RNase H1 promotes replication fork progression through oppositely transcribed regions of Drosophila mitochondrial DNA

RNase H1 promotes replication fork progression through oppositely transcribed regions of Drosophila mitochondrial DNA Received for publication, December 5, 2018, and in revised form, January 9, 2019 Published, Papers in Press, January 11, 2019, DOI 10.1074/jbc.RA118.007015 Jose M. González de Cózar ‡ , Mike Gerards ‡1 , Eveliina Teeri ‡ , Jack George ‡ , Eric Dufour ‡ , Howard T. Jacobs ‡§2 , and Priit Jõers ‡¶ From the ‡ Faculty of Medicine and Health Technology and Tampere University Hospital, FI-33014 Tampere University, Finland, § Institute of Biotechnology, FI-00014 University of Helsinki, Finland, and ¶ Institute of Molecular and Cell Biology, University of Tartu, Riia 23, 51010 Tartu, Estonia Edited by Patrick Sung Mitochondrial DNA (mtDNA) replication uses a simple core machinery similar to those of bacterial viruses and plasmids, but its components are challenging to unravel. Here, we found that, as in mammals, the single Drosophila gene for RNase H1 (rnh1) has alternative translational start sites, resulting in two polypeptides, targeted to either mitochondria or the nucleus. RNAi-mediated rnh1 knockdown did not influence growth or viability of S2 cells, but compromised mtDNA integrity and copy number. rnh1 knockdown in intact flies also produced a phenotype of impaired mitochondrial function, characterized by respiratory chain deficiency, locomotor dysfunction, and decreased lifespan. Its overexpression in S2 cells resulted in cell lethality after 5–9 days, attributable to the nuclearly localized isoform. rnh1 knockdown and overexpression produced opposite effects on mtDNA replication intermediates. The most pronounced effects were seen in genome regions beyond the major replication pauses where the replication fork needs to progress through a gene cluster that is transcribed in the opposite direction. RNase H1 deficiency led to an accumulation of replication intermediates in these zones, abundant mtDNA molecules joined by fourway junctions, and species consistent with fork regression from the origin. These findings indicate replication stalling due to the presence of unprocessed RNA/DNA heteroduplexes, potentially leading to the degradation of collapsed forks or to replication restart by a mechanism involving strand invasion. Both mitochondrial RNA and DNA syntheses were affected by rnh1 knockdown, suggesting that RNase H1 also plays a role in integrating or coregulating these processes in Drosophila mitochondria. Mitochondrial DNA (mtDNA) 3 replication utilizes a relatively simple core machinery, akin to those of bacterial viruses and plasmids. It proceeds via mechanisms that reflect this but in other respects exhibits unique features (1, 2). The mitochondrial genome of metazoans has been the most intensively studied (1), in part because of its relevance to human pathology (3). Its replication depends on a small set of well-studied proteins, including a dedicated DNA polymerase (Pol ␥ ), RNA polymerase, and DNA helicase, all related to those of the T-odd bacteriophages (4), as well as other proteins involved in DNA compaction and transcription (2). The latter notably includes mitochondrial transcription factor A (TFAM) (5, 6), which functions in both processes (7–10), several other transcription factors (11–13), the Pol ␥ accessory subunit (14–16), and the mitochondrial homologue of bacterial single-strand DNAbinding protein, mtSSB (17, 18). The full set of proteins required for mtDNA replication in vivo and their precise functions have not yet been defined. The study of mtDNA replication intermediates by EM, twodimensional neutral agarose gel electrophoresis (2DNAGE), and other methods (19–22), plus the use of cell-free replication systems in vitro (23, 24) and in organello (25, 26), has indicated a multiplicity of roles in the replication process for RNA and for transient RNA/DNA heteroduplexes. It remains unclear whether the observed heteroduplexes reflect aspects of a single mtDNA replication process or several such mechanisms operating in parallel. Furthermore, their significance, if any, for RNA synthesis is unknown. In seeking to identify all of the components of the mtDNA replication machinery, whether by genetic or biochemical means, an inherent problem arises if a relevant gene product also functions in the cell nucleus. One example that has attracted our attention is RNase H1 (27), mutants of which are associated with human disease (28, 29) and which in the mouse This work was supported by Academy of Finland Centre of Excellence Grant 272376 and Academy Professorship Grant 256615 (to H. T. J.), Estonian Research Council Grant PUT573 (to P. J.), the University of Tampere, the Tampere University Hospital Medical Research Fund, and the Sigrid Juselius Foundation. The authors declare that they have no conflicts of interest with the contents of this article. Author’s Choice—Final version open access under the terms of the Creative Commons CC-BY license. This article contains Figs. S1–S8, Table S1, and Movie S1. 1 Present address: Maastricht Center for Systems Biology (MaCSBio), Maastricht University, 6229 ER Maastricht, The Netherlands. 2 To whom correspondence should be addressed. Tel.: 358-50-341-2894; E-mail: [email protected]. 3 The abbreviations used are: mtDNA, mitochondrial DNA; Pol, polymerase; TFAM, mitochondrial transcription factor A; 2DNAGE, two-dimensional neutral agarose gel electrophoresis; NCR, noncoding region; NLS, nuclear localization signal; qPCR, quantitative PCR; Exo, exonuclease; ssDNA, single-stranded DNA; RI, replication intermediate; mTTF, mitochondrial transcription termination factor (MTERF)-related protein; bs, binding site; VDRC, Vienna Drosophila Resource Centre; Cox, cytochrome coxidase; DAPI, 4⬘,6-diamidino-2-phenylindole; qRT-PCR, quantitative RT-PCR. cro ARTICLE Author’s Choice J. Biol. Chem. (2019) 294(12) 4331–4344 4331 © 2019 González de Cózar et al. Published by The American Society for Biochemistry and Molecular Biology, Inc. at Tampere University Library on June 12, 2019http://www.jbc.org/Downloaded from is required to maintain mtDNA both in vivo and in cultured cells (30, 31). RNase H1 cleaves the RNA strand of RNA/DNA hybrids, requiring as substrate at least four consecutive ribonucleotides (32). The mammalian gene for RNase H1 encodes two variant polypeptides, which have been shown to be targeted, respectively, to the nucleus or to mitochondria, by virtue of alternate translation starts that define distinct N-terminal peptide sequences (27), with tight regulation by a short upstream ORF. In the mouse, deletion of the Rnaseh1 gene results in embryonic lethality at day 8.5 (30), a phenotype similar to that seen in mice lacking other essential components of the mtDNA replication apparatus such as Pol ␥ (33) or TFAM (5). However, this does not constitute formal proof that the lethality is due to effects on mtDNA alone, and the exact role of the enzyme in each compartment has not yet been fully defined. Moreover, the physiological function of the enzyme, if any, in the adult could not be ascertained. In mammalian mitochondria, RNase H1 has been inferred to remove RNA fragments believed to represent unprocessed primers, a process required for the completion of mtDNA replication (31), and to facilitate the separation of daughter copies (29) as well as some aspects of rRNA processing (34). In mammals, the RITOLS mode of mtDNA replication (transient RNA incorporation throughout the lagging strand; Refs. 20 and 21) also has an intrinsic requirement for the processing of intermediates containing tracts of RNA/DNA heteroduplex. In the nucleus, RNase H1 is required to eliminate persistent heteroduplexes that impair transcription (35) or impede fork progression during DNA replication (36), leading to genome instability. It has also been suggested to play roles in other processes, such as telomere maintenance (37), retroelement surveillance (19), and DNA repair (38), as well as facilitating somatic hypermutation in the immunoglobulin locus (39). The Drosophila mitochondrial genome is similar to that of vertebrates, except for two major inversions that balance the coding capacity of the two strands. The extended noncoding region (NCR) in which the replication origin is embedded (40, 41) is also unusually A ⫹T–rich (⬃95%) and contains several long repeat elements, clustered in two blocks. Drosophila mtDNA follows the unidirectional, ␪ -type replication model also seen in mammals with some minor, but intriguing differences. The initial portion of the genome appears to be replicated by strand displacement, with delayed lagging-strand synthesis (40), as proposed for mammalian mtDNA (42). However, beyond a site near the start of the coding region, replication switches to a strand-coupled mode (40). In contrast to mammals, where this can entail the incorporation of extended tracts of lagging-strand RNA, only short heteroduplexes are formed transiently at the replication fork (13) during elongation. Drosophila is a convenient model organism in which to study mtDNA maintenance because of the plethora of genetic tools available as well as the fact that the most commonly used Drosophila cell line (Schneider S2 cells) is able to survive for long periods essentially without mtDNA-encoded functions. S2 cells are also susceptible to highly effective and specific RNAi brought about by long double-stranded RNAs (dsRNAs). In an RNAi screen for Drosophila genes required for the maintenance of mtDNA nucleoids in S2 cells (43), we identified those encoding the previously identified “core set” of replication proteins, whose knockdown by RNAi resulted in depletion of mtDNA over 5 days in culture by at least 50%. In contrast, knockdown of RNase H1 (encoded by the rnh1 gene) did not result in significant mtDNA depletion over this period, nor did it cause cell death, although it did abolish the topology-dependent staining of mtDNA nucleoids by the dye Pico Green (43). Nevertheless, an earlier study identified rnh1 as an essential gene for the completion of development in the fly (44). To unravel the potential roles of rnh1 in Drosophila and in particular its relevance to mtDNA metabolism and to the physiology of the adult, we first created reporter constructs to test its subcellular localization. After confirming that it is targeted to mitochondria as well as to the nucleus, we examined the effects on cell and organismal phenotype and on mtDNA of its knockdown or overexpression. Our findings imply that RNase H1 is required to prevent or resolve conflicts between opposed mtDNA transcription and replication in addition to a role at the origin analogous to that inferred previously in mammalian mtDNA. We infer that RNase H1 is essential to ensure faithful genome maintenance and expression by removing heteroduplex from critical regions. Furthermore, these mitochondrial functions of the enzyme are inferred to be essential to the maintenance of locomotor competence and lifespan in the adult fly. Results RNaseH1 is targeted to both mitochondria and the nucleus in Drosophila Having verified that only one mRNA isoform of rnh1 mRNA is detectable in S2 cells or flies (Fig. S1B), we proceeded to investigate the subcellular localization of the encoded protein by a C-terminal epitope-tagging approach (Fig. 1, Aand B). In transient transfections, most cells showed staining in both the nucleus and mitochondria (colocalized with cytochrome coxidase subunit 4 (Cox4)), with a minority showing exclusively mitochondrial localization and a small number (fewer than 2%) exclusively nuclear. Dual targeting was confirmed by Western blotting of subcellular fractions highly enriched for either nuclear or mitochondrial markers (Fig. 1C). The N-terminal region of the rnh1 mRNA contains three AUGs, arranged similarly as in mammals (Fig. S2A). The first, shown in purple in Fig. S2A, defines a short upstream ORF unrelated to RNase H1 protein, which is considered a signature of translational regulation (45). The remaining two AUGs are in-frame with each other and potentially specify variant polypeptides that differ in their likelihood of being targeted to mitochondria according to three commonly used prediction programs (Fig. S2B). To determine whether either of these two AUGs was able to be used as a start codon and how this influenced subcellular targeting, we studied two further epitope-tagged variants with either of the methionine codons at positions 1 and 16 altered to a valine codon. The M1V variant was exclusively localized to nuclei (Fig. 2Aand S2D), whereas the M16V variant was mainly in mitochondria (Fig. 2A,S2D) with approximately one-third of the cells showing a lower, but still detectable signal in the nucleus. Deletion of the putative nuclear localization signal (Fig. S2C,⌬NLS) resulted in purely mitochondrial targeting RNase H1 and Drosophila mtDNA replication 4332 J. Biol. Chem. (2019) 294(12) 4331–4344 at Tampere University Library on June 12, 2019http://www.jbc.org/Downloaded from (Fig. 2, Aand B) despite the presence of both putative start codons. Western blotting using the intact or M16V construct detected two polypeptides, whereas M1V and ⌬NLS gave only one (Fig. S2E). Cell synchronization in G 1 by treatment with hydroxyurea for 24 h (Fig. S2F)orinG 2 by treatment with ponasterone A (Fig. S2F) revealed no significant differences in the subcellular localization of epitope-tagged RNase H1 compared with unsynchronized growing cells (Fig. 2C). RNase H1 deficiency produces mitochondrial defects The prominent mitochondrial localization of epitope-tagged RNase H1 in S2 cells prompted us to analyze more closely the effects on mitochondria of manipulating its expression. We first verified the effectiveness and persistence of rnh1 knockdown at the RNA level (Fig. S3A) and confirmed that there was no discernible effect on cell growth (Fig. 3A) or viability (Fig. 3B). Although 5 days of knockdown produced no significant mtDNA depletion (Ref. 43 and Fig. 3C), prolonged knockdown under optimal culture conditions did cause a significant drop in mtDNA copy number after 10 days (Fig. 3C). Because S2 cells are largely resistant to mitochondrial defects, including mtDNA depletion (43), and a null mutation in rnh1 is lethal in flies, we proceeded to analyze the effects of rnh1 knockdown at the whole-organism level using the ubiquiFigure 1. Subcellular localization of epitope-tagged RNase H1. A, immunocytochemistry of cells transiently transfected with RNase H1-V5, probed for the V5 epitope tag (red), Cox4 (green), and DAPI (blue), showing examples of the three types of intracellular distribution of V5-tagged RNase H1: nucleus and mitochondria (i), mitochondria only (ii), and nucleus only (iii). B, subcellular distribution of RNase H1-V5 in 100 transfected cells as indicated (mean of three experiments, error bars denote S.D.). C, Western blots of subcellular fractions from cells transfected with RNase H1-V5, highly enriched for nuclei (nuc)or mitochondria (mt) as indicated, probed simultaneously for V5 and for the markers indicated. M, molecular mass markers. Figure 2. Subcellular targeting of RNase H1 variants. A, intracellular localization of RNase H1-V5 variants in cultures of stably transfected cells exemplified in B. M1V and M16V, N-terminal methionine variants (see Fig. S2A); ⌬NLS, with the putative nuclear localization signal deleted (see Fig. S2C). C, intracellular localization of RNase H1-V5 in cells synchronized in G1 and G2 (see FACS profiles in Fig. S2E). All plotted values are means of three experiments. Error bars denote S.D. nuc, nuclei; mt, mitochondria. RNase H1 and Drosophila mtDNA replication J. Biol. Chem. (2019) 294(12) 4331–4344 4333 at Tampere University Library on June 12, 2019http://www.jbc.org/Downloaded from tously acting da-GAL4 driver. Compared with controls lacking the driver, each of two knockdown lines eclosed in normal numbers (⬎90%; Fig. S4A) and with no significant change in development time (Fig. S4B). Knockdown at the RNA level was significant in both lines tested but appeared to be more severe in RNAi line 15534 than line 109457 and more pronounced in females than males (Fig. 4A). In both lines and sexes, we observed a⬎50% mtDNA depletion (Fig. 4B), which remained stable as the flies aged (Fig. S4C). Both lines also exhibited anomalies in mitochondrial transcript levels (Fig. 4C) with increased ND1 but decreased Cox3 RNA. To clarify the latter phenomenon, we confirmed the strandedness of transcription in each of the major gene clusters (Fig. S6). Knockdown flies exhibited a severe locomotor defect (Movie S1), which was quantified by a negative geotaxis assay in flies of line 15534 (Fig. 4D) in which rnh1 knockdown was driven either ubiquitously (da-GAL4 driver) or using drivers specific for neurons (elav-GAL4), glia (nrv2-GAL4), and muscle (G14-GAL4). The neuronal and muscle-specific drivers individually produced similar but milder locomotor defects than that produced by da-GAL4, suggesting that the latter is the result of additive or synergistic effects in the different tissues. Knockdown flies also showed hallmarks of mitochondrial dysfunction, such as respiratory chain impairment (Fig. 4E) and lactate accumulation (Fig. 4F), as well as shortened lifespan (Fig. S5). RNase H1 knockdown leads to the accumulation of abnormal mtDNA species The effects of rnh1 knockdown on mtDNA and RNA levels, combined with the organismal phenotype indicative of mitochondrial dysfunction, prompted us to look more closely at the molecular effects on mtDNA and its replication intermediates. qPCR does not distinguish between different topological forms of mtDNA or even between intact and damaged or partly fragmented molecules. Therefore, we used Southern blotting of purified mtDNA, treated with various modifying enzymes, to probe its structure. The majority of the mtDNA from knockdown cells was intact, but several abnormalities were evident. First, the proportion of supercoiled molecules was decreased compared with control cells (Fig. 5A). Treatment with topoisomerase I converted the remaining molecules comigrating electrophoretically with supercoiled circles to the relaxed circular form, confirming that these molecules were indeed monomeric and supercoiled (Fig. 5A). Conversely, gyrase treatment, which converted a fraction of the relaxed circular monomers seen in control cells to the supercoiled form (Fig. 5A), had a much weaker effect on mtDNA from knockdown cells, indicating that most of it contained nicks (Fig. 5A). Mitochondrial DNA from knockdown cells also contained unique speFigure 3. Effects of rnh1 knockdown in S2 cells. A, growth curves for untreated S2 cells or cells treated with dsRNA against rnh1 (RNase H1) or an inert dsRNA against GFP as indicated. Cells were split 1:6 on days (d) 3 and 6 with addition of fresh aliquots of dsRNA (dotted lines). Values are means for three independent replicates in each case. Error bars denote S.D. B, cell viability (trypan blue exclusion) for the same cell cultures. C, mean relative mtDNA copy number for the indicated cells, normalized against the value for untreated cells in parallel cultures. Error bars denote S.D. * denotes significant difference from untreated cells (Student’s ttest, p⬍0.01, n⫽3). Cells grown for 10 days were seeded at lower density (10 5 cells/ml instead of the standard 10 6 /ml) with dsRNA added on days 0, 3, 5, and 8. RNase H1 and Drosophila mtDNA replication 4334 J. Biol. Chem. (2019) 294(12) 4331–4344 at Tampere University Library on June 12, 2019http://www.jbc.org/Downloaded from cies migrating at high molecular weight that were differentially sensitive to S1 nuclease treatment (Fig. 5B, species indicated by arrows). Following digestion with a variety of restriction enzymes cutting once in the genome, we observed novel (or greatly enhanced) species of mtDNA from rnh1 knockdown cells, some of which migrated more slowly (Fig. 5C), while others migrated faster (Fig. 5D) than linearized genomic monomers. Their mobility was consistent between sample preparations but varied according to the digest used. Some of the slow-migrating species were sensitive to the Holliday junction–specific resolvase RusA (Fig. 5E). This and their presence above the compression zone for linear molecules imply that they could represent branched molecules containing unresolved four-way junctions. Based on their mobility (Fig. 5D), the faster-migrating species seen in rnh1 knockdown cells indicate double-strand ends or fragile sites at several positions in the genome, one of which appears to lie close to the previously mapped replication origin (Refs. 40 and 41 and see Fig. 5F). This recalls previous data from mouse Rnaseh1-knockout cells (31) where fragile sites were attributed to nonremoval of RNA primers at the origin. RNase H1 knockdown produces mtDNA species consistent with fork regression To investigate further the nature of the ends mapping close to the replication origin, we hybridized agarose gel blots of the NCR-containing HindIII fragment with probes located on either side of the NCR (Fig. 6). Probe 2, located in the rDNA region downstream of the origin, detected a set of shorter fragments (Fig. 6, Aand B) with inferred termini located close to the origin and to sites upstream thereof, presumptively within each of the “four-and-a-half” copies of the repeat II element of the NCR (46, 47). None of these ⬍1nfragments or any of reciprocal size (which would be in the size range of 1–2.5 kb) were detected by probe 1, lying on the other side of the NCR, indicating that they must be derived from nascent molecules rather than from a double-strand break or fragile site. The longer ⬍1nfragments were sensitive (Fig. 6B)toS1 nuclease and to exonuclease I (Exo I), indicating that they contain 3⬘ssDNA extensions. The shortest of the bands was strongly enhanced by Exo I treatment (Fig. 6B), whereas the longest was also partially sensitive to RNase H (see Fig. 7 for interpretations). Figure 4. Effects of rnh1 knockdown in flies. Relative rnh1 transcript (A) and mtDNA copy number (mean ⫾S. D.) (B) in 2-day-old rnh1 knockdown flies and corresponding controls (i.e. with balancer in place of the da-GAL4–bearing chromosome). Data are normalized in each case against male control flies. Significance values as indicated (Student’s ttest, comparing knockdown flies with controls of the given sex and RNAi strain, n⫽4 in all cases). C, relative levels of transcripts (means ⫹S.D.) of the indicated mitochondrial genes in 2-day-old female flies of the indicated knockdown strains versus balancer controls from the same strains. All data are normalized to values from controls of strain 15534. Significance values are as indicated (Student’s ttest, comparing knockdown flies of each RNAi strain with controls of the same strain, n⫽4 in all cases). D, climbing index (54) of adult flies of the indicated sex, knocked down for rnh1 (strain 15534) using the indicated GAL4 drivers. Values are means ⫾S.D. of nine independently tested batches of flies in each case. Significant differences are indicated (from control flies of the same sex; Student’s ttest). Note that because the elav-GAL4 driver is located on chromosome X, it was present only in female progeny; the males are therefore a control for this driver. E, oxygen consumption rates of mitochondrial suspensions from 10-day-old knockdown males of da-GAL4–driven RNAi line 15334 and controls of the same line, supplied with complex I(cI)-, III (cIII)-, and IV-linked (cIV) substrates. Values are means ⫾S.D. (n⫽4, significant differences as shown between controls and knockdown flies). F, lactate levels in homogenates from 10-day-old knockdown males of da-GAL4–driven RNAi line 15334 and controls of the same line, normalized to protein content and then to the value from control flies. Values are means ⫾S.D. (n⫽3, significant differences as shown, between controls and knockdown flies). All statistical comparisons are based on Student’s ttests (unpaired, two-tailed) with pvalues denoted as ¤, hash sign (#), and * (⬍0.05, 0.01, and 0.001, respectively). con, control; cyt b, cytochrome b. RNase H1 and Drosophila mtDNA replication J. Biol. Chem. (2019) 294(12) 4331–4344 4335 at Tampere University Library on June 12, 2019http://www.jbc.org/Downloaded from A higher molecular weight fragment of apparent size 11.1 kb was observed to accumulate. 2DNAGE indicated that this material was nonlinear and digestible by RusA. Given its size and mobility, it should represent burst bubbles in which the replication fork has progressed beyond the end of the fragment but has not entered it from the other end to complete replication. Its sensitivity to RusA indicates also that it contains four-way junctions, which logically arise by fork regression from the origin, generating a chicken-foot structure that is able to branch migrate, generating the steeply descending arc observed in 2DNAGE (Fig. 6C,blue arrow). The 2DNAGE blot revealed, in addition, a “flying,” slow-moving Y-like arc (Fig. 6C,red arrows), which may signal the presence of an undigested restriction site attributable to single strandedness or RNA/ DNA hybrid, arising from failure to complete lagging-strand synthesis (20). Logically, the Exo I–sensitive extensions represent lagging strands terminating at specific sites within the repeat II region of the NCR. The structures detected in this analysis and how they may arise are depicted in Fig. 7. rnh1 knockdown leads to accumulation of specific replication intermediates We infer from the preceding experiments that mtDNA replication was severely impaired in rnh1 knockdown cells, sufficiently to result in eventual mtDNA depletion. To investigate more specifically the molecular defects caused by rnh1 knockdown, we analyzed mtDNA replication intermediates (RIs) from around the mitochondrial genome using 2DNAGE. All fragments of the genome tested showed clear deviations in the pattern of RIs in knockdown cells compared with control cells (Fig. 8), indicating replication stalling or slowing in specific genomic regions. Accumulations of RIs were prominent in the regions immediately beyond the two major replication pause sites in the direction of fork progression (Fig. 8, Band C). These pauses are also the binding sites for the mitochondrial transcription termination factor (MTERF)-related protein mTTF (48). The observed regions of RI accumulation were quite discrete, extending ⬃1.7 kb beyond the mTTF-binding site 1 (bs1) through the center of the ClaI fragment detected by probe 9 (Fig. 8B) and ⬃5kb beyond bs2 through the middle of the adjacent HindIII fragment. The latter zone exhibited internal regions of higher RI accumulation, revealed at low exposure (Fig. 8D). We confirmed that similar effects on RIs were seen in mtDNA from rnh1 knockdown flies (Fig. S7). Three other features may be noted. First, prominent junctional intermediates were detected in the ClaI fragment containing bs2 (X spike in Fig. 8C). Second, in the origin/terminus fragment, the bubble arc was weak or missing (Fig. 8A). Third, burst bubbles, in which replication forks have migrated beyond the end of the fragment but not yet entered from its other side, were enhanced relative to unreplicated mtDNA (Fig. 8A, species marked by an arrow). RNase H1 overexpression has effects on mtDNA replication opposite to those of RNase H1 knockdown To evaluate the effects of overexpression of (epitope-tagged) RNase H1, we created stably transfected cell lines using the Figure 5. rnh1 knockdown leads to the accumulation of abnormal mtDNA species. A–E, Southern blots of mtDNA (mitochondrial nucleic acid) from control S2 cells (c) or S2 cells treated with an inert dsRNA against GFP (G) or a dsRNA against rnh1 (R), probed as indicated. Migration of molecular weight markers (in kb) is indicated: dotted species in Aare extrapolated from a separate gel of control material. A, undigested DNA, either untreated (con) or treated with topoisomerase I (topo I)orDNA gyrase(gyr). Theforms inferredto bemonomeric supercoiledcircles (SC)orrelaxed circles(RC) areas indicated.B,undigested DNAtreated with(⫹)or without (⫺) S1 nuclease as shown. Arrowheads indicate molecular species unique to rnh1 knockdown cells that were sensitive to S1 nuclease. The forms inferred to be monomeric relaxed circles (RC) are as indicated. Cand E, DNA digested with restriction enzymes as shown. The forms inferred to be monomeric linears (1n) or species of higher apparent molecular weight (⬎1n) are as indicated. Samples in Ewere treated with (⫹) or without (⫺) RusA as indicated. D, DNA digested with restriction enzymesasshown.F,schematicmapofDrosophilamtDNA,indicating thelocationof relevantrestrictionsites (opencircles),thenoncoding region(bold),and theprobes used. The open arrowhead marks the location and direction of replication initiation (see Ref. 40), near which one of the double-stranded ends inferred in Dmaps. RNase H1 and Drosophila mtDNA replication 4336 J. Biol. Chem. (2019) 294(12) 4331–4344 at Tampere University Library on June 12, 2019http://www.jbc.org/Downloaded from copper-inducible expression vector pMT-V5/HisB. Upon induction, RNase H1–overexpressing cells grew normally for 4–5 days but then entered growth arrest (Fig. S8A) with loss of cell viability (Fig. S8B). A control expressing a mitochondrially targeted fluorescent protein maintained viability and grew almost at the same rate as S2 cells that had not been induced with copper, expressing only the blasticidin resistance coselection marker (Fig. S8A). Cells expressing the uniquely nuclearly targeted M1V variant entered the growth and viability crisis slightly earlier than those expressing the natural protein (Fig. S8C), whereas cells expressing the predominantly mitochondrially targeted M16V variant entered the growth crisis approximately 2 days later (Fig. S8C). Nuclear overexpression is therefore sufficient to account for the growth and cell-viability defect conferred by RNase H1. However, to avoid any interference from this phenomenon, we proceeded to analyze mtDNA RIs from cells that had been grown for only 48 h following the induction of overexpression. We observed clear alterations in the patterns of RIs detected by 2DNAGE (Fig. 9) upon overexpression of RNase H1. In most respects, the changes were opposite in nature to those produced by RNase H1 knockdown. RIs in most regions of the genome were diminished relative to the unreplicated fragment. Replication pauses were either abolished (Fig. 9B, bs1) or decreased (Fig. 9C, bs2), whereas the origin/terminus fragment and the adjacent HindIII fragment lacking pause sites were largely unaffected. Discussion In this study, we determined that RNase H1 in Drosophila,as in mammals, is dually targeted to the nucleus and to mitochondria based on alternate AUG translation starts and the presence of mitochondrial and nuclear localization signals (Figs. 1 and 2). Targeting to both compartments was confirmed by microscopy and Western blotting for both transiently and stably transfected cells. Knockdown of rnh1 in flies produced a phenotype characteristic of mitochondrial dysfunction (Figs. 4,S4, and S5) with respiratory chain deficiency, increased lactate, locomotor impairment, and curtailed lifespan. These accompanied mtDNA depletion and abnormal mitochondrial transcript levels. These findings led us to undertake a detailed study of the effects of manipulating RNase H1 expression on mtDNA repliFigure 6. rnh1 knockdown leads to fork regression. A–C, Southern blots of mtDNA (mitochondrial nucleic acid) extracted from rnh1 knockdown cells (B,C, and lanes denoted “R”inA) or control cells treated with an inert dsRNA against GFP (lanes denoted “G”inA), digested, and probed as indicated. In A, the migration of molecular weight markers of ⬍3 kb is shown to the left, and extrapolated sizes of the bands are shown to the right, based on markers of ⬎3 kb. Markers of 3 kb and above are shown to the right of B. Note that in the region below 1.5 kb, probe 2 also detected 16S rRNA. One-dimensional (Aand B) and two-dimensional (C) agarose gels are shown. ⬍1nfragments in Bare denoted by a purple arrow (nuclease-resistant) or green bar/arrows (nuclease-sensitive). In C, higher molecular weight nonlinear forms are denoted by red arrows (flying, slow-moving Y-like arc) and blue arrows (burst bubbles and an arc in the shape of an apostrophe, extending downward from them). The directions of firstand second-dimension electrophoresis are as shown. D, schematic map of the region analyzed, showing location of probes (bold gray lines), NCR (bold black line), replication origin (arrowhead), and inferred ends of ⬍1nfragments detected by probe 2 (open triangles;filled triangle for the nuclease-resistant fragment). RNase H1 and Drosophila mtDNA replication J. Biol. Chem. (2019) 294(12) 4331–4344 4337 at Tampere University Library on June 12, 2019http://www.jbc.org/Downloaded from cation intermediates. In S2 cells, rnh1 knockdown (Fig. 8) and overexpression (Fig. 9) produced reciprocal changes in mtDNA replication intermediates with knockdown resulting in the accumulation of several abnormal mtDNA species (Figs. 5 and 6), the significance of which is discussed below. Global effects of RNase H1 manipulation on cell and organismal phenotype Previous studies in Drosophila (44) and in mouse (30) implied that null mutations of the gene encoding RNase H1 are recessive-lethal. Dual targeting raises the question of whether Figure 7. Proposed interpretations of mtDNA species detected by gel electrophoresis in rnh1 knockdown cells. One of many possible scenarios is illustrated. A, portion of a hypothetical replication intermediate with uncompleted lagging strand approaching the unidirectional replication origin (O R ). A shortresidualRNA primerremains at the5⬘end oftheleading strand.B, the laggingstrand proceedsbeyond the leading-strandinitiation point asfar asspecific, reiterated termination signals in the repeat II elements of the NCR. C, impaired fork progression around the genome causes the origin structure to persist with eventual regression to form a chicken-foot structure that can branch-migrate (arc denoted by blue arrow in Fig. 6C). D, upon treatment with RusA, the four-way junctions resulting from these regressed forks are cut, generating effectively linear products. E, HindIII digestion liberates linear fragments with lagging-strand 3⬘ssDNA extensions derived from the regressed forks (green arrows in Fig. 6B). These are digestible with S1 nuclease or exonuclease I, leaving a residual double-stranded species (purple arrow in Fig. 6B). Figure 8. Knockdown of rnh1 cause accumulation of RIs beyond pause sites. A–D, 2DNAGE of four restriction fragments of Drosophila S2 cell mtDNA, probed as indicated, in material from control cells treated with an inert dsRNA against GFP and cells knocked down for rnh1 by treatment with an rnh1-specific dsRNA (denoted KD). The arrow in Adenotes burst bubbles (see text). E, schematic map of Drosophila mtDNA indicating the location of relevant restriction sites (open circles), mTTF-binding sites (bs1 and bs2; filled circles), the noncoding region (bold), and the probes used. The open arrowhead marks the location and direction of replication initiation (see Ref. 40). The directions of the firstand second-dimension electrophoresis in all gels are as indicated by the arrows. The images show relatively low exposures (low exp) to reveal fine details of the arcs of RIs. RNase H1 and Drosophila mtDNA replication 4338 J. Biol. Chem. (2019) 294(12) 4331–4344 at Tampere University Library on June 12, 2019http://www.jbc.org/Downloaded from this lethality is a nuclear or mitochondrial effect. In mouse embryos deleted for Rnaseh1, lethality coincided with drastic mtDNA depletion as for null mutations in other mtDNA maintenance genes, such as TFAM (5) or DNA polymerase ␥ (33). Using RNAi in Drosophila (Figs. 4,S4, and S5) we were able to show that RNase H1 also has a predominantly mitochondrial function in adults. This is probably the case in mammals as well: in the mouse, liver degeneration caused by hepatic Rnaseh1 knockout was accompanied by mitochondrial dysfunction (49), and a point mutation in RNASEH1 produces a recognizable mitochondrial disease phenotype in humans (28, 29). In contrast, rnh1 knockdown in S2 cells had no obvious effect on cell growth or viability (Fig. 3, Aand B). This accords with previous studies indicating that S2 cells do not depend on mitochondrial functions for survival (43) but indicates that nuclearly localized RNase H1 is also dispensable under the conditions of cell culture. In contrast, RNase H1 overexpression, specifically in the nucleus, was lethal after approximately 1 week of culture (Fig. S8). Overall, our results indicate that RNase H1 expression has to be tightly regulated in place and amount. Similarities between effects of nuclear and mitochondrial RNase H1 depletion The effects on mitochondrial RNA and DNA synthesis of rnh1 knockdown in S2 cells are similar to those previously inferred in both yeast (35, 50) and mammalian cells (36)inthe nuclear compartment where it is required for the clearance of persistent heteroduplexes. We found an accumulation of mtDNA RIs in which the fork has progressed beyond the major replication pause sites (Fig. 8) into gene clusters that are heavily transcribed in the opposite direction (Fig. S6B), implying slow movement or stalling of replication forks in these regions. Furthermore, abnormalities in transcript levels imply that transcription and/or RNA processing is also disturbed. An interference with both replication fork movement and transcription is consistent with an accumulation of unresolved heteroduplexes in cells deficient for RNase H1. This indicates a role for RNase H1 in clearing such heteroduplexes, similar to its proposed function in the nucleus and in yeast mitochondria (51). In addition, these findings could implicate RNase H1 as a component of the previously hypothesized machinery that handles conflicts between the replication and transcription machineries by regulating replication pausing at binding sites for MTERF and related proteins (13, 24). An involvement of RNase H1 with the pausing machinery is supported by the opposite effects of rnh1 knockdown and overexpression on replication pausing (Figs. 8 and 9). RNase H1 overexpression caused a decrease in pausing, similar to the effect produced by overexpression of the major mitochondrial helicase (47), whereas RNase H1 knockdown caused increased pausing and fork stalling or slowing in the regions beyond the pause sites. Improperly regulated fork progression from pause sites may result in collisions with an oncoming transcription complex, leading to fork arrest and collapse. Although its deficiency disturbed both mtDNA replication and transcription, it remains an open question whether RNase H1 functions as an integral component of the relevant machinery or as a stand-alone enzyme, processing heteroduplexes that would otherwise impair these processes. In other systems, RNase H1 has been found to interact with single strand– binding proteins (52, 53) and with gyrase (55). It will be interesting to see whether this also applies to the proteins of the mtDNA replisome in Drosophila. Fork regression and processing in RNase H1–deficient cells Fork stalling at various points in the genome, whether as a result of unresolved heteroduplex or collisions, has various consequences. First, either a restart mechanism should be required to complete replication, or else the products of aborted replication need to be degraded and replaced by new synthesis. Second, repeated stalling and restart (or degradation) should decrease the rate of completion of each round of Figure 9. Overexpression of rnh1 relieves replication pausing. A–D, 2DNAGE of four restriction fragments of Drosophila S2 cells mtDNA, probed as indicated, in material from control cells and cells overexpressing RNase H1 in the form of epitope-tagged RNase H1-V5 (denoted OE), both treated with 500 ␮ M CuSO 4 for 48 h to induce expression. E, schematic map of Drosophila mtDNA, as also shown in Fig. 8, indicating the location of relevant restriction sites (open circles), mTTF-binding sites (bs1 and bs2; filled circles), the noncoding region (bold), and the probes used. The open arrowhead marks the location and direction of replication initiation (see Ref. 40). The directions of firstand second-dimension electrophoresis in all gels are as indicated by the arrows. The images show relatively low exposures to reveal fine details of the arcs of RIs. RNase H1 and Drosophila mtDNA replication J. Biol. Chem. (2019) 294(12) 4331–4344 4339 at Tampere University Library on June 12, 2019http://www.jbc.org/Downloaded from