site (IRES)-mediated translation initiation from the same mRNA under different stress conditions that induce DNA damage, ionizing radiation and endoplasmic reticulum (ER) stress, oncogene-induced senescence and cancer. [2][3][4][5][6][7] Thus, p53 mRNA has a dual IRES structure. 8 For their function, these IRESs rely on IRES trans-acting factors (ITAFs).…”