Regulation of H-ras splice variant expression by cross talk between the p53 and nonsense-mediated mRNA decay pathways

Abstract

When cells are exposed to a genotoxic stress, a DNA surveillance pathway that involves p53 is activated, allowing DNA repair. Eukaryotic cells have also evolved a mechanism called mRNA surveillance that controls the quality of mRNAs. Indeed, mutant mRNAs carrying premature translation termination codons (PTCs) are selectively degraded by the nonsense-mediated mRNA decay (NMD) pathway. However, in the case of particular genes, such as proto-oncogenes, mutations that do not create PTCs and therefore that do not induce mRNA degradation, can be harmful to cells. In this study, we showed that the H-ras gene in the absence of mutations produces an NMD-target splice variant that is degraded in the cytosol. We observed that a treatment with the genotoxic stress inducer camptothecin for 6 h favored the production of the H-ras NMD-target transcript degraded in the cytosol by the NMD process. Our data indicated that the NMD process allowed the elimination of transcripts produced in response to a short-term treatment with camptothecin from the major proto-oncogene H-ras, independently of PTCs induced by mutations. The camptothecin effects on H-ras gene expression were p53 dependent and involved in part modulation of the SC35 splicing factor. Interestingly, a long-term treatment with camptothecin as well as p53 overexpression for 24 h resulted in the accumulation of the H-ras NMD target in the cytosol, although the NMD process was not completely inhibited as other NMD targets are not stabilized. Finally, Upf1, a major NMD effector, was necessary for optimal p53 activation by camptothecin, which is consistent with recent data showing that NMD effectors are required for genome stability. In conclusion, we identified cross talk between the p53 and NMD pathways that regulates the expression levels of H-ras splice variants.

FIG. 1.

Differential enrichment of H-ras splice variants in different cellular fractions. (A) Coamplification of H-ras splice variants containing or not containing E5. RNAs were purified from different MCF7 cellular fractions. PCR was performed to amplify tRNA^tyr or U6 snRNA or to coamplify H-ras splice variants containing or not containing E5, using primers in H-ras exons 4 and 6. (B) Characterization of the expression levels of four H-ras splice variants. RT-PCR was performed using different RNA populations and different sets of primers to coamplify specifically two splice variants, as shown in Fig. S1 in the supplemental material. (C) Nuclear E5⁺ splice variants are polyadenylated mRNAs. Polyadenylated mRNAs were prepared from total RNA isolated from C or N fractions using oligo(dT) beads. RT was performed using random or oligo(dT) primers (RP or dT, respectively). (D) Coamplification of H-ras splice variants containing or not E5. Sense primers in exon 4, 3, or 2 and the E6/7 antisense primer that overlaps the last exons, 6 and 7, were used to coamplify E5⁺ and E5⁻ splice variants. It was not possible to coamplify the E5⁺ and E5⁻ transcripts by using a sense primer in exon 1 because of its high GC content. A forward primer overlapping exons 1 and 2 (E1/2) and reverse primers overlapping exons 5 and 6 (E5/6) or exons 4 and 6 (E4/6) were used to amplify the transcripts containing or not exon 5 in separate reactions. (E) Quantification of H-ras splice variants in different RNA populations. qPCR reactions were performed using different RNA populations [isolated from total cellular extract (T), C fraction, or N fraction] to quantify the levels of all the H-ras transcripts (hras), E5⁻ splice variant, or E5⁺ splice variant as shown in Fig. S1 in the supplemental material. The value obtained for each sample in the qPCR assay was calculated per μg of RNA. Experiments were performed at least three times. Data are presented as the mean ± standard error of the mean (*, P < 0.005). (F) Characterization of the expression levels of four H-ras splice variants in MLS-1765 and HeLa cells. RT-PCR was performed using different RNA populations and different sets of primers to specifically coamplify two splice variants.

FIG. 2.

Degradation of E5⁺ in the cytosol by the NMD pathway. (A) Half-life of H-ras splice variants. Different cellular fractions were prepared from cells treated with actinomycin D for different times (hours). qPCR was performed to quantify E5⁺ and E5⁻ levels. The values at baseline (0 h) were designated 1. (B) Effect of cycloheximide on the E5⁺/E5⁻ ratio. Different cellular fractions were prepared from cells treated or not with cycloheximide for 6 h. PCR was performed to amplify PTGS2 mRNA and to coamplify H-ras E5⁺ and E5⁻ splice variants. (C) Quantification of H-ras splice variant levels in the presence of cycloheximide. Cells were treated with cycloheximide for different times (hours). qPCR was performed to quantify E5⁺ and E5⁻ levels. The values at baseline (0 h) were designated 1. (D) Effect of cycloheximide on the cytosolic and nuclear levels of E5⁺ splice variant. C and N fractions were prepared from cells treated with cycloheximide for different times (hours). qPCR was performed to quantify the cytosolic and nuclear E5⁺ levels. The values obtained at baseline (0 h) in fractions C and N were designated 1. (E) Effects of siUpf1, siUpf2, siBTZ, siRNPS1, and the control siGAPDH on H-ras splice variant levels. MCF7 cells were transfected with different siRNAs as indicated. qPCR was performed to quantify E5⁺ and E5⁻ levels. The values obtained in the control experiments (siGAPDH lanes) were designated 1. (F) Effects of siUpf1, siUpf2, siBTZ, siRNPS1, and the control siGAPDH on the cytosolic and nuclear levels of E5⁺ splice variant. C and N fractions were prepared from cells transfected with different siRNAs as indicated. qPCR was performed to quantify the cytosolic and nuclear E5⁺ levels. The values obtained in the control experiments (siGAPDH lanes) were designated 1. (G) Effect of wortmannin on E5⁺ splice variant levels. Cells were treated or not with wortmannin for 4 h. Different fractions were prepared, and qPCR was performed to quantify E5⁺ levels. Each value obtained in the presence of wortmannin was divided by the value obtained in the corresponding control experiment (i.e., cells not treated with wortmannin). Experiments were performed at least three times. Data are presented as the mean ± standard error of the mean (*, P < 0.05). (H) Effects of siUpf1, siBTZ, and the control siGAPDH on the p21^H-ras protein level. Protein extracts were prepared from cells transfected with different siRNAs, as indicated, to perform a WB analysis of H-ras and actin protein levels with anti-H-ras (αH-ras) and antiactin (αActin) antibodies.

FIG. 3.

Differential regulation of H-ras splice variant expression by topoisomerase inhibitors. (A) Effects of topoisomerase inhibitors on H-ras splice variant levels. MCF7 cells were treated or not with CPT, etoposide, or doxorubicin for 6 h. C and N fractions were prepared and used to quantify E5⁺ and E5⁻ levels (upper and middle panels, respectively). The values obtained in the absence of treatment in fraction C were assigned 1 to highlight the differences between fractions C and N. PCR was performed to coamplify both E5⁻ and E5⁺ splice variants (lower panel). (B) Effect of CPT on p21^H-ras protein levels. MCF7 cells were treated or not with CPT for 6 h. Protein extracts were used for WB analysis of H-ras and actin protein levels. (C) Effect of CPT on the H-ras E5⁺i6⁺/E5⁺ ratio. Different cellular fractions were prepared from cells treated or not with CPT for 6 h. RT-PCR was performed using a sense primer (E4/5) overlapping E4 and E5 and an antisense primer in E7 to coamplify E5⁺i6⁺ and E5⁺ splice variants. (D) Kinetics of CPT effects in the cytosolic RNA population. C fractions were prepared from cells treated with CPT for different times or cells treated with CPT for 6 h followed by CPT withdrawal and cell incubation for different times (6 + 2 h, 6 + 4 h, and 6 + 18 h). The levels of E5⁻ and E5⁺ splice variants were quantified by qPCR. The values at baseline (0 h) for each splice variant were designated 1. (E) Kinetics of CPT effects in the nuclear RNA population. Panel E is the same as panel D using N fractions (upper panel). PCR was performed to coamplify E5⁻ and E5⁺ splice variants (lower panel). (F) Time course analysis of the E5⁺(C) and E5⁺(N) levels between 10 and 24 h of CPT treatment. Experiments were performed at least three times. Data are presented as the mean ± standard error of the mean (*, P ≤ 0.05).

FIG. 4.

Role of p53 in CPT effects on H-ras splice variant levels. (A) Effect of actinomycin D on CPT-mediated effects. Cells were pretreated or not with actinomycin D for 2 h and then treated or not with CPT for 2 more hours. qPCR was performed to quantify E5⁺ levels using RNAs prepared from N fractions. The value at baseline (no treatment) was designated 1. (B) Effect of cycloheximide on p53 protein levels in response to CPT. MCF7 cells were pretreated or not with cycloheximide for 30 min and then treated or not with CPT for 6 h. WB analysis was performed with p53 and actin as a control on the same membrane. αP53, anti-p53 antibody; αActin, antiactin antibody. (C) Effect of cycloheximide on nuclear E5⁺ levels in response to CPT. MCF7 cells that were pretreated or not with cycloheximide for 2 h were treated or not with CPT for 2 h. Shown is the quantification by qPCR of E5⁺ levels using RNAs prepared from nuclear fractions. The value at baseline (no treatment) was designated 1. (D) Roles of p53 in CPT-mediated effects on nuclear E5⁺ splice variant levels. qPCR was performed to quantify E5⁺ in the nuclear RNA population. The value at baseline (lane siGAPDH in the absence of CPT) was designated 1. (E) Addition of fresh medium simultaneously with CPT stimulated the CPT effect on H-ras splice variants. qPCR was performed to quantify the E5⁺ and E5⁻ levels in the nuclear extracts prepared from MCF7 cells plated 24 h before addition of CPT alone or CPT with fresh medium. (F) Recruitment of p53 on the endogenous H-ras gene. MCF7 cells were treated or not with CPT for 2 h. A ChIP assay using a control antibody and a p53 antibody was performed. The purified DNA was used in PCR (upper panel) and qPCR (lower panel). (G) CPT favored the production of the E5⁺ splice variant in MLS-1675 cells but not in HeLa cells. qPCR was performed to quantify the E5⁺ and E5⁻ levels in nuclear extracts prepared from MCF7, MLS, or HeLa cells treated with CPT for 6 h. (H) CPT increased the E5⁺/E5⁻ ratio in MLS-1675 cells but not in HeLa cells. PCR was performed to coamplify E5⁻ and E5⁺ splice variants from nuclear extracts prepared from MCF7, MLS, or HeLa cells treated with CPT for 6 h. (I) P53 was poorly stabilized in HeLa cells treated with CPT. Protein extracts were prepared from MCF7 and HeLa cells treated or not with CPT for 6 h 1 day after transfection with a p53 or a control expression vector. p53 protein was almost not detected in HeLa cells treated or not with CPT but was strongly expressed after transfection with a p53 expression vector. (J) p53 overexpression increased the levels of the H-ras E5⁺ splice variant. Total RNAs were prepared from MCF7 and HeLa cells treated or not with CPT for 6 h 1 day after transfection with a p53 or a control expression vector. qPCR was performed to measure the levels of the H-ras E5⁺ splice variant. (K) p53 overexpression did not alter the levels of the H-ras E5⁻ splice variant. qPCR was performed to measure the levels of the H-ras E5⁻ splice variant. The same experimental conditions as those described in panel I were used. (L) p53 was stabilized in MCF7 cells treated with CPT or doxorubicin (Doxo) for 18 h. Protein extracts were prepared from MCF7 cells treated or not with CPT or doxorubicin for 18 h or from MCF7 cells transfected or not with a p53 expression vector. (M) Effects (fold) of topoisomerase inhibitors and p53 overexpression on H-ras splice variant levels. MCF7 cells were treated or not with CPT or doxorubicin for 18 h or transfected or not with a p53 expression vector. Nuclear fractions were prepared and used to quantify E5⁺ and E5⁻ levels. (N) Recruitment of p53 on the endogenous H-ras gene following p53 overexpression or in response to CPT treatment but not in response to doxorubicin treatment. MCF7 cells were treated or not with CPT or doxorubicin for 18 h or transfected or not with a p53 expression vector. A ChIP assay using a control antibody and a p53 antibody was performed. The purified DNA was used in qPCR.

FIG. 5.

Role of SC35 in CPT effects on H-ras splice variant levels. (A) Roles of SC35, A1, and p53 in H-ras splice variant levels. qPCR was performed to quantify E5⁺ and E5⁻ levels in the nuclear (N) RNA populations (upper panel). The values obtained in the control experiments (siGAPH lanes) were assigned as 1. PCR was performed on the nuclear RNA population to coamplify E5⁺ and E5⁻ splice variants (lower panel). (B) WB analysis of SC35. Protein extracts obtained from cells transfected with siSC35, siP53, or siGAPDH were prepared and used for WB analysis of SC35, with actin as a control on the same membrane. αSC35, anti-SC35 antibody; αActin, antiactin antibody. (C) Roles of p53 and SC35 in the CPT-mediated effects on H-ras splicing. Cells transfected with different siRNAs were treated or not with CPT for 6 h. qPCR was performed to quantify E5⁺ and E5⁻ levels using RNAs prepared from N fractions. In each set of experiments (siGAPDH, siP53, and siSC35), the CPT effects on the E5⁺ splice variant were compared with the CPT effects on the E5⁻ splice variant. (D) Opposite effects of siP53 and siSC35 on the nuclear E5⁺/E5⁻ ratio. N fractions were prepared from cells transfected with different siRNAs and treated or not with CPT for 6 h. PCR was performed to coamplify E5⁺ and E5⁻ splice variants using an antisense primer overlapping the last exon-exon junction and a sense primer in exon 4 (H-rasE4) or in exon 2 (H-rasE2). (E) Exogenous HA-SC35 protein was overexpressed in HeLa cells but not in MCF7 cells. Protein extracts were prepared from MCF7 and HeLa cells transfected with an HA-SC35 or a control expression vector. HA-SC35 protein was highly expressed compared to the endogenous SC35 protein in HeLa cells but not in MCF7 cells. (F) SC35 overexpression decreased the E5⁺/E5⁻ ratio. Total RNAs were prepared from MCF7 and HeLa cells transfected with a HA-SC35 or a control expression vector. qPCR was performed to measure the levels of the H-ras E5⁺ and H-ras E5⁻ splice variants. In each set of experiments, the effects on the E5⁺ splice variant were compared with the effects on the E5⁻ splice variant. (G) Effect of CPT on SC35 protein levels. MCF7 cells were treated or not with CPT for 6 h and 18 h. WB analysis of SC35 was performed, using actin as a control. (H) Effect of CPT on the expression of SR proteins. MCF7 cells were treated or not with CPT for 18 h. WB analysis of SR proteins was performed, using the mAb104 antibody and actin as a control. (I) Effect of different genotoxic stress inducers on p53 and SC35 protein levels. MCF7 cells were treated or not with CPT, hCPT, cisplatin (CDDP), or doxorubicin (DOX) for 6 h. WB analysis of p53 and SC35 protein levels was performed. (J) Effect of different genotoxic stress inducers on H-ras E5⁺ and E5⁻ nuclear levels. MCF7 cells were treated or not with CPT, hCPT, cisplatin, or doxorubicin for 6 h. qPCR was performed to measure the nuclear levels of the H-ras E5⁺ and H-ras E5⁻ splice variants. PCR was performed to coamplify E5⁺ and E5⁻ splice variants by using an antisense primer overlapping the last exon-exon junction and a sense primer in exon 4 (H-rasE4) or in exon 2 (H-rasE2). Experiments were performed at least three times. Data are presented as the mean ± standard error of the mean (*, P ≤ 0.05).

FIG. 6.

(A) Effects of CPT and p53 overexpression on the E5⁺/E5⁻ ratio. MCF7 cells were treated or not with CPT for 18 h or transfected with a p53 expression vector. qPCR was performed to quantify E5⁺ and E5⁻ levels using RNAs prepared from N fractions. The CPT effects on the E5⁺ splice variant were compared with the CPT effects on the E5⁻ splice variant. Similarly, the effects of p53 overexpression on the E5⁺ splice variant were compared with the effects of p53 overexpression on the E5⁻ splice variant. (B) Cotransfection of p53 and SC35 expression vectors. Total RNAs were prepared from HeLa cells transfected or not with p53 and SC35 expression vectors as indicated. qPCR was performed to measure the levels of the H-ras E5⁺ splice variant. The values in the control experiments (transfection of control expression vectors) were designated 1. (C) p53 overexpression increased the levels of the H-ras E5⁺ splice variant in both the N and C fractions. N and C fractions were prepared from MCF7 and HeLa cells transfected or not with a p53 expression vector. qPCR was performed to measure the levels of the H-ras E5⁺ splice variant. (D) Cycloheximide treatment did not further increase the expression levels of the H-ras E5⁺ splice variant made in cells transfected with a p53 expression vector. Total RNAs were prepared from HeLa cells transfected or not with a p53 expression vector as indicated and treated or not with cycloheximide 3 h before harvesting the cells. qPCR was performed to measure the levels of the H-ras E5⁺ splice variant. The values in the control experiments (transfection of control expression vectors in absence of cycloheximide) were designated 1. (E) Effect of cycloheximide and p53 overexpression on the expression levels of various NMD-target splice variants in HeLa cells. Total RNAs were prepared from HeLa cells transfected or not with a p53 expression vector as indicated and treated or not with cycloheximide 3 h before harvesting the cells. (F) Effect of cycloheximide and CPT treatment on the expression levels of various NMD-target splice variants in MCF7 cells. Total RNAs were prepared from MCF7 cells treated or not with cycloheximide for 6 h or with CPT for 18 h as indicated.

FIG. 7.

p53 and SC35 regulate a subset of NMD-target splice variants in an opposite manner. (A) Effect of cycloheximide on various SV^NMD/SV^CDS ratios. Different cellular fractions were prepared from cells treated or not with cycloheximide for 6 h and used to coamplify SV^NMD and SV^CDS produced from different genes. The SV^NMD are marked by an arrow on the right. (B) Effect of CPT on different nuclear SV^NMD/SV^CDS ratios. N fractions were prepared from cells treated or not with CPT for 6 h and used to coamplify SV^NMD and SV^CDS produced from different genes. (C) Effects of siP53 and siSC35 on different nuclear SV^NMD/SV^CDS ratios in the presence of CPT. N fractions were prepared from cells transfected with siP53, siSC35, or siGAPDH and treated or not with CPT for 6 h and used to coamplify SV^NMD and SV^CDS produced from different genes. (D) Effects of siSC35 on different nuclear SV^NMD/SV^CDS ratios in the absence of CPT. N fractions were prepared from cells transfected with siSC35 or siGAPDH and used to coamplify SV^NMD and SV^CDS produced from different genes. (E) SC35, SF2/ASF, SRp40, and SRp55 binding motifs in alternatively spliced exons. The search of the SC35, SF2/ASF, SRp40, and SRp55 binding motifs was done using ESEfinder within cassette exons generating or not a PTC (categories “NMD” and “no NMD,” respectively). For each category, a sequence was constituted by assembling the sequences of each corresponding cassette exon after each other. A score was calculated by dividing the number of sites by the length of the analyzed sequence as described in Materials and Methods.

FIG. 8.

Upf1 is required for optimal cellular response to CPT. (A) Effects of siUpf1 and siBTZ on E5⁺ levels induced by CPT. Cells transfected with siGAPDH, siUpf1, or siBTZ were treated or not with CPT for 6 h. The E5⁺ level was quantified by qPCR. (B) Effects of siUpf1 and siBTZ on the cytosolic E5⁺ levels induced by CPT. Panel B is the same as panel A using C fractions. (C) Effects of siUpf1 and siBTZ on the nuclear E5⁺ levels induced by CPT. Panel C is the same as panel A using N fraction. (D) Effects of siUpf1 and siBTZ on the nuclear E5⁺/E5⁺ ratio modulated by CPT. PCR was performed to coamplify E5⁺ and E5⁻ using an antisense primer overlapping the last exon-exon junction and a sense primer in exon 4 (H-rasE4) or in exon 2 (H-rasE2). (E) Effects of siUpf1 on p53 protein and mRNA levels in response to CPT. MCF7 cells transfected with siGAPDH, siUpf1, siBTZ, or siP53 were treated or not with CPT for 6 h. WB analysis of p53 and actin (upper panel) and amplification of the p53 mRNA (lower panel) were performed. (F) Effect of wortmannin on p53 protein levels in response to CPT. MCF7 cells were pretreated or not with wortmannin for 30 min and then treated or not with CPT for 6 h. WB analysis of p53 and actin as a control was performed. (G) Effects of siUpf1 and siP53 on different nuclear SV^NMD/SV^CDS ratios modulated by CPT. N fractions were prepared from cells transfected with siUpf1, siP53, or siGAPDH and treated or not with CPT for 6 h and used to coamplify different SV^NMD and SV^CDS. (H) Effect of siUpf1 on p21^waf1 mRNA expression levels in response to CPT. Quantification of p21^waf1 mRNA levels by qPCR using an RNA preparation from MCF7 cells transfected with siGAPDH, siUpf1, or siP53 and treated or not with CPT for 6 h. The value obtained with siGAPDH in the absence of CPT was designated 1. Experiments were performed at least three times. Data are presented as the mean ± standard error of the mean. (I) Outcome of the activation of the H-ras locus by p53 in response to CPT. The H-ras gene produces different splice variants that have different fates and that result in the production of different protein isoforms. In this report, we demonstrated that CPT treatment increased the synthesis of the unproductive E5⁺ splice variant (degraded by the NMD process) through the stabilization of p53 protein, which activated the H-ras gene, and through the alteration of SC35 splicing factor, which promoted exon 5 inclusion. It can be anticipated that the impacts on the H-ras locus of p53 or other p53 family proteins may have different outcomes, depending on the nature of the genotoxic stress that activates the p53 pathway, on the cellular context, and, at the molecular levels, on alternative splicing regulation.