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

Article Screen for mitochondrial DNA copy number maintenance genes reveals essential role for ATP synthase Atsushi Fukuoh 1,2,3,† , Giuseppe Cannino 1,† , Mike Gerards 1 , Suzanne Buckley 1 , Selena Kazancioglu 1 , Filippo Scialo 1 , Eero Lihavainen 4 , Andre Ribeiro 4 , Eric Dufour 1 & Howard T Jacobs 1,5,* Abstract The machinery of mitochondrial DNA (mtDNA) maintenance is only partially characterized and is of wide interest due to its involvement in disease. To identify novel components of this machinery, plus other cellular pathways required for mtDNA viability, we implemented a genome-wide RNAi screen in Drosophila S2 cells, assaying for loss of fluorescence of mtDNA nucleoids stained with the DNA-intercalating agent PicoGreen. In addition to previously characterized components of the mtDNA replication and transcription machineries, positives included many proteins of the cytosolic proteasome and ribosome (but not the mitoribosome), three proteins involved in vesicle transport, some other factors involved in mitochondrial biogenesis or nuclear gene expression, >30 mainly uncharacterized proteins and most subunits of ATP synthase (but no other OXPHOS complex). ATP synthase knockdown precipitated a burst of mitochondrial ROS production, followed by copy number depletion involving increased mitochondrial turnover, not dependent on the canonical autophagy machinery. Our findings will inform future studies of the apparatus and regulation of mtDNA maintenance, and the role of mitochondrial bioenergetics and signaling in modulating mtDNA copy number. Keywords complex V; DNA replication; mitochondrial biogenesis; mitochondrial DNA; mitophagy; nuclease; nucleoid; reactive oxygen species Subject Categories Methods & Resources; Metabolism DOI 10.15252/msb.20145117 | Received 12 January 2014 | Revised 2May 2014 | Accepted 2May 2014 Mol Syst Biol. (2014)10:734 Introduction Eukaryotes that use mitochondrial oxidative phosphorylation (OXPHOS) to generate ATP maintain a separate mitochondrial genome (mtDNA), encoding a subset of OXPHOS protein subunits, together with some components of the machinery of intramitochondrial protein synthesis. The maintenance and expression of mtDNA is otherwise dependent on nuclear-coded gene products, constituting a separate apparatus for genome maintenance and gene expression within the cell (McKinney & Oliveira, 2013). The core machinery of mtDNA replication is broadly conserved among eukaryotes. DNA replication is assumed to depend on the only DNA polymerase consistently found in mitochondria, DNA polymerase c(PolG; Kaguni, 2004), a member of the family A DNA polymerases (Ito & Braithwaite, 1991). Its closest prokaryotic homologue is the phage T7 DNA polymerase, and it is assumed to function in concert with the mitochondrial helicase Twinkle (Spelbrink et al, 2001), a homologue of phage T7 helicase-primase (gp4). Twinkle is absent from yeast, where other helicases are involved in mtDNA replication. Maintenance of mtDNA requires also mitochondrial transcription factors A (mt-TFA or TFAM), needed for mtDNA compaction and transcription (Larsson et al, 1998; Kang & Hamasaki, 2005; Campbell et al, 2012), and B (mt-TFB2, TFB2M; Matsushima et al, 2004). Three other proteins are essential for mtDNA maintenance, namely mtSSB, the mitochondrial single-stranded DNA-binding protein (Maier et al, 2001), RNase H1 (Cerritelli et al, 2003), and, in some organisms, a second DNA polymerase, PrimPol, that additionally has primase activity (Garcı ´a-Go ´mez et al, 2013). A number of other proteins are required to maintain normal mtDNA copy number or topology in different organisms (Contamine & Picard, 2000; Copeland, 2012). These include the DNA-binding AAA protein ATAD3A (He et al, 2007), some enzymes of nucleotide metabolism and transport (Saada, 2004), proteins with roles in mitochondrial membrane dynamics (Jones & Fangman, 1992; Wong et al, 2000; Elachouri et al, 2011; Vielhaber et al, 2013), chaperones (Ciesielski et al, 2013), exonucleases (Kornblum et al, 2013), proteases (Herlan et al, 2003; Matsushima et al, 2010; Sesaki et al, 2003), and even cytoskeletal proteins (Reyes et al, 2011). Mitochondria also contain 1BioMediTech and Tampere University Hospital, University of Tampere, Tampere, Finland 2Department of Clinical Chemistry and Laboratory Medicine, Kyushu University Graduate school of Medical Sciences, Fukuoka, Japan 3Department of Medical Laboratory Science, Junshin Gakuen University, Fukuoka, Japan 4Department of Signal Processing, Tampere University of Technology, Tampere, Finland 5Research Program of Molecular Neurology, University of Helsinki, Helsinki, Finland *Corresponding author. Tel: +358 3 3551 7731,+358 50 341 2894; E-mail: howard.t.jacob[email protected] † These authors equally contributed to this work. ª2014 The Authors. Published under the terms of the CC BY 4.0license Molecular Systems Biology 10:734 |2014 1 Published online: June 21, 2014 topoisomerases, ligases, and other nucleases, although their specific roles in mtDNA metabolism are unclear. While a crude DNA synthetic machinery can be reconstituted in vitro from a minimal set of these proteins, the full complement of proteins required for faithful mtDNA replication in vivo remains to be determined. Some components of the mtDNA maintenance machinery are shared with the nuclear compartment, including RNase H1 (Cerritelli et al, 2003) and many proteins implicated in base-excision repair (Alexeyev et al, 2013). Mostly, these are synthesized in two or more isoforms routed to different cellular compartments, for example, via differential splicing, alternative translational start sites (Suzuki et al, 2010), or ambiguous targeting signals (Karniely & Pines, 2005). mtDNA is packaged together with TFAM and some other proteins into discrete intramitochondrial structures of variable composition, called nucleoids, by analogy with those of bacteria (Spelbrink, 2010; Bogenhagen, 2012). They contain a number of replication proteins whose functional roles are poorly understood, as well as proteins implicated in other cellular processes, including metabolic enzymes and chaperones, and proteins involved in intramitochondrial protein synthesis (Hensen et al, 2014). The apparatus of mitochondrial translation has been functionally implicated in mtDNA maintenance in yeast (Contamine & Picard, 2000), though not in metazoan cells (Storrie & Attardi, 1972). The importance of mtDNA maintenance for cell physiology and homeostasis is underscored by the finding that its dysfunction leads to diverse types of human disease, including both infantile and lateonset pathologies, showing a bewildering variety of tissue specificities (Shadel, 2008; Ro ¨tig & Poulton, 2009; Ylikallio & Suomalainen, 2012). Loss of mitochondrial genome integrity or fidelity is also associated with aging (Oliveira et al, 2010; Bratic & Larsson, 2013). Identifying the full set of gene products involved in faithful mtDNA maintenance is thus of broad interest and importance. To this end, we implemented a genome-wide (blinded) screen of Drosophila S2 cells, using dsRNA-based RNA interference (RNAi), taking advantage of the fact that S2 cells tolerate loss of mtDNA and continue to grow within the time scale of a typical experiment, despite decreased OXPHOS capacity. Furthermore, mtDNA nucleoids may be identified in these cells on the basis of fluorescence signal from the topology-dependent DNA-intercalating dye PicoGreen (Ashley et al, 2005). Effects on mtDNA copy number were then probed further using quantitative PCR (QPCR), with additional experiments conducted on the cellular phenotypes produced by knockdown of specific genes identified in the screen, notably those encoding subunits of ATP synthase, in order to test aspects of the mechanisms by which they may act. Results and Discussion Implementation and outcome of the primary screen We set out to screen a genome-wide Drosophila dsRNA library in S2 cells, scoring for disappearance of the PicoGreen signal of mtDNA nucleoids as indicative of genes required for mtDNA maintenance. In initial trials, we found it difficult to pick out the nucleoid signal against background cytoplasmic fluorescence. Using a dsRNA against the PolG catalytic subunit (tamas) as a positive control, and a dsRNA directed against GFP as a negative control, we established a protocol whereby it was possible reliably to score (by eye) the disappearance of nucleoid signal (see Fig 1). This involved applying the test dsRNA for 5 days in a 96-well plate format, with addition on day 3 of a dsRNA directed against TFAM. Although prolonged incubation with TFAM dsRNA itself led to mtDNA depletion, the shorter-term treatment conversely enhanced the nucleoid signal in negative control cells, whereas it was decreased to very low levels in cells treated with the positive control dsRNA. The primary screen, conducted blind, was successful in identifying as positives most of the known factors involved in mtDNA metabolism (Table 1, category 1), giving confidence in its validity. In total, 105 dsRNA targets were initially judged as positive (Supplementary Table S1), of which almost half were recorded also as leading to cell death in a fraction of the cells. Consistent with previous studies (Ra ¨met et al, 2002; Boutros et al, 2004), a further 276 targets (Supplementary Tables S1 and S2) gave massive cell death but no specific loss of nucleoid signal and were considered to represent essential genes that could not be studied further. Finally, an additional 132 targets were judged to give an abnormal outcome without complete loss of PicoGreen nucleoid signal (Supplementary Tables S1 and S3), but were not analyzed further, although many fell into similar categories or pathways as those on the positives list. The specificity and knockdown efficiency of this procedure has previously been documented (Clemens et al, 2000; Kleino et al, 2005). Knockdown was here verified at the RNA level by qRT-PCR for 17 specific targets (see SI). We compared subjective judgment against a computational method to measure punctate fluorescence intensity (details to be published elsewhere). The latter gave many false positives due to variable background fluorescence, as well as false negatives due to cell debris. Based on rescreening, we judged the manual method to be superior (see SI for details), and we set criteria for defining positives as described below. Rescreening to identify definitive positives Positives were considered as confirmed if three positive but no negative findings were obtained. Those where a negative or ambiguous finding was recorded were retained only where three times as many clearly positive findings were obtained upon exhaustive rescreening (double asterisks in column H of Table 1) otherwise they were considered as false positives (double asterisks in column G of Supplementary Table S4). Five targets that did not give consistently positive findings during rescreening were noted to give rise to multiple splice variants, encoding at least one polypeptide predicted to be mitochondrially localized. For these, we tested dsRNAs targeted specifically on the relevant splice variants, confirming several additional positives (Table 1, green background), whereas dsRNAs targeted against other splice variants or the entire gene were judged negative (Supplementary Table S4, blue background). Of the original 105 positives, 83 were retained, 20 were reassigned as negative, and one was reassigned as abnormal (pink background in Supplementary Table S3). One was discarded because the dsRNA detected a pseudogene of a gene already in the list, another because a revised gene model combined it with another Molecular Systems Biology 10:734 |2014 ª2014 The Authors Molecular Systems Biology mtDNA maintenance and ATP synthase Atsushi Fukuoh et al 2 Published online: June 21, 2014 positive, while another was subsequently re-annotated as two separate genes (but shown as a single entry in Table 1). Positive findings were also obtained for specific splice isoforms of three of the negatives. Thus, the confirmation of 86 out of 106 initial targets indicates a false-positive rate of 18%. One additional positive (CG5794) was unexpectedly identified by a dsRNA nominally targeted against a different gene. The positives fell into seven distinct classes: mitochondrial DNA replication or transcription, cytosolic translation, the proteasome, ATP synthase, mitochondrial dynamics or biogenesis, nuclear gene expression, and a seventh, miscellaneous category. Although the primary negatives were not rescreened systematically, some that fell into similar functional pathways as definitive positives were re-evaluated using the same criteria. Eleven were promoted to the positives list (yellow background in Table 1), including, for example, most other subunits of ATP synthase. This implies that the initial screen may have missed as many positives as were actually retained, implying a false-negative rate of up to 1%. The overall results of the screen are summarized in Fig 2. The final number of definitive positives was 97, counting only once those with >1 positive splice variant. Copy number of mtDNA For genes on the definitive positives list, we carried out QPCR to assess changes in mtDNA copy number after 5 days of dsRNA treatment (without concomitant TFAM knockdown), normalized against a single-copy nuclear DNA standard. Based on this assay (see Table 1, column G, raw data in Supplementary Table S5), we classified the positives as showing substantial (++,≤60%) or modest (+) mtDNA depletion, no significant copy number change (0), or an increase in mtDNA (). Although these classes seem arbitrary, we found that RNAi knockdown of well-characterized components of the mtDNA replication machinery all gave values in the 20-60% range (++). Surprisingly, we identified only 6 new genes from the screen whose knockdown produced a comparably severe mtDNA depletion. Three of these encode subunits of ATP synthase. The others were CG5794, encoding a de-ubiquitinating enzyme with unknown substrate(s), TweedleY, previously identified as a cuticular protein (Guan et al, 2006), and pointed, a well-studied Ets family transcription factor (Klaes et al, 1994; Morimoto et al, 1996). The fact that knockdown GFP negative control mtDNA helicase (CG5924) positive (control) tamas (CG8987) positive control CG31380 negative CG6413 (Dis3) positive CG2028 (CkIα α ) abnormal (fewer dots) Figure 1. Screening of Drosophila dsRNA library by PicoGreen nucleoid fluorescence in S2cells. Micrographs of S2cells stained with PicoGreen, following 5days of treatment with the dsRNA indicated. GFP and tamas (Polg, CG8987) were used as negative and positive controls, respectively. In rescreening, CG5924 was also used as a positive control. Both were detected in the blinded screen as positives. Other images show a typical negative (CG31380), a typical positive (CG6413) and a typical case of a target classed as abnormal, in this case CG2028 (CkIa), which showed a decreased number of nucleoid signals per cell. Images are optimized for brightness and contrast but with no other manipulations. Scale bar indicates 50 lm. ª2014 The Authors Molecular Systems Biology 10:734 |2014 Atsushi Fukuoh et al mtDNA maintenance and ATP synthase Molecular Systems Biology 3 Published online: June 21, 2014 Table 1. Definitive positives Category number Category name CG number Official name or symbol Human orthologue Other or common name(s) or putative function mtDNA depletion >3rescreenings Note 1mtDNA replication and transcription CG3910 mtTFB2TFB2M mt transcription factor B2+* CG4217 TFAM TFAM mtTFA, mt transcription factor A ++ CG4337 mtSSB SSBP1mt single-stranded DNA-binding protein ++ CG4644 mtRNApol POLRMT mt RNA polymerase ++ CG5924 CG5924 PEO1Twinkle DNA helicase ++ CG6815 belphegor ATAD3A, B, C AAA domain containing protein 30** CG7175 mTerf5mt transcription termination factor 5++ CG8729 rnh1RNASEH1Ribonuclease H10** Weak positive, diminished nucleoid signal seen in some cells, also some cell death CG8987 tamas POLG DNA polymerase ccatalytic subunit ++ CG18124 mTTF mt transcription termination factor ++ CG33650 DNApol-c35 POLG2DNA polymerase caccessory subunit, CG8969 +** 2Cytosolic translation CG1821 RpL31 RPL31 Large subunit – CG3203 RpL17 RPL17 Large subunit – CG3751 RpS24 RPS24 Small subunit nt CG3922 RpS17 RPS17, RPS17L Small subunit – CG3997 RpL39 RPL39, RPL39L Large subunit 0 CG4111 RpL35 RPL35 Large subunit nt CG4759 RpL27 RPL27 Large subunit nt CG7283 RpL10Ab RPL10A Large subunit nt CG7490 RpLP0RPL0Large subunit nt CG7622 RpL36 RPL36 Large subunit –* CG7726 RpL11 RPL11 Large subunit nt * Cell death not substantial in some repeats CG8922 RpS5a RPS5Small subunit nt * CG9282 RpL24 RPL24 Large subunit nt * CG9677 Int6EIF3E eIF3, Initiation factor 3, subunit E nt * Abnormal distribution of some residual nucleoid signal within cells CG11522 RpL6RPL6Large subunit Molecular Systems Biology 10:734 |2014 ª2014 The Authors Molecular Systems Biology mtDNA maintenance and ATP synthase Atsushi Fukuoh et al 4 Published online: June 21, 2014 Table 1(Continued) Category number Category name CG number Official name or symbol Human orthologue Other or common name(s) or putative function mtDNA depletion >3rescreenings Note 3Proteasome CG1341 Rpt1PSMC2,5,6ATPase regulatory subunit 2(or 5or 6)+ CG5266 Pros25 PSMA2Core a-type subunit 2+** CG9324 Pomp POMP Proteasome maturation protein – CG9327 Pros29 PSMA4Core a-type subunit 4+* CG10149 Rpn6PSMD11 Non-ATPase regulatory subunit 11 + CG16916 Rpt3PSMC4ATPase regulatory subunit 4+ CG18174 Rpn11 PSMD14 Non-ATPase regulatory subunit 14 0 ** Severe cell death: few cells remained after 5days 4ATP synthase CG2968 l(1)G0230 ATP5DdSubunit of F1(stalk) + CG3321 CG3321 ATP5I Subunit e of Fo, dimerization/bending ++ CG4307 Oscp ATP5O OSCP subunit of F1/stator arm ++ ** CG4412 ATPsyn-Cf6ATP5J Coupling factor 6, subunit of Fo/stator arm + CG6105 l(2)06225 ATP5L, ATP5L2Subunit g of Fo, dimerization/bending + CG7610 ATPsyn-cATP5C1cSubunit of F1(stalk) +* CG6030 ATPsyn-d ATP5H Subunit d of Fo, stator arm +* CG8189 ATPsyn-b ATP5F1Subunit b of Fo, stator arm ++ * CG11154 ATPsyn-bATP5B Core bsubunit of F1+** 5Mitochondrial biogenesis and dynamics CG3114 erect wing NRF1Nuclear respiratory factor 1homologue 0** CG6338 Ets97D GABPA Nuclear respiratory factor 2,asubunit +** CG6512 CG6512 AFG3L2, SPG7m-AAA protease subunit 0* CG8479 opa1-like OPA1Dynamin-related protein required for inner mt membrane fusion +* CG9809 spargel PPARGC1A, B, PPRC1 PPAR ccoactivator +** Weak positive, diminished number of nucleoid signals remaining in some cells CG14981 maggie TOMM22 Tomm 22 subunit of outer mt membrane translocase + 6Nuclear gene expression CG1057 MED31 MED31 Mediator complex subunit 31, trancriptional elongation 0 CG1554 RpII215 POLR2A RNA polymerase II 215kD subunit + CG1810 mRNA-cap RNGTT mRNA capping enzyme 0 CG1874 Not1CNOT1CCR4-NOT transcription complex subunit 10 ** Weak positive, diminished number of nucleoid signals remaining in some cells, some cell death also evident ª2014 The Authors Molecular Systems Biology 10:734 |2014 Atsushi Fukuoh et al mtDNA maintenance and ATP synthase Molecular Systems Biology 5 Published online: June 21, 2014 Table 1(Continued) Category number Category name CG number Official name or symbol Human orthologue Other or common name(s) or putative function mtDNA depletion >3rescreenings Note CG2163 Pabp2PABPN1, PABPN1L Nuclear poly(A)-binding protein – CG3162 LS2Novel U2AF-related regulator of differential splicing 0 CG3675 Art2PRMT6Protein arginine methyltransferase 2+ CG6525 pps SPOCD1, PHF3 Protein partner of snf, regulator of alternative splicing of Sex lethal +* Diminished number of nucleoids remained in many cells CG7626 Spt5SUPT5H Transcription elongation factor SPT5(DSIF complex) 0 CG9591 omd INTS5Integrator complex subunit 5, snRNA processing factor +* Very few cells remained, after 5days of treatment CG9748 belle DDX3X,Y,; DDX4 RNA helicase, implicated in X-chromosome dosage compensation 0 CG10955 Rtf1RTF1Component of RNA polymerase II-associated (PAF1) complex, transcriptional elongation 0** CG11990 hyrax CDC73 Component of RNA polymerase II-associated (PAF1) complex, transcriptional elongation 0* CG17183 MED30 MED30 Mediator complex subunit 30, trancriptional elongation 0** CG17358 Taf12 TAF12 TATA box-binding protein-associated factor 12 + CG17603 Taf1TAF1, TAF1L TATA box-binding protein-associated factor 1+ 7Miscellaneous (other or unknown) CG3539 SLY-1 homologue SCFD1ER to Golgi vesicle transport 0** CG4268 Pitslre CDK11A Cyclin-dependent kinase superfamily, regulator of autophagy – CG5794 CG5794 USP34 Ubiquitin-specific peptidase (deubiquitinating enzyme) ++ * CG6413 Dis3DIS3Exosome complex exoribonuclease –* CG7368 CG7368 Zn finger protein –** CG8021 CG8021 SLIRP RNA-binding protein, regulation of mt RNA levels 0 CG9007 upSET SETD5, MLL5 Zn finger protein, related to trithorax and histone lysine methlytransferases – CG9397-H jing AEBP2Zn finger transcription factor, role in morphogenesis –** Was clearly positive in 5out of 7trials Molecular Systems Biology 10:734 |2014 ª2014 The Authors Molecular Systems Biology mtDNA maintenance and ATP synthase Atsushi Fukuoh et al 6 Published online: June 21, 2014 Table 1(Continued) Category number Category name CG number Official name or symbol Human orthologue Other or common name(s) or putative function mtDNA depletion >3rescreenings Note CG9797 CG9797 Zn finger protein 0** Weak positive, diminished number of nucleoid signals remaining in some cells CG10042 MBD-R2Methyl-DNA-binding protein 0Some small nucleoid signals remained in some cells. Another dsRNA for this gene gave consistently negative findings CG10144 CG10144 VPS8Vesicle sorting to lysosomes +** Nucleoid signals of diminished size or intensity seen in some cells, few cells remained after 5d CG10395 CG10395 Zn finger protein 0 CG10582 Sin POLR3E Sex-lethal interactor, possible alternative splicing, proposed subunit of RNA polymerase III 0* CG10582-C Sin Putative mitochondrially targeted isoform of Sin 0** Clear positive in 3trials, but also some inconsistent findings CG12242 GstD5Glutathione S-transferase superfamily (+) Depletion just outside the border of significance, due to large variance CG13203-C CG13203 Putative mitochondrially targeted isoform of protein with unknown function –** CG13779 Sem1Possible endopeptidase 0** CG14084 Bet1BET1ER to Golgi vesicle transport 0* CG14247 CG14247 Unknown function 0* CG14634 CG14634 Unknown +** CG15231 IM4Immune-induced molecule, peptide hormone? +* CG15343 CG15343 Pyridoxamine 50-phosphate oxidase-like 0* CG15793 Dsor1MAP2K1,2,5MAP kinase kinase 0* CG17077 pointed ETS1,2Ets transcription factor ++ * CG31258 Cenp-C Centromere-binding, kinetochore function nt * ª2014 The Authors Molecular Systems Biology 10:734 |2014 Atsushi Fukuoh et al mtDNA maintenance and ATP synthase Molecular Systems Biology 7 Published online: June 21, 2014 Table 1(Continued) Category number Category name CG number Official name or symbol Human orthologue Other or common name(s) or putative function mtDNA depletion >3rescreenings Note CG31079 Unknown (gene model later withdrawn from Flybase) nt Predominantly induced cell death, few cells remaining seemed positive CG32085 CG32085 FBXL16 Putative ubiquitin ligase 0 CG32561, 2 xmas-1, xmas-2MCM3AP (xmas-2) Putative protein acetyltransferase involved in DNA replication (xmas-2) 0** Clear positive in 4trials, but also some inconsistent findings CG32570 TwdlY Suggested cuticular protein ++ * CG32652 CG32652 Unknown +* CG34415 mute Muscle wasted, chromatin protein, histone locus body 0** Weak positive, diminished nucleoid signal seen in some cells, cell death seen only in some trials CG33546 gfzf GST-containing Zn finger protein, putative mitotic checkpoint protein 0 CG42666-G CG42666 REXO1Exoribonuclease, predicted mitochondrially targeted isoform nt ** Weak positive, diminished number of nucleoid signals remained in some cells, dsRNA for entire gene was an inconsistent weak positive; see also Table S1. isoform-specific primers also target Adar, CG12598 CG42666-D CG42666 REXO1Exoribonuclease, predicted mitochondrially targeted isoform 0* Weak positive, diminished number of nucleoid signals remained in some cells, dsRNA for entire gene was an inconsistent weak positive; see also Table S2 CG42281 bunched Signal transduction in response to growth factor (dpp) binding – =not in original positives list; =positive isoform with putative mt targeting; =gene model withdrawn from Flybase. Positives also showing substantial cell death are shown in underline and italic. >3rescreenings: Positives that required many rounds of rescreening for final validation. * = 4rounds; ** ≥8rounds. Molecular Systems Biology 10:734 |2014 ª2014 The Authors Molecular Systems Biology mtDNA maintenance and ATP synthase Atsushi Fukuoh et al 8 Published online: June 21, 2014 of many genes resulted in loss of PicoGreen signal without major changes in mtDNA copy number suggests that indirect effects may be common, for example, affecting DNA topology, nucleoid architecture, membrane potential or cellular dye uptake. Positives implicated in mtDNA metabolism The positives include most of the proteins with known roles in mtDNA replication or transcription, notably the five shown previously to be essential for mtDNA maintenance in Drosophila (Goto et al, 2001; Maier et al, 2001; Iyengar et al, 2002; Matsushima et al, 2004; Humphrey et al, 2012). The list comprises the two subunits of PolG, the catalytic subunit of the mitochondrial RNA polymerase, mtSSB, the Drosophila homologue of the Twinkle helicase, transcription factors TFAM and mtTFB2M, mTERF family members mTTF and mTerf5 (Jo ˜ers et al, 2013), plus rnh1 (RNaseH1) and belphegor (homologue of mammalian ATAD3). All gave significant mtDNA depletion except for rnh1 and belphegor. DNA ligase III (lig3, CG17227) and mTERF family members mTerf3 and CG15390 (homologue of mammalian MTERF4) were consistently negative. DNA ligase III was previously reported as dispensable for nuclear DNA repair but essential for mtDNA maintenance in human cells (Ruhanen et al, 2011) and mouse (Puebla-Osorio et al, 2006; Gao et al, 2011). Our data imply that it is redundant to at least one other mtDNA ligase in Drosophila. Similar arguments may apply to the absence of any topoisomerase, gyrase, recombinase, resolvase or helicase (other than Twinkle). Our study suggests that few dedicated components of the mtDNA replication apparatus remain to be identified, but this does not exclude factors with overlapping roles in other cell compartments. In mammalian mitochondria, the transcriptional apparatus is considered essential for both leadingand lagging-strand synthesis (Clayton, 1982; Fuste et al, 2010). RNase H1, also required for mtDNA maintenance in mouse (Cerritelli et al, 2003) and human cells (Ruhanen et al, 2011), might be involved in primer removal, but this typically also needs other helicases and nucleases such as Fen1 (CG8648) and Dna2 (CG2990), both implicated in mtDNA replication in mammalian cells (Duxin et al, 2009; Kazak et al, 2013). Their absence from the positives list is unsurprising, however, since both also function in the nucleus. One positive from the miscellaneous category, CG8021, appears to be a Drosophila homologue of SLRP, a mammalian protein involved in mitochondrial mRNA stabilization and processing (Sasarman et al, 2010; Chujo et al, 2012). The mammalian ATAD3 family has been implicated in nucleoid organization (He et al, 2007), mitochondrial protein synthesis (He et al, 2012), regulation of apoptosis (Huang et al, 2011) and autophagy (Chen et al, 2011), cholesterol trafficking (Rone et al, 2012), mitochondrial dynamics (Gilquin et al, 2010) and stress resistance (Hoffmann et al, 2012). Loss of PicoGreen nucleoid signal with only a minor drop in mtDNA copy number may indicate that belphegor functions also in diverse pathways and that its effects on nucleoids and mtDNA may be indirect. Nucleases other than RNase H1 have been shown or suggested to have roles in mtDNA metabolism in various organisms, including EXOG (Tann et al, 2011), EndoG (McDermott-Roe et al, 2011) and yeast Exo5 (Burgers et al, 2010). Exo5 has no Drosophila NEGATIVE POSITIVE ABNORMAL CELL DEATH Primary screen 105 276 132 15,506 16,019 dsRNAs Rescreen DEFINITIVE POSITIVES 1 83 + 3 with splice variants 11 Total = 97 10 Figure 2. Overall results of the screen. Schematic diagram illustrating number of dsRNAs analyzed, numbers of primary positives, negatives and other classes, and the results of rescreening. ª2014 The Authors Molecular Systems Biology 10:734 |2014 Atsushi Fukuoh et al mtDNA maintenance and ATP synthase Molecular Systems Biology 9 Published online: June 21, 2014 Bogenhagen, 2012). The involvement of cV, the proteasome, and some key genes for mitochondrial dynamics and quality control suggests that mtDNA copy number is dependent on a balance between mitochondrial turnover and biogenesis, in which specific stresses, notably mitochondrial ROS production and impaired cytosolic protein turnover, may be crucially important. ATP synthase was inferred to be a key player in homeostatic maintenance of mitochondria, and thus in the amount of mtDNA and its gene products. Mutations in ATP synthase in fungi are already known to play a determining role in whether mtDNA loss can be tolerated (Contamine & Picard, 2000; Lefebvre-Legendre et al, 2003), or even facilitated (Giraud & Velours, 1997; Lai-Zhang et al, 1999; Contamine & Picard, 2000), but with membrane potential implicated as a key parameter (Duvezin-Caubet et al, 2006; Wang et al, 2007). In S2 cells, excess ROS production was better correlated with the strength and kinetics of mtDNA depletion, but this could be an epi-phenomenon. The fact that membrane potential ‘per mitochondrion’ (Supplementary Fig S4D) was restored to its starting value suggests that its disturbance may yet prove to be the primary inducer. Excess ROS has elsewhere been proposed to lead to mtDNA depletion under pathological conditions (Larosche et al, 2010; Quinzii et al, 2013), and pathological defects in ATP synthase associated with ROS overproduction (Baracca et al, 2007) may downregulate mitochondrial functions (Wojewoda et al, 2010, 2011) and even lead to mtDNA loss (Vergani et al, 1999; Turner et al, 2005). ROS overproduction has been widely suggested both to provoke mtDNA damage, but also to result from it. However, a recent report showed that unrepaired damage leads to mtDNA depletion without increased ROS (Shokolenko et al, 2013), and the role of ROS in producing somatic mtDNA mutations in the PolgA mutator mouse is disputed (Trifunovic et al, 2005; Dai et al, 2010). Thus, the increased mitochondrial ROS seen when cV is knocked down is more logically a cause than a consequence of mtDNA depletion. Furthermore, although ROS may provoke strand breakage, interfering directly with mtDNA replication (Han & Chen, 2013), our data instead suggest that ROS activates mitochondrial turnover before widespread DNA damage would be sustained, as occurs in mammalian cells under TNFasignaling (Nagakawa et al, 2005; Vadrot et al, 2012). However, an opposing pathway has also been suggested, in which ROS over-production promotes mitochondrial biogenesis, not turnover (MorenoLoshuertos et al, 2006, 2011). A key aim of future research will be to identify the sensor molecule(s) integrating changes in mitochondrial ROS (or membrane potential) with other metabolic signals, in order to modulate mitochondrial biogenesis and turnover. One possibility consistent with our data is that ATP synthase itself is that sensor. Materials and Methods Cell maintenance Drosophila S2 cells (Invitrogen) were cultured under standard conditions, in Schneider0s medium (Sigma) and diluted 1:6 every 3-4 d. In selected experiments, various drugs were added or glucose was replaced with the same concentration of galactose. S2 cells stably expressing Ciona intestinalis AOX were generated by co-transfection with a plasmid conferring hygromycin resistance. Screening of Drosophila dsRNA library and fluorescence microscopy of nucleoids S2 cells were seeded into 96-well plates and treated over 5 days with 0.6–1.2 lg of dsRNA from the library (Open BioSystems), alongside positive and negative controls in each plate, as described previously (Jo ˜ers et al, 2013). Nucleoids were visualized by fluorescence microscopy after staining with Quant-iT TM PicoGreen dsDNA reagent (7.5 ll/ml, Invitrogen). Larger-scale dsRNA treatments for mtDNA copy number evaluation and analysis of cellular parameters were performed essentially as previously (Jo ˜ers et al, 2013). Nucleic acid isolation and QPCR Total RNA was isolated from S2 cells as previously (Jo ˜ers et al, 2013). For DNA isolation, cells from a single well of a 24-well plate were processed by a procedure determined to give consistent results irrespective of cell density, involving SDS lysis, proteinase K digestion, isopropanol precipitation and overnight resuspension at 55°C (see SI). For larger-scale experiments, DNA was prepared from 1.5 ×10 6 cells cultured in 6-well plates, as previously (Jo ˜ers et al, 2013). Mitochondrial DNA copy number was assessed by QPCR using primers against COXII or 16S rRNA (for mtDNA) and RpL32 (nuclear DNA, single-copy, for normalization). Transcript levels were estimated relative to that of RpL32 by a similar procedure, but using cDNA as template. Measurements of mitochondrial function Mitochondrial membrane potential, ROS level, and content per cell were determined by flow cytometry (Cannino et al, 2012) of cells stained, respectively, with 200 mM tetramethylrhodamine methyl ester (TMRM), 2.5 lM MitoSox TM (Invitrogen), or either 200 nM 10-nonyl acridine orange (NAO) or 40 nM MitoTracker Green FM (Life Technologies). Oxygen consumption of living cells (Cannino et al, 2012) was measured using a Clark-type electrode (Hansatech Oxyterm system). Analyses of lysosomal and mitochondrial content Lysosome content per cell was measured by flow cytometry of cells stained with 50 nM LysoTracker Red DND-99 (Life Technologies). MitoTracker Green FM (Molecular Probes) and LysoTracker Red DND-99 were used for live cell imaging by confocal microscopy of mitochondria and lysosomes, respectively, with spot-area calculation (ImageJ) and image deconvolution (SVI, Huygens software). Western blotting Post-nuclear extracts resolved by SDS-PAGE were electroblotted and probed using standard methods (essentially as Fernandez-Ayala et al, 2009; see SI). Primary antibodies used were against NDUFS3 (Abcam, mouse), 1:10,000), ATP5A (Abcam, mouse, 1:1,000), and GAPDH (Everest Biotech, goat, 1:2,000), with appropriate horseradish peroxidase-conjugated secondary antibodies. Visualization Molecular Systems Biology 10:734 |2014 ª2014 The Authors Molecular Systems Biology mtDNA maintenance and ATP synthase Atsushi Fukuoh et al 16 Published online: June 21, 2014 used the ECL system (Amersham Biosciences) according to the manufacturer’s protocols. Statistical analyses Comparisons between populations were performed using unpaired two-tailed Student’s t-tests or analyses of variance when more than two samples were compared, with Bonferroni-corrected post hoc t-test. For further details, see Supplementary Materials and Methods. Data availability Original images from the primary screen are deposited at: http:// dx.doi.org/10.5061/dryad.v55p5. Supplementary information for this article is available online: http://msb.embopress.org Acknowledgements This work was supported by funding from the Academy of Finland, Tampere University Hospital Medical Research Fund, and the Sigrid Juselius Foundation. AF was supported by FY 2008 Researcher Exchange Program between JSPS and Academy of Finland. We thank Tea Tuomela, Outi Kurronen, Hanna Ojala, and Eveliina Kaulio for technical assistance, and Susanna Valanne, Mika Rämet, Ian Holt, Cory Dunn, Brendan Battersby, Anu Suomalainen, and Laurie Kaguni for useful discussions and advice. Author contributions AF initiated the project, conducted the primary screen, and supervised the implementation and interpretation of the secondary screening. GC conducted the functional analysis of ATP synthase knockdown. 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EMBO J 25:3618 –3626 License: This is an open access article under the terms of the Creative Commons Attribution 4.0 License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. ª2014 The Authors Molecular Systems Biology 10:734 |2014 Atsushi Fukuoh et al mtDNA maintenance and ATP synthase Molecular Systems Biology 21 Published online: June 21, 2014