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RNA-mediated response to heat shock inmammalian cellsIlya Shamovsky1,2, Maxim Ivannikov1, Eugene S. Kandel3, David Gershon2,4 & Evgeny Nudler1

The heat-shock transcription factor 1 (HSF1) has an importantrole in the heat-shock response in vertebrates by inducing theexpression of heat-shock proteins (HSPs) and other cytoprotectiveproteins1. HSF1 is present in unstressed cells in an inactivemonomeric form and becomes activated by heat and other stressstimuli. HSF1 activation involves trimerization and acquisition ofa site-specific DNA-binding activity2,3, which is negatively regu-lated by interaction with certain HSPs4–6. Here we show that HSF1activation by heat shock is an active process that is mediated by aribonucleoprotein complex containing translation elongationfactor eEF1A and a previously unknown non-coding RNA thatwe term HSR1 (heat shock RNA-1). HSR1 is constitutivelyexpressed in human and rodent cells and its homologues arefunctionally interchangeable. Both HSR1 and eEF1A are requiredfor HSF1 activation in vitro; antisense oligonucleotides or shortinterfering (si)RNA against HSR1 impair the heat-shock responsein vivo, rendering cells thermosensitive. The central role of HSR1during heat shock implies that targeting this RNA could serve asa new therapeutic model for cancer, inflammation and otherconditions associated with HSF1 deregulation.

HSF1 activation has been proposed to occur spontaneouslythrough relief of negative regulation imposed by chaperones6–8.Activated HSF1 binds to the heat shock element (HSE), a consensussequence in HSP promoters composed of inverted NGAAN repeats9,and rescues the RNA polymerase II elongation complex frompromoter-proximal arrest10,11. A large portion of the inactive HSF1in unstressed cells is believed to be complexed with HSP90, P23 andan immunophilin12–14. HSP90 has emerged as a primary candidatefor the role of repressor of both HSF oligomerization and transcrip-tional competence5. Some data, however, point to the existence ofauxiliary cellular factors that regulate HSF1 activation. The tempera-ture threshold of HSF1 activation in vivo during heterologousexpression is reprogrammed to that of the host cell12,15 and is higherin motor neurons and lower in T lymphocytes than in othermammalian cells16,17. Moreover, the fast HSF1 activation kinetics inresponse to heat shock18 is difficult to reconcile with a simplediffusion-controlled mechanism (Supplementary Information).

To identify putative auxiliary factors involved in HSF1 activation,we looked at proteins from whole-cell lysates retained on covalentlyimmobilized HSF1. HSF1 was expressed in E. coli and purified asa glutathione S-transferase (GST) fusion protein. The purificationprocedure was optimized to minimize spontaneous trimerization ofHSF1 (see Supplementary Information). A polypeptide of ,45 kDawas retained on HSF1-Sepharose after incubation with a lysate fromheat-shocked, but not untreated, BHK-21 (Fig. 1a) or HeLa cells(Supplementary Fig. 1). The bound polypeptide was identified byMALDI-TOF (matrix-assisted laser desorption/ionization–time offlight) as translation elongation factor eEF1A. Retention of eEF1Awas temperature-sensitive, as incubation at 43 8C released most of

the bound eEF1A (Fig. 1a). No eEF1A binding was observed ifbovine serum albumin (BSA)-Sepharose was used in place ofHSF1-Sepharose.

We next examined whether the eEF1A-containing fraction elutedby heat from HSF1-Sepharose affected HSF1 function in vitro.Notably, the eEF1A fraction activated endogenous HSF1 in the lysateof unstressed cells as shown by an electromobility shift assay (EMSA)(Fig. 1b). Furthermore, this fraction induced DNA-binding activityof recombinant HSF1 (Fig. 1c). The effect was dose-dependent withrespect to both the amount of HSF1 (or cell lysate, SupplementaryFig. 1c) and eEF1A fraction (Fig. 1c). In our system, heating lysatefrom unstressed cells in the absence of the eEF1A fraction did notyield any detectable HSF1 activity (Fig. 1b, lane 1, see SupplementaryInformation). The induced HSF1 DNA-binding activity was specific,as it was sensitive to both an anti-HSF antibody and an excessof unlabelled HSE oligonucleotide (Fig. 1b, lanes 6, 7 and 8, 9).Activation of purified HSF1 by the eEF1A fraction was accompaniedby trimerization of HSF as confirmed by protein–protein cross-linking (Fig. 1d).

Co-immunoprecipitation of eEF1A and HSF1 from a whole-celllysate (Fig. 1e) revealed the presence of a small amount of HSF1–eEF1A complexes in unstressed cells (lanes 1–4) with the amountincreasing during heat-shock treatment, reaching a plateau after30 min of heat shock (lanes 6, 7) and returning to the initial level aftera 1-h recovery at 37 8C (lane 8). These results, together with elution ofeEF1A from HSF1-Sepharose at 43 8C (Fig. 1a), indicate that eEF1Ahas a higher affinity for the HSF1 trimer than the monomer.

Release of HSF1 from the inhibitory multichaperone complexduring heat shock4 could also contribute to the greater availability ofHSF1 for eEF1A. Moreover, free eEF1A should accumulate in the cellunder heat-shock conditions owing to general translational shut-down19 and cytoskeleton collapse20. These events would ensure theavailability of eEF1A molecules capable of forming a complex withHSF1, and thus link the major cellular perturbations caused by heatshock with HSF1 activation.

Our initial attempts to activate HSF1 with eEF1A isolated fromrat liver or HeLa cells were unsuccessful, suggesting that the eEF1Afraction eluted from HSF1-Sepharose contained unidentified com-ponent(s) required for HSF1 activation. As eEF1A normally bindsaminoacyl-tRNA, we sought to determine whether this interactionhas a role in HSF1 activation. As shown in Fig. 2a, HSF1 binding toDNA was strongly inhibited in vitro by pre-incubation of the celllysate with RNase A. The effect was specific, as addition of excesstRNA before treatment with RNase protected HSF1 activity. Similarresults were obtained using micrococcal nuclease in the presence ofCa2þ (not shown). These data prompted us to search for a specificRNA in the eEF1A fraction that might participate in HSF1 activation.This RNA, which we term HSR1, was detected as a single bandmigrating at,2 kb on denaturing polyacrylamide gel electrophoresis

LETTERS

1Department of Biochemistry, New York University School of Medicine, New York, New York 10016, USA. 2Faculty of Biology, Technion-Israel Institute of Technology, Haifa32000, Israel. 3The Cleveland Clinic Foundation Lerner Research Institute, Cleveland, Ohio 44195, USA. 4Redox Pharmaceutical Corp, Greenvale, New York 11548, USA.

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(PAGE) (Fig. 2b). The band was sensitive to RNase A, but not toDNase (Fig. 2b). Addition of purified HSR1 (but not total RNA) to alysate of heat-shocked BHK cells treated sequentially with micro-coccal nuclease (to remove endogenous HSR1) and EGTA (toinactivate the nuclease) restored the DNA-binding activity of HSF1(Supplementary Fig. 2a).

We cloned HSR1 from BHK and HeLa cells, sequenced it, andfound it to be a ,600-nucleotides (nt) long RNA without a poly(A)tail (see Supplementary Information). Sequence comparison ofHSR1 from BHK and HeLa cells revealed a high degree of homology,with only a 4-nt difference. Northern blot analysis showed that HSR1is constitutively expressed in BHK and HeLa cells, and its level seemsunaffected by heat shock (Supplementary Fig. 2b).

For the reconstitution experiment shown in Fig. 2c, recombinantmouse HSF1 was used along with eEF1A purified from rat liver21.Both HSR1 isolated from heat-shocked BHK or HeLa cells (lanes 7, 8)and HSR-T3 (in vitro synthesized HSR1, lane 10) activated HSF1when added together with purified eEF1A. Neither component alonewas capable of activating HSF1 (lanes 2–6). The slight stimulatoryeffect of isolated eEF1A (lane 2) on HSF1 activation resultedfrom residual amounts of co-purified HSR1, and was eliminatedby RNase A treatment (not shown). Crosslinking with ethyleneglycol-bis-succinimidylsuccinate (EGS) confirmed that HSR-T3-mediated activation was accompanied by HSF1 trimerization (seeSupplementary Fig. 2c). In contrast, HSR-T7 (antisense HSR) failedto induce HSF1 binding to DNA (Fig. 2c, lane 9) and suppressed theactivating effect of HSR-T3 (lane 11). We conclude that HSR1 andeEF1A are necessary and sufficient for HSF1 activation in vitro.

Notably, the mobility of HSR-T3 depended on the conditions towhich it was exposed before PAGE analysis. Both Mg2þ and elevatedtemperature caused in vitro synthesized HSR to migrate similarly toHSR1 isolated from heat-shocked cells (Fig. 2d, compare lanes 2 and3, 4). This conformational change of HSR1 may be associated withtriggering HSF1 activation, as only the slow-migrating form of HSR1

was retained on HSF1-Sepharose (Fig. 2b). Polymerase chain reac-tion (PCR) analysis of genomic DNA using HSR1-specific primersproduced a single product of ,600 nt, ruling out RNA processing asthe cause of variability in HSR1 mobility and indicating that thecloned HSR fragment indeed represents full-length HSR1.

To obtain evidence of in vivo complex formation between HSF1,eEF1A and HSR1, we extracted RNA from immunoprecipitatescorresponding to lanes 5 and 7 of Fig. 1e and analysed them byPCR with reverse transcription (RT–PCR). A greater amount ofHSR1 was precipitated from heat-shocked cells compared with thecontrol cells (lanes 3 and 4, Fig. 2e), indicating that the amount ofHSR1–eEF1A complexes in vivo increased upon heat shock.

To delineate essential functional domains within HSR1, we used aset of 15 overlapping 45-mer antisense DNA oligonucleotides cover-ing the entire HSR1 sequence (Fig. 2f). Each oligonucleotide wastested in the reconstituted system for its ability to suppress HSF1activation by HSR1–eEF1A. The results of this experiment identifiedtwo domains in HSR1 that were essential for HSF1 activation: fouroligonucleotides—1HSR and 2HSR, which span 84 nt at the 5

0terminus

of HSR1; and 5HSR and 6HSR, which correspond to nucleotides157–240—inhibited HSF1 activation by more than 90% (Fig. 2f).

We used the inhibitory oligonucleotide 6HSR to suppress the heat-shock response in vivo. BHK cells were transfected with either 6HSR ora control double-stranded (ds) oligonucleotide (6HSR/anti-6HSR),and subjected to heat-shock treatment. HSF1 activation was mon-itored by EMSA (Fig. 3a) and HSP72 synthesis was assessed byimmunoblotting after recovery of the cells for 16 h at 37 8C forfollowing heat shock (lower panel). We found that the 6HSR oligo-nucleotide, but not the 6HSR/anti-6HSR ds-oligonucleotide, inhibitedHSF1 activation in vivo in addition to the production of HSP72 inresponse to heat shock (Fig. 3a). Consistently, 6HSR compromised theinduction of thermotolerance, demonstrated by a reduction in cellsurvival following a lethal heat-shock challenge (Fig. 3b). Transfectionwith 6HSR resulted in more than 80% lethality after 60 min of heat

Figure 1 | Identification of an HSF1-activating fraction containing eEF1A.a, Fractionation of a lysate of heat-shocked BHK cells on HSF1-Sepharose.Lane 1, whole-cell lysate of heat-shocked BHK cells; lane 2, supernatant afterincubation of BHK lysate with HSF1 Sepharose; lane 3, HSF1-Sepharosebeads incubated with the lysate and washed; lanes 4, HSF1-Sepharose beadsafter three successive rounds of elution at 43 8C; lanes 5–7, proteins releasedfrom HSF1-Sepharose after successive elutions at 43 8C. b, The eEF1A-containing fraction activates HSF1 in a lysate from unstressed cells. Analiquot of a whole-cell lysate of unstressed BHK cells (20 mg) was incubatedin the absence (lanes 1, 5) or in the presence (lanes 2–4) of fractions 5–7 fromFig. 1a and analysed by EMSA. ‘43 8C’ indicates that the lysate was heat-shocked for 15 min before EMSA (lane 1). Lanes 6–9 contain fraction 5.‘HSE’ (lanes 6, 7) indicates the inclusion of an excess of unlabelled HSEoligonucleotide. ‘Ab’ indicates the presence of a monoclonal anti-HSF1

antibody that causes a supershift (lane 9). c, In vitro activation ofrecombinant HSF1 by the eEF1A fraction. Increasing amounts of purifiedrecombinant mouse HSF1 were incubated in the absence (2) or presence ofthe eEF1A fraction and analysed by EMSA. d, The eEF1A fraction inducestrimerization of purified recombinant HSF1. Purified recombinant mouseHSF1 was incubated in the absence (2) or presence of the eEF1A-containingfraction and the crosslinking reagent EGS. Proteins were then separated bySDS–PAGE, transferred to a nitrocellulose membrane, and probed with ananti-HSF1 antibody. e, Formation of an eEF1A–HSF1 complex in vivo. BHKcells were heat-shocked (HS) for the times indicated and whole-cell lysateswere analysed by immunoprecipitation with an anti-eEF1A antibodyfollowed by immunoblotting with anti-HSF1. ‘60R’ indicates 60-minrecovery after 60-min heat shock.

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shock at 45 8C, whereas the control anti-6HSR oligonucleotide had nosignificant effect on cell viability (Fig. 3b). Similar results wereobtained with HeLa cells (not shown).

We obtained independent evidence for an essential role of HSR1 inthe heat-shock response in vivo from RNA interference (RNAi)experiments. On the basis of data obtained using antisense oligonu-cleotides, we constructed vectors expressing siRNA against differentdomains of HSR1 (siRNAHSR). The effect of siRNA was monitored bythe heat-shock-induced expression of a plasmid-derived Renillaluciferase (RLuc) reporter fused to an inducible human HSP70promoter (see Methods). We co-transfected this construct intoHeLa cells together with RNAi vectors and measured changes inRLuc activity induced by heat shock. Whereas RLuc activity wasinduced ,200-fold by heat-shock treatment followed by recovery at37 8C, the siRNAHSR corresponding to the 6HSR antisense oligo-nucleotide strongly inhibited heat-shock induction of RLuc(Fig. 3c). Notably, a mutant construct carrying a single G ! Csubstitution in the siRNA sequence had a significantly smaller effecton RLuc induction by heat shock.

Finally, we generated HeLa cell lines that stably expressed siRNAHSR.These cells were transiently transfected with the RLuc reporterplasmid, and heat-shock induction of RLuc activity was monitored.We found that cells expressing siRNAHSR or full-length antisense

HSR1, but not green fluorescent protein (GFP), were deficientin their ability to induce RLuc activity after a 2-h heat shock at43 8C (Fig. 3d). Consistently, heat-shock induction of HSF1 DNA-binding activity was severely impaired in siRNAHSR, but not inGFP-expressing cells (Fig. 3d, inset). Predictably, cells stablyexpressing siRNAHSR failed to acquire thermotolerance afterheat-shock pre-conditioning (Fig. 3e). Taken together, these datashow that HSR1 is essential for the heat-shock response inmammalian cells.

It has been argued that the inhibitory complex of HSF1 withHSP90 and other chaperones dissociates as a result of competitionbetween denatured cellular proteins and HSF for binding toHSP904,12,13,22. Here we demonstrate that HSF1 activation duringheat shock is enhanced and perhaps triggered by specific cellularfactors, including translation elongation factor eEF1A and a pre-viously unknown untranslated RNA, HSR1 (Supplementary Fig. 4).HSF1 activation in vitro can be achieved in a reconstituted systemwith eEF1AþHSR1. Although purified HSF1 has been reported totrimerize spontaneously in vitro in response to elevated temperature,pH and other stimuli23–25, our data suggest that the concentrationthreshold at which spontaneous HSF activation occurs26 in vitroexceeds the effective intracellular HSF1 concentration, particularlyduring the first moments of heat-shock treatment. Nonetheless, the

Figure 2 | HSR1-mediated activation of HSF1. a, Inhibition of HSF1activation in a whole-cell lysate of heat-shocked (HS) BHK cells by RNase A.The lysate (10 mg total protein) was treated with 500 ng RNase A for 1 h at37 8C with or without excess tRNA. The amount of HSF1 in each lane wasmonitored by immunoblotting (lower panel). b, HSR1 from the eEF1Afraction. Silver-stained denaturing 4 % PAGE of the RNA isolated frompooled eEF1A-containing fractions (left panel). Where indicated, HSR1samples were treated for 30 min at 25 8C with DNase I (10 U) or RNase A(100 ng) before loading onto the gel (right panel). c, Reconstitution of theHSF1-activating complex. Schematic on the top shows HSR1 cDNA flankedby T7 and T3 promoters. Lower panel shows EMSA of recombinant HSF1incubated with pure eEF1A and HSR1, which was isolated either from HeLa-or BHK-activating fractions (lanes 7, 8) or synthesized by in vitrotranscription using T7 or T3 polymerase (lanes 9–11). Quantification ofHSF1 activation is presented as the fold increase relative to a backgroundcontrol (lane 1). d, Electrophoretic mobility of in vitro synthesized andendogenous HSR1. HSR1 isolated from BHK cells (lane 1) or synthesized byin vitro transcription (T3, lanes 2–4) was incubated in Mg2þ free buffer (2)

(lane 4) or in buffer containing 4 mM Mg2þ (lanes 1–3) and subjected toelectrophoresis on a 1 % agarose gel. The sample in lane 2 was heated at 43 8Cbefore loading. M, RNA ladder (nt). e, Co-immunoprecipitation (IP) ofHSR1 with an anti-eEF1A antibody. IP was performed as described in thelegend to Fig. 1e. RNA was isolated from a precipitate prepared from heatshocked (HS, lane 3) or control (lane 4) cells and subjected to RT–PCR usingHSR1-specific primers. Lane 1, no reverse transcription step (2RT). Lane 2,in vitro transcribed HSR1 (HSR1-T3) was used in place of HSR1 isolatedfrom the immunoprecipitate. f, Mapping functional domains of HSR1.Fifteen overlapping 45-mer HSR1 antisense DNA oligonucleotides coveringthe entire length of HSR1 were screened for their ability to inhibit HSF1activation in the reconstituted system. Recombinant HSF1 (,10 nM) wasactivated in the presence of purified eEF1A (0.01 mg ml21) and HSR1-T3(0.4 mM). Where indicated, an antisense oligonucleotide was added to a finalconcentration of 10mM. Changes in HSF1 activation were quantified aftersetting the activity of HSF1 in the absence of oligonucleotide at 100 %(lane 2) and without eEF1A at 0 % (lane 1). Note that some oligonucleotidespotentiate HSF1 activation (for example, oligonucleotide no. 10).

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chaperone-based repression of HSF1 and the activation mechanismdescribed here are likely to coexist (Supplementary Fig. 4). Thus,HSR1–eEF1A complexes could capture HSF1 released from theHSP90 complex and assist its assembly into trimers and/or increasethe stability of HSF1 trimers.

Our data assign a new function to eEF1A as a co-activator of HSF1and suggest that this ubiquitous and extremely conserved protein hasa multifaceted role in the response to heat shock. Heat shock leads totwo major physiological perturbations in the cell—translationalshutdown and cytoskeleton collapse. These events might cause therelease of eEF1A, which then becomes available for interaction withHSR1 and HSF1 to initiate the heat-shock response (SupplementaryFig. 4). Thus, eEF1A may provide a functional link betweenaberrations in general cellular processes and the stress response.

Recently, non-coding RNAs have been implicated in a widespectrum of regulatory functions27,28. Here we describe a previouslyuncharacterised non-coding RNA that is essential for heat-shockresponse activation and that could potentially serve as a thermo-sensor. Indeed, the concept of an RNA thermosensor, albeit with adifferent mode of action, has already been described in bacteria29.HSR1 may represent an attractive pharmacological target in variouspathological conditions associated with HSF1 activation, includinginflammation, ischaemia/reperfusion and cancer30.

METHODSCell culture, heat-shock treatment and lysate preparation. HeLa and BHK-21cells were grown at 37 8C in a 5 % CO2 atmosphere in DMEM medium

containing 10 % fetal bovine serum (FBS) and a DMEM/F-12 (1:1) mixturecontaining 10 % newborn calf serum (NBCS), respectively. Growth mediacontained 2 mM glutamine and an antibiotic/antimycotic cocktail (penicillin/streptomycin/fungizone) (Invitrogen). Heat shock of cultured cells was per-formed by submerging tightly closed, parafilm-sealed screw-cap flasks in a waterbath adjusted to 43 8C or 45 8C as indicated. Control cells were maintained intightly closed flasks in an incubator at 37 8C. Whole-cell lysate was prepared bythree cycles of freezing in liquid nitrogen/thawing at ambient temperature inHEDG buffer (20 mM HEPES-NaOH, pH 7.8, 0.5 mM EDTA, 0.5 mM dithio-threitol (DTT), 10 % glycerol and protease inhibitor cocktail (Complete,Roche)) containing 0.42 M NaCl at a ratio of 100ml per confluent 75-cm2

flask followed by centrifugation at 25,000g for 15 min. The lysate was dividedinto small aliquots, flash-frozen and stored at 270 8C. Total protein concen-tration was ,3–5 mg ml21.Protein expression and purification. The pGex-2T plasmid carrying mouseHsf1 cDNA fused to amino-terminal GST was a gift from K. Sarge. E. coli BL-21cells were transformed with pGex-2THSF1, grown until they reached anabsorbance of ,0.6 at 600 nm (A600) and induced with 0.1 mM IPTG for 5 hat 28 8C. For details of the purification procedure and HSF1 immobilization onSepharose, see Supplementary Methods. eEF1A was purified from rat liveressentially as described in ref. 21, and is described in detail in the SupplementaryMethods.HSF-Sepharose pull-down experiments and isolation of HSR1. For theisolation of eEF1A fraction, whole-cell lysate was prepared as described aboveand diluted with HEDG buffer to give a final concentration of 0.21 M NaCl. Abed volume of 10 ml HSF1-Sepharose beads per 75-cm2 flask of confluent cellswas added and mixed at 4 8C for 4 h. Beads were collected by centrifugation at500g for 5 min and washed three times with ten bed volumes of HEDGcontaining 0.21 M NaCl. Elution was performed by incubating the beads with

Figure 3 | Inhibition of the heat-shock response in vivo by targeting HSR1.a, Effect of 6HSR antisense oligonucleotide transfections on HSF1 activationand Hsp expression in BHK cells. Cells were transfected with 100 nM ofeither 6HSR antisense oligonucleotide (lane 2), an equal amount of thecorresponding double-stranded oligonucleotide (ds6HSR; lane 3) or weremock-transfected (lane 1). Transfected cells were heat-shocked for 1 h at43 8C, a whole-cell lysate was immediately prepared from half of the cells and20 mg of the total protein was analysed by EMSA for HSF1 activity. Theremaining half of the cells were allowed to recover at 37 8C for 16 h before awhole-cell lysate was analysed by immunoblotting using an anti-HSP72(inducible HSP70) antibody. Control (lane 5), lysate of unstressed cells. HS(lane 4), lysate of untransfected heat-shocked cells. Upper panel showsquantification of EMSA data. Error bars represent s.d. from 3 independentexperiments. b, Sensitizing BHK cells to heat shock with the 6HSR

oligonucleotide. Cells were transfected with 100 nM 6HSR or anti-6HSR, orwere mock-transfected (2). Thermotolerance was induced by incubationfor 1 h at 43 8C followed by 15 h recovery at 37 8C. Lethal heat shock (45 8C)was performed for the indicated time periods. Cell viability was determinedby MTS assay 16 h after lethal heat shock. Data for 6HSR and anti-6HSR

oligonucleotides were normalized to the efficiency of transfection. Similarresults were obtained with HeLa cells. Data show mean ^ s.e.m. from 6

independent experiments. c, Inhibition of heat shock promoter activationby siRNA against HSR1. RLuc activity was measured in lysates of BHK cellstransiently transfected with a mixture of RLuc reporter plasmid, a b-galinternal control, and an siRNA construct expressing siRNA against HSR1(siHSR) or its mutant derivative (siHSR: C ! G). C, control lysate ofunstressed cells. Similar results were observed with HeLa cells. Error barsrepresent s.d. from 4 independent experiments. d, Heat-shock response instably transfected cells expressing siRNA against HSR1. RLuc activity wasmeasured in lysates of HeLa cell lines that stably express either GFP (HS),siRNA against HSR1 (siHSR) or full-length antisense HSR1 (aHSR). Each ofthese cell lines was transiently transfected with an RLuc reporter plasmidand a b-gal internal control. C, lysate of unstressed cells. The HSF1 activityin the lysates of GFP- (HS) and siHSR- expressing cells is shown in the inset.Error bars represent s.d. from 4 independent experiments. e, Inhibition ofthermotolerance by siRNA against HSR1. Cells stably expressing GFP(control, C) or siRNA against HSR1 (siHSR) were subjected to a lethal heatshock for 2 h at 46 8C with (dark grey) or without (light grey) pre-conditioning at 43 8C for 1 h followed by 16 h recovery at 37 8C. Cell viabilitywas determined by the MTS-based assay. Data show mean ^ s.e.m. from 3independent experiments.

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one bed volume of HEDG containing 0.21 M NaCl at 43 8C for 30 min withoccasional mixing. Three successive elutions were performed and the fractionswere analysed by SDS–PAGE. To isolate HSR1, eEF1A-containing fractions werepooled, supplemented with 12.5 mM EDTA and 0.25% SDS and treated with6,000 units of proteinase K (Fermentas) for 30 min at 50 8C. RNA was thenextracted twice with phenol-chloroform and precipitated with isopropanol.EMSA and chemical crosslinking. EMSA was performed as described in ref. 2with minor modifications. Binding reactions contained 10–20mg protein (fromwhole-cell lysate) or 10 nM purified recombinant HSF1, 20 mM HEPES-NaOH(pH 7.9), 100 mM NaCl, 1.5 mM MgCl2, 0.5 mM EDTA, 2 mM DTT, 10 % (v/v)glycerol, 1 mg ml21 BSA, 2.5mg poly(dI-dC), and 1.25 ng of 32P-end-labelledHSE oligonucleotide (5 0 -GCCTCGAATGTTCGCGAAGTTTCG-3 0 ) in a finalvolume of 40ml. Reactions were incubated for 20 min at room temperature(22–25 8C), mixed with 4 ml of 0.02 % bromophenol blue in 50 % glycerol, loadedonto a 4 % polyacrylamide gel, and run in 25 mM Tris, 190 mM glycine, 1 mMEDTA (pH 8.3) at 50 mA. The gel was dried and analysed using a phosphor-imager. Recombinant HSF1 (10 nM) was incubated with eEF1A and HSR1 at theindicated concentrations. The crosslinking reagent EGS (Pierce) was added to0.2–0.5 mM in the same buffer used for EMSA for 30 min at room temperature.Reactions were quenched with 75 mM glycine and processed forimmunoblotting.Transfections and cell viability assay. The oligonucleotides (IDT) used fortransfection, 6HSR (5 0 -AGTCCTCACACCACTCCAACGTCTGCTTATGGCGGCGGATTCAAC-3

0) and anti-6HSR (5

0-GTTGAATCCGCCGCCATAAGCA

GACGTTGGAGTGGTGTGAGGACT-30), were phosphothioate-modified at

the 30

and 50

ends to increase their stability. In the experiment shown inFig. 3a, 6HSR oligonucleotide or 6HSR/anti-6HSR ds-oligonucleotide (0.56mMfinal concentration) were used to transfect BHK cells using Superfect reagent(Qiagen) according to the manufacturer’s instructions. The next day, cells wereheat-shocked for 1 h at 43 8C and a whole-cell lysate was prepared to determineHSF1 activity. In some experiments, cells were allowed to recover at 37 8Covernight after heat shock to allow synthesis of HSPs. Next, a whole-cell lysatewas prepared and analysed by immunoblotting using anti-HSP72 antibodies(Stressgen) that specifically recognize only the inducible form of HSP70. In theexperiment depicted in Fig. 3b, cells were transfected using Oligofectaminereagent (Invitrogen) at a final oligonucleotide concentration of 100 nM, accord-ing to the manufacturer’s instructions. Cell viability was determined using theMTS reagent-based CellTiter 96 AQueous non-radioactive cell proliferationassay (Promega, Supplementary Methods). For siRNA experiments, cells weretransfected in 12-well plates with a mixture of 100 ng pMLucHSP70, 50 ngpSV40bGal (Supplementary Methods) and 250 ng siRNA constructusing Effectene reagent (Qiagen), according to the manufacturer’s instructions.Cells were incubated with the transfection complexes for 24 h, heat-shocked for 2 h at 43 8C and allowed to recover at 37 8C for 16 h. SeeSupplementary Methods for details of the generation of stably transfected celllines. Transfection with RLuc and b-gal reporter plasmids, heat-shock treatment,and reporter gene activity assays were performed as described above for transienttransfection.

Received 31 August; accepted 15 December 2005.

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Supplementary Information is linked to the online version of the paper atwww.nature.com/nature.

Acknowledgements We thank M. Schick and E. Avetissova for technicalassistance and N. J. Cowan for critical reading of the manuscript. This work wassupported by grants from the NIH and the Edward Mallinckrodt Jr Foundation(E.N.).

Author Information Reprints and permissions information is available atnpg.nature.com/reprintsandpermissions. The authors declare no competingfinancial interests. Correspondence and requests for materials should beaddressed to E.N. (evgeny.nudler@med.nyu.edu) or D.G.(dgershon@redoxpharm.com).

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