HUMANMUTATION 29(1),65^73,2008

RESEARCH ARTICLE

Does the Nonsense-Mediated mRNA DecayMechanism Prevent the Synthesis of TruncatedBRCA1, CHK2, and p53 Proteins?

Olga Anczukow,1 Mark D. Ware,1 Monique Buisson,1 Almoutassem B. Zetoune,1

Dominique Stoppa-Lyonnet,2 Olga M. Sinilnikova,1,3 and Sylvie Mazoyer1�

1Laboratoire de Genetique Moleculaire, Signalisation et Cancer UMR5201 CNRS, Lyon, France, Universite Lyon 1, Lyon, France; 2Service deGenetique Oncologique, Institut Curie, Paris, France; 3Unite Mixte de Genetique Constitutionnelle des Cancers Frequents, Hospices Civils deLyon/Centre Leon Berard, Lyon, France

Communicated by Richard Wooster

The nonsense-mediated mRNA decay (NMD) mechanism is an evolutionarily conserved process ensuring thedegradation of transcripts carrying premature termination codon(s). NMD is believed to prevent the synthesisof truncated proteins that could be detrimental to the cell. However, although numerous studies have assessedthe efficiency of this mechanism at the mRNA level, data are lacking in regard to whether NMD fulfills itsexpected goal at the protein level. In this study, we have investigated whether endogenous alleles of breastcancer predisposing genes carrying nonsense codons were able to produce detectable amounts of truncatedproteins in lymphoblastoid cell lines. A total of 20 truncating BRCA1 mutations were analyzed, along with the1100delC CHEK2 and the 770delT TP53 mutations. All the studied alleles triggered NMD, the amount ofmutant transcript ranging from 16 to 63% of that of the wild-type species. We found that BRCA1 and CHK2truncated proteins could not be detected, even when NMD was inhibited. This suggests that BRCA1 and CHK2truncated proteins are highly unstable. Conversely, the p53 protein encoded by the 770delTallele is as abundantas the wild-type protein, as removal of the C-terminal p53 domain leads to a stabilized mutant protein, whoseabundance is markedly increased when NMD is inhibited. Therefore, our results show that it is not possible toinfer the presence of truncated proteins in cells from carriers of a truncated mutation without experimentalverification, as each case is expected to be different. Hum Mutat 29(1), 65–73, 2008. rr 2007 Wiley-Liss, Inc.

KEY WORDS: BRCA1; CHK2; p53; nonsense-mediated mRNA decay; truncated protein; breast cancer predisposition

INTRODUCTION

The nonsense-mediated mRNA decay (NMD) pathway recog-nizes and degrades mRNAs that contain a premature terminationcodon (PTC) [Conti and Izaurralde, 2005]. PTCs can arise intranscripts as a consequence of mutations, errors in transcription,faulty or alternative splicing, or programmed rearrangements.In mammals, NMD distinguishes normal termination codons fromPTCs on the basis of their location relative to exon–exon junctions(EEJs). During a pioneering round of translation, the positions ofEEJs are indicated by the presence of the exon–exon junctioncomplex (EJC), a multiprotein complex deposited approximately20–24 nucleotides (nt) upstream of exon–exon boundaries duringthe process of mRNA splicing. Transcripts bearing terminationcodons 450 nt upstream of the last EEJ are considered prematureand are targeted for degradation while those with terminationcodons located o50 nt from the last EEJ or lacking a downstreamEEJ (i.e., the termination codon occurs in the last exon) avoiddetection [reviewed in Conti and Izaurralde, 2005].

NMD is evolutionarily conserved and is one of many quality-control processes that have evolved to ensure the exclusiveproduction of functional proteins. Its aim is to prevent thesynthesis of C-terminally truncated proteins that could havedominant-negative functions and interfere with normal cellular

pathways. NMD efficiency appears to vary depending on thegenes, the position of the PTCs, and possibly the tissue type. Inprevious studies, we showed that the level of mutant transcripts inlymphoblastoid cell lines (LCLs) established from mutationcarriers ranged from 18 to 70% of their normal counterparts inthe case of BRCA1 (MIM] 113705) [Perrin-Vidoz et al., 2002]and from 30 to 71% for BRCA2 (MIM] 600185) [Ware et al.,2006]. Similar figures were obtained in studies focusing onmutated endogenous alleles such as MLH1 (MIM] 120436)[Tournier et al., 2004], IDS (MIM] 309900) [Lualdi et al., 2006],

Published online 10 August 2007 in Wiley InterScience (www.interscience.wiley.com).

DOI10.1002/humu.20590

The Supplementary Material referred to in this article can beaccessed at http://www.interscience.wiley.com/jpages/1059-7794/suppmat.

Received 27March 2007; accepted revisedmanuscript 4 June 2007.

Grant sponsors: le ComiteŁ DeŁ partemental du RhoŒ ne et de la Loirede la Ligue contre le Cancer; Institut Curie ‘‘Programme Incitatif etCoopeŁ ratif: GeŁ neŁ tique et Biologie des Cancers du Sein’’.

�Correspondence to: Sylvie Mazoyer, Laboratoire de GeŁ neŁ tiqueMoleŁ culaire, Signalisation et Cancer UMR5201 CNRS, FaculteŁ deMeŁ decine,8 avenue Rockefeller,69373 LYON cedex 08, France.E-mail: sylvie.mazoyer@recherche.univ-lyon1.fr

rr 2007 WILEY-LISS, INC.

and COL6A2 (MIM] 120240) [Zhang et al., 2002]; in the case ofHNF-1b (MIM] 189907) [Harries et al., 2005] and JAGGED1(MIM] 601920) [Boyer et al., 2005], higher levels of degradationwere obtained for some mutations.

Therefore, it seems that NMD does not completely eradicatePTC-containing transcripts in most cases. This raises the questionof whether PTC1 transcripts that escape degradation by NMDcould produce detectable amounts of truncated proteins. Thereare several well-studied examples of human phenotypes that aremodulated by NMD [Khajavi et al., 2006]. As a striking example,PTC mutations in the SOX10 gene (MIM] 602229) give rise totwo distinct peripheral neuropathy diseases, depending on whethertranscripts with PTCs escape or are degraded by NMD. As alltested truncated Sox10 proteins have been shown to befunctionally equivalent in vitro [Inoue et al., 2004], this impliesthat NMD, when it can operate, does result in a marked decreaseor even a total disappearance of truncated Sox10 proteins.However, this may vary depending on the protein.

In this work, we aimed to determine if the PTC-containingalleles of three breast cancer predisposing genes, BRCA1, CHEK2(MIM] 604373), and TP53 (MIM] 191170), allowed the synthesisof truncating proteins in LCLs from carriers.

MATERIALSANDMETHODSCell Culture

Human LCLs were established by Epstein-Barr virus immorta-lization of patient’s blood lymphocytes. Cells were maintained inRPMI 1640 medium (Invitrogen, Cergy Pontoise, France)supplemented with 10% fetal calf serum (VWR, Fontenay sousBois, France) and 1% penicillin-streptomycin (Invitrogen) in a 5%CO2 incubator at 371C. The mutations identified in one allele ofeither the BRCA1, CHEK2, or TP53 genes are describedaccording to GenBank accession number U14680.1, DQ893133.1,and BT019622.1, respectively. For NMD inhibition, lymphoblas-toid cells were treated for 6 hr with puromycin or wortmannin(Sigma Aldrich, St Quentin Fallavier, France) at a finalconcentration of 100 and 10 mM, respectively. For inhibition ofprotein degradation, cells were treated for 6 hr with either MG132,chloroquine, N-acetyl-leu-leu-norleucinal (ALLN), or N-acetyl-leul-leu-normethional (ALLM) (Sigma Aldrich) at a finalconcentration of 50mM for the former and 100 mM for the othersor mock-treated with the solvent alone (Me2SO).

InVitroTranscription-Translation

Fragments coding for BRCA1-ter 507 and BRCA1-ter 845 wereamplified from cDNA using Platinum Taq (Invitrogen) witha common forward primer including a T7 promoter sequence50-CGCTAATACGACTCACTATAGGAACAGACCACCATGGATTTATCTGCTCTTCGCG-30 and the following reverse primer,50-TCTCCTTTTACGCTTTAATTTATTTG-30 or 50-TATGCTTGTTTCCCGACTGTG-30, respectively.

After purification of the PCR fragments with the MiniElutePCR Purification kit (Qiagen, Courtaboeuf, France), DNAconcentration was measured, and 300 ng was used for in vitrotranscription-translation with the TNT Coupled ReticulocyteLysate System (Promega, Charbonnieres, France) according to themanufacturer’s protocol.

Minigene Constructs

CHEK2 minigenes. The CHEK2 minigenes (either wild-type or carrying the 1100delC mutation) containing the entirecoding sequence of the CHEK2 gene (exon 2 to exon 15) with or

without intron 14 have been constructed as described in theSupplementary Methods (available online at http://www.inters-cience.wiley.com/jpages/1059-7794/suppmat).

BRCA1minigenes. The sequence corresponding to exon 10to exon 12 of the BRCA1 gene with intron 10 was introduced inthree steps into the pEGFP-C1 vector (Clontech, Saint-Germain-en-Laye, France) as described in the Supplementary Methods.

The BRCA1 wild-type (WT)-hemagglutinin (HA) minigene(PCDNA3b) has been described previously [Scully et al., 1997]and was a kind gift of Pr. Jean Feunteun (Institut Gustave Roussy,Paris, France). A BRCA1 minigene containing exon 1 to 12,described previously [Buisson et al., 2006], was used to introducethe 2594delC and 3875del4 mutations by site-directed mutagenesis.

All primers were produced by MWG (Ebersberg, Germany). Allenzymes were purchased at New England Biolabs (Ozyme, StQuentin-en-Yvelines, France). All fragments were gel purifiedusing GeneClean Turbo (Qbiogen, Illkirch, France), and con-trolled by sequencing (Genome Express, Meylan, France).

TransientTransfections

The plasmids used for the transfections were prepared with thePhoenIX midiprep kit (Qbiogen). HeLa cells were grown at 371Cin Dulbecco’s modified Eagle’s medium (Invitrogen) supplementedwith 10% fetal calf serum (VWR) and 1% penicillin-streptomycin(Invitrogen). They were seeded at 1� 106 cells per 100-mm-diameter petri dish 8 hr before transfection, performed by thecalcium phosphate precipitation method with 2mg of any BRCA1minigene and 1mg of the WT b-globin gene [Thermann et al.,1998], and collected 48 hr later.

StabilityAnalysis

HeLa cells were seeded at 250,000 cells per well in six-wellplates. They were transiently transfected as described above with2 mg of BRCA1 plasmid per well, and were treated 24 hrposttransfection with cycloheximide (Sigma-Aldrich) at a finalconcentration of 100mg/ml.

Allele-Speci¢cTranscript Expression

Single-nucleotide primer extension was executed as described inthe Supplementary Methods with the ABI Prism SNaPshotMultiplex Kit (Applied Biosystems, Foster City, CA) [Wareet al., 2006].

PCR-TP53 i9 Variant

The TP53 i9 variant was coamplified with the WT TP53transcript in a 35-cycle PCR using Platinum Taq (Invitrogen) andthe following primers 50-GCGCACAGAGGAAGAGAATC-30

(exon 8) and 50-CCTCATTCAGCTCTCGGAAC-30 (exon 10),according to the manufacturer’s protocol. Fragments wereseparated on a 2% agarose gel and stained with ethidium bromide.

gDNA and RNA Extraction

gDNA was isolated from 5� 106 frozen lymphoblastoid cellsusing a QIAamp DNA Minikit (Qiagen) according to themanufacturer’s instructions. Total RNA was isolated from5� 106 transfected HeLa cells or 5� 106 lymphoblastoid cellsusing the Nucleospin RNA II kit (Macherey-Nagel, Hoerdt,France) according to the manufacturer’s instructions.

RNA Analysis

Total RNA extracted from transfected HeLa cells was separatedon denaturing formaldehyde agarose gel (1 mg per well) and blotted

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onto Hybond-N membrane (Amersham, Orsay, France). Afragment corresponding to nucleotides 2142–4216 of the BRCA1cDNA sequence (U14680.1) within exon 11, nucleotides 75–464of the b-globin coding sequence (NM_000518.4) corresponding toexons 1 to 3, or nucleotides 820–1858 of the CHEK2 codingsequence (NM_007194.3) corresponding to exon 6 to 15 wasgenerated by PCR using plasmids or cDNAs originating fromcontrol LCLs as templates and labeled with 32P with the Prime-ItII Random Primer Labeling Kit (Stratagene, Amsterdam, TheNetherlands). Hybridization was carried out overnight with b-globin and either BRCA1 or CHEK2 probes at 651C in Churchbuffer (0.5 M Na-phosphate, pH 7, 7% sodium dodecyl sulfatebuffer [SDS], 10% bovine serum albumin [BSA]). Blots werewashed and exposed to Hyperfilms (Amersham); autoradiogramswere then quantified using Fluor-S (Biorad, Marnes-la-Coquette,France).

Western Blot Analysis

Transfected cells or LCLs were washed with phosphate-bufferedsaline and lysed in RIPA buffer (50 mM Tris pH 8, 150 mM NaCl,1% NP40, 0.5% sodium deoxycholate, 0.1% SDS) supplementedwith Complete Protease Inhibitor Cocktail Tablets (Roche, Rosny-sous-Bois, France). Equal amounts of total proteins as measuredby Bradford assay were loaded on a 6% SDS-PAGE for BRCA1analysis or a 10% SDS-PAGE for CHK2 and p53 analysis,transferred to polyvinylidene fluoride (PVDF) membrane (Milli-pore, Molsheim, France) and blocked in 5% milk in Tween 20-TBS(50 mM Tris pH 7.5, 150 mM NaCl, 0.05% Tween 20). Blots wereincubated overnight in their respective antibodies. BRCA1 OP92monoclonal antibody (N-terminal epitope; Oncogene ResearchProducts/Calbiochem VWR), p53 D07 monoclonal antibody(N-terminal epitope; Dako, Trappes, France), and HA monoclonalantibody (Covance Research/Eurogentec, Seraing, Belgium) wereused as per the manufacturer’s recommendations. Peroxidase-conjugated AffiniPure goat anti-mouse or anti-rabbit immunoglo-bulin G (IgG) antibodies (Jackson ImmunoResearch Laboratories,Suffolk, England) were used for detection via Lumilight Westernblotting substrate (Roche). p53 pAB122 (C-terminal epitope) andhUpf1 antibodies were kind gifts from Dr. Thibault Voeltzel

(Centre Leon Berard, Lyon, France) and Dr. Niels Gehring(University of Heidelberg, Germany), respectively.

Immunoprecipitation Experiments

A total of 25� 106 LCLs or 10� 106 transfected HeLa cellswere washed with phosphate-buffered saline and lysed in Tris LysisBuffer (50 mM Tris pH 8.0, 150 mM NaCl, 2 mM EDTA pH 8.0)supplemented with 1% NP40 and Complete Protease InhibitorCocktail Tablets (Roche). Lysates containing equal amounts ofproteins were precleared by incubating with 40 ml of agarose A/Gbeads (Tebu-Bio, Le Perray en Yvelines, France) for 1 hr at 41C.Immunoprecipitation was performed with the precleared lysateand 5ml of CHK2 MC672 polyclonal rabbit antibody (N-terminalepitope) for 1 hr 30 min at 41C, then 40 ml of agarose A/G beadswere added and incubated for 45 min at 41C. Beads were washedfive times with Tris Lysis Buffer, then proteins were eluted byboiling 5 min at 951C in SDS loading buffer with 100mM DTT,and analyzed by Western blot as described above with the CHK2A-12 monoclonal antibody (N-terminal epitope; Santa Cruz/Tebu-Bio). CHK2 MC672 antibody was a kind gift from Dr. Junjie Chen(Mayo Clinic College of Medicine, Rochester, MN).

RESULTSAnalysis of Proteins Expressed FromBRCA1PTC1

Alleles

In a previous study, we assessed the relative amount oftranscripts encoded by BRCA1 alleles harboring 30 differenttruncating mutations in LCLs established from carriers frombreast/ovarian cancer families [Perrin-Vidoz et al., 2002]. We havefound that NMD is triggered by most PTC1 alleles; the amount ofmutant transcripts ranged from 18 to 70% of their normalcounterparts. To determine if the remaining BRCA1 mutanttranscripts could allow expression of detectable amounts oftruncated proteins, we analyzed whole-cell lysates obtained fromthe same LCLs by Western blot analysis with an anti-BRCA1antibody directed against an N-terminal epitope. Figure 1 showsthat the BRCA1 WT protein can be visualized in all the samples,while our analysis of six cell lysates from BRCA1 truncating

FIGURE 1. Analysis of BRCA1proteins expressed in LCLs carrying a heterozygousBRCA1mutation. Protein lysates were analyzed byWesternblot.Thenameof themutation (traditional nomenclature; seeSupplementaryTable S1for correspondencewith the systema-tic nomenclature), and thepositionof terminationcodons are indicated.The amount ofmutant vs.WT transcripts (%MU/WTmRNA)as determined in Perrin-Vidoz et al. [2002] is indicated. Prediction of the expected molecular weight (MW) of mutant BRCA1 wascarried out using the Compute pI/Mw software (www.expasy.org/tools/pi_tool.html). Bands corresponding toWT BRCA1 proteinand in-vitro translated (IVT) ‘‘ter845’’and ‘‘ter503’’BRCA1truncated proteins are indicatedwith arrows.

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mutation carriers detected no obvious truncated BRCA1 proteins.As the BRCA1 antibody did weakly cross-react with many proteinsin the whole cell lysates, we generated in vitro translated BRCA1truncated proteins that corresponded to the expected productsfrom the 1623del5 and 2594delC alleles, ter503 and ter845,respectively, for use as comigration controls. None of the cross-reacting bands observed in lysates from LCLs carrying thesemutations appeared to comigrate with the in vitro generatedtruncated proteins (Fig. 1), indicating that these proteins are notnormally produced despite the fact that mutant transcripts werenot totally degraded. We have subsequently analyzed whole-celllysates from 14 other LCLs carrying BRCA1 mutations (Supple-mentary Table S1) with similar results: no obvious truncatedBRCA1 proteins are expressed under normal conditions (data notshown).

Analysis of Proteins Expressed From CHEK2 PTC1

Alleles

We next analyzed LCLs from carriers of the c.1100delCmutation in the CHEK2 gene. The 1100delC mutation introducesa PTC, ter381, in exon 10, and is followed by five exon–exonjunctions; it is therefore expected to trigger NMD. Low amounts of1100delC carrying transcripts (16–25% of the WT transcripts)were indeed measured in LCLs established from five carriers (Fig.2A). Pretreatment of the LCLs with puromycin, a translationinhibitor frequently used to block NMD, led to a roughly three-fold increase of the amount of the 1100delC transcript as istypically observed with other PTC1 transcripts. Protein analysisperformed by immunoprecipitation/Western blot with two anti-CHK2 antibodies directed against N-terminal epitopes revealedthe WT CHK2 protein in lysates extracted from WT and1100delC-carrying LCLs, but failed to reveal any truncatedCHK2 protein in the latter (Fig. 2B). CHEK2 minigenesexhibiting the WT sequence or containing the 1100delC mutationwere transiently transfected in HeLa cells. Analysis of the CHK2proteins in HeLa lysates, using the same procedure as before,showed the presence of a truncated protein of the expected size,ter381, only in the cells transfected with the minigene carrying the1100delC mutation (Fig. 2B). This showed that the failure todetect a truncated CHK2 protein by Western blot in LCLs fromcarriers of the 1100delC mutation was not due to a technicalproblem, but rather indicates that the endogenous CHK2truncated protein is typically not detectable in LCLs.

NMD Inhibition byWortmannin

To determine if the absence or the undetectable level of BRCA1and CHK2 truncated proteins in LCLs is entirely due to NMD, weset about to inhibit this pathway and to assess the consequences atthe protein level. We first tried to inhibit NMD by RNAinterference, as the use of translation inhibitors (e.g., puromycin)typically used for this purpose would have been counterproductiveby preventing the analysis of proteins. However, we did notsucceed to introduce significant amounts of siRNAs directedagainst hUpf1 or hUpf2, two key factors of NMD [Mendell et al.,2002], into LCLs despite numerous attempts using classictransfection reagents, siRNA transfection reagents, or electro-poration. Previously, a novel mode of inhibiting NMD viachemical inhibition of the hSMG-1 kinase by wortmannin wasreported [Usuki et al., 2004; Yamashita et al., 2001]. hSMG-1 is acritical component of the NMD pathway responsible for thephosphorylation of hUpf1 that, in turn, induces remodeling of themRNA surveillance complex recognizing PTCs [Yamashita et al.,

2001]. We first checked that the use of wortmannin indeedstabilized CHEK2 1100delC transcripts: we found that there wasroughly a three-fold increase in the amount of PTC1 transcriptswhen hSMG-1 was inhibited, comparable to our results obtainedwith puromycin (Supplementary Fig. S1A). Similar results wereseen with BRCA1 PTC1 transcripts (data not shown). Theincreased stability of CHEK2 PTC1 transcripts was not, however,followed by an increase in the amount of the correspondingtruncated protein, as it was still impossible to detect CHK2-ter381(Supplementary Fig. S1B). Likewise, we could not visualize anytruncated BRCA1 protein in the presence of wortmannin (datanot shown), despite our examination of 11 LCLs carrying differentBRCA1 truncating mutations (Supplementary Table S1).

Truncated Protein Degradation Mechanism

Despite the fact that NMD had been inhibited, we did notsucceed in detecting truncated proteins in LCLs carrying PTC1

mutations in the BRCA1 or the CHEK2 genes. We thus wonderedif truncated proteins could be specifically degraded by an as-yetuncharacterized mechanism that would act as a failsafe system tocounteract the lack of efficiency of the NMD pathway. If thismechanism were to exist, two successive processes would take

FIGURE 2. Analysis ofmRNA andprotein expression in LCLs car-rying the CHEK2 1100delC mutation at the heterozygous state.A: Relative abundance of mutant vs.WT CHEK2 transcripts ex-pressed in untreated or puromycin (PURO)-treated LCLs, usingthe SNaPshot technique. Normalizationwas performed by divid-ing the observed values by those obtained for the correspondinggDNA. Normalized values are representative of six experiments.Fivedi¡erent LCLswere analyzed, threeof themestablished fromindividuals belonging to the same family (Family 3569). B: Im-munoprecipitation andWestern blot analysis of the CHK2 pro-tein in WT or 1100delC-carrying LCLs, and in HeLa cellstransiently transfectedwith theWTormutatedCHEK2minigene.Bands corresponding to WT CHK2 protein and ectopicallyexpressed CHK2-ter381 truncated proteins are indicated witharrows.

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place: the first one aimed to degrade PTC1 transcripts, thefollowing to degrade truncated proteins translated from thesetranscripts despite NMD. We reasoned that this truncated proteindegradation mechanism was likely to be linked to NMD tospecifically target truncated proteins, as this seemed the simplestway to achieve their recognition. We also postulated that thismechanism did not rely on the kinase activity of hSMG-1, asotherwise its inhibition by wortmannin would have resulted in anincrease in the amount of truncated proteins. To test the existenceof such a mechanism, we compared the amount of truncatedproteins expressed from mutated minigenes (Fig. 3A and D)containing or lacking an intron 30 of the PTC (i.e., able or unableto trigger NMD, respectively). We showed (Fig. 3B and E) thatwhile the amount of BRCA1 and CHEK2 PTC1 transcripts wasmarkedly reduced as compared to the amount of the correspond-ing WT transcripts when an intron was present 30 to the PTC,there was no significant difference in the WTand PTC1 transcriptamounts in the absence of this 30 intron. It should be noted thatthe removal of the downstream intron, in both the BRCA1 andthe CHEK2 minigenes, led to a marked reduction in the amountof transcripts. This situation has been observed frequently and maybe the result of reduced nuclear export of the transcripts due tothe presence of one fewer EJC. As seen in Figure 3C and F, theamounts of the BRCA1-ter200 or CHK2-ter381 proteins wereproportional to the amount of the corresponding PTC1

transcripts, regardless of whether these transcripts were subjectedto NMD or not. If an NMD-linked truncated protein degradation

mechanism were to exist, we would expect truncated proteins tobe less abundant when produced from NMD-sensitive transcriptsthan when produced from NMD-resistant species. Therefore, ourfindings do not favor the existence of a protein degradationmechanism specifically targeted against truncated proteins andrelying on the NMD pathway for their recognition.

Study of the Stability of BRCA1and CHK2 TruncatedProteins

We next hypothesized that truncated proteins generated fromtranscripts that escaped the effects of NMD might be unstable dueto proteolysis. To test this theory we examined what effect specificinhibitors of the proteasome and lysosome pathways had ontruncated protein expression in LCLs bearing PTC1 mutations.Treatment with both wortmannin, to inhibit NMD, and thelysosome inhibitor chloroquine resulted in the identification of lowbut detectable levels of two BRCA1 truncated proteins, ter1563and ter1163 (Fig. 4A), while similar treatment with wortmanninand the proteasome inhibitor ALLN had no effect (data notshown). Conversely, we did not detect the BRCA1 ter1546truncated protein in the LCL bearing the 4688del4 mutation(Fig. 4A), nor did we detect any BRCA1 truncated protein infive more LCLs (Supplementary Table S1) analyzed under thesame conditions (data not shown). We further tested otherprotease inhibitors such as MG132 and ALLM in conjunctionwith wortmannin with similar negative results, although these

FIGURE 3. Analysis of mRNA and proteins expressed from transfected BRCA1 and CHEK2 minigenes.Transient transfections wereperformed inHeLa cells with (A^C) BRCA1minigenes exhibiting theWTor containing the 717G4Tmutation (systematic nomencla-ture c.598G4Taccording toGenBank accession numberU14680.1with numbering starting at theAof the ¢rst ATG),with orwithout(Di10) downstream intron 10; (D^F) CHEK2 minigenes exhibiting theWTor containing the c.1100delC mutation, with or without(Di14) downstream intron14. A,D: Minigene constructs used for transfection. Exons are represented by boxes, introns by lines, andthe cytomegalovirus (CMV) promoter (pCMV) by a gray box.The start codons (ATG) and the stop codons (TAAorTGA) are indicated.The positions of the PTC generated by the 717G4Tor1100delCmutations are indicated. B,E: Northern blot analysis. b-globin tran-scripts expressed fromacotransfectedvectorwere also analyzed,which allowscompensation for variations in transfectione⁄ciency.The normalized amount of transcriptswas thenexpressed as a percentageof the amount of normalizedWTmRNA, and is representa-tive of three experiments. C,F: Western blot analysis of the proteins, using anti-GFP or anti-HA antibodies for detection of theGFP-BRCA1or theHA-CHK2 fusion proteins, respectively.

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compounds were effective to stabilize p53 in the same lysates (datanot shown). Interestingly, neither the combination of wortmanninwith proteasomal nor lysosomal inhibitors resulted in the detectionof the truncated CHK2-ter381 protein (Fig. 4B).

To compare the stability of WT and truncated proteins,HeLa cells transfected with BRCA1 minigenes harboringeither one of two truncating mutations (2594delC-ter845 and3875del4-ter1262; systematic nomenclature c.2475delC andc.3756_3759del4, respectively, according to GeneBank accessionnumber U14680.1 with numbering starting at the A of the firstATG) or the WT-tagged BRCA1 sequence were treated withcycloheximide for various length of time to block new proteinsynthesis. The steady-state levels of the ectopically expressedBRCA1 polypeptides were then monitored and compared to thatof the endogenous hUpf1 protein, which did not display anydecrease of signal even after 24 hr of cycloheximide treatment(data not shown). WT BRCA1 levels decreased only after 4 hr of

treatment, while BRCA1-ter845 and BRCA1-ter1262 levelsdecreased after as little as 1 hr of treatment, and showed a higherrate of degradation compared to WT-BRCA1 (SupplementaryFig. S2).

Analysis of Proteins ExpressedFrom aTP53 PTC1 Allele

Under normal conditions, p53, which contains a ubiquitinationdomain at its C-terminal end, is maintained at a low steady-statelevel through proteasome-mediated degradation. Following varioustypes of stress, ubiquitination is inhibited; p53 accumulates andinduces growth arrest or apoptosis. Truncating mutations in theTP53 gene are expected to both lead to a decrease in the amountof transcripts because of NMD and to an increase in the stability ofthe protein product, due to the absence of the ubiquitinationdomain. We therefore wondered whether truncated p53 proteinscould be detected in cells carrying a germline truncating mutation.To answer this question, we undertook to analyze a LCL carryingthe c.770delT mutation in exon 9 of the TP53 gene [Mazoyeret al., 1994], which introduces a PTC (ter344) 67 nt upstream ofthe last exon–exon junction, in exon 10. We first examinedwhether this mutation triggered NMD; indeed, the amount of themutant transcript was 25% that of the WT species and could beincreased two- to three-fold in the presence of puromycin orwortmannin (data not shown). Western blot analysis with anantibody directed against an N-terminal epitope revealed, inaddition to the WT p53 protein, a truncated form of �45 kDa, theexpected size of p53-ter344 (Fig. 5A). The intensity of the bandcorresponding to p53-ter344 is similar to the intensity of the bandcorresponding to WT p53 in untreated or mock-treated cellscarrying the 770delT mutation. The inhibition of NMD bywortmannin led to a marked increase in the intensity of p53-ter344 in these cells (Fig. 5A). Unexpectedly, a band of the samesize was also revealed in control cells lacking a TP53 mutation,although at a much lower level. Similarly to mutant cells, NMDinhibition led to a marked increase of the intensity of this band incontrol cells. Western blot analysis of the same lysates using anantibody that recognizes a p53 C-terminal epitope allowed thedetection of WT p53 only (Fig. 5A). Therefore, the 45-kDa bandcontains a C-terminally truncated p53 protein that couldcorrespond to p53-ter344 in mutant LCLs. In the case of controlLCLs, we theorized that this C-terminally truncated protein couldcorrespond to p53 variant b [Bourdon et al., 2005]. This variant isencoded by p53i9, a transcript produced by alternative splicing ofthe intron 9 that contains a PTC at codon 342 [Flaman et al.,1996]. When we analyzed TP53 transcripts by RT-PCR using aforward primer in exon 8 and a reverse primer in exon 10, we coulddetect, in addition to the fragment generated by the canonicaltranscript, a larger fragment in both mutant and control LCLs(Fig. 5B). The intensity of this fragment was enhanced when cellswere treated with NMD inhibitors wortmannin or puromycin, aswould be expected if it resulted from the amplification of a PTC1

transcript. Sequencing of this fragment revealed that it corre-sponded to p53i9.

It is expected that the faster-migrating band revealed with thep53 N-terminal antibody in the wortmannin-treated cells carryingthe 770delT mutation in fact corresponds to a mixture ofp53-ter342 and p53-ter344. In support of this notion, the signalratio of this band to the WT protein band is higher in those cells ascompared to wortmannin-treated cells lacking a TP53 mutation(Fig. 5A). It should be noted that we cannot be certain that theobserved increase in the level of p53 truncated proteins in thepresence of wortmannin is only attributable to the inhibition of

FIGURE 4. Analysis of proteins in LCLs carrying either one ofthree di¡erent BRCA1 truncating mutations or the CHEK21100delC mutation, in condition of proteasome or lysosome in-hibition, along with NMD inhibition. A:Western blot analysis ofthe BRCA1 protein in (mock)-treated or wortmannin (WORT)-treated and chloroquine (CHL)-treated cells. B: Immunoprecipi-tation andWestern blot analysis of theCHK2 protein.Cells wereeither mock-treated or treated with wortmannin (WORT), chlor-oquine (CHL), wortmannin and chloroquine (WORT1CHL),ALLN, or wortmannin and ALLN (WORT 1 ALLN). Predictedactivity (1) and inhibition (^) ofNMD, lysosomeandproteasomepathways are indicated.

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NMD, as this chemical compound inhibits many kinases and istherefore expected to impair several signaling pathways.

DISCUSSION

The NMD mechanism is aimed to prevent the synthesis oftruncated proteins that could be harmful by interfering withnormal cellular processes. Surprisingly, although the efficiency ofNMD has been assessed at the mRNA level in the caseof numerous truncating mutation-carrying genes responsible forvarious diseases, less attention has been given to the amountof truncated proteins actually present in the cells of the patients.The large number of previous studies aimed at elucidating themolecular mechanisms of NMD has relied on the analysis oftransfected minigenes, and the putative resulting proteins haverarely been investigated. In fact, the question of whether NMDactually fulfils its goal (i.e., preventing the synthesis of truncatedproteins) has never been raised directly. Instead, assumptionsprevail; depending on the publication, NMD is either consideredto eradicate truncated proteins or is completely ignored andtruncated proteins are expected to be systematically produced. Toillustrate this point, one need only consider the numerousattempts to draw phenotype–genotype correlations for cancerpredisposing genes, where most germline mutations are of thetruncating variety. Typically in these studies, the position of themutations along the coding sequence is the only molecularparameter considered, while the production of truncated proteinsis taken as a given. However, when one examines the literature, it

is rather difficult to find examples of truncated proteins expressedfrom NMD-sensitive PTC1 transcripts, despite the fact that PTC1 transcripts have been observed at levels 20 to 70% of those oftheir WTcounterparts. Regardless of the extent of NMD measuredin the cells of patients, Western blot analysis or immunoprecipita-tion experiments have not detected truncated XPC protein(MIM] 278720) [Khan et al., 2006], BubR1 protein (MIM]602860) [Matsuura et al., 2006], or, in mice tissues, truncatedgalactosylceramidase protein (MIM] 606890) [Lee et al., 2006].Very low amounts of truncated collagen VI a2 chain (MIM]120240) were detected in one patient with Ullrich’s disease(MIM] 254090) [Zhang et al., 2002]. However, three otherpatients expressed no detectable amounts [Usuki et al., 2004;Zhang et al., 2002] and no correlation could be drawn betweenthe extent of NMD and the presence of a truncated protein[Zhang et al., 2002]. The potential generation of truncatedproteins from NMD-sensitive transcripts is clearly an important,yet underappreciated, facet of NMD that needs to be specificallyaddressed.

We first examined the expression of truncated proteins incells established from individuals carrying a heterozygousgermline mutation that introduces a PTC in either of two breastcancer predisposing genes, BRCA1 and CHEK2. Our interestin BRCA1 mutations stemmed from the fact that we hadpreviously shown that the amount of BRCA1 mutant transcriptswere reduced in LCLs of carriers as compared to their WTcounterparts [Perrin-Vidoz et al., 2002]. We also chose to analyzethe CHEK2 1100delC mutation associated with a moderatebut consistent increase in breast cancer risk [CHEK2 BreastCancer Case–Control Consortium, 2004; Nevanlinna andBartek, 2006] because it is frequently characterized in theliterature as resulting in the abolition of the kinase function ofCHK2, a notion based on results obtained with an ectopicallyexpressed truncated protein [Wu et al., 2001]. However, uponexamination, we noted that this mutation fulfills all the necessarycriteria to trigger NMD, thus prompting us to determine whetherthis truncated CHK2 protein could be detected in cells fromcarriers of 1100delC.

We were unable to detect any BRCA1 or CHK2 truncatedprotein in LCLs of patients bearing truncating mutations, evenwhen PTC 1 transcript levels were increased three- to four-fold byinhibiting hSMG-1, and consequently, NMD. These results, anddata from the literature, led us to hypothesize that truncatedproteins that were translated from PTC1 transcripts which escapeNMD were further prevented from being detrimental to the cellsby an uncharacterized degradation process. It appeared to us thatthe simplest way for such a mechanism to specifically targettruncated proteins would be to rely on at least one componentof the NMD machinery. For example, the presence of anEJC downstream of a stop codon could trigger the recruitmentof specific proteases and lead to the degradation of the nascentpolypeptides. We thus compared the amount of truncated BRCA1or CHK2 proteins synthesized from transcripts resistant orsensitive to NMD, but we observed no significant differences intheir expression. This result and those on TP53 mutations (seebelow) led us to abandon the hypothesis of a NMD-linked,truncated protein degradation mechanism.

In the case of two BRCA1 mutations, minute amounts oftruncated proteins could be detected in LCL lysates when NMDand lysosome protein degradation pathways were simultaneouslyinhibited. However, lysosomal or proteasomal inhibition inconjunction with inhibition of NMD did not yield similar resultin our other mutant BRCA1 carrier LCLs. Despite the exponential

FIGURE 5. Analysis of proteins and mRNA expression in LCLscarrying theTP53 770delT mutation at the heterozygous state.A: Western blot analysis of p53 protein expressed in mutated(TP53 770delT) orWT LCLs using an antibody directed againstan N-terminal or a C-terminal epitope. Cells were untreated,mock-treated, or wortmannin (WORT)-treated to inhibit NMD.Bands corresponding to the WT p53 protein and to p53 trun-cated proteins are indicated with arrows. Those correspondingto p53-ter344 and p53-ter342 proteins are comigrating. B: RT-PCR analysis of canonical and alternatively splicedTP53 tran-script isoforms expressed in mutated (TP53 770delT) or WTLCLs. Cells were either untreated, mock-treated, wortmannin(WORT)-treated, or puromycin (PURO)-treated to inhibitNMD.

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Human Mutation DOI 10.1002/humu

growth in published data concerning the BRCA1 protein, still verylittle is known about how BRCA1 degradation is conducted andregulated. BRCA1 degradation in cell lines expressing low orundetectable levels of this protein has been found to be mediatedprimarily by a cathepsin-like protease or protease(s) that can beinhibited by lysosomal inhibitors such as chloroquine [Blagosk-lonny et al., 1999]. However, it has been shown that the cellcycle–dependent pattern of BRCA1 expression is determined inpart by ubiquitin-dependent proteasomal degradation [Choudhuryet al., 2004]. Our inability to stabilize substantial amounts ofendogenous truncated BRCA1 polypeptides by inhibiting thelysosome or the proteasome jointly with NMD is puzzling. Suchpolypeptides can be detected in HeLa cells when overexpressed bytransient transfection; we found, nevertheless, that they werenoticeably less stable than ectopically expressed WT BRCA1,although the difference was not tremendous. Further work isneeded to understand how BRCA1 truncated proteins aredegraded in cells of carriers of a germline mutation.

While this work was being conducted, a functional analysis ofthe CHEK2 1100delC allele in heterozygous individuals waspublished that showed that the protein encoded by this mutantallele, CHK2-ter381, was not detectable in LCLs, even when theproteasome was inhibited with ALLN [Jekimovs et al., 2005], inaccordance with our findings. In their publication, Jekimovs et al.[2005] suggested that the truncated CHK2 protein is nottranslated, a hypothesis not supported by our results showing thatthe CHEK2 mutant transcripts are subjected to NMD. Indeed, ifthe CHEK2 1100delC mutant transcripts were not translated,they would not be degraded by NMD, as NMD is dependent upontranslation.

It has been shown by two different groups that the ectopicallyexpressed CHK2-ter381 protein was much less stable than WTCHK2 [Sodha et al., 2006; Staalesen et al., 2004], which couldexplain why we cannot detect it. Here again, further work isneeded to understand how this CHK2 truncated protein isdegraded in cells of carriers of the 1100delC mutation.

Given the absence of detection of truncated BRCA1 and CHK2proteins in cells from carriers of germline mutations, and to findout if these observations reflected a typical scenario, we proceededto examine a third breast cancer predisposing gene, TP53. The p53protein is one of the best-studied substrates of the ubiquitinproteasome degradation pathway, and its ubiquitination domainresides in the C-terminal end of the protein. We thus reasonedthat TP53 truncating mutations were the most likely to give rise tostable truncated proteins. Indeed, despite the fact that TP53transcripts carrying a PTC were degraded by NMD to the sameextent as CHEK2 transcripts, and more efficiently than mostBRCA1 PTC1 transcripts, a p53 C-terminally truncated proteinwas readily detectable in cells from carriers of the 770delTgermline mutation. We observed that the amount of steady-statep53-ter344 was nearly identical in normal conditions and largelysuperior when NMD was inhibited to that of WT p53. This impliesthat although NMD does greatly diminish the amount of themutant TP53 transcript and subsequently, of the p53-ter344protein, it was not efficient enough to prevent its accumulationdue to the truncated protein’s increased stability. Conversely, thep53 isoform (p53b) that results from the translation of thealternatively spliced TP53 transcript containing an additional 133-bp exon derived from intron 9 [Flaman et al., 1996] is efficientlydownregulated by NMD, as it is barely detectable when NMD isnot inhibited. The amount of TP53 PTC1 transcripts producedby alternative splicing is much lower than the amount ofPTC1 transcripts expressed from the 770delT allele, which may

also explain why there is such a difference in the level of thecorresponding proteins. p53b has been shown to bind differentiallyto promoters and to have the ability to enhance p53 target geneexpression in a promoter-dependent manner [Bourdon et al.,2005]. It has been suggested that certain p53 isoforms, includingp53b, may be abundant and active enough to regulate theactivities of a specified pool of posttranslationally modified p53 innormal cells and/or in tumors [Bourdon et al., 2005]. It is expectedthat NMD efficiency will be an important factor in the regulationof the amount of p53b.

In conclusion, our analysis on the expression of truncatedproteins translated from transcripts subjected to NMD showedthat the ability to detect truncated proteins did not correlate withthe extent of NMD, but rather seemed to depend entirely uponthe proteins’ stability and upon the amount of PTC1 transcriptsthat are NMD substrates. The mechanism of protein qualitycontrol and elimination of misfolded proteins in the cytoplasm isstill poorly understood, although the control of protein half-life bydegradation has emerged as a major cellular regulatory process.

Therefore, the answer to the question of whether NMDsucceeds in preventing the synthesis of truncated proteins is notunequivocal, as each case is expected to be different. This meansthat experimental investigation will systematically be necessary todetermine whether truncated proteins are present in cells fromcarriers of a truncated mutation.

Once believed to function solely against newly-arising nonsensemutations, NMD is now emerging as a pathway that governsgeneral cellular gene regulation, notably alternative splicing [Lewiset al., 2003; Mendell et al., 2004]. Our results strengthen thisconcept by showing that when leading to stable truncated proteinswith a potential dominant-negative effect, alternative TP53transcripts that contain PTCs are more likely to be neutralizedby NMD, given their low abundance, than PTC1 transcriptsproduced from mutant TP53 alleles.

ACKNOWLEDGMENTS

We thank the family members who collaborated in this study.We also thank H.T. Lynch for a long-term collaboration on theBRCA1 families; C. Bonnardel and C. Snyder for their expertassistance; C. Houdayer and V. Moncoutier for the CHEK2mutant cell lines; M. Leone for performing the SnaPshotexperiments; and Q. Wang and C. Navarro for the TP53 mutantcell line. We are indebted to J. Feunteun for the generous gift ofthe BRCA1 WT-HA minigene, and to T. Voeltzel, J. Chen, andN. Gehring for the generous gifts of the p53 pAB122, CHK2MC672, and hUpf1 antibodies, respectively. O.A. was supportedby a fellowship from the Comite Departemental de Saone-et-Loirede la Ligue contre le Cancer, and M.D.W. was supported by theAssociation pour la Recherche contre le Cancer.

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