Multiple Roles of the Splicing Complex SF3B in DNA end Resection and Homologous Recombination

Depósi o de in es igación de la Uni e sidad de Se illa h ps://idus.us.es/ “This is an Accep ed Manusc ip o an a icle published by Else ie in: DNA REPAIR on 2018, a ailable a : h ps://doi.o g/10.1016/j.dna ep.2018.04.003” 1 Mul iple oles o he splicing complex SF3B in DNA end esec ion and homologous ecombina ion Rosa io P ados-Ca ajal1,2, Ana López-Saa ed a1,2, C is ina Cepeda-Ga cía2, Sonia Jimeno1,2 and Pablo Hue as1,2 1 Depa amen o de Gené ica, Uni e sidad de Se illa, Se illa, 41080, Spain 2 Cen o Andaluz de Biología Molecula y Medicina Regene a i a-CABIMER, Uni e sidad de Se illa-CSIC-Uni e sidad Pablo de Ola ide, Se illa, 41092, Spain Co espondence o P. Hue as ([email p o ec ed]) Tel: (+34) 954 467 667 Fax: (+34) 954 461 664 2 ABSTRACT The app op ia e epai o DNA double s and b eaks is c i ical o genome main enance. Thus, se e al cellula pa hways collabo a e o o ches a e a coo dina ed esponse. These include he epai o he b eaks, which could be achie ed by di e en mechanisms. A key p o ein in ol ed in he egula ion o he epai o b oken ch omosomes is C IP. He e, we ha e ound new pa ne s o C IP in ol ed in he egula ion o DNA b eak epai h ough a ec ing DNA end esec ion. We ocus on he splicing complex SF3B and show ha i s deple ion impai s DNA end esec ion and hampe s homologous ecombina ion. Func ionally, SF3B con ols C IP unc ion a , as leas , wo le els: by a ec ing C IP mRNA le els and con olling C IP ec ui men o DNA b eaks, in a way ha equi es ATM-media ed phospho yla ion o SF3B2 a se ine 289. Indeed, o e exp ession o C IP escues he esec ion de ec caused by SF3B down egula ion. S ikingly, o he SF3B deple ion pheno ypes, such as impai ed homologous ecombina ion o cellula sensi i i y o DNA damaging agen s, a e independen o C IP le els, sugges ing a mo e gene al ole o SF3B in con olling he esponse o ch omosome b eaks. 3 INTRODUCTION Li ing o ganisms a e exposed o mul iple physical and chemical h ea s ha migh comp omise hei su i al. Especially ele an a e hose challenges ha jeopa dize he in eg i y o he genome as hey will no only a ec he cu en gene a ion bu migh endange he u u e p ogeny. Du ing e olu ion, a e y in ica e esponse has e ol ed o sa egua d genomic in o ma ion [1, 2]. This in ol es he s imula ion o DNA epai mechanisms, bu also he coo dina ion o all cellula e en s h ough he ac i a ion o he DNA damage checkpoin s [1]. Pa icula ly challenging is he epai o DNA Double S and B eaks (DSBs), due o he simul aneous ha m o bo h s ands [1]. Di e en o he healing o any o he ype o lesion, DSBs canno be mended by using a complemen a y s and. So, he epai o b oken ch omosomes is indeed a complica ed a ai . Two close DNA agmen s migh be simply e-joined wi h li le o no p ocessing, a mechanism named Non-Homologous End-Joining (NHEJ; [3]). Such p ocedu e is as , highly e icien and ene ge ically cheap, hus is he me hod o choice o he majo i y o he epai e en s in highe euka yo es. Howe e , he lack o a p oo eading ac i i y migh esul in he e oneous joining o DNA segmen s ha o iginally we e no adjacen , con ibu ing o he appea ance o g oss ch omosomal ea angemen s [4]. As a consequence, mo e complex DSB epai mechanisms coexis wi hin he cells. DNA b eaks can be p ocessed, a s ep known as DNA end esec ion, which consis s on he deg ada ion o one s and o he DNA in a 5’-3’ pola i y ha lea es a ail o p o uding ssDNA [5]. ssDNA is immedia ely coa ed by he p o ec ing complex RPA [5]. This esec ed DNA can be engaged in he pai ing wi h a homologous sequence elsewhe e in he genome, p omp ing he epai mechanisms collec i ely known as Homologous Recombina ion (HR; [6]). HR migh ensue in di e en ashions [6], bu all o hem sha e he same ini ial s ep, he a o emen ioned DNA end esec ion. Thus, esec ed DNA is an obliga o y subs a e in all HR pa hways and, a he same ime, e ec i ely blocks NHEJ [6, 7]. Al hough adi ionally HR has been conside ed an e o - ee epai pa hway, now i is clea ha unscheduled HR migh also inc ease he appea ance o g oss ch omosomal ea angemen s [4]. Thus, he e is a igh egula ion o he choice be ween DSB epai pa hways in esponse o cell s a us o minimize genomic ins abili y upon he occu ence o b eaks on he DNA. A c i ical ac o o such egula ion is C IP. This ac o ac s as a molecula swi ch ha ac i a es DNA end esec ion, hence homologous ecombina ion, when he cellula en i onmen is suppo i e o his ype o epai [8]. C IP, oge he wi h he MRN complex, ini ia es esec ion and con o ms he 4 minimal esec ion machine y [9, 10]. Al hough C IP i sel displays a nuclease ac i i y [11, 12], i is s ill deba ed how much i con ibu es o he ac ual p ocess. In addi ion o i s ole in DNA epai , C IP has been in ol ed in se e al p ocesses, including i s own ansc ip ion [13]. To assess he gene al sui abili y o HR, C IP collec s cellula in o ma ion h ough pos - ansla ional modi ica ions and p o ein-p o ein in e ac ions [8]. Indeed, di e en pa ne s o C IP a e known o play p o- o an i- esec ion oles [8, 14-16]. Recen ly, a c oss alk be ween he DNA Damage Response (DDR) and he RNA me abolism a di e en le els has become appa en . RNA is in ol ed in acili a ing he ec ui men o checkpoin p o eins like 53BP1 o damaged ch oma in [17]. Mo eo e , highly ansc ibed genes a e epai ed di e en ly o o he ch oma in egions, and he nascen RNA migh play a ole on his speci ici y [18]. Indeed, i has been ecen ly shown ha RNA splicing changes globally upon challenging DNA wi h a DNA damage sou ce, and such al e na i e splicing equi es he ac i a ion o he DDR o ake place [19]. This DNA damage-dependen al e na i e splicing is con olled by BRCA1 [19], a known pa ne o C IP in ol ed in DNA esec ion modula ion [13, 14]. This modula ion implies ha BRCA1 in e ac s speci ically upon appea ance o DNA damage wi h se e al p o eins such as SF3B1 [19], one ou o he se en subuni s o he co e SF3B splicing complex [20]. This complex is c i ical o co ec RNA splicing, as con ac s wi h he nascen mRNA a o nea he b anch si e [21]. Addi ionally, se e al subuni s o he complex ha e been al eady ound in genome wide sc eens o ac o s in ol ed in DNA epai , a ec ing homologous ecombina ion [22], con olling genome s abili y [23] o as subs a es o he checkpoin kinases [24, 25]. He e we ha e isola ed addi ional pa ne s o C IP. Among hem, we ha e ound h ee subuni s o he SF3B splicing complex. We ha e analysed he ole o his complex in he epai o DSBs. Func ionally, SF3B is equi ed o p ope DNA end esec ion and homologous ecombina ion. Indeed, SF3B egula es C IP mRNA le els bu also he ec ui men o C IP o DSBs. S ikingly, SF3B ole on he epai o DSBs exceeds i s egula ion o C IP, as cells deple ed o SF3B bu o e exp essing C IP a e p o icien o DNA end esec ion bu hype sensi i e o DNA damage and de ec i e o homologous ecombina ion. RESULTS AND DISCUSSION Isola ion o no el C IP biochemical in e ac o s 5 As men ioned, C IP is egula ed, among o he signals, by p o ein-p o ein in e ac ions [8]. In o de o isola e new pa ne s o C IP, we pe o med andem a ini y pu i ica ion om U2OS cells exp essing a GFP and iple FLAG- agged e sion o his p o ein. Fi s , we used an an i-FLAG esin o immunop ecipi a e GFP-3XFLAG-C IP complexes ha we e subsequen ly elu ed wi h an excess o 3XFLAG pep ide. Then, an an i-GFP esin was used o u he pu i y he p o ein samples (Figu e 1A). The inal p ecipi a ed we e esol ed in an SDS-PAGE (Figu e 1A). C IP sel -in e ac s [26], hus endogenous C IP was used o ollow he quali y o he immunop ecipi a ion p o ocol. P o eins immunop ecipi a ed exclusi ely om cells exp essing he usion p o ein and absen on con ol cells we e de ec ed using Mass Spec ome y. We isola ed nine p o eins p e iously unknown o be C IP in e ac ion pa ne s, including h ee subuni s o he SF3B complex, and wo p e iously epo ed in e ac o s, a membe o he hnRNPU amily and CCAR2 [15, 27] (Table 1). C IP has di e en unc ions wi hin he cell [8, 16]. To s udy he ele ance o hose new C IP in e ac ing p o eins in DNA end esec ion, we analysed he o ma ion o ssDNA upon ea men wi h ionizing adia ion (IR) on cells deple ed o all he no el in e ac ing p o eins. Ini ially, we chose SF3B3 as a ep esen a i e example o he SF3B complex. The oles o p o eins o he hnRNPU amily and CCAR2 on DNA end esec ion ha e been published elsewhe e [15, 27]. By using RPA oci as a p oxy o DNA esec ion, we ound wo dis inc ca ego ies o C IP in e ac ing p o eins in ela ionship wi h DNA damaged-induced end p ocessing (Figu e 1B): hose ha do no a ec i (RBM10, SFPQ) and hose ha , like C IP i sel , a e equi ed o esec ion (SF3B3, PRMT5, DHX9, DHX15, KIF11). Deple ion e iciencies a e shown in supplemen a y igu e S1A. In e es ingly, PRMT5 has been e y ecen ly disco e ed o mobilize he an i- esec ion ac o 53BP1 om damaged ch oma in and s imula e homologous ecombina ion [28], in ag eemen wi h a ole p omo ing esec ion. I is no ewo hy ha om all 11 p o eins we ha e de ec ed, 8 co espond o RNA- ela ed ac o s. In p inciple, a possibili y was ha we pu i ied C IP-complexes mo e in ol ed in epai -independen C IP oles, such as ansc ip ional egula ion [13, 29]. Howe e , deple ion o he majo i y o hem hampe ed DNA esec ion, poin ing ou o a ole on modula ing DNA epai di ec ly o indi ec ly. Mo eo e , e en SFPQ, which did no educe DNA esec ion when down egula ed, is a known in e ac o o Rad51, Rad51 pa alogues and he Ku complex, and has been shown o a ec di ec ly hei oles in DSB epai [30-33]. Such a high incidence o RNA- ela ed p o eins in e ac ing wi h a known DNA epai ac o is no 6 su p ising. In ac , mRNA p ocessing ac o s a e commonly en iched in many genome- wide sc eenings aimed o ind new ac o s in ol ed in he DDR and he main enance o genomic in eg i y [15, 22, 23, 34] and many RNA- ela ed p o eins ha e been de ec ed as a ge s o he DNA damage-induced pos - ansla ional modi ica ions [24, 25, 35, 36]. Thus, in ecen yea s, accumula ed g owing e idence e ealed he impo ance o RNA- ela ed ac o s o he DDR and DNA epai [37, 38]. F om ha poin onwa ds, we decided o ocus on he ole o he SF3B complex in DNA end esec ion and he esponse o DNA damage. No only we ound h ee dis inc subuni s o he same complex in e ac ing wi h C IP, bu also all h ee ha e been p e iously ound in global sc eenings o a ec homologous ecombina ion and epai [22] and as di ec a ge s o he DNA Damage Response (DDR) [24, 25, 39, 40]. In o de o alida e he ela ionship o C IP wi h his complex, we i s immunop ecipi a ed p o eins wi h a GFP- ap using samples om cells bea ing a GFP- C IP o GFP as a con ol and immunoblo ing o SF3B1 and SF3B2. Albei some unspeci ic binding o hese p o eins, we con i med an en ichmen in bo h subuni s o he SF3B complex in GFP-C IP cells (Figu e 1C). Then, we pe o med he ecip ocal expe imen by using an i-SF3B1 and an i-SF3B2 an ibodies om immunop ecipi a ions wi h samples om cells ha bou ing GFP-C IP o GFP cons uc s (Figu e 1D and 1E, espec i ely). As seen in hose igu es, bo h SF3B1 and SF3B2 eadily immunop ecipi a ed GFP-C IP and, o a lesse ex en , endogenous C IP. Bo h SF3B complex and C IP a e ela ed wi h RNA me abolism, splicing and ansc ip ion, espec i ely. Thus, we wonde ed i he in e ac ion be ween SF3B and C IP migh be media ed o s abilized by RNA. None heless, ea men wi h RNase nei he abolished no se e ely educed SF3B1-C IP co-immunop ecipi a ion (Figu e 1F). To de e mine he subcellula localiza ion o his in e ac ion, we pe o med a p oximi y liga ion assay (PLA). We obse ed ha C IP in e ac s wi h SF3B2 inside he nucleus (Figu e 1G). Deple ion o ei he C IP o SF3B componen s abolished he PLA signal, suppo ing he speci ici y o he assay (Figu e 1G). In ag eemen wi h ou da a, SF3B1 has been shown o in e ac also wi h BRCA1 [19], a C IP well es ablished pa ne [8, 14, 16]. A s a k di e ence be ween his s udy and ou s is he DNA damage dependency. Whe eas SF3B1 and BRCA1 only in e ac upon he exposu e o he cul u es o DNA damage [19], C IP and SF3B seem o in e ac e en in he absence o DNA damage in ou hands. Howe e , an inc ease in C IP and SF3B in e ac ion could be obse ed upon DNA damage (Figu e 1G). Mo eo e , such inc ease was abolished when he checkpoin was 7 inac i a ed by inhibi ion o ATM (Supplemen a y Figu e S1B). Su p isingly, he majo i y o such in e ac ion did no occu a he si es o DSB, labelled wi h GFP- MDC1, a p o ein ec ui ed ea ly o DSBs [41](Figu e 1H). These indings suppo ed a cons i u i e in e ac ion o C IP and SF3B, which is s imula ed by he p esence o damaged ch oma in bu mos ly akes place, and mos likely ha e a unc ional meaning, ou side he ac ual damaged DNA. The SF3B complex is in ol ed in DNA end esec ion and ecombina ion C IP is a mul i unc ional p o ein wi h oles in ansc ip ion, eplica ion, DNA epai , e c [8, 16]. In o de o in es iga e i he SF3B complex migh be a ec ing DNA ecombina ion, and speci ically, DNA end esec ion, we deple ed SF3B1 and SF3B2 subuni s o he complex using siRNA a ge ed agains hem (see Supplemen a y Figu e S1C and S1D o deple ion e iciency). To be su e ha he obse ed pheno ypes we e no caused by o - a ge e ec s, we also complemen ed SF3B2 deple ion wi h a siRNA- esis an FLAG-SF3B2 cons uc (Supplemen a y Figu e S1D). Following he o ma ion o RPA-coa ed ssDNA as a eadou , we obse ed ha upon challenging he cells wi h ionizing adia ion (IR), and no ma e he subuni a ge ed, a educ ion o he le els o SF3B complex leaded o an impai men o DNA end esec ion (Figu e 2A and 2B). As expec ed, ec opic exp ession o siRNA- esis an wild ype FLAG- agged SF3B2 was able o ully complemen his esec ion impai men (Figu e 2B). As men ioned, he subuni s o he SF3B complex su e DNA damage-dependen pos - ansla ional modi ica ions, including an ATM-dependen phospho yla ion o SF3B2 a se ine 289 [25]. Thus, we wonde ed i such modi ica ion migh a ec DNA end esec ion. Indeed, exp ession o siRNA esis an FLAG-SF3B2-S289A mu an did no comple ely escue he esec ion de ec obse ed upon deple ion o endogenous SF3B2 (Figu e 2B), a guing he impo ance o such se ine in he p ocess. The e o e, i sugges s a double con ibu ion o SF3B2 o he p ocess, one media ed by his phospho yla ion an ano he independen o i . No su p isingly, based on his de ec on DNA end esec ion, DSB epai pa hways we e a ec ed upon deple ion o SF3B subuni s. In ag eemen wi h a esec ion impai men when siRNAs agains SF3B1 o SF3B2 we e used, ecombina ion was s ongly educed (Figu es 2C and 2D). NHEJ was no signi ican ly changed upon SF3B1 down egula ion (Figu e 2E) bu mildly inc eased upon SF3B2 deple ion (Figu e 2F), sugges ing he main e ec o he complex was h ough HR. Ec opic exp ession o siRNA esis an wild 8 ype FLAG-SF3B2 escued he e ec o SF3B2 deple ion (Figu es 2D and 2F). On he con a y, he S289A mu an o SF3B2 was s ill comp omised o homologous ecombina ion (Figu e 2D). These e ec s on DNA esec ion and HR we e no caused by an accumula ion o cells in G1 phase (Supplemen a y Figu es S2A and S2B). Thus, we conclude ha he SF3B complex plays a ole on DNA esec ion and in he epai o DSBs, mainly in HR. SF3B con ols he abundance o C IP RNA binding p o eins ha e been shown o a ec he esponse o DNA damage a se e al le els: hey can ha e RNA-independen unc ion and di ec oles in DNA epai [30-33, 38], o hey can a ec he RNA me abolism, including mRNA s abili y, splicing o ansla ion [22, 37, 38]. In ac , i has been es ablished ha mRNA splicing con ols he abundance o speci ic epai p o eins [19, 37, 42]. As he in e ac ion be ween SF3B and C IP seems o occu mainly ou side he ac ual si es o damaged DNA (Figu e 1H), we wonde ed i he pheno ypes obse ed upon SF3B deple ion migh be pa ially o o ally ela ed o changes on he abundance o speci ic epai ac o s. Conside ing he e ec on DNA end esec ion, he in e ac ion wi h C IP and he ac ha C IP con ols i s own exp ession [29] we analysed he abundance o C IP mRNA in he absence o SF3B2. Indeed, deple ion o SF3B2, educed he amoun o C IP mRNA, and such e ec was escued by he exp ession o a siRNA esis an e sion o he gene (Figu e 3A). S ikingly, cells exp essing he S289A mu an we e p o icien in C IP exp ession (Figu e 3A), despi e being de ec i e in DNA end esec ion and HR (Figu e 2B and 2D). Mo eo e , o e exp ession o SF3B2 ele a es he abundance o C IP ansc ip , measu ed by quan i a i e PCR (Figu e 3A). This lowe abundance o C IP mRNA caused a educ ion in he abundance o he p o ein, ha was complemen ed by ec opic exp ession o FLAG-SF3B2 (Figu e 3B and 3C). In p inciple, such dec ease could be a di ec consequence o low mRNA o due o he p oduc ion o an al e na i e-spliced a ian o he p o ein wi h inc eased u no e . In his case, lowe C IP p o ein le els we e no due o a dec ease in he p o ein s abili y, analysed by a cycloheximide chase (Figu e 3C), sugges ing ha SF3B complex con ols C IP abundance a he le el o mRNA. Mo eo e , he S289A mu an , in conco dance wi h he mRNA esul s, showed no mal accumula ion o C IP p o ein. Thus, his mu an in an ATM- a ge si e beha es as a sepa a ion o unc ion mu an , de ec i e in DNA end esec ion and HR bu wi hou a ec ing C IP exp ession. In ag eemen , ou da a sugges ha he educ ion o C IP 15 analysed wi h a BD FACSA ia wi h he BD FACSDi a So wa e 5.0.3. Fou di e en pa ame e s we e conside ed: side sca e (SSC), o wa d sca e (FSC), blue luo escence (407 nm iole lase BP, Fil e 450/40) and g een luo escence (488 nm blue lase BP Fil e 530/30). Finally, he numbe o g een cells om a leas 10,000 e en s posi i es o blue luo escence (in ec ed wi h he I-SceI–BFP cons uc ) was sco ed. The a e age o bo h duplica es was calcula ed o each sample o e e y expe imen . To acili a e he compa ison be ween expe imen s, his a io was no malized wi h shRNA o siRNA con ol. A leas ou comple ely independen expe imen s we e ca ied ou o each condi ion and he a e age and s anda d de ia ion o all ou ep esen ed. MTT cell iabili y assay Cell iabili y was e alua ed using a comme cially a ailable MTT cell g ow h assay ki (Millipo e), acco ding o he manu ac u e 's ins uc ions. B ie ly, U2OS cells exp essing GFP o GFP-C IP plasmid we e ans ec ed wi h he indica ed siRNAs. 24h a e , 7,000 cells we e seeded in o a 96-well pla e, ea ed wi h 0.5 µM e oposide o DMSO. 42h la e , he samples we e incuba ed o 4h a a 37º C a e addi ion o MTT solu ion. A e emo ing he medium and MTT solu ion, o mazan was solubilized in 100 μl HCl in isop opanol 0.4 N. Cell iabili y was calcula ed measu ing abso bance a 570 nm and 630 nm as desc ibed in he manu ac u e p o ocol and no malized wi h con ol. Expe imen s we e epea ed independen ly h ee imes. RT-qPCR RNA was ex ac ed using he RNeasy Mini Ki (Quiagen) ollowing he manu ac u e ’s ins uc ions om cells 48 h a e ans ec ion wi h siRNA o in ec ion wi h he indica ed shRNA (Supplemen a y Tables S1 and S2). Then, RNA was quan i ied and i s quali y was checked by esol ing 10% o he sample in a 1% aga ose gel. Re o ansc ip ion was ca ied ou using Quan iTec Re e se T ansc ip ion Ki (Quiagen) acco ding o he manu ac u e ’s p o ocol. RT-qPCR eac ions we e pe o med mixing 10 µl o SYBR G een (BIO-RAD) wi h 100 ng cDNA and 10 µl o p ime mix (Supplemen a y Table S3). RT-qPCR expe imen s we e ca ied ou using iplica e echnical eplicas in an ABI 7500FAST. The e iciency o each p ime pai eac ion was p e iously calcula ed using genomic DNA. The C o each sample was no malized o β-Ac in and ela i ized o con ol. Expe imen s we e epea ed ou imes. Cell cycle analysis by low cy ome y 16 Cells we e ha es ed, washed wi h PBS and esuspended in ice-cold PBS. E OH (70%) was added d opwise while o exing a low speed, and cells we e hen ixed a 4ºC o e nigh . Cells we e washed wi h PBS and ea ed wi h 250 µg/ml RNase A (Sigma) and 10 µg/ml p opidium iodide dilu ed in PBS (Fluka). Cells we e incuba ed a 37ºC o 20 min and analyzed using a FACSCALIBUR (BD Biosciences). Immuno luo escence mic oscopy U2OS cells we e in ec ed wi h len i i us ha bou ing he indica ed shRNA o ans ec ed wi h he men ioned siRNAs. A e 48 h, cells we e i adia ed (10 Gy) o mock ea ed, incuba ed 1 h o oci o ma ion and collec ed. Fo RPA oci s udies, co e slips we e ea ed o 5 min on ice wi h p e-ex ac ion bu e (25 mM T is.HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 3 mM MgCl2, 300 mM suc ose and 0.2% T i on X-100). Then, cells we e ixed wi h 4% pa a o maldehyde (w/ ) in PBS o 15 min. A e ixa ion, cells we e washed h ee imes wi h PBS and blocked o a leas 1 h wi h 5% FBS dilu ed in PBS. Cells we e incuba ed wi h he adequa e p ima y an ibodies (Supplemen a y Table S4) dilu ed in 5% FBS in PBS o 2 h a oom empe a u e, washed h ice wi h PBS, and hen incuba ed wi h seconda y an ibodies (Supplemen a y Table S5) dilu ed in 5% FBS in PBS o 1 h a oom empe a u e. Cells we e hen washed wice wi h PBS, and co e slips we e moun ed wi h Vec ashield moun ing medium (Vec o Labo a o ies) con aining 4,6-diamidino-2-phenylindole (DAPI) and analysed using a LEICA mic oscope. A leas 200 cells we e sco ed pe sample. Expe imen s we e epea ed independen ly a leas ou imes. Immuno-FISH Fo immuno-FISH, U2OS19p igh 13 cells we e ixed in 4% pa a o maldehyde o 15 min, pe meabilized in 0.5% i on o 15 min, blocked in 3% BSA in PBS 0.1% ween and incuba ed wi h p ima y and seconda y an ibodies (Supplemen a y Tables S4 and S5) o 1 h each. Cells we e hen pos - ixed in 4% o maldehyde o 20 min, and he FISH p o ocol was ca ied ou as p e iously desc ibed [46]. Immuno-FISH samples we e analysed using a con ocal mic oscopy. A leas 100 cells we e sco ed pe sample. Expe imen s we e epea ed independen ly h ee imes. Immunoblo ing Ex ac s we e p epa ed in Laemmli bu e (4% SDS, 20% glyce ol and 125 mM T is- HCl, pH 6.8), and p o eins we e esol ed by SDS-PAGE and ans e ed o PVDF memb anes (Millipo e), ollowed by immunoblo ing. Wes e n blo analysis was ca ied 17 ou using he an ibodies lis ed in supplemen a y ables S4 and S5. Resul s we e isualized using an Odyssey In a ed Imaging Sys em (Li-Co ). Cycloheximide chase Cells we e exposed o esh medium con aining 150 μg/ml o cycloheximide (Sigma- Ald ich) 48 h pos ans ec ion o in ec ion o inhibi u he p o ein syn hesis. Cells we e collec ed immedia ely (0h), 4 h o 8 h a e he cycloheximide addi ion and p o ein samples we e p epa ed as desc ibed in he immunoblo ing sec ion. S a is ical analysis S a is ical signi icance was de e mined wi h a pai ed -s uden o ANOVA es s, as indica ed in he igu e legends, using he PRISM so wa e (G aphpad So wa e Inc). S a is ically signi ican di e ences we e labelled wi h one, wo o h ee as e isks i p <0.05, p <0.01 o p <0.001, espec i ely. ACKNOWLEDGEMENTS This wo k was unded by a R+D+I g an om he Spanish Minis y o Economy and Compe i i i y (SAF2013-43255-P) and an ERC S a ing G an (DSBRECA). AL-S is he ecipien o a FPI ellowship om he Spanish Minis y o Economy and Compe i i i y, and RP-C is unded wi h an FPU ellowship om he Spanish Minis y o Educa ion. CABIMER is suppo ed by he egional go e nmen o Andalucia (Jun a de Andalucía). REFERENCES [1] A. Ciccia, S.J. Elledge, The DNA damage esponse: making i sa e o play wi h kni es, Molecula cell, 40 (2010) 179-204. [2] S.P. Jackson, J. Ba ek, The DNA-damage esponse in human biology and disease, Na u e, 461 (2009) 1071-1078. [3] A.J. Da is, D.J. 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TABLES Table 1: Mass spec ome y da a o C IP complexes P o ein Aliases Gene Molecula Weigh Unique pep ides C IP RBBP8, RIM, SCKL2, JWDS RBBP8 101.942 37 SF3B1 SAP155, MDS, Hsh155, PRPF10, PRP10 SF3B1 145.830 34 RBM10 DXS8237E, KIAA0122, GPATCH9, GPATC9, TARPS, ZRANB5 RBM10 103.533 31 SF3B3 SF3b130, SAP130, STAF130, KIAA0017, RSE1 SF3B3 135.577 27 SF3B2 SF3B145, SAP145, Cus1 SF3B2 100.228 26 PRMT5 HRMT15L, SKB1Hs, INP72, JBP1 PRMT5 72.684 25 DHX15 DDX15, DBP1, HRH2, PRPF43, PRP43 DHX15 90.933 24 SFPQ PSF, PPP1R140, POMP100 SFPQ 76.149 23 DHX9 DDX9, NDHII, NDH2, LKP, RHA DHX9 140.958 22 23 CCAR2 KIAA1967, DBC1, P30DBC, NET35 CCAR2 102.902 18 KIF11 KNSL1, TRIP5, MCLMR, EG5, HKSP KIF11 119.159 15 hnRNPU SAF-A, P120 HNRNPU 90.584 14 In bold, p e iously desc ibed C IP in e ac o s FIGURES LEGENDS Figu e 1. Disco e y o he SF3B complex as a physical and unc ional in e ac o o C IP A, P o ein samples om cells ha bou ing GFP-3XFLAG-C IP o con ol cells we e subjec ed o wo-s ep a ini y pu i ica ion. Fi s , complexes we e isola ed wi h an i- FLAG beads and elu ed using 3XFLAG pep ide. Then, a second pu i ica ion was pe o med wi h an i-GFP esin. A wes e n blo displaying he endogenous (whi e iangle) and GFP-3XFLAG-C IP (solid iangle) bands a each s ep o pu i ica ion is shown. B, DNA esec ion p o iciency, measu ed as he pe cen age o RPA oci-posi i e cells upon 10 Gy o ionizing adia ion, in cells down egula ed o he indica ed genes. The a e age and s anda d de ia ion o a leas h ee independen expe imen s is shown. A S uden - es compa ing wi h con ol shNT (Non-Ta ge shRNA) was pe o med and one, wo o h ee as e isk(s) deno es s a is ical signi icance a p<0.05, p<0.01 o p<0.001, espec i ely. C, Cells bea ing a GFP-C IP usion o GFP as a con ol we e used o immunop ecipi a ion using a GFP- ap. Immunop ecipi a ed samples (IP) and p o ein inpu s (Inpu ) we e esol ed in SDS-PAGE and blo ed wi h an ibodies agains he indica ed p o eins. D, P o ein samples om U2OS cells ha bou ing GFP-C IP we e used o immunop ecipi a ion wi h an an ibody agains SF3B1 (B1) o a con ol IgG (C), esol ed in SDS-PAGE and blo ed wi h he indica ed an ibodies. E, Same as D, bu in cells bea ing GFP o GFP-C IP cons uc s and wi h an an ibody agains SF3B2 (IP-SF3B2). F, Same as D, bu du ing immunop ecipi a ion, RNase A was added o no as designa ed in he igu e. G, P oximi y Liga ion Assay be ween C IP and SF3B2. PLA signal was sco ed using an ibodies agains C IP and he SF3B2 subuni in unpe u bed cells (undamaged) o cells exposed o 10 Gy o ionizing adia ion and le o eco e o 1 h (IR). To es ablish he speci ici y o he signal, he expe imen was epea ed wi h deple ion o ei he C IP o SF3B2. shNT deno es an shRNA wi h no a ge in he human genome. The quan i ica ion ( igh ) and depic i e images (le ) o one ep esen a i e expe imen , ou o ou wi h simila esul s, is shown. Median PLA oci is ep esen ed 24 wi h a ed line. H, PLA expe imen using SF3B2 and C IP an ibodies in i adia ed cells (10 Gy) ha bou ing a GFP-MDC1 cons uc o isualize DNA b eaks. Figu e 2. Deple ion o SF3B subuni s hampe s DNA end esec ion A, Cells ans ec ed wi h siRNA agains SF3B1 (siSF3B1) o a con ol sequence (siNT) we e exposed o 10 Gy o ionizing adia ion (IR). An hou la e , cells we e collec ed and RPA was de ec ed by immuno luo escence. Images o a ep esen a i e expe imen and a plo o he a e age and s anda d de ia ion o h ee independen expe imen s a e shown. O he de ails as in igu e 1B. B, Same as A, bu cells ans ec ed wi h a siRNA agains SF3B2 (siSF3B2) o con ol (siNT) bea ing a siRNA- esis an FLAG-SF3B2, FLAG-SF3B2-S289A o FLAG plasmid, as indica ed. S a is ical signi icance was measu ed wi h an ANOVA es . C, Classical ecombina ion assay using he DR-GFP epo e in cells deple ed o SF3B1. A scheme o he epo e is shown on he le . Induc ion o a DSB using I-SceI meganuclease will ende GFP posi i e cells when gene con e sion occu s using he dono epea (iGFP). Such e en could happen in amolecula ly (as shown) o wi h a dono sequence loca ed in he sis e ch oma id (no shown). The e iciency o classical ecombina ion was calcula ed as he pe cen age o GFP posi i e cells in esponse o I-SceI exp ession upon down egula ion o he indica ed genes, no malized wi h he con ol. S a is ical signi icance was es ed using a -S uden es as desc ibed in me hods. D, Same as C, bu in cells s ably exp essing FLAG, siRNA- esis an FLAG-SF3B2 o FLAG-SF3B2-S289A and ans ec ed wi h a siRNA agains SF3B2 o a con ol sequence. S a is ical signi icance was de e mined wi h a 2-way ANOVA es . E, Same as C, bu using he NHEJ epo e EJ5-GFP. In his case, wo I-SceI-induced DSBs could be epai ed by conse a i e (le ) o mu agenic ( igh ) NHEJ g an ing he accumula ion o unc ional GFP. F, Same as D bu in cells bea ing he NHEJ epo e EJ5-GFP. Figu e 3. The SF3B complex con ols C IP exp ession A, The amoun o C IP mRNA was calcula ed by RT-qPCR in cells s ably exp essing a agged e sion o siRNA- esis an wild ype SF3B2 (FLAG-SF3B2), SF3B2 mu an (FLAG-SF3B2-S289A) o he emp y ec o (FLAG) and ans ec ed wi h a siRNA agains SF3B2 (siSF3B2) o a con ol sequence (siNT). The ba s ep esen he a e age and s anda d de ia ion o h ee independen expe imen s. S a is ical analysis as in igu e 1B. C, P o ein abundance and s abili y we e de e mined ea ing he cells wi h FLAG-SF3B2 - Dox + Dox P ados-Ca ajal e al Figu e 5 A C IPFISHDAPI - Dox + Dox FLAG B 0 20 40 60 80 Cells wi h C IP oci a he a ay (%) FLAG FLAG-SF3B2 FLAG-SF3B2-S289A *** *** *** *** *** siSF3B2 -Dox +Dox FLAG-SF3B2-S289A - Dox + Dox P ados-Ca ajal e al Figu e 6 A shNT shSF3B2 shC IP shNT shSF3B2 shC IP 0 20 40 60 80 * *** * * *** GFP % RPA oci-posi i e cells GFP-C IP 0 20 40 60 80 * ** ** % RPA oci-posi i e cells shNT shSF3B2 shNT shSF3B2 shC IP shNT RPADAPI shSF3B2shNT shNT shNT shC IP shSF3B2 BRPA DAPI shC IP shSF3B2 shNT GFP GFP-C IP RPA DAPI C HR e ficiency no malized o con ol siNT siSF3B2 siC IP 0.0 0.5 1.0 1.5 FLAG FLAG-C IP ** ** ** *** ** D siNT siSF3B2 0 50 100 150 Su i al (%) GFP GFP-C IP ** * * 1 Supplemen a y Figu e legends: Supplemen a y Figu e S1. Down egula ion e iciency o a ge ed genes A, U2OS cells we e ansduced wi h he indica ed shRNAs and cul u ed o 48 h. Then, p o eins we e ex ac ed using Laemmli bu e , esol ed in SDS-PAGE and ans e ed o PVDF memb anes. Rep esen a i e blo s upon incuba ion wi h an ibodies agains he speci ied p o eins a e shown. shNT deno es a non- a ge shRNA con ol. B, PLA signal using SF3B2 and C IP an ibody as indica ed in igu e 1G, bu in cells p e ea ed o 1 hou be o e i adia ion wi h ATM inhibi o (ATMi) o DMSO. C, same as A bu in cells deple ed o SF3B1 using siRNA. D, P o ein samples om cells bea ing he siRNA esis an wild ype FLAG-SF3B2, he FLAG-SF3B2-S289A mu an o FLAG emp y ec o and ans ec ed wi h siRNA agains he endogenous o m SF3B2 (siSF3B2) o a non- a ge ed con ol (siNT) we e p epa ed, esol ed by SDS-PAGE and blo ed o he indica ed an ibodies Supplemen a y Figu e S2. Cell cycle e ec o SF3B1 and SF3B2 deple ion A, Cell cycle dis ibu ion upon ans ec ion wi h he indica ed siRNAs. The a e age and s anda d de ia ion o h ee independen expe imen a e plo ed. B, Same as A bu cells s ably exp essing FLAG, siRNA- esis an wild ype FLAG-SF3B2 o FLAG-SF3B2- S289A mu an and ans ec ed wi h he indica ed siRNAs. Supplemen a y Figu e S3. S289 phospho yla ion does no a ec C IP-SF3B2 in e ac ion PLA was pe o med as desc ibed in Figu e 1G in cells down egula ed o endogenous SF3B2 and bea ing a siRNA- esis an e sion o wild ype SF3B2, an S289A mu an o he emp y FLAG ec o as a con ol. S a is ical di e ences we e calcula ed using an ANOVA es . Supplemen a y Figu e S4. FLAG-C IP o e exp ession Cells exp essing FLAG-C IP o he emp y FLAG ec o , as indica ed, we e ans ec ed wi h siRNAs agains C IP, SF3B2 o a con ol sequence. Then, p o ein samples we e ob ained as desc ibed in me hods, esol ed in SDS-PAGE and blo ed wi h he indica ed an ibodies. Supplemen a y ables 2 Supplemen a y Table S1: shRNAs used in his s udy. Ta ge gene TCR Numbe ID Sequence (5’-3’) Non Ta ge SHC016V 01031212MN - CCGGCAACAAGATGAAGA GCACCAACTC GAGTTGGTGCTCTTCATCT TGTTGTTTTT C IP TRCN0000318738 NM_002894. 2-3041s21c1 CCGGCAGAAGGATGAAGG ACAGTTTCTC GAGAAACTGTCCTTCATC CTTCTGTTTTTG SF3B2 TRCN0000314606 NM_006842. 2-1133s21c1 CCGGGCTGATGTTGAGAT TGAGTATCTCG AGATACTCAATCTCAACA TCAGCTTTTTG SF3B3 TRCN0000000069 NM_012426. x-3898s1c1 CCGGAGGAGGGTGACTGG ATAATTACTC GAGTAATTATCCAGTCAC CCTCCTTTTTTG RBM10 TRCN0000233276 NM_005676. 3-491s21c1 CCGGGACATGGACTACCG TTCATATCTCGAGATATG AACGGTAGTCCATGTCTTT TTG 3 SFPQ TRCN0000001084 NM_005066. x-1855s1c1 CCGGGCGCCAAAGAGAGG AAAGTTACTCGAGTAACT TTCCTCTCTTTGGCGCTTT TT DHX9 TRCN0000001210 NM_001357. x-515s1c1 CCGGGAAGGATTACTACT CAAGAAACTCGAGTTTCT TGAGTAGTAATCCTTCTTT TT DHX15 TRCN0000000006 NM_001358. x-2632s1c1 CCGGGTTGGTTCGATAAT GGCCTTTCTCGAGAAAGG CCATTATCGAACCAACTTT TT KIF11 TRCN0000116497 NM_004523. 2-3927s1c1 CCGGGCTTGAGCTTAACA TAGGTAACTCGAGTTACC TATGTTAAGCTCAAGCTTT TTG PRMT5 TRCN0000107086 NM_006109. 2-1577s1c1 CCGGGCCCAGTTTGAGAT GCCTTATCTCGAGATAAG GCATCTCAAACTGGGCTT TTTG Supplemen a y Table S2: siRNAs used in his s udy. Ta ge Re e ence Supplie Sequence (5’-3’) 4 gene Non Ta ge D-001810-10-20 Dha macon UGGUUUACAUGUCGACUAA, UGGUUUACAUGUUGUGUGA, UGGUUUACAUGUUUUCUGA and UGGUUUACAUGUUUUCCUA (mix) C IP Hue as and Jackson 2009 Sigma GCUAAAACAGGAACGAAUC SF3B1 D-020061-07- 0005 Dha macon CGAGUUUGCUUGGUCAGAA SF3B2 HSC.RNAI.N00 6843.12.6 IDT GGACUUGUCAUUUCAUGUUCUUATT Supplemen a y Table S3: P ime s used in his s udy. P ime s Applica ion Sequence (5’-3’) Fw Ac B qPCR ACGAGGCCCAGAGCAAGA R Ac B qPCR GACGATGCCGTGCTCGAT Fw C IP qPCR AGAAATTGGCTTCCTGCTCAAG R C IP qPCR GAAAACCAACTTCCCAAAAATTCTC SF3B2 UP SF3B2 cloning AAGCTCAAGCTTATGGATGACCC SF3B2 LOW SF3B2 cloning TTCGCGGCCGCCTAAACTTGAAC Fw G418 SF3B2 cloning GTCAGTGATATCCCCTGATAAATGCTTCAA R G418 SF3B2 cloning GTCCAGTGATATCCGAAATCTCGTGATGG Supplemen a y Table S4: P ima y An ibodies used in his s udy. 5 IF, immuno luo escence. IF-FISH, Immuno-FISH. WB, Wes e n blo ing. PLA, P oximi y liga ion assay. IP, Immunop ecipi a ion. Ta ge p o ein Applica ion Supplie Re e ence -Tubulin WB Sigma T9026 -Ac in WB Abcam ab8227 γ-H2AX IF, IF-FISH Cell Signaling 2577L γ-H2AX IF Millipo e 05-636 BRCA1 WB San a C uz sc-6954 C IP WB, PLA, IF-FISH R. Bae 14.1 DHX9 WB Be hyl Labo a o ies A300-855A-1 DHX15 WB Be hyl Labo a o ies A300-118A-1 FLAG WB, IF-FISH Sigma F3165-0.2MG GFP WB San a C uz Sc-8334 GFP IF Roche 11 814 460 001 KIF11 WB No us NB100-78467 PRMT5 WB Abcam ab31751 RBM10 WB Be hyl Labo a o ies A301-006A RPA32 WB, IF Abcam ab2175 SF3B1 WB, IP MBL D221-3 SF3B2 WB, PLA, IP P o ein ech 10919-1-AP SF3B3 WB Abcam ab3608 SFPQ WB Abcam ab38148 Supplemen a y Table S5: Seconda y an ibodies used in his s udy. IF, immuno luo escence. IF-FISH, Immuno-FISH. WB, Wes e n blo ing. An ibody Applica ion Supplie Re e ence 6 Alexa Fluo 594 goa an i-mouse IF In i ogen A11032 Alexa Fluo 488 goa an i- abbi IF In i ogen A11034 Alexa Fluo 568 goa an i- abbi IF-FISH In i ogen A11011 Alexa Fluo 647 goa an i-mouse IF-FISH In i ogen A21235 Alexa Fluo 568 donkey an i-mouse IF-FISH Molecula p obes A10037 Fluo escein an i-Bio in IF-FISH Vec o SP-3040 IRDye 800 CW Donkey an i-goa IgG (H+L) WB Li-Co 926-32214 IRDye 680RD goa an i-mouse IgG (H+L) WB Li-Co 926-68070 IRDye 800CW goa an i- abbi IgG (H+L) WB Li-Co 926-32211 P ados-Ca ajal e al Supplemen a y Figu e S1 A shNT shRBM10 RBM10 α-Tubulin shNT shSFPQ SFPQ shNT shDHX9 DHX9 shNT shDHX15 DHX15 shNT shKIF11 KIF11 shNT shPRMT5 PRMT5 α-Tubulinα-Tubulin α-Tubulin α-Tubulin α-Tubulin shNT shSF3B3 SF3B3 α-Tubulin shNT shC IP C IP α-Tubulin FLAG-SF3B2 Endogenous SF3B2 α-Tubulin C D SF3B1 β-Ac in siNT siSF3B1 siC IP siNT siSF3B2 FLAG FLAG-SF3B2 FLAG-SF3B2-S289A FLAG FLAG-SF3B2 FLAG-SF3B2-S289A B 0 50 100 150 200 PLA signal ela i e o con ol No damage IR *** ** ** *** * DMSO ATMi P ados-Ca ajal e al Supplemen a y Figu e S2 AB siNT siSF3B1 0 50 100G1 S G2/M % Cells siNT siSF3B2 siNT siSF3B2 siNT siSF3B2 0 50 100G1 S G2 FLAG % Cells FLAG-SF3B2 FLAG-SF3B2- S289A