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Cell Cycle. Aug 1, 2011; 10(15): 2561–2567.
Published online Aug 1, 2011. doi:  10.4161/cc.10.15.16347
PMCID: PMC3180194

The RNA surveillance protein SMG1 activates p53 in response to DNA double-strand breaks but not exogenously oxidized mRNA

Abstract

DNA damage, stalled replication forks, errors in mRNA splicing and availability of nutrients activate specific phosphatidylinositiol-3-kinase-like kinases (PIKKs) that in turn phosphorylate downstream targets such as p53 on serine 15. While the PIKK proteins ATM and ATR respond to specific DNA lesions, SMG1 responds to errors in mRNA splicing and when cells are exposed to genotoxic stress. Yet, whether genotoxic stress activates SMG1 through specific types of DNA lesions or RNA damage remains poorly understood. Here, we demonstrate that siRNA oligonucleotides targeting the mRNA surveillance proteins SMG1, Upf1, Upf2 or the PIKK protein ATM attenuated p53 (ser15) phosphorylation in cells damaged by high oxygen (hyperoxia), a model of persistent oxidative stress that damages nucleotides. In contrast, loss of SMG1 or ATM, but not Upf1 or Upf2 reduced p53 (ser15) phosphorylation in response to DNA double strand breaks produced by expression of the endonuclease I-PpoI. To determine whether SMG1-dependent activation of p53 was in response to oxidative mRNA damage, mRNA encoding green fluorescence protein (GFP) transcribed in vitro was oxidized by Fenton chemistry and transfected into cells. Although oxidation of GFP mRNA resulted in dose-dependent fragmentation of the mRNA and reduced expression of GFP, it did not stimulate p53 or the p53-target gene p21. These findings establish SMG1 activates p53 in response to DNA double strand breaks independent of the RNA surveillance proteins Upf1 or Upf2; however, these proteins can stimulate p53 in response to oxidative stress but not necessarily oxidized RNA.

Key words: DNA double strand breaks, nonsense-mediated mRNA decay (NMD), oxidative stress, phosphatidylinositiol-3-kinase-like kinases (PIKKs), RNA damage

Introduction

The phosphatidylinositol-3-kinase-like kinases (PIKK) play an essential role in coordinating the cellular response to a variety of stresses, including nucleotide damage. Four of 6 PIKKs are involved in the response to DNA damage. The ataxia-telangiectasia mutated (ATM) protein and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) respond primarily to DNA double strand breaks, while the ataxia-telangiectasia and Rad3-related (ATR) kinase is activated by single stranded DNA and stalled replication forks.13 The human suppressor with morphogenetic effect on genitalia (SMG1) is also activated by genotoxic stress, but is better known for its ability to respond to errors in mRNA splicing.4,5 Recruitment and activation of the PIKKs requires interaction with various protein partners. ATM binds Nbs1 in the Mre11-Rad50-Nbs1 (MRN) complex found at sites of DNA strand breaks,68 while DNA-PKcs binds the heterodimer Ku70/80 bound to broken ends of DNA.9 ATR binds ATRIP, which in turn recognizes replication protein A (RPA) bound to single stranded DNA.10 Although the partners necessary for SMG1 to respond to DNA damage are unclear, SMG1 facilitates nonsense-mediated mRNA decay (NMD) in conjunction with the SURF and exon-junction-complex (EJC) proteins on mRNAs containing a pre-termination codon (PTC).1113

Once activated, the PIKKs phosphorylate serine/threonine residues on effector proteins that maintain DNA integrity by regulating the cell cycle, DNA repair and apoptosis. Phosphorylation of p53 on serine 15 (ser15) is a common target and one that is of particular importance for cell cycle arrest, apoptosis and senescence and aging.1417 Serine 15 phosphorylation increases both protein stability and transcriptional activity of p53, therefore activating p53 signaling.14,18,19 Since genotoxic stress can create different types of DNA lesions or other forms of molecular damage, the coordinated activation of PIKKs responding to different lesions ensures proper activation of downstream targets. For instance, ATM is rapidly activated in response to ionizing radiation while ATR responds later, perhaps reacting to replication fork collapse or transcription stalling on damaged DNA.1,20 Similarly, chronic exposure to hyperoxia stimulates p53 (ser15) phosphorylation via the sequential activation of SMG1 followed by ATM.5 In contrast, another study suggested ATR and not ATM was required to phosphorylate p53 (ser15) during hyperoxia.21 However, in other studies, the use of dominant-negative and siRNA oligonucleotides targeting ATR dispute a role for ATR in stimulating p53 during hyperoxia.5,22 The differences between these observations have yet to be clarified. Regardless, analogous to the sequential activation of ATM and ATR in cells exposed to ionizing radiation, the early activation of SMG1 implies that hyperoxia causes some type of RNA damage recognized by SMG1. One candidate is oxidized RNA, which has been shown to be 10–20 times more sensitive to oxidation than DNA.2325 The late activation of ATM is likely a response to DNA double strand breaks produced during exposure.2629

Since oxidative stress can damage all molecules, models have been created to test the cellular response to specific forms of damage. For instance, the cellular response to DNA double strand breaks has been studied using expression of unique or rare cutting endonucleases.6,30,31 Likewise, translation inhibition of oxidized mRNA has been studied in cells transfected with oxidized mRNAs.32,33 Here, we use these models to investigate the ability of damaged RNA to activate p53 and the role of mRNA surveillance proteins in activation of p53 in response to DNA double strand breaks.

Results

DNA damage stimulates SMG1 and ATM-dependent phosphorylation of p53 serine 15.

A549 cells were transfected with siRNA oligonucleotides targeting SMG1, ATM or luciferase as a non-targeting control. Cells were then exposed to room air for =24 h or hyperoxia for 24 and 48 h. As previously reported in reference 5, hyperoxia stimulated phosphorylation of p53 (ser15) within 24 h that was suppressed by SMG1, but not ATM knockdown (Fig. 1A). By 48 h, p53 (ser15) phosphorylation was inhibited by both SMG1 and ATM knockdown. Consistent with RNA surveillance proteins contributing to p53 activation, loss of Upf1 or Upf2 also reduced phosphorylation of p53 (ser15) during hyperoxia (Fig. 1B and C). Similar results were observed in HCT116 colon carcinoma cells (data not shown), suggesting this is not a response unique to one cancer cell line. siRNA knockdown of Upf1 did not affect p53 mRNA expression (data not shown).

Figure 1
Hyperoxia activates SMG1-Upf and ATM-dependent phosphorylation of p53 serine 15. A549 cells were transfected with siRNA oliognucleotides targeting (A) SMG1, ATM, (B) Upf1, (C) Upf2 or luciferase (Luc) as a non-targeting control, and then exposed to room ...

Because hyperoxia is a strong clastogenic agent,26,27 it likely activates ATM by causing DNA double strand breaks. Whether DNA double strand breaks can also activate SMG1 is unknown. To test this, DNA double strand breaks were created by infecting cells with retroviruses expressing the eukaryotic homing endonuclease I-PpoI fused to a mutant estrogen response domain (ERT) required for nuclear translocation and increased stability in response to tamoxifen.6 Treatment of infected cells with tamoxifen for 24 h stimulated expression of hemagglutinin antigen (HA)-tagged I-PpoIERT, phosphorylation of p53 (ser15) and p53 abundance (Fig. 2A). I-Ppol localization to the nucleus likely protects it against degradation, resulting in increased protein levels with tamoxifen.6 siRNA oligonucleotides targeting SMG1 and ATM attenuated p53 ser(15) phosphorylation in response to I-PpoIERT (Fig. 2B and C). This was a response to DNA damage because p53 (ser15) phosphorylation was unaffected by loss of SMG1 or ATM in cells cultured without tamoxifen. The fold change of p53 (ser 15) expression between the luciferase control and SMG1 or ATM knockdown samples in the absence of tamoxifen was 1.29 ± 0.3 (p = 0.19, n = 3) and 0.84 ± 0.45 (p = 0.45, n = 3), respectively. Despite involvement of SMG1, loss of Upf1 or Upf2 did not affect the phosphorylation of p53 (ser15) in response to DNA double strand breaks.

Figure 2
DNA double strand breaks activate SMG1 and ATM. A549 cells were infected with retrovirus expressing HA-tagged I-PpoIERT restriction enzyme and then in the absence or presence of tamoxifen (1 µM). (A) Cell lysates were immunoblotted for indicated ...

Exogenously oxidized mRNA does not activate p53.

Since RNA is a more sensitive target of oxidation than DNA2325 and SMG1 is known to play a role in a type of RNA surveillance, the early activation of SMG1 during hyperoxia may be in response to oxidized RNA. To test this, GFP mRNA with a poly A+ tail was transcribed in vitro, and oxidized as described in the Materials and Methods. The oxidation reaction was quenched by ethanol precipitation and RNA integrity evaluated by agarose gel electrophoresis. Although the majority of RNA migrated as a single band of expected full-length, a smear that was indicative of strand breaks was readily observed with the two highest levels of oxidation (Fig. 3A). The RNA was then transfected into A549 cells and its presence detected 24 h later by semi-quantitative RT-PCR. Comparable amounts of GFP mRNA were detected at all oxidation levels when using primers that amplified an internal 200 bp sequence while less RNA was detected at the two highest oxidation conditions when using primers that amplify the entire cDNA (Fig. 3B). The amplification of GFP in all conditions using internal primers but not full-length primers suggests that highly oxidized GFP mRNA enters the cell similarly to un-oxidized mRNA and then is degraded to smaller fragments. To assess the effects of mRNA oxidation on protein synthesis, the expression of GFP was investigated. Consistent with an earlier report showing that oxidized mRNA is poorly translated,32 expression of GFP protein by protein gel blot or immunostaining (data not shown) was markedly reduced in cells transfected with RNA that was oxidized under conditions 2 and 3 (Fig. 3C).

Figure 3
Transfected oxidized mRNA does not activate p53. (A) Integrity of non-oxidized (condition 0) or oxidized (condition 1–3) GFP mRNA. (B) GFP mRNA oxidized under conditions 0–3 was transfected into cells and RT-PCR was performed on total ...

Based upon the number of cells with green fluorescence, transfection efficiency of non-oxidized mRNA was approximately 85% (data not shown). Despite this relatively high transfection efficiency, oxidized GFP mRNA did not stimulate p53 (ser 15) phosphorylation or p53 abundance by protein gel blot or by immunostaining (data not shown) when compared with transfection with unoxidized mRNA (Fig. 3D and E). Also, oxidized mRNAs did not stimulate expression of the p53-target gene p21Cip1/Waf1 (Fig. 3F). In contrast and as a control for the assay, p21 mRNA was stimulated 8-fold in cells exposed to hyperoxia.

Discussion

Cells are constantly exposed to intrinsic and extrinsic sources of reactive oxygen species that damage DNA, RNA, proteins and lipids. While much attention has focused on how cells recognize and repair DNA damage, less is known about how they respond to RNA damage. RNA is more susceptible to oxidation than DNA, perhaps because it is single stranded, its bases are not hydrogen bonded, and it is less likely to be protected by proteins.2325 Hence, our previous observation that SMG1, a PIKK that plays a role in RNA surveillance and was activated during hyperoxia earlier than ATM, suggested that it would respond to oxidized RNA. Here, using hyperoxia as a model to induce chronic oxidative and genotoxic stress, we show that two additional proteins involved in NMD, Upf1 and Upf2, are also necessary for full activation of p53, suggesting further interplay between RNA and DNA damage surveillance pathways. However, unlike SMG1, Upf1 and Upf2 were not required to activate p53 in response to DNA double strand breaks created by expression of the endonuclease I-PpoI. SiRNA knockdown of Upf2 did however increase mRNA levels of the endogenous NMD target BAG1, confirming that this approach was sufficient to affect endogenous NMD activity (data not shown). Although Upf1 and Upf2 are necessary for NMD, our findings provide strong evidence that SMG1 responds to DNA double strand breaks independent of its role in NMD. They also imply that SMG1 responds to DNA double strand breaks by interacting with a different set of proteins, such as those required to activate ATM or DNA-PKcs.69

Our studies support a separate role for NMD factors in p53 activation during chronic oxidative stress that is distinct from their ability to mediate NMD. Indeed, Upf1 has numerous NMD-independent roles, some of which directly involve genomic maintenance. These include roles in other RNA decay pathways,34 RNA splicing,35 as well as chromatin stability, S-phase entry,36 and telomere stability.37 Further, Rodriguez-Gabriel et al.38 demonstrated that Upf1 stabilizes mRNAs encoding catalase and glutathione peroxidase, specifically during oxidative stress in yeast. Hence, additional studies are needed to fully reveal the role of NMD proteins in the early exposure to hyperoxia or oxidative stress in general.

During NMD, SMG1 and Upf1 track along the RNA with the ribosome until they encounter and bind to proteins in the EJC, which is the signal that recruits decay factors to the RNA. The EJC complex is present on aberrantly spliced mRNAs because a PTC stops translation before the EJC proteins can be removed by the tracking ribosome. Similarly to the presence of a PTC inhibiting complete translation of mRNA transcripts, ribosomes stall on oxidized RNA resulting in inefficient translation and translation of truncated proteins.33,39 Oxidized RNAs have been shown to accumulate in diseases associated with oxidative stress, including Alzhiemer disease, emphysema and a mouse model of emphysema caused by exposure to cigarette smoke.4043 However, it is unknown whether the accumulation of these RNAs affects the disease state or is simply a manifestation of the disease. One possibility is that oxidized RNA could act as substrate to bring proteins together to activate signaling cascades, similarly to a DNA double strand break joining the MRN complex and ATM.6,8 For instance, if the ribosome stalls before reaching the last exon/intron junction, the SURF complex and EJC can interact. During NMD, this leads to recruitment of decay factors. However, the interaction of these proteins could also initiate p53 signaling either alone or in combination with other factors. For example, the presence of YB1 or hPNPase, proteins that bind oxidized RNA,44,45 might alter the activity of the proteins in these complexes and signal to p53 activation.

Our inability to activate p53 when cells are transfected with oxidized mRNA disputes this hypothesis. However, there are limitations to this conclusion. Although the transfection efficiency of oxidized mRNA was high, the amount of damaged RNA per cell may not have been sufficient to activate a detectable damage response. The oxidized mRNA did not contain an intron/exon junction that is required for the interaction of all the NMD factors. Further, the oxidized mRNA experiments were conducted entirely in A549 cell lines; therefore, these results could be specific to this cancer cell line and not representative of in vivo biology. Despite these limitations, our inability to activate p53 with oxidized mRNA is consistent with another study showing that electroporation of RNase A into ML-1 cells was also unable to stimulate p53.46

In conclusion, the present study provides strong evidence that DNA double strand breaks can stimulate SMG1-dependent and NMD-independent phosphorylation of p53 (ser15). SMG1 therefore plays independent roles in responding to DNA and RNA damage. Since it also protects cells against TNFα mediated cytotoxity,47 SMG1 appears to play a broader role in protection against cellular stresses than other members of the PIKK family. Further, we identify a role for two other NMD factors in oxidant-mediated regulation of p53, albeit, whether the observed regulation was a result of changes in NMD itself remains to be determined. Understanding how the various members of the PIKK and NMD families respond to specific types of damage could prove useful for designing targeted approaches for treating disease.

Materials and Methods

Cell culture.

Human lung adenocarcinoma cell line A549 (American Type Culture Collection) was cultured in room air (with 5% CO2) or hyperoxia (95% O2 and 5% CO2).48 Cells were transfected with 100 nM siRNA oligomers using lipofectamine (Invitrogen). Cells were washed twice with Hank's Balanced Salt Solution (HBSS) and media was replenished 6 h after transfection. The sense siRNA targeting oligonucleotides were:

SMG1: 5′-CCA GGA CAC GAG GAA ACU GTT-3′

ATM: 5′-GCG CCU GAU UCG AGA UCC UUU TT-3′

Upf1: 5′-GAU GCA GUU CCG CUC CAU UTT-3′ or smart pool (Dharmacon)

Upf2: 5′-GGC UUU UGU CCC AGC CAU CTT-3′ or 5′-AAC AAC AGC CCU UCC CAG AAU CTT-3′

Luciferase (luc): 5′-CGU ACG CGG AAU ACU UCG ATT-3′.

Virus production and infection.

Retrovirus expressing an HA tagged, tamoxifen-inducible PpoI restriction enzyme was produced using the Phoenix system (www.stanford.edu/group/nolan/retroviral_systems/phx.html). In brief, pBABE-HA-ERpPoI6 and pCMV-VSVG were transfected into phoenix ampho cells using calcium phosphate. Media was collected 48 h later, filtered (0.45 µm) and used to infect cells for 24 h. Cells were washed and media was replenished with that containing tamoxifen (1 µM) (Sigma) or ethanol vehicle.

Transcription in vitro and oxidation of GFP mRNA.

Full-length green fluorescent protein (GFP) cDNA containing a poly A+ tail was created by transcription in vitro of the pGEM4Z/GFP/A64 plasmid using the mMessage machine kit (Ambion). The mRNA was then oxidized in a 10 mM Hepes solution using 3 µM H2O2, and either 2 µM, 20 µM or 40 µM of both FeCl2 and ascorbic acid for one hour at 37°C (adapted from Tanaka et al.). RNA was then ethanol precipitated and re-suspended in DEPC treated water (Sigma). Cells were transfected with 1 µg/ml of GFP mRNA using Lipofectamine 2000 (Invitrogen). The proportion and intensity of cells with green fluorescence was quantified using an LSR II flow cytometer (Becton, Dickinson and Company).

Protein gel blot analysis.

Cells were washed with HBSS, harvested in lysis buffer and sonicated at 20% with a stepped microtip (Sonics Vibra Cell).48 Protein concentrations were determined using BCA assay (Thermo Scientific) and samples were separated using SDS-PAGE before transferring to polyvinylidene difluoride membrane (Pall Life Sciences). All primary antibodies were diluted in 5% milk in Tris buffered saline with 0.1% tween (TBS-T) at the following concentrations: anti-actin (1:10,000, Sigma Aldrich), p53 (DO1) (1:1,000, Novacastra Laboratories), p53 phospho-Ser 15 (1:1,000, Cell Signaling) or EGFP (1:5,000, Clontech). Secondary HRP conjugated antibodies (Southern Biotech) were incubated in 5% milk in TBS-T (1:5,000). HRP was detected using the ECL Plus Protein gel blotting Detection kit (GE Healthcare) and developed using Blue Sensitive film (Laboratory Products Sales). Images were scanned using an EPSON 3200 scanner and quantified using ImageJ software (NIH).

Reverse transcriptase (RT) and real time PCR.

RNA was collected using trizol (Invitrogen). Reverse transcription was performed using iScript cDNA synthesis kit (Biorad). The following primers were used to amplify cDNA or used for real-time PCR using sybrgreen incorporation with a Biorad iQ5 instrument.

GFP internal: 5′-GCA GCA CGA CTT CTT CAA GTC C-3′ and 5′-ATC TTG AAG TTC ACC TTG ATG C-3′

GFP full-length: 5′-ATG GTG AGC AAG GGC GAG GAG CTG-3′ and 5′-CTT GTA CAG CTC GTC CAT GCC GAG-3′

p21: 5′-CTG GAG ACT CTC AGG GTC GAA-3′ and 5′-GGA TTA GGG CTT CCT CTT GGA-3′

HPRT: 5′-CCT GGC GTC GTG ATT AGT GAT GAT-3′ and 5′-AGC AAG ACG TTC AGT CCT GTC CAT-3′.

Statistical analyses.

Values are represented as means ± standard deviations. Significance was determined by ANOVA using Fisher's procedure post hoc analysis in Stat View (Adept Scientific). p < 0.05 was considered significant.

Acknowledgments

This work was funded in part by National Institutes of Health (NIH) Grant HL-67392 and HL-01247. NIH Training Grant HL-66988 supported J. Gewandter. We thank Dr. Michael Kastan for sharing the pBABE-HA-ER-I-PpoI plasmid and Dr. Craig Jordan for sharing the pGEM4Z/GFP/A64 plasmid.

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