Two internal ribosome entry sites mediate the translation of p53 isoforms

Abstract

The p53 tumour suppressor protein has a crucial role in cell-cycle arrest and apoptosis. Previous reports show that the p53 messenger RNA is translated to produce an amino-terminal-deleted isoform (DeltaN-p53) from an internal initiation codon, which acts as a dominant-negative inhibitor of full-length p53. Here, we show that two internal ribosome entry sites (IRESs) mediate the translation of both full-length and DeltaN-p53 isoforms. The IRES directing the translation of full-length p53 is in the 5'-untranslated region of the mRNA, whereas the IRES mediating the translation of DeltaN-p53 extends into the protein-coding region. The two IRESs show distinct cell-cycle phase-dependent activity, with the IRES for full-length p53 being active at the G2-M transition and the IRES for DeltaN-p53 showing highest activity at the G1-S transition. These results indicate a novel translational control of p53 gene expression and activity.

Figure 1

p53 5′-untranslated regions mediate cap-independent translation. (A) Nucleotide sequence of p53 5′-untranslated region (5′UTR) and part of the protein-coding region. The 134 nt canonical 5′UTR is underlined by a solid line, whereas the 251 nt 5′UTR for ΔN-p53 is underlined by a dotted line. (B) In vitro translation of capped and uncapped monocistronic RNAs in the presence or absence of exogenously added cap analogue. The band intensities were quantified and are represented graphically.

Figure 2

p53 5′-untranslated region sequences mediate internal ribosome entry site-dependent translation. (A) Schematic representation of bicistronic plasmids used in transient transfections. (B(i)) Transfection of bicistronic plasmids pRnullF, pRp53(+39)F and pRp53(−1)F containing a 264 nt unrelated sequence, p53 5′UTR (+39) and (−1) sequences, respectively, in the intercistronic space into HeLa cells. (ii) Transfection of bicistronic plasmids containing p53 5′UTR (+39) and (−1) downstream of the ΔEMCV sequence. The control bicistronic plasmid pRΔEF contained only the ΔEMCV sequence. Transfection efficiencies were normalized by co-transfecting with a β-galactosidase plasmid. The luciferase activities for Fluc and Rluc are shown separately as fold increase compared with that from control, taken as 1. (C(i,ii)) Transfection of the same set of bicistronic plasmids into H1299 cells. The data are represented as in (B). (D) Transfection of bicistronic plasmids containing p53 5′UTR (+39) or (−1) sequences in the intercistronic place and the ΔEMCV sequence upstream of Rluc into HeLa cells. The Fluc and Rluc activities from the ΔEMCV-containing plasmids are shown as fold increase or decrease with respect to the corresponding controls, taken as 100.

Figure 3

p53 bicistronic plasmid does not show splicing or cryptic promoter activity. (A) Reverse transcription–PCR analysis, using two sets of primers P1/P2 and P3/P4, of RNA extracted from pRp53(+39)F bicistronic plasmid-transfected (T) and untransfected (UT) HeLa cells. Lane 5 shows reverse transcriptase-negative control, whereas lanes 6 and 7 show PCR products amplified from the bicistronic DNA. (B) Northern blot of total RNA extracted from HeLa cells transfected with pRp53(+39)F (lane 1), pRp53(−1)F (lane 2), pR(coxsackievirusB3-IRES)F (lane 3) DNAs and in vitro-synthesized Fluc RNA (lane 4) using a ³²P-labelled riboprobe corresponding to Fluc. (C) Transfection of bicistronic plasmids pBS-Rp53(+39)F and pBSRnullF lacking eukaryotic promoters into HeLa cells in the absence and presence of infection by VTF7-3. The luciferase activity values are indicated above the respective bars. (D) Transfection of HeLa cells with capped bicistronic RNAs containing the p53(+39) and (−1) 5′UTRs. Fluc/Rluc ratios are shown as fold increase compared with that from a control bicistronic RNA lacking p53 sequences.

Figure 4

Cell cycle-dependent p53 internal ribosome entry site activity in G2/M synchronized cells. (A) Flow cytometric analysis of HeLa cells collected at different time points after being synchronized at G2/M phase by nocodazole treatment. The percentage of cells in S phase at each time point is indicated. (B) Luciferase assay of cells transfected with p53(+39) and (−1) bicistronic constructs and synchronized at G2/M phase at various time points after release. Fluc and Rluc activities at each time point are expressed as fold of the activity obtained from non-synchronized, transfected cells taken as control. The data mean±s.d. from three independent experiments.