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The translation initiation factor DAP5 promotes IRES-driven translation of p53 mRNA

Abstract

Translational regulation of the p53 mRNA can determine the ratio between p53 and its N-terminal truncated isoforms and therefore has a significant role in determining p53-regulated signaling pathways. Although its importance in cell fate decisions has been demonstrated repeatedly, little is known about the regulatory mechanisms that determine this ratio. Two internal ribosome entry sites (IRESs) residing within the 5’UTR and the coding sequence of p53 mRNA drive the translation of full-length p53 and Δ40p53 isoform, respectively. Here, we report that DAP5, a translation initiation factor shown to positively regulate the translation of various IRES containing mRNAs, promotes IRES-driven translation of p53 mRNA. Upon DAP5 depletion, p53 and Δ40p53 protein levels were decreased, with a greater effect on the N-terminal truncated isoform. Functional analysis using bicistronic vectors driving the expression of a reporter gene from each of these two IRESs indicated that DAP5 preferentially promotes translation from the second IRES residing in the coding sequence. Furthermore, p53 mRNA expressed from a plasmid carrying this second IRES was selectively shifted to lighter polysomes upon DAP5 knockdown. Consequently, Δ40p53 protein levels and the subsequent transcriptional activation of the 14-3-3σ gene, a known target of Δ40p53, were strongly reduced. In addition, we show here that DAP5 interacts with p53 IRES elements in in vitro and in vivo binding studies, proving for the first time that DAP5 directly binds a target mRNA. Thus, through its ability to regulate IRES-dependent translation of the p53 mRNA, DAP5 may control the ratio between different p53 isoforms encoded by a single mRNA.

Introduction

The tumor suppressor p53 has a major role in maintaining genomic integrity and inhibiting cell transformation. It acts in response to a variety of cellular stresses such as DNA damage, oncogene activation, mitotic impairment and oxidative stress, resulting in DNA repair, cell-cycle arrest, senescence or apoptosis.1, 2 The mechanisms that determine which functional arm of p53 will dominate are not fully understood. Alternative translation initiation at an internal codon has been described, which produces an N-terminal truncated isoform, referred to as ΔN-p53 or Δ40p53 (refs 3–5). This isoform lacks the first 39 amino acids that contain the binding site for the E3 ubiquitin ligase Mdm2 and most of the first transactivation domain (TA1), but retains the second transactivation domain (TA2), the DNA-binding domain and the oligomerization domain. Full-length p53 and Δ40p53 differ in their pattern of activation of downstream target genes,6, 7 and can lead to different biological outcomes under the same stress conditions. For example, during endoplasmic reticulum (ER) stress induction, p53 leads to G1 arrest while Δ40p53 leads to G2 arrest.8

The p53 pathway is often activated during conditions such as ER stress or apoptosis that involve global inhibition of cap-dependent translation. The cell maintains continuous p53 protein synthesis under these conditions by means of two internal ribosome entry site (IRES) elements residing within the p53 mRNA.9, 10, 11 The first IRES, located in the 5’UTR, was shown to regulate p53 translation in response to etoposide-induced DNA damage.11 A second IRES, located within the coding sequence between the translation initiation codons of p53 and Δ40p53, was shown to regulate Δ40p53 translation in response to ER stress.8, 9

DAP5 (also named p97 and NAT1) is a member of the eIF4G protein family identified simultaneously by four independent studies.12, 13, 14, 15 The homology to eIF4G is restricted to the central segment, which corresponds to the region that binds to eIF4A and eIF3, essential elements of the translation initiation complex and ribosome recruitment. Interestingly, the N-terminal part of eIF4G, which mediates the interaction with the mRNA cap structure by direct binding to eIF4E, is completely missing from DAP5, suggesting that DAP5 has evolved as a gene separate from eIF4G to mediate IRES-driven translation of cellular mRNAs.12, 16 Supporting this conjecture, DAP5 was shown to positively regulate IRES-driven translation of c-Myc, Apaf-1, XIAP, c-IAP/HIAP2, CDK1 and Bcl2 under stress conditions and in non-stressed cells.17, 18, 19, 20, 21 DAP5 itself contains an IRES element in its 5’UTR, thus creating a positive auto-regulatory loop enabling its continuous translation under conditions when cap-dependent translation is compromised.18, 22 A previous report demonstrated that DAP5 can be immunoprecipitated from cells together with its target CDK1 mRNA.21 However, it remained undetermined whether DAP5 binds directly to its target mRNA, or whether the interaction occurs via accessory proteins such as IRES trans-acting factors (ITAFs).

We now report the identification of p53 mRNA as a novel target of DAP5. DAP5 drives the translation of full-length p53 and Δ40p53 with stronger effects on the latter. This is documented here by assessing the outcome of DAP5 depletion on the levels of both the endogenous and the ectopically expressed proteins. By performing bicistronic functional studies we find that DAP5 knockdown exerts a differential effect on translation from the two p53 IRESs, selectively inhibiting translation from the second IRES both under stress conditions and in non-stressed cells. The strong effects of DAP5 on translation from the second IRES are also documented here by measuring the shift of these IRES carrying mRNA transcripts in polysomal profiles and by measuring the induction of 14-3-3σ mRNA, which is a downstream transcriptional target of Δ40p53. Furthermore, we show that purified recombinant DAP5 binds to the p53 IRES elements, providing the first evidence for a direct interaction of DAP5 with its target RNA.

Results

DAP5 knockdown reduces Δ40p53 protein levels and to a lesser extent the full-length p53 protein

First, we set out to investigate the effect of siRNA-mediated knockdown of DAP5 on p53 isoforms expressed from a wild-type construct (p53wt+5′UTR) in H1299 cells. Three conditions were examined: untreated cells, thapsigargin treatment to induce ER stress and doxorubicin treatment to induce DNA damage. To facilitate the identification of the Δ40p53 isoform on western blots, we compared the detection by the CM1 Ab, which recognizes both isoforms of p53, to the DO-1 monoclonal antibody, which is specific to the N-terminus of p53 and therefore does not detect Δ40p53. While a mild effect was observed for full-length p53, the Δ40p53 was significantly reduced in response to DAP5 knockdown in all three conditions (Figure 1a).

Figure 1
figure1

DAP5 knockdown reduces expression of Δ40p53 and p53 protein levels. (a) H1299 cells were co-transfected with p53wt+5′UTR plasmid and siRNA corresponding to DAP5 or control siRNA for 48 h. Exogenous p53 was detected with CM-1 polyclonal antibody (left panel, recognizing both isoforms) or DO-1 monoclonal antibody (right panel, does not recognize Δ40p53). p53wt+5′UTR plasmid contains p53 ORF and 134 nt of the native 5′UTR. (b) A549 cells were transfected with siRNA corresponding to DAP5 or control siRNA for 48 h and endogenous p53 was detected with the CM-1 or DO-1 antibodies. Cells extracts were analyzed on a single gel so that the DO-1 blot and CM-1 blot are made from an identical gel and membrane.

Next, we examined the effect of DAP5 knockdown on the endogenous expression of both p53 and Δ40p53 isoforms in A549 cells. Knocking down DAP5 reduced the expression of Δ40p53 in both untreated cells, and in cells treated with thapsigargin. In both these cases, DAP5 knockdown had no apparent effect on the expression of the full-length p53 (Figure 1b). During doxorubicin treatment, the expression of both p53 and Δ40p53 was reduced by DAP5 knockdown (Figure 1b). Together, these results suggest that DAP5 can regulate the expression of both p53 isoforms with greater effect on Δ40p53. This is true for endogenous as well as exogenously expressed protein in unstressed cells and during stress conditions.

DAP5 protein specifically interacts with the p53 IRES elements

To investigate whether DAP5 interacts directly with the p53 IRES elements, RNA-protein UV crosslinking studies were performed. Recombinant His-DAP5 was expressed and purified from bacteria (Figure 2a) and incubated with α32P-UTP-radiolabeled RNA probe corresponding to the first or second IRES of p53. As a negative control for non-specific binding, radiolabeled RNA probes corresponding to the multiple cloning site of the pcDNA3.1 plasmid or the HCV IRES were used. Resolution of the crosslinked protein–RNA complex on SDS gels indicated that DAP5 interacted with both p53 IRESs (Figure 2b; Supplementary Figure S1). To study the specificity of this interaction, the binding of DAP5 to the radiolabeled p53 IRES was competed with 250- and 500-fold molar excess of either unlabeled self or the HCV IRES, which does not interact with DAP5 (Supplementary Figure S1). Unlabeled p53 IRES RNA, but not the HCV IRES, could compete out the binding of DAP5 to both IRESs (Figure 2c, lanes 4–5 vs 6–7 and lanes 10–11 vs 12–13). These experiments established that DAP5 protein can directly bind to the p53 IRES elements.

Figure 2
figure2

DAP5 protein binds both p53 IRESs. (a) GelCode staining of recombinant bacterially produced His-DAP5 protein. *Degradation product. (b) RNA-protein UV crosslinking. 32P-UTP labeled RNAs corresponding to the first IRES, second IRES and non-specific sequence (pcDNA3.1 multiple cloning site (MCS)) were incubated with increasing concentrations of DAP5 protein. Proteins were resolved by SDS–PAGE and visualized by phosphorimaging. (c) Cold competition assay. Interaction of DAP5 protein with 32P-UTP-labeled p53 IRESs RNA was competed out with increasing concentrations of unlabeled self or HCV IRES. (d) RT–PCR analysis of RNA extracted from ribonucleoprotein complexes immunoprecipitated from H1299 cells transfected with pRp53(1st IRES)F or pRp53(2nd IRES)F bicistronic RNA using anti-DAP5, anti-PTB antibodies or IgG pre-immune serum. Primers corresponding to the first or second IRES were used for the assay and on the original plasmid as template (last lane) to obtain the expected size of RT–PCR product.

Next, we investigated whether DAP5 interacts with p53 IRES elements in cells. H1299 cells were transfected with bicistronic RNA carrying the first or second IRES. Then, RNA–protein complexes were immunoprecipitated with anti-DAP5 antibody. The ITAF PTB, which was shown to bind p53 IRESs,23 served as a positive control. RT–PCR analysis of the immunoprecipitate-associated RNA showed the presence of the RNA corresponding to the first or second IRES (Figure 2d), which was absent from RNA–protein complexes immunoprecipitated by the IgG pre-immune serum. Together, these results demonstrate that DAP5 can specifically interact with the first and the second IRES elements both in vitro and in cells.

DAP5 knockdown preferentially inhibits translation from the second p53 IRES residing in the coding region of p53

To determine whether DAP5 participates in IRES-mediated translation of p53 mRNA in a more direct way, the effect of DAP5 knockdown on translation from both the first and the second IRESs was assessed using a dual luciferase reporter assay system. Bicistronic plasmids containing the first or second IRES of p53 were transfected into H1299 cells (Figure 3a). These IRESs do not have any splicing or cryptic promoter activity, hence the Rluc and Fluc cistrons reside within a single transcript.10 Relative IRES activity was calculated by measuring the Fluc/Rluc luminescence ratio and was compared with a plasmid that does not contain an IRES. As previously reported,23 both sequences exhibited IRES activity, resulting in a >7-fold increase in the Fluc/Rluc ratio as compared with the control plasmid (Figure 3b). Next, DAP5 expression was knocked down, resulting in a reduction of >90% in the steady-state protein levels (Figure 3c). The activity of the transfected bicistronic plasmids in these DAP5-depleted cells was then measured relative to control cells transfected with HcRed siRNA. Notably, under these conditions DAP5 knockdown led to a reduction in IRES-mediated translation from the second IRES while translation from the first IRES was not significantly affected (Figure 3d).

Figure 3
figure3

DAP5 knockdown inhibits translation from the second p53 IRES. (a) Schematic representation of the bicistronic constructs. pRp53(1st IRES)F contains the first IRES of p53, corresponding to the last 134 nt in the 5’UTR. pRp53(2nd IRES)F contains the second IRES of p53, corresponding to the first 117 nt of the coding sequence. pRF contains only the Renilla and Firefly cistrons. (b) Relative IRES activity is represented by the Fluc/Rluc ratio for all constructs transfected into H1299 cells. (c) Western blot of H1299 cells transiently transfected with siDAP5 or siHcRed (control) for 48 h. DAP5 protein levels are normalized to actin in the graph below. (d) Relative IRES activity in H1299 cells co-transfected with siDAP5 or siHcRed together with bicistronic plasmids. For ER stress induction, cells were treated with 0.1 μM thapsigargin for 16 h. The data represent the average of four independent experiments ±s.d. *P<0.05.

Recently, it has been shown that during ER stress, the second IRES can promote cap-independent translation of the Δ40p53 isoform, leading to G2 arrest.8 To investigate the role of DAP5 in IRES-mediated p53 translation during ER stress, cells were treated with thapsigargin for 16 h before measuring IRES activity. This resulted in 30% and 70% increase in IRES-mediated translation from the first and second IRES, respectively (Figure 3d). As in non-stressed cells, DAP5 knockdown again led to a reduction in IRES-mediated translation from the second IRES, but not from the first IRES (Figure 3d).

DAP5 knockdown reduces Δ40p53 protein levels expressed from the second IRES

Although DAP5 has the potential to bind both p53 IRESs, the observation that DAP5 preferentially promotes translation from the second IRES, together with its stronger effect on Δ40p53 protein expression led us to thoroughly investigate the regulation of this isoform. To this end, we examined the outcome of DAP5 depletion on the expression of the Δ40p53 protein from a plasmid carrying the second IRES element (Δ40p53+IRES). To minimize cap-dependent ribosomal readthrough, the plasmid contains a hairpin structure upstream of the first ATG. A plasmid expressing the coding sequence of Δ40p53 in a cap-dependent manner (Δ40p53 ΔIRES) served as a control (Figure 4a). Both plasmids lack the 5′UTR and therefore do not contain the first IRES of p53. DAP5 knockdown reduced the relative Δ40p53 protein levels by 80% when expressed from the IRES-containing construct (Figure 4b). However, DAP5 knockdown had no significant effect when Δ40p53 was expressed in a cap-dependent manner (no IRES). To exclude the possibility that the observed differences in Δ40p53 protein levels were due to changes in mRNA expression, p53 mRNA levels were measured by quantitative RT–PCR (qRT–PCR). As shown in Figure 4c, similar mRNA levels were present in cells transfected with either siDAP5 or control siRNA.

Figure 4
figure4

DAP5 knockdown reduces Δ40p53 protein levels expressed from the second IRES. (a) Schematic representation of p53 plasmids. The Δ40p53+IRES plasmid contains a hairpin structure to minimize cap-dependent ribosomal readthrough. (b) Western blot of H1299 cells co-transfected with p53 constructs and siRNA corresponding to DAP5 or HcRed as control. Graphs show Δ40p53 expression levels normalized to GAPDH. (c) p53 mRNA levels as measured by qRT–PCR and a western blot from the same experiment showing Δ40p53 protein levels. (d) Western blot of H1299 cells co-transfected with pcDNA3 empty vector or Δ40p53+IRES construct and siRNA corresponding to DAP5 or HcRed as control (left). 14-3-3σ and p21 mRNA levels as measured by qRT–PCR (right). (e) Western blot of H1299 cells co-transfected with p53β constructs and siRNA corresponding to DAP5 or control siRNA.

The reduction in Δ40p53 expression due to DAP5 knockdown also impaired its ability to induce transcription of downstream target genes. 14-3-3σ was recently reported as a preferential Δ40p53 target gene.8 As expected, expressing Δ40p53 from the Δ40p53+IRES plasmid resulted in a 14-fold increase in 14-3-3σ mRNA (Figure 4d). Remarkably, DAP5 knockdown greatly compromised the induction of 14-3-3σ mRNA, confirming that this increase is DAP5 dependent. To assess DAP5 specificity, we also checked p21 mRNA expression levels. The p21 promoter is activated preferentially by p53 compared with Δ40p53 and therefore serves as a mean to differentiate between the transcriptional outputs of these two isoforms.7 The expression of the full-length p53 from the plasmid (may represent residual cap-dependent translation) resulted in 8-fold increase in p21 mRNA (Figure 4d). Unlike 14-3-3σ, this induction was hardly affected by DAP5 knockdown.

To determine whether the effect of DAP5 on translation from the second IRES is common to other p53 N-truncated isoforms, we examined the effect of DAP5 depletion on the expression of p53β and its N-truncated isoform Δ40p53β, translated from the same internal codon as Δ40p53.24 Interestingly, DAP5 knockdown strongly reduced the expression of Δ40p53β while the expression of p53β isoform remained unchanged (Figure 4e). A similar reduction was observed for Δ40p53γ when it was expressed from a plasmid carrying the p53γ isoform. In that case, DAP5 knockdown led to a decrease in Δ40p53γ and p53γ protein levels (Supplementary Figure S2).

DAP5 knockdown impairs Δ40p53 translation without affecting cap-dependent translation and global polysomal profile

To test more directly whether the effects of DAP5 knockdown result from selective inhibition of translation initiation from the p53 second IRES, the effect of DAP5 depletion on the polysomal distribution of the mRNA expressed from the Δ40p53+IRES plasmid was assessed. In three independent experiments, DAP5 knockdown did not change the overall polysomal profile compared with the control cells (Figure 5a). Hence, there was no global disassembly of the heavy polysomes. The distribution of p53 mRNA in the different polysome fractions was examined by qRT–PCR. GAPDH mRNA served as a control, since GAPDH protein levels remained unchanged in DAP5-depleted cells (Figure 4b–d). As seen in Figure 5b, DAP5 knockdown induced a shift of p53 mRNA to lighter polysomes. In contrast, the distribution pattern of GAPDH mRNA was hardly affected (Figure 5c). Thus, DAP5 depletion specifically reduces Δ40p53 translation efficiency.

Figure 5
figure5

DAP5 regulates p53 mRNA translation. (a) H1299 cells were co-transfected with Δ40p53+IRES construct and siRNA corresponding to DAP5 or HcRed as control for 48 h, and the polysomal profile was analyzed. 40S, 60S, 80S and polysomes are indicated. (b, c) Twenty-four fractions were collected from each gradient in (a) and p53 (b) or GAPDH (c) mRNA distributions in polysomes (fractions 13–24) were measured by qRT–PCR. Percent values indicate the proportion of mRNA in fractions 13–20 and 21–24 out of the total amount in fractions 13–24.

Discussion

We report here that p53 mRNA is a novel direct target of DAP5. DAP5, previously shown to function as a mediator of cap-independent translation, positively regulates p53 translation from the second IRES element located within its coding region. Several findings indicate that the effects of DAP5 knockdown on Δ40p53 steady-state protein levels are exerted at the level of translation initiation. First, p53 mRNA levels were not significantly affected by DAP5 depletion. Second, DAP5 knockdown did not reduce Δ40p53 levels when Δ40p53 was expressed from a construct lacking the second IRES, indicating that DAP5 has no effect on protein stability. Third, experiments using a bicistronic plasmid revealed that DAP5 knockdown reduces the functional activity of the second IRES. Finally, and most importantly, while DAP5 depletion in H1299 cells did not affect the overall polysomal profile, in line with earlier findings in other cells,21 nor the distribution of GAPDH mRNA, p53 mRNA was clearly shifted toward lighter polysomal fractions, attesting to its reduced translation efficiency in response to DAP5 depletion.

The downstream effects of Δ40p53 induction on target gene activation, and the resulting biological outcome, have been previously demonstrated.6, 7, 25 For example, it was shown that during ER stress, Δ40p53 upregulation directs the cellular response toward G2 arrest rather than G1 arrest, by induction of 14-3-3σ mRNA, a transcriptional target of Δ40p53 (ref. 8). However, little is known about the upstream mechanisms governing IRES-driven translation of the Δ40p53 isoform. In the current study, we showed that DAP5 is important for IRES-dependent Δ40p53 induction, as well as for the resulting elevation of 14-3-3σ mRNA. Therefore, activation of the second p53 IRES by DAP5 is likely to contribute to p53-mediated cell fate decisions by elevating Δ40p53 expression.

Most of the reported cellular IRES elements are located in the 5’UTR of the mRNA. Very few IRESs residing within the coding region have been described so far. One example is the translation of p58PITSLRE, a short isoform of the PITSLRE protein kinase, which is initiated at an internal in-frame AUG and is mediated by an internal IRES present in the coding sequence of PITSLRE mRNA.26 Interestingly, a similar example was also reported for the HIV-2 virus. The HIV-2 Gag protein and two additional shorter isoforms, encoded from two internal AUG codons, are produced by an IRES element that lies downstream to the authentic AUG initiation site.27 It was suggested that while the IRESs located in the 5’UTR ensure the continued synthesis of the same protein that is produced by cap-dependent translation, internal IRESs initiate the synthesis of alternative translation products.8 DAP5 therefore may possess a dual role as mediator of IRES-dependent translation: in addition to supporting the translation of stress response genes under conditions where cap-dependent translation is inhibited, it can also promote translation of shorter isoforms such as Δ40p53 and Δ40p53β, both in stressed and non-stressed cells in which the full-length isoform is translated in a canonical cap-dependent mechanism. Our finding that DAP5 knockdown attenuates translation from the second IRES of p53 also in non-stressed cells supports this notion.

Until now, the nature of the interaction between DAP5 and its targets has been unclear. By performing RNA binding analysis with purified recombinant DAP5, we show for the first time that this interaction is direct and specific.

Interestingly, in vitro and in vivo studies indicate that DAP5 can bind both p53 IRESs with similar affinity. This stands in contrast to the preferential functional effects toward the second IRES in cells. As shown here, in some cases the first IRES was not influenced by DAP5 depletion, and in others the depletion did cause a reduction in the expression of full-length p53 but to a smaller extent than Δ40p53 reduction (Figure 1). These observations may indicate that, in some circumstances, DAP5 has the potential to mediate translation from the first IRES as well. It has been shown that different stress conditions evoke distinct changes in IRES-mediated translation.19 ITAFs enhance or inhibit translation from IRESs by affecting their structural conformation and recognition by the translation machinery. The accessibility of ITAFs changes through the cell cycle as well as under stress, enabling tighter translational regulation. Thus, the observation that DAP5 binds the first IRES but fails to enhance its activity in some cases (Figure 3d) may imply the involvement of additional factors that function in trans that are missing in these cellular contexts, or, alternatively, the specific presence of factors that act as repressors of the first IRES. Along these lines, one could speculate that the previously shown translocation of the ITAF PTB to the cytoplasm in response to DNA damage23 might be essential for the ability of DAP5 to enhance translation from the first IRES specifically during DNA damage.

In conclusion, our findings indicate that DAP5 has a critical role in regulating different p53 isoforms, at least under some stress conditions. In the future, it will be of interest to further explore the consequences of this novel regulatory interaction.

Materials and methods

Plasmid constructs

Bicistronic constructs of the first and second IRESs of p53, and the HCV IRES (18–383) were previously described.10, 23, 28, 29 Δ40p53+IRES, Δ40p53ΔIRES and p53wt+5’UTR constructs were kindly provided by Dr Robin Fahraeus (Paris, France). pcDNA3-p53β and pSV-p53γ constructs were kindly provided by Dr Jean-Christophe Bourdon (Dundee, Scotland).

Cell culture, transfection and drug treatment

H1299 and A549 human lung carcinoma cells were cultured in Dulbecco's modified Eagle's medium (Biological Industries, Beit Haemek, Israel) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 50 units/ml penicillin, and 50 μg/ml streptomycin (Invitrogen, Grand Island, NY, USA). For RNA interference, siRNA oligonucleotides corresponding to DAP5 (eIF4G2), HcRed or On-Target Plus control siRNA (Dharmacon, Chicago, IL, USA) were used. All DNA and siRNA transfections were performed with Lipofectamine 2000 Transfection Reagent (Invitrogen) for 48 h. For western blot and mRNA expression analysis, 3 × 105 cells were plated per 6 cm culture dish before co-transfection with 0.5 μg DNA and 20 nM siRNA. For pSV-p53γ, H1299 cells were transfected in 60 mm dish with 7.5 μg plasmid DNA and 20 nM siDAP5. Four hours post transfection, zVAD (Calbiochem, Merck, NJ, USA) was added to a final concentration of 25 μM. For dual luciferase assays, 2.5 × 104 cells were plated per well in 24-well plates before co-transfection with 30 ng DNA and 20 nM siRNA. For polysomal profiling, 1.2 × 106 cells were plated per 14 cm culture dish before co-transfection with 1.5 μg DNA and 20 nM siRNA. When indicated, cells were treated with 0.1 μM thapsigargin, 1 μM doxorubicin or an equal volume of DMSO for 16 h.

Western blot analysis

Cells were lysed in PLB buffer (100 mM NaCl; 0.1 M phosphate pH 7.5; 0.1% SDS; 1% Triton X-100; 1% DOC) supplemented with protease inhibitor cocktail (Sigma-Aldrich, St Louis, MO, USA) and phosphatase inhibitor cocktail (Sigma-Aldrich). A total of 10 or 200 μg protein (for ectopically expressed or endogenous Δ40p53, respectively) was resolved by 10% SDS–PAGE, transferred onto nitrocellulose membranes, and incubated with antibodies against DAP5 (anti-NAT1 from BD, San Jose, CA, USA), GAPDH (Millipore, Billerica, MA, USA), actin (A3854, Sigma-Aldrich), p53 DO-1 monoclonal antibody (sc-126, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or p53 polyclonal antibody (CM-1, Novocastra, Wetzlar, Germany, or Dr R Fahraeus). Secondary antibodies (HRP-conjugated goat anti-mouse or anti-rabbit antibodies, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) were detected by SuperSignal enhanced chemiluminescence (Pierce, Rockford, IL, USA) and Immobilon Western Systems chemiluminescence (EMD Millipore).

Isolation of total RNA, RT–PCR and quantitative real-time PCR

Total RNA was extracted from cells using RNeasy Plus mini kit (Qiagen, Valencia, CA, USA) according to manufacturer’s instructions. RT–PCR was performed using random hexamers (Invitrogen) with Superscript II reverse transcriptase (Invitrogen). Primers used for qRT–PCR: p53: Fw: 5′-IndexTermTGGGCTTCTTGCATTCTGG-3′, Rev: 5′-IndexTermGCTGTGACTGCTTGTAGATGGC-3′, 14-3-3σ: Fw: 5′-IndexTermGCCTATAAGAACGTGGTGGGC-3′, Rev: 5′-IndexTermCCTCGTTGCTTTTCTGCTCAA-3′, p21: Fw: 5′-IndexTermGGCAGACCAGCATGACAGATT-3′, Rev: 5′-IndexTermGCGGATTAGGGCTTCCTCTT-3′ GAPDH: Fw: 5′-IndexTermGTCGGAGTCAACGGATTTGG-3′, Rev: 5′-IndexTermAAAAGCAGCCCTGGTGACC-3′.

Dual luciferase assay

Renilla and Firefly luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA), according to manufacturer's protocol, with a Veritas Luminometer (Promega).

Polysomal profile

Cells were incubated for 15 min with 0.1 mg/ml cycloheximide (CHX) and then resuspended in LBA buffer (20 mM Tris, pH 7.5, 45 mM KCl, 10 mM MgCl, 0.1 mg/ml CHX, 1 mM DTT, 0.5 mg/ml heparin, 2.5 μl/ml RNasin, protease inhibitor cocktail (Sigma-Aldrich), phosphatase inhibitor cocktail (Sigma-Aldrich) and lysed in the presence of 1.2% Triton X-100 and 1.2% sodium deoxycholate. 10 OD units (260 nm) from each sample were separated on 10–50% sucrose gradients. In all, 0.5 ml fractions were collected, and 30 μl 10% SDS and 1 ml of ethanol was added to each fraction. RNA was extracted from each fraction using TRI reagent (Sigma-Aldrich) according to manufacturer’s protocol. Equal volumes from fractions 13 to 24 were used to perform RT–qPCR analysis.

RNA-protein UV crosslinking

Recombinant His-DAP5 was produced by the Israel Structure Proteomic Center at the Weizmann Institute as described before.30 The [α32P] labeled RNAs were incubated with increasing concentrations of purified recombinant His-DAP5. RNA–protein complexes were UV crosslinked and digested with RNase A as described previously.23 For the competition assays, calculated molar excess of unlabeled self and non-self RNAs were added along with the components of the reaction mixture and 850 ng DAP5 before UV crosslinking. The complexes were separated on 10% SDS-polyacrylamide gels followed by phosphorimaging.

Immunoprecipitation of ribonucleoprotein (RNP) complexes in vivo

Capped bicistronic RNAs were synthesized from ApaI linearized and Klenow polymerase-treated pRp53(1st IRES)F or pRp53(2nd IRES)F constructs using RiboMax kit (Promega) as per manufacturer’s instructions. H1299 cells at 85–90% confluence 16 h post seeding were transfected with these RNAs. After 8 h, the cells were lysed with polysome lysis buffer (100 mM KCl, 5 mM MgCl2, 10 mM HEPES-pH 7.0, 0.05% NP-40, 1 mM DTT, 100 U/ml RNasin, 1% protease inhibitor cocktail). Supernatants were pre-cleared with protein-G sepharose beads (Fast-Flow, Sigma-Aldrich) for 30 min at room temperature. Pre-cleared lysates containing equal total protein (500 μg) were incubated with anti DAP5- (anti-NAT1 from BD), anti PTB- (SH-54, Calbiochem) or rabbit IgG-preimmune serum saturated protein-G sepharose beads overnight at 4 °C. RNP complexes conjugated to antibody-bound beads were spun down at 8000 r.p.m. for 2 min, washed four times in ice-cold polysomal lysis buffer and spun down similarly. RNP complexes were precipitated followed by proteinase-K (Promega) treatment for 30 min at 55 °C. RNA was extracted using TRI reagent (Sigma-Aldrich) as per manufacturer’s protocol and treated with DNase-I (Promega) at 37 °C for 20 min. Finally with phenol-chloroform precipitated RNA, RT–PCR was performed using IRES-specific reverse primer and Superscript II reverse transcriptase (Invitrogen). cDNA was used for semi-quantitative PCR using IRES-specific primers. The primer sequences for first IRES are as follows: p53-10-27-F-IP: 5′-IndexTermACCGTCCAGGGAGCAGGT-3′, p53-108-124-R-IP: 5′-IndexTermCGGAAGGCAGTCTGGCT-3′. The primer sequences for second IRES are as follows: p53-138-154-F-IP: 5′-IndexTermGAGGAGCCGCAGTCAGA-3′, p53-238-251-R-IP: 5′-IndexTermTGCTTGGGACGGCA-3′.

Statistical analysis

Data are expressed as mean±s.d. Statistical significance was determined using two-sided Student’s t-test. The criterion for statistical significance was P<0.05.

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Acknowledgements

We gratefully acknowledge Dr Robin Fahraeus and Dr Jean-Christophe Bourdon for the p53 cDNA constructs, the Israel Structure Proteomic Center (ISPC) at the Weizmann Institute for supplying recombinant DAP5 protein and Sylvia Wilder for technical help. This work was supported by grants from the Flight Attendant Medical Research Institute (to AK and MO), the European Union FP7 APO-SYS (to AK), the Indo French Centre for the Promotion of Advanced Research (to SD), the Department of Biotechnology, India (to SD), and a pre-doctoral fellowship from the Council of Scientific and Industrial Research, India (to DK). AK and MO are incumbents of the Helena Rubinstein Chair of Cancer Research and the Andre Lwoff Chair in Molecular Biology, respectively.

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Correspondence to A Kimchi.

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Supplementary Information accompanies the paper on the Oncogene website

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Weingarten-Gabbay, S., Khan, D., Liberman, N. et al. The translation initiation factor DAP5 promotes IRES-driven translation of p53 mRNA. Oncogene 33, 611–618 (2014). https://doi.org/10.1038/onc.2012.626

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Keywords

  • p53
  • Δ40p53
  • DAP5
  • IRES
  • translational control

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