Screen for mitochondrial DNA copy number maintenance genes reveals essential role for ATP synthase

A icle Sc een o mi ochond ial DNA copy numbe main enance genes e eals essen ial ole o ATP syn hase A sushi Fukuoh 1,2,3,† , Giuseppe Cannino 1,† , Mike Ge a ds 1 , Suzanne Buckley 1 , Selena Kazancioglu 1 , Filippo Scialo 1 , Ee o Liha ainen 4 , And e Ribei o 4 , E ic Du ou 1 & Howa d T Jacobs 1,5,* Abs ac The machine y o mi ochond ial DNA (m DNA) main enance is only pa ially cha ac e ized and is o wide in e es due o i s in ol e- men in disease. To iden i y no el componen s o his machine y, plus o he cellula pa hways equi ed o m DNA iabili y, we implemen ed a genome-wide RNAi sc een in D osophila S2 cells, assaying o loss o luo escence o m DNA nucleoids s ained wi h he DNA-in e cala ing agen PicoG een. In addi ion o p e iously cha ac e ized componen s o he m DNA eplica ion and ansc ip- ion machine ies, posi i es included many p o eins o he cy osolic p o easome and ibosome (bu no he mi o ibosome), h ee p o eins in ol ed in esicle anspo , some o he ac o s in ol ed in mi ochond ial biogenesis o nuclea gene exp ession, >30 mainly uncha ac e ized p o eins and mos subuni s o ATP syn hase (bu no o he OXPHOS complex). ATP syn hase knockdown p ecipi a ed a bu s o mi ochond ial ROS p oduc ion, ollowed by copy numbe deple ion in ol ing inc eased mi ochond ial u no e , no dependen on he canonical au ophagy machine y. Ou indings will in o m u u e s udies o he appa a us and egula ion o m DNA main enance, and he ole o mi ochond ial bioene ge ics and signaling in modula ing m DNA copy numbe . Keywo ds complex V; DNA eplica ion; mi ochond ial biogenesis; mi ochon- d ial DNA; mi ophagy; nuclease; nucleoid; eac i e oxygen species Subjec Ca ego ies Me hods & Resou ces; Me abolism DOI 10.15252/msb.20145117 | Recei ed 12 Janua y 2014 | Re ised 2May 2014 | Accep ed 2May 2014 Mol Sys Biol. (2014)10:734 In oduc ion Euka yo es ha use mi ochond ial oxida i e phospho yla ion (OXPHOS) o gene a e ATP main ain a sepa a e mi ochond ial genome (m DNA), encoding a subse o OXPHOS p o ein subuni s, oge he wi h some componen s o he machine y o in ami o- chond ial p o ein syn hesis. The main enance and exp ession o m DNA is o he wise dependen on nuclea -coded gene p oduc s, cons i u ing a sepa a e appa a us o genome main enance and gene exp ession wi hin he cell (McKinney & Oli ei a, 2013). The co e machine y o m DNA eplica ion is b oadly conse ed among euka yo es. DNA eplica ion is assumed o depend on he only DNA polyme ase consis en ly ound in mi ochond ia, DNA polyme ase c(PolG; Kaguni, 2004), a membe o he amily A DNA polyme ases (I o & B ai hwai e, 1991). I s closes p oka yo ic homologue is he phage T7 DNA polyme ase, and i is assumed o unc ion in conce wi h he mi ochond ial helicase Twinkle (Spelb ink e al, 2001), a homologue o phage T7 helicase-p imase (gp4). Twinkle is absen om yeas , whe e o he helicases a e in ol ed in m DNA eplica ion. Main enance o m DNA equi es also mi ochond ial ansc ip ion ac o s A (m -TFA o TFAM), needed o m DNA compac ion and ansc ip ion (La sson e al, 1998; Kang & Hamasaki, 2005; Campbell e al, 2012), and B (m -TFB2, TFB2M; Ma sushima e al, 2004). Th ee o he p o eins a e essen ial o m DNA main enance, namely m SSB, he mi ochond ial single-s anded DNA-binding p o ein (Maie e al, 2001), RNase H1 (Ce i elli e al, 2003), and, in some o ganisms, a second DNA polyme ase, P imPol, ha addi ionally has p imase ac i i y (Ga cı ´a-Go ´mez e al, 2013). A numbe o o he p o eins a e equi ed o main ain no mal m DNA copy numbe o opology in di e en o ganisms (Con amine & Pica d, 2000; Copeland, 2012). These include he DNA-binding AAA p o ein ATAD3A (He e al, 2007), some enzymes o nucleo ide me abolism and anspo (Saada, 2004), p o eins wi h oles in mi ochond ial memb ane dynamics (Jones & Fangman, 1992; Wong e al, 2000; Elachou i e al, 2011; Vielhabe e al, 2013), chape ones (Ciesielski e al, 2013), exonucleases (Ko nblum e al, 2013), p o eases (He lan e al, 2003; Ma sushima e al, 2010; Sesaki e al, 2003), and e en cy oskele al p o eins (Reyes e al, 2011). Mi ochond ia also con ain 1BioMediTech and Tampe e Uni e si y Hospi al, Uni e si y o Tampe e, Tampe e, Finland 2Depa men o Clinical Chemis y and Labo a o y Medicine, Kyushu Uni e si y G adua e school o Medical Sciences, Fukuoka, Japan 3Depa men o Medical Labo a o y Science, Junshin Gakuen Uni e si y, Fukuoka, Japan 4Depa men o Signal P ocessing, Tampe e Uni e si y o Technology, Tampe e, Finland 5Resea ch P og am o Molecula Neu ology, Uni e si y o Helsinki, Helsinki, Finland *Co esponding au ho . Tel: +358 3 3551 7731,+358 50 341 2894; E-mail: howa d. .jacob[email p o ec ed] † These au ho s equally con ibu ed o his wo k. ª2014 The Au ho s. Published unde he e ms o he CC BY 4.0license Molecula Sys ems Biology 10:734 |2014 1 Published online: June 21, 2014 opoisome ases, ligases, and o he nucleases, al hough hei speci ic oles in m DNA me abolism a e unclea . While a c ude DNA syn he ic machine y can be econs i u ed in i o om a minimal se o hese p o eins, he ull complemen o p o eins equi ed o ai h- ul m DNA eplica ion in i o emains o be de e mined. Some componen s o he m DNA main enance machine y a e sha ed wi h he nuclea compa men , including RNase H1 (Ce i elli e al, 2003) and many p o eins implica ed in base-excision epai (Alexeye e al, 2013). Mos ly, hese a e syn hesized in wo o mo e iso o ms ou ed o di e en cellula compa men s, o example, ia di e en ial splicing, al e na i e ansla ional s a si es (Suzuki e al, 2010), o ambiguous a ge ing signals (Ka niely & Pines, 2005). m DNA is packaged oge he wi h TFAM and some o he p o eins in o disc e e in ami ochond ial s uc u es o a iable composi ion, called nucleoids, by analogy wi h hose o bac e ia (Spelb ink, 2010; Bogenhagen, 2012). They con ain a numbe o eplica ion p o eins whose unc ional oles a e poo ly unde s ood, as well as p o eins implica ed in o he cellula p ocesses, including me abolic enzymes and chape ones, and p o eins in ol ed in in ami ochond ial p o ein syn hesis (Hensen e al, 2014). The appa a us o mi ochond ial ansla ion has been unc ionally implica ed in m DNA main enance in yeas (Con amine & Pica d, 2000), hough no in me azoan cells (S o ie & A a di, 1972). The impo ance o m DNA main enance o cell physiology and homeos asis is unde sco ed by he inding ha i s dys unc ion leads o di e se ypes o human disease, including bo h in an ile and la e- onse pa hologies, showing a bewilde ing a ie y o issue speci ici ies (Shadel, 2008; Ro ¨ ig & Poul on, 2009; Ylikallio & Suomalainen, 2012). Loss o mi ochond ial genome in eg i y o ideli y is also associa ed wi h aging (Oli ei a e al, 2010; B a ic & La sson, 2013). Iden i ying he ull se o gene p oduc s in ol ed in ai h ul m DNA main enance is hus o b oad in e es and impo ance. To his end, we implemen ed a genome-wide (blinded) sc een o D osophila S2 cells, using dsRNA-based RNA in e e ence (RNAi), aking ad an age o he ac ha S2 cells ole a e loss o m DNA and con inue o g ow wi hin he ime scale o a ypical expe imen , despi e dec eased OXPHOS capaci y. Fu he mo e, m DNA nucle- oids may be iden i ied in hese cells on he basis o luo escence signal om he opology-dependen DNA-in e cala ing dye Pico- G een (Ashley e al, 2005). E ec s on m DNA copy numbe we e hen p obed u he using quan i a i e PCR (QPCR), wi h addi ional expe imen s conduc ed on he cellula pheno ypes p oduced by knockdown o speci ic genes iden i ied in he sc een, no ably hose encoding subuni s o ATP syn hase, in o de o es aspec s o he mechanisms by which hey may ac . Resul s and Discussion Implemen a ion and ou come o he p ima y sc een We se ou o sc een a genome-wide D osophila dsRNA lib a y in S2 cells, sco ing o disappea ance o he PicoG een signal o m DNA nucleoids as indica i e o genes equi ed o m DNA main enance. In ini ial ials, we ound i di icul o pick ou he nucleoid signal agains backg ound cy oplasmic luo escence. Using a dsRNA agains he PolG ca aly ic subuni ( amas) as a posi i e con ol, and a dsRNA di ec ed agains GFP as a nega i e con ol, we es ablished a p o ocol whe eby i was possible eliably o sco e (by eye) he disappea ance o nucleoid signal (see Fig 1). This in ol ed applying he es dsRNA o 5 days in a 96-well pla e o ma , wi h addi ion on day 3 o a dsRNA di ec ed agains TFAM. Al hough p olonged incuba ion wi h TFAM dsRNA i sel led o m DNA deple ion, he sho e - e m ea men con e sely enhanced he nucleoid signal in nega i e con ol cells, whe eas i was dec eased o e y low le els in cells ea ed wi h he posi i e con ol dsRNA. The p ima y sc een, conduc ed blind, was success ul in iden i y- ing as posi i es mos o he known ac o s in ol ed in m DNA me abolism (Table 1, ca ego y 1), gi ing con idence in i s alidi y. In o al, 105 dsRNA a ge s we e ini ially judged as posi i e (Supple- men a y Table S1), o which almos hal we e eco ded also as lead- ing o cell dea h in a ac ion o he cells. Consis en wi h p e ious s udies (Ra ¨me e al, 2002; Bou os e al, 2004), a u he 276 a ge s (Supplemen a y Tables S1 and S2) ga e massi e cell dea h bu no speci ic loss o nucleoid signal and we e conside ed o ep esen essen ial genes ha could no be s udied u he . Finally, an addi- ional 132 a ge s we e judged o gi e an abno mal ou come wi hou comple e loss o PicoG een nucleoid signal (Supplemen a y Tables S1 and S3), bu we e no analyzed u he , al hough many ell in o simila ca ego ies o pa hways as hose on he posi i es lis . The speci ici y and knockdown e iciency o his p ocedu e has p e i- ously been documen ed (Clemens e al, 2000; Kleino e al, 2005). Knockdown was he e e i ied a he RNA le el by qRT-PCR o 17 speci ic a ge s (see SI). We compa ed subjec i e judgmen agains a compu a ional me hod o measu e punc a e luo escence in ensi y (de ails o be published elsewhe e). The la e ga e many alse posi i es due o a iable backg ound luo escence, as well as alse nega i es due o cell deb is. Based on esc eening, we judged he manual me hod o be supe io (see SI o de ails), and we se c i e ia o de ining posi- i es as desc ibed below. Resc eening o iden i y de ini i e posi i es Posi i es we e conside ed as con i med i h ee posi i e bu no nega i e indings we e ob ained. Those whe e a nega i e o ambigu- ous inding was eco ded we e e ained only whe e h ee imes as many clea ly posi i e indings we e ob ained upon exhaus i e esc eening (double as e isks in column H o Table 1) o he wise hey we e conside ed as alse posi i es (double as e isks in column G o Supplemen a y Table S4). Fi e a ge s ha did no gi e consis en ly posi i e indings du ing esc eening we e no ed o gi e ise o mul iple splice a ian s, encoding a leas one polypep ide p edic ed o be mi ochond ially localized. Fo hese, we es ed dsRNAs a ge ed speci ically on he ele an splice a ian s, con i ming se e al addi ional posi i es (Table 1, g een backg ound), whe eas dsRNAs a ge ed agains o he splice a ian s o he en i e gene we e judged nega i e (Supplemen a y Table S4, blue backg ound). O he o iginal 105 posi i es, 83 we e e ained, 20 we e eas- signed as nega i e, and one was eassigned as abno mal (pink backg ound in Supplemen a y Table S3). One was disca ded because he dsRNA de ec ed a pseudogene o a gene al eady in he lis , ano he because a e ised gene model combined i wi h ano he Molecula Sys ems Biology 10:734 |2014 ª2014 The Au ho s Molecula Sys ems Biology m DNA main enance and ATP syn hase A sushi Fukuoh e al 2 Published online: June 21, 2014 posi i e, while ano he was subsequen ly e-anno a ed as wo sepa- a e genes (bu shown as a single en y in Table 1). Posi i e indings we e also ob ained o speci ic splice iso o ms o h ee o he nega- i es. Thus, he con i ma ion o 86 ou o 106 ini ial a ge s indica es a alse-posi i e a e o 18%. One addi ional posi i e (CG5794) was unexpec edly iden i ied by a dsRNA nominally a ge ed agains a di e en gene. The posi i es ell in o se en dis inc classes: mi o- chond ial DNA eplica ion o ansc ip ion, cy osolic ansla ion, he p o easome, ATP syn hase, mi ochond ial dynamics o biogenesis, nuclea gene exp ession, and a se en h, miscellaneous ca ego y. Al hough he p ima y nega i es we e no esc eened sys ema i- cally, some ha ell in o simila unc ional pa hways as de ini i e posi i es we e e-e alua ed using he same c i e ia. Ele en we e p omo ed o he posi i es lis (yellow backg ound in Table 1), including, o example, mos o he subuni s o ATP syn hase. This implies ha he ini ial sc een may ha e missed as many posi i es as we e ac ually e ained, implying a alse-nega i e a e o up o 1%. The o e all esul s o he sc een a e summa ized in Fig 2. The inal numbe o de ini i e posi i es was 97, coun ing only once hose wi h >1 posi i e splice a ian . Copy numbe o m DNA Fo genes on he de ini i e posi i es lis , we ca ied ou QPCR o assess changes in m DNA copy numbe a e 5 days o dsRNA ea - men (wi hou concomi an TFAM knockdown), no malized agains a single-copy nuclea DNA s anda d. Based on his assay (see Table 1, column G, aw da a in Supplemen a y Table S5), we classi- ied he posi i es as showing subs an ial (++,≤60%) o modes (+) m DNA deple ion, no signi ican copy numbe change (0), o an inc ease in m DNA (). Al hough hese classes seem a bi a y, we ound ha RNAi knockdown o well-cha ac e ized componen s o he m DNA epli- ca ion machine y all ga e alues in he 20-60% ange (++). Su p is- ingly, we iden i ied only 6 new genes om he sc een whose knockdown p oduced a compa ably se e e m DNA deple ion. Th ee o hese encode subuni s o ATP syn hase. The o he s we e CG5794, encoding a de-ubiqui ina ing enzyme wi h unknown subs a e(s), TweedleY, p e iously iden i ied as a cu icula p o ein (Guan e al, 2006), and poin ed, a well-s udied E s amily ansc ip ion ac o (Klaes e al, 1994; Mo imo o e al, 1996). The ac ha knockdown GFP nega i e con ol m DNA helicase (CG5924) posi i e (con ol) amas (CG8987) posi i e con ol CG31380 nega i e CG6413 (Dis3) posi i e CG2028 (CkIα α ) abno mal ( ewe do s) Figu e 1. Sc eening o D osophila dsRNA lib a y by PicoG een nucleoid luo escence in S2cells. Mic og aphs o S2cells s ained wi h PicoG een, ollowing 5days o ea men wi h he dsRNA indica ed. GFP and amas (Polg, CG8987) we e used as nega i e and posi i e con ols, espec i ely. In esc eening, CG5924 was also used as a posi i e con ol. Bo h we e de ec ed in he blinded sc een as posi i es. O he images show a ypical nega i e (CG31380), a ypical posi i e (CG6413) and a ypical case o a a ge classed as abno mal, in his case CG2028 (CkIa), which showed a dec eased numbe o nucleoid signals pe cell. Images a e op imized o b igh ness and con as bu wi h no o he manipula ions. Scale ba indica es 50 lm. ª2014 The Au ho s Molecula Sys ems Biology 10:734 |2014 A sushi Fukuoh e al m DNA main enance and ATP syn hase Molecula Sys ems Biology 3 Published online: June 21, 2014 Table 1. De ini i e posi i es Ca ego y numbe Ca ego y name CG numbe O icial name o symbol Human o hologue O he o common name(s) o pu a i e unc ion m DNA deple ion >3 esc eenings No e 1m DNA eplica ion and ansc ip ion CG3910 m TFB2TFB2M m ansc ip ion ac o B2+* CG4217 TFAM TFAM m TFA, m ansc ip ion ac o A ++ CG4337 m SSB SSBP1m single-s anded DNA-binding p o ein ++ CG4644 m RNApol POLRMT m RNA polyme ase ++ CG5924 CG5924 PEO1Twinkle DNA helicase ++ CG6815 belphego ATAD3A, B, C AAA domain con aining p o ein 30** CG7175 mTe 5m ansc ip ion e mina ion ac o 5++ CG8729 nh1RNASEH1Ribonuclease H10** Weak posi i e, diminished nucleoid signal seen in some cells, also some cell dea h CG8987 amas POLG DNA polyme ase cca aly ic subuni ++ CG18124 mTTF m ansc ip ion e mina ion ac o ++ CG33650 DNApol-c35 POLG2DNA polyme ase caccesso y subuni , CG8969 +** 2Cy osolic ansla ion CG1821 RpL31 RPL31 La ge subuni – CG3203 RpL17 RPL17 La ge subuni – CG3751 RpS24 RPS24 Small subuni n CG3922 RpS17 RPS17, RPS17L Small subuni – CG3997 RpL39 RPL39, RPL39L La ge subuni 0 CG4111 RpL35 RPL35 La ge subuni n CG4759 RpL27 RPL27 La ge subuni n CG7283 RpL10Ab RPL10A La ge subuni n CG7490 RpLP0RPL0La ge subuni n CG7622 RpL36 RPL36 La ge subuni –* CG7726 RpL11 RPL11 La ge subuni n * Cell dea h no subs an ial in some epea s CG8922 RpS5a RPS5Small subuni n * CG9282 RpL24 RPL24 La ge subuni n * CG9677 In 6EIF3E eIF3, Ini ia ion ac o 3, subuni E n * Abno mal dis ibu ion o some esidual nucleoid signal wi hin cells CG11522 RpL6RPL6La ge subuni Molecula Sys ems Biology 10:734 |2014 ª2014 The Au ho s Molecula Sys ems Biology m DNA main enance and ATP syn hase A sushi Fukuoh e al 4 Published online: June 21, 2014 Table 1(Con inued) Ca ego y numbe Ca ego y name CG numbe O icial name o symbol Human o hologue O he o common name(s) o pu a i e unc ion m DNA deple ion >3 esc eenings No e 3P o easome CG1341 Rp 1PSMC2,5,6ATPase egula o y subuni 2(o 5o 6)+ CG5266 P os25 PSMA2Co e a- ype subuni 2+** CG9324 Pomp POMP P o easome ma u a ion p o ein – CG9327 P os29 PSMA4Co e a- ype subuni 4+* CG10149 Rpn6PSMD11 Non-ATPase egula o y subuni 11 + CG16916 Rp 3PSMC4ATPase egula o y subuni 4+ CG18174 Rpn11 PSMD14 Non-ATPase egula o y subuni 14 0 ** Se e e cell dea h: ew cells emained a e 5days 4ATP syn hase CG2968 l(1)G0230 ATP5DdSubuni o F1(s alk) + CG3321 CG3321 ATP5I Subuni e o Fo, dime iza ion/bending ++ CG4307 Oscp ATP5O OSCP subuni o F1/s a o a m ++ ** CG4412 ATPsyn-C 6ATP5J Coupling ac o 6, subuni o Fo/s a o a m + CG6105 l(2)06225 ATP5L, ATP5L2Subuni g o Fo, dime iza ion/bending + CG7610 ATPsyn-cATP5C1cSubuni o F1(s alk) +* CG6030 ATPsyn-d ATP5H Subuni d o Fo, s a o a m +* CG8189 ATPsyn-b ATP5F1Subuni b o Fo, s a o a m ++ * CG11154 ATPsyn-bATP5B Co e bsubuni o F1+** 5Mi ochond ial biogenesis and dynamics CG3114 e ec wing NRF1Nuclea espi a o y ac o 1homologue 0** CG6338 E s97D GABPA Nuclea espi a o y ac o 2,asubuni +** CG6512 CG6512 AFG3L2, SPG7m-AAA p o ease subuni 0* CG8479 opa1-like OPA1Dynamin- ela ed p o ein equi ed o inne m memb ane usion +* CG9809 spa gel PPARGC1A, B, PPRC1 PPAR ccoac i a o +** Weak posi i e, diminished numbe o nucleoid signals emaining in some cells CG14981 maggie TOMM22 Tomm 22 subuni o ou e m memb ane anslocase + 6Nuclea gene exp ession CG1057 MED31 MED31 Media o complex subuni 31, anc ip ional elonga ion 0 CG1554 RpII215 POLR2A RNA polyme ase II 215kD subuni + CG1810 mRNA-cap RNGTT mRNA capping enzyme 0 CG1874 No 1CNOT1CCR4-NOT ansc ip ion complex subuni 10 ** Weak posi i e, diminished numbe o nucleoid signals emaining in some cells, some cell dea h also e iden ª2014 The Au ho s Molecula Sys ems Biology 10:734 |2014 A sushi Fukuoh e al m DNA main enance and ATP syn hase Molecula Sys ems Biology 5 Published online: June 21, 2014 Table 1(Con inued) Ca ego y numbe Ca ego y name CG numbe O icial name o symbol Human o hologue O he o common name(s) o pu a i e unc ion m DNA deple ion >3 esc eenings No e CG2163 Pabp2PABPN1, PABPN1L Nuclea poly(A)-binding p o ein – CG3162 LS2No el U2AF- ela ed egula o o di e en ial splicing 0 CG3675 A 2PRMT6P o ein a ginine me hyl ans e ase 2+ CG6525 pps SPOCD1, PHF3 P o ein pa ne o sn , egula o o al e na i e splicing o Sex le hal +* Diminished numbe o nucleoids emained in many cells CG7626 Sp 5SUPT5H T ansc ip ion elonga ion ac o SPT5(DSIF complex) 0 CG9591 omd INTS5In eg a o complex subuni 5, snRNA p ocessing ac o +* Ve y ew cells emained, a e 5days o ea men CG9748 belle DDX3X,Y,; DDX4 RNA helicase, implica ed in X-ch omosome dosage compensa ion 0 CG10955 R 1RTF1Componen o RNA polyme ase II-associa ed (PAF1) complex, ansc ip ional elonga ion 0** CG11990 hy ax CDC73 Componen o RNA polyme ase II-associa ed (PAF1) complex, ansc ip ional elonga ion 0* CG17183 MED30 MED30 Media o complex subuni 30, anc ip ional elonga ion 0** CG17358 Ta 12 TAF12 TATA box-binding p o ein-associa ed ac o 12 + CG17603 Ta 1TAF1, TAF1L TATA box-binding p o ein-associa ed ac o 1+ 7Miscellaneous (o he o unknown) CG3539 SLY-1 homologue SCFD1ER o Golgi esicle anspo 0** CG4268 Pi sl e CDK11A Cyclin-dependen kinase supe amily, egula o o au ophagy – CG5794 CG5794 USP34 Ubiqui in-speci ic pep idase (deubiqui ina ing enzyme) ++ * CG6413 Dis3DIS3Exosome complex exo ibonuclease –* CG7368 CG7368 Zn inge p o ein –** CG8021 CG8021 SLIRP RNA-binding p o ein, egula ion o m RNA le els 0 CG9007 upSET SETD5, MLL5 Zn inge p o ein, ela ed o i ho ax and his one lysine me hly ans e ases – CG9397-H jing AEBP2Zn inge ansc ip ion ac o , ole in mo phogenesis –** Was clea ly posi i e in 5ou o 7 ials Molecula Sys ems Biology 10:734 |2014 ª2014 The Au ho s Molecula Sys ems Biology m DNA main enance and ATP syn hase A sushi Fukuoh e al 6 Published online: June 21, 2014 Table 1(Con inued) Ca ego y numbe Ca ego y name CG numbe O icial name o symbol Human o hologue O he o common name(s) o pu a i e unc ion m DNA deple ion >3 esc eenings No e CG9797 CG9797 Zn inge p o ein 0** Weak posi i e, diminished numbe o nucleoid signals emaining in some cells CG10042 MBD-R2Me hyl-DNA-binding p o ein 0Some small nucleoid signals emained in some cells. Ano he dsRNA o his gene ga e consis en ly nega i e indings CG10144 CG10144 VPS8Vesicle so ing o lysosomes +** Nucleoid signals o diminished size o in ensi y seen in some cells, ew cells emained a e 5d CG10395 CG10395 Zn inge p o ein 0 CG10582 Sin POLR3E Sex-le hal in e ac o , possible al e na i e splicing, p oposed subuni o RNA polyme ase III 0* CG10582-C Sin Pu a i e mi ochond ially a ge ed iso o m o Sin 0** Clea posi i e in 3 ials, bu also some inconsis en indings CG12242 Gs D5Glu a hione S- ans e ase supe amily (+) Deple ion jus ou side he bo de o signi icance, due o la ge a iance CG13203-C CG13203 Pu a i e mi ochond ially a ge ed iso o m o p o ein wi h unknown unc ion –** CG13779 Sem1Possible endopep idase 0** CG14084 Be 1BET1ER o Golgi esicle anspo 0* CG14247 CG14247 Unknown unc ion 0* CG14634 CG14634 Unknown +** CG15231 IM4Immune-induced molecule, pep ide ho mone? +* CG15343 CG15343 Py idoxamine 50-phospha e oxidase-like 0* CG15793 Dso 1MAP2K1,2,5MAP kinase kinase 0* CG17077 poin ed ETS1,2E s ansc ip ion ac o ++ * CG31258 Cenp-C Cen ome e-binding, kine ocho e unc ion n * ª2014 The Au ho s Molecula Sys ems Biology 10:734 |2014 A sushi Fukuoh e al m DNA main enance and ATP syn hase Molecula Sys ems Biology 7 Published online: June 21, 2014 Table 1(Con inued) Ca ego y numbe Ca ego y name CG numbe O icial name o symbol Human o hologue O he o common name(s) o pu a i e unc ion m DNA deple ion >3 esc eenings No e CG31079 Unknown (gene model la e wi hd awn om Flybase) n P edominan ly induced cell dea h, ew cells emaining seemed posi i e CG32085 CG32085 FBXL16 Pu a i e ubiqui in ligase 0 CG32561, 2 xmas-1, xmas-2MCM3AP (xmas-2) Pu a i e p o ein ace yl ans e ase in ol ed in DNA eplica ion (xmas-2) 0** Clea posi i e in 4 ials, bu also some inconsis en indings CG32570 TwdlY Sugges ed cu icula p o ein ++ * CG32652 CG32652 Unknown +* CG34415 mu e Muscle was ed, ch oma in p o ein, his one locus body 0** Weak posi i e, diminished nucleoid signal seen in some cells, cell dea h seen only in some ials CG33546 g z GST-con aining Zn inge p o ein, pu a i e mi o ic checkpoin p o ein 0 CG42666-G CG42666 REXO1Exo ibonuclease, p edic ed mi ochond ially a ge ed iso o m n ** Weak posi i e, diminished numbe o nucleoid signals emained in some cells, dsRNA o en i e gene was an inconsis en weak posi i e; see also Table S1. iso o m-speci ic p ime s also a ge Ada , CG12598 CG42666-D CG42666 REXO1Exo ibonuclease, p edic ed mi ochond ially a ge ed iso o m 0* Weak posi i e, diminished numbe o nucleoid signals emained in some cells, dsRNA o en i e gene was an inconsis en weak posi i e; see also Table S2 CG42281 bunched Signal ansduc ion in esponse o g ow h ac o (dpp) binding – =no in o iginal posi i es lis ; =posi i e iso o m wi h pu a i e m a ge ing; =gene model wi hd awn om Flybase. Posi i es also showing subs an ial cell dea h a e shown in unde line and i alic. >3 esc eenings: Posi i es ha equi ed many ounds o esc eening o inal alida ion. * = 4 ounds; ** ≥8 ounds. Molecula Sys ems Biology 10:734 |2014 ª2014 The Au ho s Molecula Sys ems Biology m DNA main enance and ATP syn hase A sushi Fukuoh e al 8 Published online: June 21, 2014 o many genes esul ed in loss o PicoG een signal wi hou majo changes in m DNA copy numbe sugges s ha indi ec e ec s may be common, o example, a ec ing DNA opology, nucleoid a chi- ec u e, memb ane po en ial o cellula dye up ake. Posi i es implica ed in m DNA me abolism The posi i es include mos o he p o eins wi h known oles in m DNA eplica ion o ansc ip ion, no ably he i e shown p e i- ously o be essen ial o m DNA main enance in D osophila (Go o e al, 2001; Maie e al, 2001; Iyenga e al, 2002; Ma sushima e al, 2004; Humph ey e al, 2012). The lis comp ises he wo subuni s o PolG, he ca aly ic subuni o he mi ochond ial RNA polyme ase, m SSB, he D osophila homologue o he Twinkle helicase, an- sc ip ion ac o s TFAM and m TFB2M, mTERF amily membe s mTTF and mTe 5 (Jo ˜e s e al, 2013), plus nh1 (RNaseH1) and belphego (homologue o mammalian ATAD3). All ga e signi ican m DNA deple ion excep o nh1 and belphego . DNA ligase III (lig3, CG17227) and mTERF amily membe s mTe 3 and CG15390 (homologue o mammalian MTERF4) we e consis en ly nega i e. DNA ligase III was p e iously epo ed as dispensable o nuclea DNA epai bu essen ial o m DNA main enance in human cells (Ruhanen e al, 2011) and mouse (Puebla-Oso io e al, 2006; Gao e al, 2011). Ou da a imply ha i is edundan o a leas one o he m DNA ligase in D osophila. Simila a gumen s may apply o he absence o any opoisome ase, gy ase, ecombinase, esol ase o helicase (o he han Twinkle). Ou s udy sugges s ha ew dedi- ca ed componen s o he m DNA eplica ion appa a us emain o be iden i ied, bu his does no exclude ac o s wi h o e lapping oles in o he cell compa men s. In mammalian mi ochond ia, he ansc ip ional appa a us is conside ed essen ial o bo h leading- and lagging-s and syn hesis (Clay on, 1982; Fus e e al, 2010). RNase H1, also equi ed o m DNA main enance in mouse (Ce i elli e al, 2003) and human cells (Ruhanen e al, 2011), migh be in ol ed in p ime emo al, bu his ypically also needs o he helicases and nucleases such as Fen1 (CG8648) and Dna2 (CG2990), bo h implica ed in m DNA eplica ion in mammalian cells (Duxin e al, 2009; Kazak e al, 2013). Thei absence om he posi i es lis is unsu p ising, howe e , since bo h also unc ion in he nucleus. One posi i e om he miscellaneous ca ego y, CG8021, appea s o be a D osophila homologue o SLRP, a mammalian p o ein in ol ed in mi ochond ial mRNA s abiliza ion and p ocessing (Sasa man e al, 2010; Chujo e al, 2012). The mammalian ATAD3 amily has been implica ed in nucleoid o ganiza ion (He e al, 2007), mi ochond ial p o ein syn hesis (He e al, 2012), egula ion o apop osis (Huang e al, 2011) and au o- phagy (Chen e al, 2011), choles e ol a icking (Rone e al, 2012), mi ochond ial dynamics (Gilquin e al, 2010) and s ess esis ance (Ho mann e al, 2012). Loss o PicoG een nucleoid signal wi h only a mino d op in m DNA copy numbe may indica e ha belphego unc ions also in di e se pa hways and ha i s e ec s on nucleoids and m DNA may be indi ec . Nucleases o he han RNase H1 ha e been shown o sugges ed o ha e oles in m DNA me abolism in a ious o ganisms, including EXOG (Tann e al, 2011), EndoG (McDe mo -Roe e al, 2011) and yeas Exo5 (Bu ge s e al, 2010). Exo5 has no D osophila NEGATIVE POSITIVE ABNORMAL CELL DEATH P ima y sc een 105 276 132 15,506 16,019 dsRNAs Resc een DEFINITIVE POSITIVES 1 83 + 3 wi h splice a ian s 11 To al = 97 10 Figu e 2. O e all esul s o he sc een. Schema ic diag am illus a ing numbe o dsRNAs analyzed, numbe s o p ima y posi i es, nega i es and o he classes, and he esul s o esc eening. ª2014 The Au ho s Molecula Sys ems Biology 10:734 |2014 A sushi Fukuoh e al m DNA main enance and ATP syn hase Molecula Sys ems Biology 9 Published online: June 21, 2014 Bogenhagen, 2012). The in ol emen o cV, he p o easome, and some key genes o mi ochond ial dynamics and quali y con ol sugges s ha m DNA copy numbe is dependen on a balance be ween mi ochond ial u no e and biogenesis, in which speci ic s esses, no ably mi ochond ial ROS p oduc ion and impai ed cy o- solic p o ein u no e , may be c ucially impo an . ATP syn hase was in e ed o be a key playe in homeos a ic main enance o mi ochond ia, and hus in he amoun o m DNA and i s gene p oduc s. Mu a ions in ATP syn hase in ungi a e al eady known o play a de e mining ole in whe he m DNA loss can be ole a ed (Con amine & Pica d, 2000; Le eb e-Legend e e al, 2003), o e en acili a ed (Gi aud & Velou s, 1997; Lai-Zhang e al, 1999; Con amine & Pica d, 2000), bu wi h memb ane po en- ial implica ed as a key pa ame e (Du ezin-Caube e al, 2006; Wang e al, 2007). In S2 cells, excess ROS p oduc ion was be e co ela ed wi h he s eng h and kine ics o m DNA deple ion, bu his could be an epi-phenomenon. The ac ha memb ane po en ial ‘pe mi ochond ion’ (Supplemen a y Fig S4D) was es o ed o i s s a ing alue sugges s ha i s dis u bance may ye p o e o be he p ima y induce . Excess ROS has elsewhe e been p oposed o lead o m DNA deple ion unde pa hological condi ions (La osche e al, 2010; Quinzii e al, 2013), and pa hological de ec s in ATP syn hase associa ed wi h ROS o e p oduc ion (Ba acca e al, 2007) may down egula e mi ochond ial unc ions (Wojewoda e al, 2010, 2011) and e en lead o m DNA loss (Ve gani e al, 1999; Tu ne e al, 2005). ROS o e p oduc ion has been widely sugges ed bo h o p o oke m DNA damage, bu also o esul om i . Howe e , a ecen epo showed ha un epai ed damage leads o m DNA deple ion wi hou inc eased ROS (Shokolenko e al, 2013), and he ole o ROS in p oducing soma ic m DNA mu a ions in he PolgA mu a o mouse is dispu ed (T i uno ic e al, 2005; Dai e al, 2010). Thus, he inc eased mi ochond ial ROS seen when cV is knocked down is mo e logically a cause han a consequence o m DNA deple ion. Fu he mo e, al hough ROS may p o oke s and b eakage, in e e ing di ec ly wi h m DNA eplica ion (Han & Chen, 2013), ou da a ins ead sugges ha ROS ac i a es mi o- chond ial u no e be o e widesp ead DNA damage would be sus ained, as occu s in mammalian cells unde TNFasignaling (Nagakawa e al, 2005; Vad o e al, 2012). Howe e , an opposing pa hway has also been sugges ed, in which ROS o e -p oduc ion p omo es mi ochond ial biogenesis, no u no e (Mo eno- Loshue os e al, 2006, 2011). A key aim o u u e esea ch will be o iden i y he senso mole- cule(s) in eg a ing changes in mi ochond ial ROS (o memb ane po en ial) wi h o he me abolic signals, in o de o modula e mi o- chond ial biogenesis and u no e . One possibili y consis en wi h ou da a is ha ATP syn hase i sel is ha senso . Ma e ials and Me hods Cell main enance D osophila S2 cells (In i ogen) we e cul u ed unde s anda d condi- ions, in Schneide 0s medium (Sigma) and dilu ed 1:6 e e y 3-4 d. In selec ed expe imen s, a ious d ugs we e added o glucose was eplaced wi h he same concen a ion o galac ose. S2 cells s ably exp essing Ciona in es inalis AOX we e gene a ed by co- ans ec ion wi h a plasmid con e ing hyg omycin esis ance. Sc eening o D osophila dsRNA lib a y and luo escence mic oscopy o nucleoids S2 cells we e seeded in o 96-well pla es and ea ed o e 5 days wi h 0.6–1.2 lg o dsRNA om he lib a y (Open BioSys ems), alongside posi i e and nega i e con ols in each pla e, as desc ibed p e iously (Jo ˜e s e al, 2013). Nucleoids we e isualized by luo es- cence mic oscopy a e s aining wi h Quan -iT TM PicoG een dsDNA eagen (7.5 ll/ml, In i ogen). La ge -scale dsRNA ea men s o m DNA copy numbe e alua ion and analysis o cellula pa ame e s we e pe o med essen ially as p e iously (Jo ˜e s e al, 2013). Nucleic acid isola ion and QPCR To al RNA was isola ed om S2 cells as p e iously (Jo ˜e s e al, 2013). Fo DNA isola ion, cells om a single well o a 24-well pla e we e p ocessed by a p ocedu e de e mined o gi e consis en esul s i espec i e o cell densi y, in ol ing SDS lysis, p o einase K diges- ion, isop opanol p ecipi a ion and o e nigh esuspension a 55°C (see SI). Fo la ge -scale expe imen s, DNA was p epa ed om 1.5 ×10 6 cells cul u ed in 6-well pla es, as p e iously (Jo ˜e s e al, 2013). Mi ochond ial DNA copy numbe was assessed by QPCR using p ime s agains COXII o 16S RNA ( o m DNA) and RpL32 (nuclea DNA, single-copy, o no maliza ion). T ansc ip le els we e es ima ed ela i e o ha o RpL32 by a simila p ocedu e, bu using cDNA as empla e. Measu emen s o mi ochond ial unc ion Mi ochond ial memb ane po en ial, ROS le el, and con en pe cell we e de e mined by low cy ome y (Cannino e al, 2012) o cells s ained, espec i ely, wi h 200 mM e ame hyl hodamine me hyl es e (TMRM), 2.5 lM Mi oSox TM (In i ogen), o ei he 200 nM 10-nonyl ac idine o ange (NAO) o 40 nM Mi oT acke G een FM (Li e Technologies). Oxygen consump ion o li ing cells (Cannino e al, 2012) was measu ed using a Cla k- ype elec ode (Hansa ech Oxy e m sys em). Analyses o lysosomal and mi ochond ial con en Lysosome con en pe cell was measu ed by low cy ome y o cells s ained wi h 50 nM LysoT acke Red DND-99 (Li e Technologies). Mi oT acke G een FM (Molecula P obes) and LysoT acke Red DND-99 we e used o li e cell imaging by con ocal mic oscopy o mi ochond ia and lysosomes, espec i ely, wi h spo -a ea calcula- ion (ImageJ) and image decon olu ion (SVI, Huygens so wa e). Wes e n blo ing Pos -nuclea ex ac s esol ed by SDS-PAGE we e elec oblo ed and p obed using s anda d me hods (essen ially as Fe nandez-Ayala e al, 2009; see SI). P ima y an ibodies used we e agains NDUFS3 (Abcam, mouse), 1:10,000), ATP5A (Abcam, mouse, 1:1,000), and GAPDH (E e es Bio ech, goa , 1:2,000), wi h app op ia e ho se- adish pe oxidase-conjuga ed seconda y an ibodies. Visualiza ion Molecula Sys ems Biology 10:734 |2014 ª2014 The Au ho s Molecula Sys ems Biology m DNA main enance and ATP syn hase A sushi Fukuoh e al 16 Published online: June 21, 2014 used he ECL sys em (Ame sham Biosciences) acco ding o he manu ac u e ’s p o ocols. S a is ical analyses Compa isons be ween popula ions we e pe o med using unpai ed wo- ailed S uden ’s - es s o analyses o a iance when mo e han wo samples we e compa ed, wi h Bon e oni-co ec ed pos hoc - es . Fo u he de ails, see Supplemen a y Ma e ials and Me hods. Da a a ailabili y O iginal images om he p ima y sc een a e deposi ed a : h p:// dx.doi.o g/10.5061/d yad. 55p5. Supplemen a y in o ma ion o his a icle is a ailable online: h p://msb.embop ess.o g Acknowledgemen s This wo k was suppo ed by unding om he Academy o Finland, Tampe e Uni e si y Hospi al Medical Resea ch Fund, and he Sig id Juselius Founda ion. AF was suppo ed by FY 2008 Resea che Exchange P og am be ween JSPS and Academy o Finland. We hank Tea Tuomela, Ou i Ku onen, Hanna Ojala, and E eliina Kaulio o echnical assis ance, and Susanna Valanne, Mika Räme , Ian Hol , Co y Dunn, B endan Ba e sby, Anu Suomalainen, and Lau ie Kaguni o use ul discussions and ad ice. Au ho con ibu ions AF ini ia ed he p ojec , conduc ed he p ima y sc een, and supe ised he implemen a ion and in e p e a ion o he seconda y sc eening. GC conduc ed he unc ional analysis o ATP syn hase knockdown. 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EMBO J 25:3618 –3626 License: This is an open access a icle unde he e ms o he C ea i e Commons A ibu ion 4.0 License, which pe mi s use, dis ibu ion and ep oduc- ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. ª2014 The Au ho s Molecula Sys ems Biology 10:734 |2014 A sushi Fukuoh e al m DNA main enance and ATP syn hase Molecula Sys ems Biology 21 Published online: June 21, 2014