Journal of Cell Science nvriyo alsFdrc I izaeTcho8,Npe 02,Italy 80126, Naples 80, Tecchio Piazzale II, Federico Naples of University oneuae naytohcltrlslrss(ALS) sclerosis More 2012). Kim, and (Kim (Steidinger lateral disease (PD) Alzheimer’s and disease 2011) Parkinson’s al., amyotrophic et 2010), al., et in (McLaughlin 2009; al., downregulated et (Ibaragi 2006). insults also al., environmental et It to Yoshioka and response 2005). in proliferation genetic al., cell cancer both et for role (Kishimoto and important an factors 2003) plays a angiogenic by al., stimulated of et angiogenesis ANG-mediated variety for (Xu 2002). necessary al., is rDNA it transcription et of where rRNA (Xu cells, region transcription rRNA 2012) promoter stimulates al., the al., et to et (Thiyagarajan binds proliferating (Tsuji neuronal cancer in and 1994), 2005) translocation Riordan, and nuclear (Moroianu endothelial undergoes al., et Xu ANG promote growth- 2005; to al., The 2002). et ability (Tsuji 2005). its transcription by (rRNA) al., mediated RNA et ribosomal is ANG (Tsuji of al., growth activity al., et cell stimulating et cancer (Yoshioka (Kishimoto angiogenesis and progression tumor 2005) cancer both (Tello-Montoliu stimulating promotes by cancer It 2006) human 2006). secreted of al., types vertebrate-specific, et various the is in expression of ANG upregulated 2001). member (Riordan, family fifth (RNase) ribonuclease the is ANG Introduction the to no localized and is inhibited words: of is ANG Key activity Knockdown not conditions, enzymatic tiRNA the energy. for is stress that active survival. anabolic ensure enzymatically and Under and to save remains growth nucleus prevented. cells thus to cell stressed and the is in produced RNH1 decreased RNH1 with RNA in is with associated cellular located associated rRNA not 1 is is with of unnecessary is ANG inhibitor it associated nuclear cleavage where ANG is ribonuclease/angiogenin contrast, granules ANG random By conditions, that stress Cytoplasmic production. in transcription. that here growth concentrated rRNA so report ensure is Under to and We RNH1 ANG. retained cytoplasm unknown. is by of activity is is inhibited ribonucleolytic activity ANG the regulated of and and that is activity so localization ribonucleolytic RNH1 activity with The the stress- this associated mechanism. tRNA-derived both how survival of production a controls but the into processes (RNH1) mediates translation ANG both protein stressed, for reprograms are accumulates which cells essential and When (tiRNA), translocation transcription. nuclear RNA rRNA undergoes small stimulates ANG induced it conditions, growth where Under nucleolus survival. the and in growth cell promotes (ANG) Angiogenin Summary work this to equally ` contributed authors *These 3 2 1 Pizzo Elio survival and to growth angiogenin control of localization subcellular stress- induced regulates 1 inhibitor Ribonuclease/angiogenin 4308 o:10.1242/jcs.134551 doi: 4308–4319 126, Science Cell of Journal 2013 June 20 Accepted ehoYu Wenhao uhr o orsodne( correspondence for Authors etrfrAvne imtrasfrHat aeIainIsiueo ehooyadItricpiayRsac eteo Biomaterials, on Centre Research Interdisciplinary Italy and 80126, Technology USA Naples of 02111, Cintia, Institute MA via Italian Boston, Angelo, Care Street, S. Health Washington Monte for 800 di Biomaterials Center, Universitario Advanced Medical Complesso for Tufts II, Center Institute, Federico Research Naples Oncology of Molecular University Biology, of Department 03 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2013. ncnrs oisurglto nvroscnes N is ANG cancers, various in upregulation its to contrast In nignn N1 tesgranules Stress RNH1, Angiogenin, 1, ,Cre Sarcinelli Carmen *, 2 ispeD’Alessio Giuseppe , [email protected] ; 1,2, [email protected] 1, ,JnhoSheng Jinghao *, ` n u-uHu Guo-fu and ) 2 2, aaoFusco Sabato , ` tesadi eitdb N Ymsk ta. 2009). al., et stress of (Yamasaki formation the ANG stimulate by to able mediated are by tiRNAs induced is Moreover, is tiRNA protein and of survival production reprogram stress cell The tiRNA 2008). promoting tiRNAs al., thereby by et stress, Therefore, (Thompson to inhibited anti- 2011). response not by in al., translation used is often et genes, mechanism (Ivanov pro-survival a protein eIF4G 2006), and al., weak apoptosis global al., et with et suppress (Baird translation (Ivanov binding mRNA IRES-mediated to uncapped However, and shown al., 2011). et capped Yamasaki been recent both 2011; of al., have translation the et Ivanov (Emara which of 2009; tiRNA with al., of 2009), et factor Fu production coincided 2010; the al., risk mediates et survival ANG that a neuron discovery to is in progression deficiency role 2010). a its Hu, plays and (Li ANG and diseases that neurodegenerative mutations. PD-associated survival, indicate loss-of-function the ‘loss-of-function’ clearly be neuron of only data to Most genetic the predicted These ALS. is also al., in ANG are et Es mutations identified 2007). van gene al., 2008; al., al., et mutated et et Gellera Paubel in Wu 2008; 2006; al., 2009; found al., et et (Conforti been Greenway PD 2008; and have ALS with mutations patients loss-of-function importantly, h xeso fAGsbooia ciiyfo cancer from activity biological ANG’s of extension The RNH1 blse tesidcdrlclzto fAGand ANG of relocalization stress-induced abolished 3 ai Formiggini Fabio , 3 al Netti Paolo , eerhArticle Research 3 , Journal of Cell Science otoldbt yislclzto n yisascainwith association Results its by and activity. localization but pro-survival its is RNH1. ANG by inhibited its of both activity cellular abolishes is controlled that demonstrate and results ANG our ANG of Together, nuclear Knockdown of not. that localization stress with is not ANG under so but cytoplasmic contrast, ANG ANG nuclear By with cytoplasmic associated inhibited. active is enzymatically is RNH1 is conditions, ANG growth ANG not nuclear cytoplasmic under but that ANG but that so cytoplasmic ANG growth study with nuclear associated with different this is under in RNH1 and conditions, demonstrate ANG locations We of activity conditions. subcellular ribonucleolytic different the regulates in 1989), al., et (Lee r nudryn ehns o ohcnesand cancers both for mechanism underlying and an (Yamasaki conditions are protein reprogram adverse cells survive 2008). which Anderson, to by where is foci mechanism SGs translation of cytoplasmic important Formation 2010), stored. an transiently al., are et mRNPs untranslated (Emara (SGs) granules id N ihfmooa fiiy(e ta. 99.To 1989). al., et (Lee affinity that femtomolar and 2011) al., with al., et et ANG (Haigis (Furia nucleus abundant binds and is cytoplasm that avoids both ANG in RNH1 how 2003) of is action question rRNA surveillance important Hu, an the of stimulating and activity and ribonucleolytic essential, in the is (Li Therefore, ANG ability 2012). the respectively Hu, its and and production, Li 2010; to growth tiRNA mediating related and in transcription is ANG response of stress activity biological conditions The stress RNH1 and and growth ANG under of localization subcellular Differential kDa a 50 with ANG abundant binds an that in 2003) RNH1, al., controlled that et is (Haigis hypothesized degradation protein ANG We RNA random avoided. of that is so activity compartments ribonucleolytic cellular Saxena that various 1992; the question, al., important how et another raised (Saxena is, direct results cells degradation These the 1991). in However, al., kills results et and cytosol 1989). RNA the cellular into of Vallee, protein ANG and of injection (Shapiro angiogenesis and cytoplasmic rather is SGs. but in nucleus, accumulated is the nuclear is ANG to conditions, mainly localized different stress longer is Under no is under accumulation. ANG nucleolus ANG conditions, compartments a that with growth cellular show Under paper different conditions. this to in or subcellular presented localized rRNA results either might producing The differential in conditions ANG tiRNA. stress of activity and that ultimate growth stress the control under and hypothesized ANG of growth localizations transcription under We rRNA respectively, stimulate conditions. production, properly tiRNA can is activity it and ANG that how so is cell question controlled promoting remaining a thereby However, translation, that survival. protein conceivable results of stress to reprogramming therefore response in in is tiRNAs of It example, production ANG-mediated hallmark disease. a For is neurodegenerative aggregates role. of protein resulting misfolded a stress of (ER) accumulation play reticulum cancer. from for endoplasmic to etiology and common stress a shown conditions Oxidative are stresses been oxidative pathological and has hypoxic two ANG the where diseases, neurodegenerative h iouloyi ciiyi seta o N oinduce to ANG for essential is activity ribonucleolytic The factors genetic and environmental by inflicted stresses Cellular RNH1 lescellular alters K d of , 1fM N1rgltsAGlclzto n ciiy4309 activity and localization ANG regulates RNH1 omlgot odtos oeoeosAGwsaddt the under to cultured added was were ANG nucleus cells exogenous when No the conditions. with arrows) in growth by normal Consistent detected indicated mainly conditions. 1C, was (Fig. stress ANG and and results, cytoplasm growth immunoblot the in under RNH1 and nucleus ANG of regulation converse the 1B). nucleus whereas (Fig. the cytoplasm in conditions, detected the growth was in RNH1 than under fraction more cytoplasmic fraction conditions, the stress nuclear under in the detected in was than RNH1 More ANG. of tiRNA and conditions. transcription these and rRNA with under stimulating nucleus consistent production in is the role conditions respective to stress its and ANG growth the of under in than cytoplasm localization cytoplasm the Preferential sodium the in with detected from nucleus. is stressed ANG distribution were more cells ANG (SA), When arsenite of cytoplasm. the shift fraction. is to analysis cytoplasmic a nucleus ANG the induced in more Immunoblot than stress conditions, fraction Oxidative cells conditions. nuclear growth the HeLa under in of stress that detected nucleus of showed and and levels 1A) (Fig. cytoplasm protein growth the the in examined under RNH1 first and we ANG question, this address tmltdrN rncito ae lc osv anabolic ANG- save is no to RNH1 ensure place to but takes RNH1 nucleus transcription by the rRNA inhibited in nuclear of probably stimulated amounts not trace are the is that ANG fully so is nucleolus ANG remains the stressed, and in are of ANG accumulated RNH1 cells that majority When with so transcription. the rRNA colocalized RNH1 stimulate not by to active is inhibited nucleus it not the When in therefore where stress. mainly growth is by nucleolus ANG regulated the conditions, or oppositely on growth are under dependent and are are cells cells the RNH1 of and status ANG of RNH1. localization by was inhibited been RNH1 have in would ANG of remaining nucleolus any the staining that suggesting (indicated nucleolar arrows), RNH1 (dashed of Prominent observed free arrowheads). those with and colocalized by arrows) those by images: staining (indicated merged ANG RNH1 the cytoplasmic from punctate in identified of pattern The were types staining 1D). Two punctate (Fig. cells. more RNH1 stressed a of displayed accumulation ANG nuclear cytoplasmic more and ANG stimulating 2002). of al., task et nucleolar the (Xu for that transcription is ribonuclease so rRNA a ANG inhibited, as not nucleolus, active is the remains and ANG in RNH1 clear least with thus at is associated It not conditions, nucleolus. growth the under in cytoplasm not in that nucleoli clearly colocalized image but mainly also merged nucleoplasm, are 1C, The RNH1 and nucleus. (Fig. was and the in ANG RNH1 nucleolus as that strong shows Cytoplasmic as the not arrows). was in but visible dashed not with but indicated plasma nuclear was ANG S1). of Fig. accumulation material was nucleolar (supplementary ANG detected clear much exogenous conditions, and growth If prominent normal nucleus. cytoplasm, under more cultured the the cells in the in as to detected added strongly also as was not ANG albeit the 2004). in takes (Nazar, assembly concentrated and place processing by was rRNA generated where ANG regions were perinucleolar signals Endogenous IF the ANG. all endogenous so experiments these in cells muoloecne(F a sdt eelmr eal of details more reveal to used was (IF) Immunofluorescence that to opposite is RNH1 of pattern distribution subcellular The ae oehr hs eut eosrtdta subcellular that demonstrated results these together, Taken of localization cytoplasmic more induced stress Oxidative the in detected strongly was RNH1 conditions, growth Under Journal of Cell Science N.RH A n lx-lo-8-aee otat-abtF(ab goat-anti-rabbit Alexa-Fluor-488-labeled and pAb RNH1 ANG. inl fAGadRH.Arwed niaeAGsgasnnoelpigwt N1 ahdarw niaencel.Saebr:10 bars: Scale nucleoli. indicate arrows Dashed RNH1. with non-overlapping signals ANG indicate Arrowheads RNH1. and ANG of signals N1()b muoltwt fiiyprfe A gis N n N1 epciey 2,ncepomn ula akr CD Fdtcino AN F(ab of anti-mouse goat detection Alexa-Fluor-555-labeled IF and (C,D) mAb marker. ANG nuclear conditions. a (D) stress nucleophosmin, and B23, (C) respectively. growth RNH1, under and cultured ANG cells against HeLa in pAb RNH1 affinity-purified and with Immunoblot by (B) RNH1 evstencess httert frN rncito is in accumulate transcription to RNH1 rRNA adverse more under for of survive sense to makes rate cells also for the is It waste conditions. ANG that energy growth, RNH1 avoid so for to reduced unfavorable nucleus more conditions sense the of in makes but role leaves it are major transcription, cells a ANG rRNA when that stimulate that fact to less is the ANG Given stressed, nuclear nucleus. that the phenomenon are general in a accumulated is be certain cells might to cells It limited the stresses. when not or is of cells conditions of environment stress types to the growth the when from between shifted RNH1 cytoplasm results and ER ANG and These an of S3). nucleus traffic tunicamycin, opposite Fig. the with that material treated indicate (supplementary cells inducer RNH1 HeLa and stress and, cells. in ANG of LNCaP observed cytoplasm relocalization in of was to pattern nucleus similar to nucleus a cytoplasm Moreover, from from ANG RNH1 conversely, of SA relocation with Treatment S2). induced Fig. material (supplementary cells cancer damage for possible as resources many as repair. allocate to and energy + (DMEM medium growth normal in cultured were 37 cells at HeLa conditions. conditions. SA stress stress mM and and 0.5 growth growth with under under treated cultured cells or RNH1 HeLa FBS) and of ANG fractions of cytoplasmic and localization nuclear subcellular Differential 1. Fig. 4310 dnia eut eeotie ihLCPhmnprostate human LNCaP with obtained were results Identical ora fCl cec 2 (18) 126 Science Cell of Journal ˚ o or el eefatoae n h ula n yolsi rtis(30 proteins cytoplasmic and nuclear the and fractionated were Cells hour. 1 for C 9 ) 2 eeue osanRH.Nce r tie ihDP.Arw niaeoverlapping indicate Arrows DAPI. with stained are Nuclei RNH1. stain to used were h ulou oesr httermiigAGi h nucleolus transcription. the rRNA in halt ANG to remaining the inhibited that is ensure to nucleolus the Fg A ae o8.We el eesrse ihSA with stressed were cells detected was When RNH1 8). and ANG to between association 5 no treatment, lanes extracts 2A, nuclear the (Fig. in polyclonal ANG co-immunoprecipitated no be RNH1 conditions, could these RNH1 Under by and 4). precipitated lane 2A, was be (Fig. (pAb) lane could RNH1 antibody 2A, ANG (Fig. conditions. Similarly, (mAb) stress antibody 3). cultured monoclonal cells SA-induced ANG by the and precipitated of and extracts growth ANG nuclear between under and interactions cytoplasmic the in examine RNH1 to (co- experiments co-immunoprecipitation IP) performed and we growth cytoplasmic conditions, under stress RNH1 of with and association to by activity regulated order randomly is ribonucleolytic In ANG will nuclear cell. the the it to whether controlled, detrimental well know be and not RNAs is cellular biological it degrade its if for essential However, is activity. ANG conditions of stress activity under ribonucleolytic growth nucleus The under the cytoplasm in in and RNH1 conditions with associated is ANG AB muoltaaye fAGadRH in RNH1 and ANG of analyses Immunoblot (A,B) m )wr nlzdfrAG()and (A) ANG for analyzed were g) 9 ) 2 eeue ostain to used were m m. 10% G Journal of Cell Science rcin7adRH a eetdi rcin n Fg 3B) (Fig. 7 and 6 fractions in of detected in was distribution only RNH1 detected of and was ANG 7 shift fractions. fraction different a in RNH1 was and of ANG there formation the structures, with supramolecular Accompanied high of complexes. formation mass indicating 3C,D) fractions, molecular absorbance mass (Fig. of molecular shift filtration stress high drastic the and to a induced 3A,B) gel stress (Fig. of Oxidative growth extracts the some conditions. cytoplasmic under examine used from cultured into to RNH1 and cells column ANG first assembles G-200 of profiles Superdex elution or We a on organelles chromatography structures. and cytoplasmic ANG supermolecular localizes some both RNH1 and of ANG that to staining suggesting cells, cytoplasmic stressed in punctate RNH1 shows supracomplexes 1 mass Fig. molecular and high ANG into cytoplasmic RNH1 of redistribution induces Stress cell to and repair place damage takes by for possible transcription inhibited as survival. rRNA is energy unwanted much ANG survival as nuclear no apportion cell that that of reasonable so viewpoint al., is RNH1 the et by it From (Emara survival stress, 2011). stress proposed under al., cell under et the tiRNA promoting Ivanov with of 2010; in consistent production ANG the is mediating cytoplasmic This of active. function be is should that suggesting extracts ANG ANG cytoplasmic ANG the in between nuclear detected association was RNH1 no remaining and condition, activity stress the ANG Under of inhibited. amount also trace is any but ensure of to out RNH1 1) with moves associated (Fig. only not nucleus ANG stressed, the are cells results degradation, when these Further, that RNA cell. indicate the avoid to harmful otherwise to be would its inhibited which and is RNH1 with activity associated is ribonucleolytic ANG cytoplasmic time, At growth. same cell the promoting able the thereby is transcription ANG in rRNA and stimulate RNH1 to RNH1 These by its inhibited that with 8). not so is conditions associated to activity growth ribonucleolytic under not 5 cultured are is lanes cells when rabbit ANG 2B, nucleus the (Fig. that of chain extracts was indicate heavy but nuclear the 4) results to is the 1 band lanes RNH1 in 2B, specific (Fig. the apparent of extracts above cytoplasmic analyses wit band the Immunoblot The performed in nu by co-IP co-IP. were and by analyzed for analyses 1–4) was used Immunoblot (lanes materials fractions pAb. cytoplasmic the nuclear RNH1 the of and affinity-purified and 10% cytoplasmic or fractionated had of mAb, were lanes purity ANG control Cells conditions. IgG, The Input (B). stress mouse IgG. pAb. conditions and non-immune ANG stress growth a or oxidative under with pAb hour)-induced cytoplasm precipitated RNH1 1 and were mM, 5–8) nucleus (0.5 (lanes the SA fractions in and RNH1 (A) and conditions ANG growth between Interaction 2. Fig. N1rgltsAGlclzto n ciiy4311 activity and localization ANG regulates RNH1 h niesetu rmfatos2t n N1from both 3D). RNH1 (Fig. of and mass distribution molecular 7 high widespread of a to structures suggesting various 2 in 6, fractions proteins across to detected from 2 was spectrum fractions ANG cells, entire generated stressed fractions the of the cytoplasm In the cytoplasm. existed the from RNH1 in and forms ANG monomeric that as indicating conditions growth under Fg D.I h w el oae naselected a SGs the in of located some cells in two found the only was In RNH1 4D). but (Fig. SGs. 4C) all (Fig. not that SGs but indicating some RNH1, in of colocalized free were were RNH1 contain that and not SGs did ANG are that SGs there no with RNH1 but were with ANG overlapped There overlapped arrows). them (red) of dashed all 4B, to induce SGs not (Fig. interesting but green), PABP-containing is stresses 4A, It the (Fig. SGs. ANG all oxidative in RNH1 that SGs and that note ANG both both in demonstrated arrowheads). of present 4B, localization be (Fig. results to organelles non-SG found These and also arrows) the was 4B, in RNH1 (Fig. that SGs. organelles staining cytoplasmic not arrowheads), in ANG present 4A, are also (Fig. punctate is PABP ANG the that with is indicating colocalize It of not SGs. some did to PABP localized cytoplasm that and is notable ANG ANG that of also indicating (Buchan to colocalization arrows), SG 4A, specific showed of (Fig. mAb 2009), marker a a Parker, (PABP), and and protein ANG binding an to with polyadenine located cells specific HeLa are SA-stressed RNH1 pAb on and IF affinity-purified ANG Double stress. RNH1 whether stress under examine under and SGs to in cytoplasm us ANG the led that in 1), (Fig. patterns and staining 3), punctate (Fig. is display that structures high itself findings to mass RNH1 The ANG and produced. ANG molecular whether cytoplasmic is of unknown tiRNA relocation induce where is stresses and it SGs in However, Yamasaki located 2010; 2009). al., of et al., formation (Emara et stress-induced tiRNA of potentiate production to through SGs shown SGs been in has RNH1 and ANG ANG of assembly Stress-induced dse ros.Termiig3 G l otie ANG contained ANG all contain SGs not did 34 SGs remaining countable 35 The the arrows). of (dashed 1 only 4C, Fig. ofclmcocp losoe htAGwspeeti all in present was ANG that showed also microscopy Confocal b tblnadPN rm1 ftemtra sdfrco-IP. for used material the of 1% from PCNA and -tubulin eaclswr utrdudrnormal under cultured were cells HeLa z scinin -section clear h Journal of Cell Science nest fterdfursec rmteacpo RH)was (RNH1) acceptor the from the fluorescence in red 29.3 the 5A), green was of of (Fig. (ANG) intensity SGs. intensity donor stressed the the cytoplasm, from not in the for fluorescence in were interest used associated of cells region was marked When to physically (FRET) us purpose. transfer are prompted this energy SGs resonance they in Fluorescence RNH1 whether and ANG examine both of Localization stress and growth under conditions RNH1 and ANG between cytoplasmic transfer energy resonance Fluorescence SGs 4F, ANG-positive (Fig. of arrows). SGs that by than indicated RNH1-positive 4E, lower and (Fig. TIA1-positive was of arrows) in positive percentage by observed both indicated the were were RNH1 Again, and that SGs. (Anderson ANG Granules marker for 2009). SG negative another Kedersha, TIA1, and type. with cell certain colocalization a their to limited SGs not in is RNH1 and and phenomenon the ANG general of a localization similar in is material that Very (supplementary suggesting co-immunoprecipitated cells 2). S4), LNCaP Fig. (Fig. with not cells obtained were stressed were results from extracts that RNH1 finding cytoplasmic stress our and under with These consistent ANG different. RNH, ANG cytoplasmic with is associated the be occupancy of not will some SG that that their are suggest cells suggested of when results results SGs extent in These located the are (arrows), free. stressed, RNH1 remaining and RNH1 RNH1 The ANG both were SGs. contained although of (67%) 4D 33% SGs Fig. in 24 presence in a RNH1 from SGs an are of representing shown countable 8 Data only immunoblot. 24 However, kDa). by coverage. (15 RNH1 the ANG RNaseA and 97% and ANG a kDa) for experiment. representing (45 analyzed each (arrows), ovalbumin were from kDa), C) obtained filtration (105 and were gel antibody A results to compact in Identical subjected a numbers repeats. and kDa), by four extracted (148 (indicated of were Herceptin eluates experiment fractions include the representative Cytosolic which from (C,D). markers, fractions hour size selected 1 of (B,D), for volume cm SA elution complexes. (30 mM the mass column 0.5 indicate molecular G-200 by high Superdex induced into a RNH1 stress on and oxidative chromatography ANG under cytosolic or of (A,B) assemble induces conditions stress Oxidative 3. Fig. 4312 oaiaino N n N1i G a ofre by confirmed was SGs in RNH1 and ANG of Localization ora fCl cec 2 (18) 126 Science Cell of Journal 6 6 m 5m) h lto rflswr eodda il bobneuisa 6 m(,) h arrows The (A,C). nm 260 at units absorbance milli as recorded were profiles elution The ml). 25 cm, 1 . nt n the and units 7.2 ups fgnrtn iN opooecl uvvlunder survival cell the Therefore, promote for to there. activity tiRNA located ribonucleolytic stress. generating both its of were retains purpose results they ANG These in the though associated cytoplasmic increase. physically of even not were 0.8% RNH1 SGs photobleaching and mere ANG after that a The indicated and 5B). representing (Fig. before acceptor, observed 75.4 respectively, was was donor units, transfer the of energy ANG intensity both fluorescence no contained normal that RNH1, under SGs cultured selected and were the In cells and conditions. when growth ANG cytoplasm between the existed in fluorophore interaction RNH1 acceptor physical to a that fluorophore 32.0 suggesting donor from became transfer 10.1 fluorescence energy of increase donor an the representing of 5.3 intensity to fluorescence 76.4 ubradtesz fSswssgiiatyrdcdcompared reduced significantly was the SGs both of but RNH1 size and S5B,C) the ANG that Fig. and both material number of and (supplementary amount SGs trace nucleus in a S5A, RNH1 still the Fig. was There material to arrows). (supplementary dashed back nucleolus relocated from feature disappeared ANG significant hours. most cultured 3 that The cells the 1C). of is Fig. (Fig. for that conditions to material growth similar (supplementary in very medium be only cells IF to found recovered growth double was and in by S5A) RNH1 then full examined and and first hour in ANG was 1 of for recover localization SA Subcellular mM to 0.5 with allowed stressed were cells from HeLa recovered cells in RNH1 stress and ANG of Localization 6 . nt,rsetvl.Atrpoolahn fred of photobleaching After respectively. units, 3.5 6 . nt 69 fteoiia) the original), the of (6.9% units 0.4 eaclswr utrdudrnra growth normal under cultured were cells HeLa 6 .% hrfr,teewsan was there Therefore, 1.2%. 6 . n 76.0 and 9.1 6 . units, 7.6 6 9.6 Journal of Cell Science E,adb N1adPB D rTA F.Dse rosidct G htcnanol AP(,)o I1(,) mgsi n eetknwt SP5 10 with bars: taken were Scale D microscope. and confocal C in 410 Images LSM (E,F). Zeiss TIA1 a or (A,D) with PABP taken only were contain that F SGs and indicate E arrows Dashed in (F). Images TIA1 microscope. or confocal (D) PABP Leica and RNH1 by and (E), of series ih05m Afr1hu,fxdwt %prfradhd n emaiie ih05 rtnX10()ad()o ie nmtao at methanol in fixed or (D) and (C) incubated X-100 organelles. were cells Triton cytoplasmic HeLa 0.5% non-SG (F). with and TIA1 permeabilized and SG RNH1 and in and paraformaldehyde (E), signals 4% TIA1 RNH1 and with indicate ANG fixed (D), arrowheads hour, PABP and and 1 Arrows RNH1 for (C), PABP. SA PABP and and mM RNH1 ANG 0.5 between of with IF IF double Double of image (B) Confocal SGs. (C–F) not are that organelles re dnr N)adrd(cetr N1 loecnei h eetdRIwsrcre.Terdfursec a hnbece t52n o second bot 5 of for nm intensity 592 The at analyses. bleached FRET then 5 for was used bars: fluorescence was Scale red images The again. recorded. Z-section recorded was the was with ROI of detected fluorescence selected were panel red the RNH1 center and in and The fluorescence green ANG images RNH1) merged. X-100. the confocal (acceptor, Triton were and fluorescence red 0.5% and red with and and wavelength permeabilized ANG) Green nm) and (donor, antibodies. paraformaldehyde (488 green secondary 4% Cy3-labeled excitation with and single fixed Alexa-Fluor-488- a (B), with conditions. hour under visualized stress and 1 and respectively, for pAb, growth SA and under mM mAb RNH1 0.5 with and treated ANG or cytoplasmic (A) between Immuno-FRET 5. Fig. SGs. in RNH1 and ANG at methanol of Detection 4. Fig. Z sciniae eetknadtecne ae a eetdfraayi.Arw niaeSsta eesandbt o N n AP()o TIA1 or (C) PABP and ANG for both stained were that SGs indicate Arrows analysis. for selected was panel center the and taken were images -section 2 20 ˚ o 0mnts elnce eesandwt AI rosidct N inl nS.Arwed niaesann fAGi cytoplasmic in ANG of staining indicate Arrowheads SG. in signals ANG indicate Arrows DAPI. with stained were nuclei Cell minutes. 10 for C A obeI fAGadPB.HL el eeicbtdwt . MS o oradwr ie by fixed were and hour 1 for SA mM 0.5 with incubated were cells HeLa PABP. and ANG of IF Double (A) m m. N1rgltsAGlclzto n ciiy4313 activity and localization ANG regulates RNH1 eaclswr utrdi omlgot conditions growth normal in cultured were cells HeLa m m. 2 20 ˚ eetaken were EF.A (E,F). C s, h Journal of Cell Science aasonaefo ersnaieeprmn ffu eet.Identical repeats. repeat. four each A. of from in experiment obtained collected representative were fractions a results gradient from analyses alternate are Immunoblot from shown (C) PABP Data EB. and with RNH1 stained ANG, and of gels agarose 1% RNA in (B) fractions. separated 50 gradient fraction. sucrose each the in of (60–15%). nm content gradient 260 at sucrose the profiles a and Absorbance fractionated in (A) were ultracentrifuged Cells times were hours. three fractions 3 washed for cytoplasmic were conditions cells growth the experiment, in and recovery cultured exchange the and medium In by hour). removed 1 was SA, SA mM (0.5 under conditions RNH1 stress oxidative and ANG conditions. cytoplasmic growth of various localization Dynamic 6. Fig. from that to The similar cells. very stressed was from cells was generated stress-recovered mass absorbance from fractions UV molecular profile the the high where in 9 the higher and much in 3 noticed fractions filtration between was gel region recovery conditions from growth stress stress between obtained difference and and significant results stress most the the growth, chromatography, to in cells Similar of cultured shows conditions. extracts been 6A cytoplasmic Fig. the have gradient. of that sucrose profiles nucleolus a gradient sucrose using the the by studied in further was ANG transcription. rRNA of stimulating in activity activity its so enabling ribonucleolytic resumed, nucleolus and and the cytoplasm the from the dissembled that disappeared to SGs RNH1 both stress, Specifically, relocated nucleus. RNH1 from that and indicate recovered ANG results These therefore were 4). (Fig. cells cells stressed when the in that with 4314 h aeo yolsi N n N1i eoee cells recovered in RNH1 and ANG cytoplasmic of fate The ora fCl cec 2 (18) 126 Science Cell of Journal m eaclswr utrdudrgot and growth under cultured were cells HeLa rmeeyohrfato ftegain were gradient the of fraction other every from l 6,rsetvl,o htbfr h ramn Fg 8B), (Fig. treatment the before that and 57% was of transfectants Sh35 respectively, number and the hours, scramble 26%, that 4 in for cells SA shows mM viable 0.5 of 8B,C with stress. treated Fig. oxidative were under cells stress. cells When the of of SA viability sensitivity decreased the to knockdown examined cells efficiency next We knockdown knockdown 8A). the Fig. extents to 7A, the positively (Fig. and correlated growth 8A). cell inhibition (Fig. inhibited of control constructs scramble shRNA the four in All levels with compared reduced SGs in These when ANG arrows). of impaired dashed relocalization was by stress-induced that (indicated indicate nucleolus ANG results Instead, the 7C). in (Fig. found PABP with was colocalized was ANG no but niaigta eoeypoeso el rmsrs is stress normal from their to cells RNH1 of and ANG process of return recovery a panels), three with a bottom accompanied 6C, that (Fig. stress that from indicating fractions recovered note mass cells molecular to lower the interest in in gradient detected significant sucrose were of RNH1 mass is and ANG molecular It of high confirmed. growth to was relocation RNH1 fractions Stress-induced under and were structures. ANG fractions RNH1 both supramolecular and mass in ANG were molecular cytoplasmic RNH1 these not that and lower was confirming ANG it the conditions, Both that in eluted. PABP indicating were found cells, 3–11, SGs stressed where fractions In fractions in cytoplasm. indicating the enriched fraction, every in were in distribution density even equal an was roughly similar PABP a conditions, growth in are Under detected 6C). immunoblot ribosomes distributed (Fig. by also PABP confirmed of were and was analysis SGs hypothesis that This SGs likely fractions. is these of in it al., 2009), size et al., et (Bevilacqua the (Souquere polyribosomes Because stress of that 2010). finding abundance decreased early is an the with RNA sucrose agreement decreases total the in that the cells, stressed notable is the is in where in 3–9) It 6B rRNA) (fractions eluted. Fig. region were (mainly the ribosomes conditions. marks RNA which growth fractions, cellular gradient under of cultured distribution cells the eoaiaino N n N1i yai n srestored is and dynamic is lifted. RNH1 is stress-induced stress and when that ANG indicate of results relocalization These patterns. localization F N a on ob ooaie ihPB nSsin 4A. SGs Fig. in in shown cells PABP in HeLa However, untransfected with from different with so colocalized seen not pattern 7B), be the (Fig. to cells transfected found by shRNA examined scramble was was further 97% ANG ANG of for localization a IF. cells and selected HeLa SA transfected was with with shRNA stressed and were Sh35 expression and level, control RNH1 Scramble protein study. RNH1 down in knocking decrease in RNH1-specific efficient 8.6 shRNA, 100, 12.9 were 37 scramble and and 35 a them 34, 33, encoding shRNA of lentivirus four to RNH1 7A. with of constructs, Fig. in intensity shown relative shRNA as The efficiently IF. different expression by RNH1 down localization five knocked ANG in the changes down Among resultant knock the lentivirus-mediated to examined used (shRNA) and we RNA stress, SGs hairpin to to small exposed ANG of were localization cells on RNH1 when of effect behavior the survival understand and To growth cell alters and of Knockdown elpoieainin proliferation Cell 6 .,rsetvl.Tu,coe3 S3)wsmost was (Sh35) 35 clone Thus, respectively. 6.9, RNH1 RNH1 kokoncls G eefre normally formed were SGs cells, -knockdown RNH1 RNH1 mar oaiaino N nSGs in ANG of localization impairs a downregulated. was kokonclswssignificantly was cells -knockdown b tblni h el infected cells the in -tubulin 6 .,18.2 0.1, RNH1 6 .,3.0 4.8, expression RNH1 RNH1 6 0.7 - Journal of Cell Science h el eetetdwt MS o or h ecnaeof percentage 15.9 11.3 the was hour, transfectants and 1 Sh35 for and scramble SA in mM cells apoptotic 1 with treated were (6.2 cells transfectants the control scramble 11.8 in was percentage that transfectants The 1.9-fold Sh35 conditions. in 8E) cells (Fig. apoptotic in stress of increased and 8D) significantly (Fig. apoptosis growth Cell 2005). RNH1 al., red nuclei et apoptotic stains of (Ribble it integrity so the compromised when is only stains membrane cells and plasma enters cells EB intact whereas permeates green was nuclei AO staining all cells. (AO) apoptotic stain Orange to Acridine used and (EB) bromide ethidium ujce o05m Afr1hu n tie o N n AP rosidct ooaiaino N n APi oto el.Arwed indicate Arrowheads cells. control in PABP and ANG of colocalization indicate Arrows PABP. and ANG for over stained RNH1 and in of hour ANG intensity 1 of band for staining relative SA nucleoli of mM analyses 0.5 ImageJ to in panel, subjected ANG Right of Immunoblots. localization panel, Subcellular Left (B,C) cells. HeLa lentivirus-infected shRNA eesnilfrteclua ucin fANG. cell of as functions might cellular level well RNH1 the proper as for a essential that ANG that be suggesting indicate of survival, results localization and in growth the these than changed together, (15%) Sh35 knockdown Taken more cells and still (9%). control were scramble cells there in both that cells Sh35 apparent in viable is and occurred treatment it death However, scramble mM of transfectants. 2 cell At of 8C). hour massive (Fig. Viability 1 respectively SA, 60%, control. and after 81% was scramble transfectants obvious in the was rate than Again, to death death increased 8C). (Fig. was cell cell concentration in SA mM, 1 the increase When cells. 37% knockdown a representing 1.5 of presence the in selected were cells Infected control. scramble and shRNA specific of Knockdown 7. Fig. oeaieteefc of effect the examine To kokonclswe hywr utrdbt under both cultured were they when cells -knockdown 6 .% epciey ersniga4%ices in increase 41% a representing respectively, 4.9%, RNH1 RNH1 RNH1 kokonclswr oesensitive more were cells -knockdown lessbellrlclzto fAGudrstress. under ANG of localization subcellular alters RNH1 kokoncls cl as 10 bars: Scale cells. -knockdown ncdw ncl apoptosis, cell on knockdown RNH1 kokonclsudroiaiesrs.Clsifce ihsrml oto B n h5()lniiu were lentivirus (C) Sh35 and (B) control scramble with infected Cells stress. oxidative under cells -knockdown RNH1 6 6 .% hc is which 3.7%, .%.When 2.7%). -knockdown 6 RNH1 m RNH1 .Smlrrslswr bandi el netdwt h3adSh34. and Sh33 with infected cells in obtained were results Similar m. 4.5% - N1rgltsAGlclzto n ciiy4315 activity and localization ANG regulates RNH1 eut ugs hticesdaotssmgtb esnfor reason a be might in survival apoptosis and growth increased decreased that suggest results when apoptosis SA-induced Discussion oaodwseo eore npouiguncsayrRNA. unnecessary producing in therefore stress resources under of are It cells waste when avoid for nucleus mRNAs. leave to to anti-apoptotic ANG energy for sense of IRES- makes anabolic because alter protein not translations purpose do save the but this mediated translation meets protein to conditions, global tiRNA suppress of adverse tiRNAs decreased production in The produce is survival. to are ability rate its cells by translation mediated pro- When The is meet rRNA. ANG tiRNA. to for is of nucleus cells which the function growing rDNA, in of survival demand mainly of metabolic is region high ANG promoter the purpose, why the reason this to the For bind probably transcription. to by rRNA needs mediated promoting ANG is in ANG activity of of functions its function survival growth-stimulating and The growth growth the ANG. a of be regulation normal might of conditions subcellular growth mechanism in different Differential under ANG ANG are cytoplasm. of of to localization shift cells a nucleus induced from when both localization stress mainly ER nucleus is and ANG Oxidative cell. the conditions. the of to status growth dynamic localized the is on ANG dependent of is localization and subcellular that RNH1 show and results ANG Our of localization cellular Dynamic eaclswr netdwt LO1lniia atce encoding particles lentiviral pLKO.1 with infected were cells HeLa m /lprmcn A N1lvl nsrml oto n specific and control scramble in levels RNH1 (A) puromycin. g/ml RNH1 RNH1 a nce on These down. knocked was kokoncells. -knockdown b -tubulin. RNH1 - Journal of Cell Science i.8 ncdw fRH nHL el eraegot n nrae estvt ostress. 0.5 to of sensitivity presence increases the in and FBS growth 10% decrease + cells DMEM HeLa in in cultured were RNH1 transfectants of Knockdown 8. Fig. 4316 N1 u lodsbeisitrcinwt N ncytoplasm. in ANG with interaction its from disable cytoplasmic of extracted also amount but fractions the reduce RNH1, nuclear only and not and the Stresses bound ANG cells. in stressed that be associated showed trace will results were any be nucleolus co-IP RNH1 that Indeed, the to RNH1. in so RNH1 by remained conditions inhibited nuclear ANG stress for of under reasonable amount nucleolus is in It located noticed. of localization was nucleolar RNH1 prominent significantly, More stressed. cell. the so of RNH1 to status cytoplasm by healthy the inhibited proper in likely a occurs degradation maintain most RNA is unfavorable completely cytoplasm no be that amount the will small in the activity ANG conditions, its of growth RNH1, under by Therefore, that enzymatic bound inhibited. assume both reasonably is FRET inhibit can ANG We to and when ANG. shown of been co-IP activity has angiogenic and both and 1989) affinity al., by sub-femtomolar et a shown (Lee with ANG to as binds is RNH1 ANG RNH1, experiments. cytoplasmic time, with same the associated RNH1 At nuclear by conditions. inhibited of not growth is function as under transcription ANG normal rRNA stimulating with Thus, in associated experiments. ANG not co-IP conditions is by growth it under shown However, RNH1 unclear. nuclear (Furia currently nucleus of the role is was in The detected al., 2011). RNH1 been et al., also et conditions. (Haigis had protein it growth though cytoplasmic even a 2003) under be to nucleus considered previously the in located E Aidcdaotss el eetetdwt MS o oradsandwt BadA.Tenmessoni – r means are D–E in counted. shown were numbers cells The (green) AO. live and and EB (red) with apoptotic stained Both and AO. hour fields. and 1 microscopic wer EB for five numbers with SA in Cell stained mM concentrat counted time. and different 1 indicated cells with FBS with apoptotic the treated 10% treated of for and + means were hours percentage SA are DMEM Cells 48 mM A–C in for apoptosis. in 0.5 medium shown cultured SA-induced with growth Data were (E) assay. treatment in Cells MTT cultured to by conditions. were subjected determined Cells growth were then survival. numbers under and cell Cell of hour. hours, response 1 48 dose for for SA SA medium (C) assay. growth MTT in by cultured determined were Cells survival. cell upiigfnigi htRH a on ob mainly be to found was RNH1 that is finding surprising A vnmr N1wsfudi h ulu hnclswere cells when nucleus the in found was RNH1 more Even ora fCl cec 2 (18) 126 Science Cell of Journal m /lAG elnmeswr eemndwt ole one.()Tm oreof course Time (B) counter. Coulter a with determined were numbers Cell ANG. g/ml htAGi oae nSsi tesdclsadta this not that but some and in found cells also was stressed almost RNH1 in SA. in detected by was SGs induced ANG SG RNH1. in every by regulated located is significant is localization is It ANG SG. the high is One that to structures cytoplasm. supramolecular the fractions these the of to mass within class nucleus molecular structures the low supramolecular from from molecular only also not but ANG cytoplasm of stress traffic under ANG induces of Stress localization SG regulates RNH1 ietnstergltr ciiiso N nsiuaigcell stimulating survival. in cell ANG promoting stress, is in of tiRNA or activities under growth where regulatory RNH1 place cytoplasm the with interacting likely fine-tunes that in the demonstrated results is These RNH1 produced. cytoplasm event, that from to any suggesting lead In dissociates complex. may RNH1–ANG which ANG from 1992), ANG ANG of of Hofsteenge, structure with dissociation 3D and contacts the (Fominaya alters important drastically RNH1 1997), forms al., Cys which et of (Papageorgiou of derivatization or none agents Oxidation residues, reducing RNase. active of complex, fully the releasing absence complexes dissociates RNH1–RNase rapidly the of p-hydroxymercuribenzoate Treatment with disulfide in 1977). no al., activity and et (Blackburn residues loses tiRNA Cys and free of bonds, 32 Cys purpose its ANG contains to RNH1 the attributable that oxidation, residues. so to for to sensitive RNH1 stress is activity RNH1 for under production. ribonucleolytic reasonable cytoplasm its is in it regains ANG view, from of point dissociate functional a From 6 ..o rpiae farpeettv xeiet D elapoptosis Cell (D) experiment. representative a of triplicates of s.d. A elgot.Sal cabeadRH shRNA RNH1 and scramble Stable growth. Cell (A) 6 ..o the of s.d. osof ions e Journal of Cell Science r rmrl opsdo h tle 8 preinitiation SGs 48S 2008). Kedersha, and stalled (Anderson factors subunits the ribosome initiation small the of to and bound mRNA composed containing SGs cytoplasmic complexes active. primarily enzymatically Therefore, be RNH1, would RNH1. SGs and are in with localized ANG is associated both that free. ANG contained not RNH1 was that was but SGs ANG ANG contained those SGs in of subset Moreover, a So SGs. all ncne ramn LeadRie,20;Rtok n Raines, and Rutkoski design 2008; Raines, purposes to 2008). and therapeutic (Lee for put treatment RNH1 cancer bind been from in not the have do cells into that efforts protect RNase entry cytotoxic extensive to gain that 2003) Indeed, RNase al., cytoplasm. non-cytosolic et by serve caused (Haigis to damage ‘sentry’ thought first and cellular was physiological RNH1 as well- its evolving. the are with RNH1, roles Compared of pathological 2011). properties al., regulation et biochemical immune Wit defined de to ranging 2008; repair al., functions DNA et cellular (Bella been to protein–protein diverse regulation have with in cell-cycle that proteins from and involved many 1995) in often Deisenhofer, found and are (Kobe that interactions conserved 2007). a al., domain and ANG repeats et leucine-rich structure (Johnson well seven 1996), of RNase1 composed Deisenhofer, and is been RNH1 1997) and al., (Kobe et have in (Papageorgiou RNaseA and 1993) X- Deisenhofer, with RNH1 and (Kobe high-resolution complex RNH1 free of of of structures characterization ray properties 2003). including al., et documented, biochemical (Haigis accounting content proteins protein cellular cytosolic The abundant total most of 0.08% the for of one survival is and RNH1 proliferation cell RNH1 regulates the RNH1 cellular by the regulated both are that ANG of demonstrate level. function results and these localization event, any 2012). al., In and when et ribosome-bound (Saikia and is states the active aminoacyltRNA-synthetase-bound between tiRNA the is frequently synthesis where more tiRNA protein transit place that this tRNAs when finding the higher recent for the is is with has production consistent candidate polysome be ANG will where the it was possible subunit produced, If 60S structure One the detected. or of been stress. polysome the type is to structure(s) this subjected to in were ANG with increased colocalization of are show significantly because structures not Localization P-bodies did these not markers ANG. of probably P-body are nature is with they The staining but level SGs. present at not RNH1 unknown are that the structure when that shown 2009). increased al., been be et has is (Yamasaki would it downregulated But production production tiRNA. produced tiRNA tiRNA ANG ANG in SGs. where where decreased SGs in place be the to of ANG is expected SGs of Knockdown that localization unlikely tRNA. is ANG it microRNA- role cleaves what SGs, be with matter in No potentially integrated pathways. plays thus cellular be diverse mechanism may in involved silencing SGs translational in RNA-induced function induced located the contain of that ANG also conceivable is therefore of SGs is It that It 2009). complexes. note silencing Kedersha, to including and interest and functions kinases particular (Anderson nucleases, diverse molecules helicases, with signaling RNA proteins proteins, other RNA-binding contain also N a lolclzdi te lse fsuperamolecular of classes other in localized also was ANG RNH1 RNH1 kokonclswe they when cells -knockdown kokonclsi twere it if cells -knockdown RNH1 rsial reduced drastically N1rgltsAGlclzto n ciiy4317 activity and localization ANG regulates RNH1 1poesn,teeycnrbtn ocne rgeso (Kim progression pri-miR- cancer mediate to to contributing 2007) shown thereby been al., RNH1 also processing, et of has 21 function RNH1 (Monti regulatory survival. homeostatic the cell to in redox antioxidant contribute thereby and also reported might ANG, 2003) cause The effects of al., also survival. localization et and could cellular (Cui growth regulate cells cell to stressed controlling is in RNH1 of SGs of Failure to stressed. of survival are localize decreased cells to to when nucleolus ANG the rRNA in be produce to ANG continually viewpoint, for survival counterproductive cell certainly is the is it From transcription process. rRNA costly survival. energetically in decreased an for not reason but of the nucleolus be the localization might in cellular localized when is abnormal ANG SGs that to fact related The ANG. probably is stress N ignssadta G r nadtoa ralternative or metabolized. additional or generated an are micro are in is RNAs role micro SGs it a where SGs, that that play place in and and RNH1 found RNase and biogenesis all a ANG RNA are that is complex ANG speculate RISC that to facts tempting the the and of RNH1 view ANG, In 2011). al., et rcino 50 of fraction 1 MHPS H74 MMgCl mM gradients 5 sucrose 7.4, 60–15% onto pH layered HEPES, were mM cells (10 the of fractions cytoplasmic The ultracentrifugation gradient Sucrose 6 from fraction cytoplasmic The chromatography filtration Gel MgCl mM 1.5 8.0, pH KCl, HEPES, mM mM 10 10 in resuspended and containing trypsin-EDTA by detached were Cells hours. co-IP 3 and for extracts medium Cell growth in cultured washed and were times cells To and three indicated. removed DMEM otherwise was as with SA or 5 containing hour with medium 1 treated experiments, for recovery were SA cells oxidative mM stress, induce ER 0.5 To induce with FBS. treated 10% cells were with LNCaP cells supplemented FBS. stress, 1640 10% RPMI with in supplemented maintained DMEM were in maintained were cells HeLa treatment and cultures Cell Methods and Materials epciey o oI xeiet,tectpamcadncerfatosfrom fractions of nuclear and analyses cytoplasmic the immunoblot nuclear and experiments, 4 cytoplasmic by co-IP the For by of examined respectively. purity obtained The was was fraction. nuclear fraction fractions the Cytoplasmic as 0.1%. designated was adding of by concentration lysed 1000 were at final Cells centrifugation minutes. a 10 to for ice NP-40 on incubated and cocktail inhibitors 800rpm vrih t4 at overnight r.p.m. 38,000 g,AGmb2-F rafnt-uiidRH A 17a 4 at R127 pAb RNH1 affinity-purified or 26-2F, 5 mAb with incubated ANG fractions, IgG, equal three into divided were odtos copne iha nraei elapoptosis. cell in increase of an survival with Decreased accompanied conditions, apoptotic of percentage RNH1 when The doubled apoptosis. in cells increased to growth for reasons attributed the cell of One manner. decreased dose-dependent a in proliferation yU bobnea 6 m N otnswr nlzdb grs gel agarose by analyzed were contents Immunoblot. by RNA analyzed nm. were 260 RNH1 monitored and and at pump ANG peristaltic electrophoresis. absorbance a using tubes UV the of by bottom the from collected were h laewsmntrda 6 m olce n02 lfatosadanalyzed and fractions ml Immunoblot. 0.25 by in contents collected RNH1 nm, and 260 ANG cm at for (30 monitored column was eluate G-200 The Superdex a to applied or.Temxuewscnrfgd ahdadaaye yImnbo for Immunoblot respectively. by R127, analysed and and R113 washed with centrifuged, RNH1 was and ANG mixture The hours. 2 a ie ih50 with mixed for was taken was sample each 6 10 ehv on htkokonof knockdown that found have We 6 el eedltdi lo 0m EE otiig01 P4.A NP-40. 0.1% containing HEPES mM 10 of ml 1 in diluted were cells losgiiatydcesdcl uvvludrstress under survival cell decreased significantly also RNH1 m a ae n sda nu oto.Termiigmaterials remaining The control. input as used and taken was l m g kokonclswr ujce oS stress SA to subjected were cells -knockdown f5%PoenAGSpaoeb oaiga 4 at rotating by A/G-Sepharose Protein 50% of l o iue.Teple a isle nRP ufrand buffer RIPA in dissolved was pellet The minutes. 5 for RNH1 RNH1 b ˚ tblnadPN nlss h eann solution remaining The analysis. PCNA and -tubulin RNH1 naBcmnS 1rtr h rdetfractions gradient the rotor, 41 SW Beckman a in C 6 10 kokoncls hs ao role major a Thus, cells. -knockdown a nce on ncdw of Knockdown down. knocked was 6 kokonclsaantoxidative against cells -knockdown eaclswsdltdi 200 in diluted was cells HeLa RNH1 2 n 0 MKl.Atrcnrfgto at centrifugation After KCl). mM 300 and 2 m . MDT n 1 and DTT, mM 0.5 , /ltncmcnfr2 or.I the In hours. 24 for tunicamycin g/ml 6 kokonclscnbe can cells -knockdown m 5m)eulbae nPBS. in equilibrated ml) 25 cm, 1 RNH1 m fnnimn mouse non-immune of g b tblnadPCNA, and -tubulin niie cell inhibited ˚ .Five C. m 6 fPSand PBS of l proteinase m ˚ from l for C Journal of Cell Science eiaqa . ag . audr . acoi . un .L,Wn,C,Zhu, C., Wang, L., C. Yuan, F., Gaccioli, M., Majumder, X., Wang, E., Bevilacqua, el,J,Hnl,K . cwn .A n oel .C. S. Lovell, and A. P. McEwan, M. L., Holcik, K. and Hindle, G. J., R. Bella, Korneluk, M., Turcotte, D., S. Baird, nesn .adKdrh,N. Kedersha, and P. Anderson, rrLsr(8/6/4)wsue.Bn as5055ad50ad60was 610 and 555. 590 Fluor and Alexa nm, 410, 510–525 and 546 LSM 488 pass Zeiss and Fluor For Band nm Alexa 546. 488 used. for Fluor was at used Alexa (488/568/647) and set 560– 488 Laser was and Fluor ArKr 500–530 the laser Alexa pass Leica, HeNe for band SP5 respectively, by and revealed 650, For laser was 410. emission ion LSM Fluorescence argon respectively. Zeiss the by of and Leica lambda SP5 microscope laser-scanner nesn .adKdrh,N. Kedersha, and P. Anderson, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.134551/-/DC1 online available material Supplementary after release for PMC NS065237 in R01 Deposited months. numbers G.-f.H.]. 12 to [grant CA105241 Health and R01 of University and Institutes of Ministry National Italian the by part by in supported was work This interpreted Funding and experiments. analyzed performed manuscript. ideas, the E.Y. conceived wrote and G.-f.H. G.-f.H. P.N. results. and F.F., G.D. S.F., E.P., J.S., C.S., E.P., contributions Author assistance. and technical Fusco and Giuseppina discussions Giudice, helpful Del for Rita Yang Monti, Hailing M. Daria thank We Acknowledgements trypsinized, were hour) 1 for mM (1 al., treatment 4 et SA with (Ribble washed described after and as or staining pelleted AO before and Cells EB by 2005). identified cells were apoptotic cells of Apoptotic staining AO and EB human of set A RNH1 as calculated was FRET of Efficiency fluorescence. 488 Fluor ( Alexa cytoplasm of performed increase in were the Measurements ROI by photo-bleaching. was measured on Cy3 bleaching was (Cy3) after acceptor FRET intensity The nm. nm. fluorescence 592 500–530 was at 488) at detected Fluor performed and (Alexa nm donor 488 The at used. excited was method photo-bleaching acceptor The FRET at methanol in fixed were and coverslips on cultured were Cells microscopy confocal and Immunofluorescence 4318 ld n oee ihcoverslips. with covered and slide odtos oesr erdcblt n eiblt fAeaFur488 Fluor Alexa of reliability and reproducibility ensure To conditions. el otasetdwt hN netdpK. (5.8 pLKO.1 inserted shRNA HEK293T with in packaged co-transfected were particles cells Lentiviral Biosystems. Open from purchased to photo-bleached was Cy3 measurements, fluorescence I B n ie ih5 with mixed and PBS netdwt etvrsi h rsneo 10 of presence the in lentivirus with infected (1.8 pMD2.G n .%Ten2.Teatbde sdwr N A 62 (5 26-2F mAb NaCl ANG mM were 150 8.0, used (2 pH antibodies R113 Tris-HCl, (2 pAb The mM 10 ANG 20. in affinity-purified Tween BSA 0.1% 3% with and blocked minutes, 10 Bi B)a 37 at PBS) in EB uoyi-eitn el eeslce n h ee fRH a xmndby examined was RNH1 of level the analysis. and Immunoblot selected were cells Puromycin-resistant 8-ojgtdga nirbi rAeaFur55cnuae otanti-mouse goat Alexa-Fluor-555-conjugated or F(ab anti-rabbit goat 488-conjugated 1 #ab2712, cat (Abcam, DA . odn .E,Shue,D,Kumn .J tal. et J. R. stress. Kaufman, osmotic during D., apoptosis to Scheuner, balance 285 E., the tips L. phosphorylation Jordan, X., eetstructure. repeat IRES. ice.Sci. Biochem. m r loecneitniisbfr n fe photo-bleaching. after and before intensities fluorescence are /l,PB A 01 Acm a a62,1 #ab6125, cat (Abcam, 10E10 mAb PABP g/ml), 9 ) 17098-17111. , 2 knockdown 110 iuin.Cnoa irsoywspromdwt confocal a with performed was microscopy Confocal dilution). (1:1000 RNA ora fCl cec 2 (18) 126 Science Cell of Journal 12 m 33 )adpcaigpamdpPX(4.4 psPAX plasmid packaging and g) 1755-1785. , RNH1 ˚ el o.Lf Sci. Life Mol. Cell. 141-150. , o 0mnts tie el eepae namicroscope a on placed were cells Stained minutes. 20 for C seii hN lndi LO1lniia etrwas vector lentiviral pLKO.1 in cloned shRNA -specific m m fteE/Odemxue(100 mixture dye EB/AO the of l n /l.Tescnayatbde sdwr Alexa-Fluor- were used antibodies secondary The g/ml). 5 ˚ B.Teclswr eupne n100 in resuspended were cells The PBS. C 0 ne rwhadi Gudrsrs ( stress under SG in and growth under 10) 20) tesgaue:teToo N triage. RNA of Tao the granules: Stress (2008). 20) tesgranules. Stress (2009). 65 m 2307-2333. , /l,afnt-uiidRH A R127 pAb RNH1 affinity-purified g/ml), m /lplbeefr4 hours. 48 for polybrene g/ml E 5 ur Biol. Curr. ( I DA m m m 20) h leucine-rich The (2008). m /l n I1mAb TIA1 and g/ml) ) neoeplasmid envelope g), ) eaclswere cells HeLa g). 2 /lec fA and AO of each g/ml , 20) erhn for Searching (2006). I 0 fisinitial its of 10% D )/ 21) eIF2alpha (2010). 19 I D R397-R398. , .Bo.Chem. Biol. J. where 2 m 20 lof4 m ˚ I Trends g/ml), for C D n 5 and ˚ 7) C u . eg . i,Q,Sn . i,Y,Zu . ig . u,Z n Zheng, and Z. Sun, R., Xing, Calı J., M., Zhu, Moscato, A., Y., Furia, Tie, F., Sun, Q., Liu, J., Feng, H., Fu, rewy .J,Adre,P . us . ni,S,Csmn . oah,C., Donaghy, S., Cashman, S., Ennis, C., Russ, M., P. A., Andersen, J., Ratti, M. C., Greenway, Bragato, B., Castellotti, N., Ticozzi, C., Colombrita, C., Gellera, J. Hofsteenge, and M. J. Fominaya, Hu, N., Kedersha, S., Tisdale, N., Dawra, T., Hickman, P., Ivanov, M., M. Emara, eWt . og . u,L n hs,A. C. 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