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• W2128741337 abstract "RNADNA hybrids form in the nuclei and mitochondria of cells as transcription-induced R-loops or G-quadruplexes, but exist only in the cytosol of virus-infected cells. Little is known about the existence of RNA:DNA hybrids in the cytosol of virus-free cells, in particular cancer or transformed cells. Here, we show that cytosolic RNA:DNA hybrids are present in various human cell lines, including transformed cells. Inhibition of RNA polymerase III (Pol III), but not DNA polymerase, abrogated cytosolic RNA:DNA hybrids. Cytosolic RNA:DNA hybrids bind to several components of the microRNA (miRNA) machinery-related proteins, including AGO2 and DDX17. Furthermore, we identified miRNAs that are specifically regulated by Pol III, providing a potential link between RNA:DNA hybrids and the miRNA machinery. One of the target genes, exportin-1, is shown to regulate cytosolic RNA:DNA hybrids. Taken together, we reveal previously unknown mechanism by which Pol III regulates the presence of cytosolic RNA:DNA hybrids and miRNA biogenesis in various human cells. DNA hybrids form in the nuclei and mitochondria of cells as transcription-induced R-loops or G-quadruplexes, but exist only in the cytosol of virus-infected cells. Little is known about the existence of RNA:DNA hybrids in the cytosol of virus-free cells, in particular cancer or transformed cells. Here, we show that cytosolic RNA:DNA hybrids are present in various human cell lines, including transformed cells. Inhibition of RNA polymerase III (Pol III), but not DNA polymerase, abrogated cytosolic RNA:DNA hybrids. Cytosolic RNA:DNA hybrids bind to several components of the microRNA (miRNA) machinery-related proteins, including AGO2 and DDX17. Furthermore, we identified miRNAs that are specifically regulated by Pol III, providing a potential link between RNA:DNA hybrids and the miRNA machinery. One of the target genes, exportin-1, is shown to regulate cytosolic RNA:DNA hybrids. Taken together, we reveal previously unknown mechanism by which Pol III regulates the presence of cytosolic RNA:DNA hybrids and miRNA biogenesis in various human cells. RNA:DNA hybrids can occur during transcription and replication of DNA (1.Aguilera A. García-Muse T. R loops: from transcription byproducts to threats to genome stability.Mol. Cell. 2012; 46: 115-124Abstract Full Text Full Text PDF PubMed Scopus (640) Google Scholar). The DNA primase generates short RNA:DNA fragments during replication of the lagging strand (2.Komissarova N. Becker J. Solter S. Kireeva M. Kashlev M. Shortening of RNA:DNA hybrid in the elongation complex of RNA polymerase is a prerequisite for transcription termination.Mol. Cell. 2002; 10: 1151-1162Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 3.Kireeva M.L. Komissarova N. Waugh D.S. Kashlev M. The 8-nucleotide-long RNA:DNA hybrid is a primary stability determinant of the RNA polymerase II elongation complex.J. Biol. Chem. 2000; 275: 6530-6536Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Short hybrids also form during the transcription of DNA by RNA polymerases. In contrast, long RNA:DNA hybrids can occur during stalling of the RNA polymerase or during replication of mitochondria DNA (1.Aguilera A. García-Muse T. R loops: from transcription byproducts to threats to genome stability.Mol. Cell. 2012; 46: 115-124Abstract Full Text Full Text PDF PubMed Scopus (640) Google Scholar). Stalling of the RNA polymerases can lead to the formation of R-loops, which consist of long RNA:DNA hybrids and the displaced non-template DNA strand. Long RNA:DNA hybrids also occur in G-quadruplexes, which promote class switch recombination in B-cells (5.Maizels N. Dynamic roles for G4 DNA in the biology of eukaryotic cells.Nat. Struct. Mol. Biol. 2006; 13: 1055-1059Crossref PubMed Scopus (362) Google Scholar). Recent evidence suggests that R-loops and G-quadruplexes may occur more frequently than previously assumed and interfere with gene expression and threaten genome stability (6.Wahba L. Amon J.D. Koshland D. Vuica-Ross M. RNase H and multiple RNA biogenesis factors cooperate to prevent RNA:DNA hybrids from generating genome instability.Mol. Cell. 2011; 44: 978-988Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 7.Biffi G. Tannahill D. McCafferty J. Balasubramanian S. Quantitative visualization of DNA G-quadruplex structures in human cells.Nat. Chem. 2013; 5: 182-186Crossref PubMed Scopus (1427) Google Scholar8.Wahba L. Gore S.K. Koshland D. The homologous recombination machinery modulates the formation of RNA-DNA hybrids and associated chromosome instability.eLife. 2013; 2: e00505Crossref PubMed Scopus (132) Google Scholar). Although many studies have focused on the generation of nuclear RNA:DNA hybrids, it is unclear how nuclear RNA:DNA hybrids are resolved, and their role in diseases related to genomic instability, such as cancer, is not understood. We recently discovered the presence of ssDNA and dsDNA in the cytosol of B-cell lymphoma cells (9.Lam A.R. Le Bert N. Ho S.S. Shen Y.J. Tang M.L. Xiong G.M. Croxford J.L. Koo C.X. Ishii K.J. Akira S. Raulet D.H. Gasser S. RAE1 ligands for the NKG2D receptor are regulated by STING-dependent DNA sensor pathways in lymphoma.Cancer Res. 2014; 74: 2193-2203Crossref PubMed Scopus (108) Google Scholar). Inhibition of ATM (ataxia telangiectasia-mutated) and ATR (ataxia telangiectasia- and Rad3-related) kinases, which initiate the DNA damage response (DDR), 3The abbreviations used are: DDRDNA damage responsePol IIIRNA polymerase IIImiRNA/miRmicroRNADMSOdimethyl sulfoxideCOX IVcytochrome c oxidaseBis-Tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. leads to the disappearance of cytosolic DNA. Conversely, the levels of cytosolic ssDNA and dsDNA increase in response to DNA damage, suggesting that constitutive nuclear DNA damage and the ensuing DDR induce the presence of cytosolic ssDNA and dsDNA in B-cell lymphoma cells. Cytosolic DNA in B-cell lymphoma cells activates STING-dependent DNA sensor pathways, leading to the expression of ligands for the activating immune receptor NKG2D (natural killer group 2, member D) (9.Lam A.R. Le Bert N. Ho S.S. Shen Y.J. Tang M.L. Xiong G.M. Croxford J.L. Koo C.X. Ishii K.J. Akira S. Raulet D.H. Gasser S. RAE1 ligands for the NKG2D receptor are regulated by STING-dependent DNA sensor pathways in lymphoma.Cancer Res. 2014; 74: 2193-2203Crossref PubMed Scopus (108) Google Scholar). Delocalized DNA is important for innate immune recognition of pathogens, and recent reports suggest that TLR9 and the NLRP3 inflammasome sense pathogen-derived RNA:DNA hybrids in dendritic cells (10.Theofilopoulos A.N. Kono D.H. Beutler B. Baccala R. Intracellular nucleic acid sensors and autoimmunity.J Interferon Cytokine Res. 2011; 31: 867-886Crossref PubMed Scopus (55) Google Scholar11.Jounai N. Kobiyama K. Takeshita F. Ishii K.J. Recognition of damage-associated molecular patterns related to nucleic acids during inflammation and vaccination.Front. Cell. Infect. Microbiol. 2012; 2: 168PubMed Google Scholar, 12.Kailasan Vanaja S. Rathinam V.A. Atianand M.K. Kalantari P. Skehan B. Fitzgerald K.A. Leong J.M. Bacterial RNA:DNA hybrids are activators of the NLRP3 inflammasome.Proc. Natl. Acad. Sci. U.S.A. 2014; 111: 7765-7770Crossref PubMed Scopus (69) Google Scholar13.Rigby R.E. Webb L.M. Mackenzie K.J. Li Y. Leitch A. Reijns M.A. Lundie R.J. Revuelta A. Davidson D.J. Diebold S. Modis Y. MacDonald A.S. Jackson A.P. RNA:DNA hybrids are a novel molecular pattern sensed by TLR9.EMBO J. 2014; 33: 542-558Crossref PubMed Scopus (114) Google Scholar). However, whether RNA:DNA hybrids exist in the cytosol of non-infected cells is unknown. DNA damage response RNA polymerase III microRNA dimethyl sulfoxide cytochrome c oxidase 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. RNA polymerase III (Pol III) is the largest RNA polymerase, consisting of 17 subunits, including a DNA-binding site (14.Dieci G. Conti A. Pagano A. Carnevali D. Identification of RNA polymerase III-transcribed genes in eukaryotic genomes.Biochim. Biophys. Acta. 2013; 1829: 296-305Crossref PubMed Scopus (59) Google Scholar, 15.Dieci G. Fiorino G. Castelnuovo M. Teichmann M. Pagano A. The expanding RNA polymerase III transcriptome.Trends Genet. 2007; 23: 614-622Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar16.Lorenzen K. Vannini A. Cramer P. Heck A.J. Structural biology of RNA polymerase III: mass spectrometry elucidates subcomplex architecture.Structure. 2007; 15: 1237-1245Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). It catalyzes the transcription of genes required for transcription and RNA processing, such as tRNAs, ribosomal 5 S rRNA, and U6 snRNAs. It also transcribes short interspersed elements and repeated elements in the human genome (14.Dieci G. Conti A. Pagano A. Carnevali D. Identification of RNA polymerase III-transcribed genes in eukaryotic genomes.Biochim. Biophys. Acta. 2013; 1829: 296-305Crossref PubMed Scopus (59) Google Scholar). Pol III expression is regulated by oncogene products, tumor suppressors such as p53, and Pol III-associated transcription factors (17.Gjidoda A. Henry R.W. RNA polymerase III repression by the retinoblastoma tumor suppressor protein.Biochim. Biophys. Acta. 2013; 1829: 385-392Crossref PubMed Scopus (25) Google Scholar, 18.Marshall L. White R.J. Non-coding RNA production by RNA polymerase III is implicated in cancer.Nat. Rev. Cancer. 2008; 8: 911-914Crossref PubMed Scopus (86) Google Scholar19.Veras I. Rosen E.M. Schramm L. Inhibition of RNA polymerase III transcription by BRCA1.J. Mol. Biol. 2009; 387: 523-531Crossref PubMed Scopus (34) Google Scholar). Consistent with these observations, Pol III activity is increased in many cancers, including melanomas, myelomas, and carcinomas (20.Schwartz L.B. Sklar V.E. Jaehning J.A. Weinmann R. Roeder R.G. Isolation and partial characterization of the multiple forms of deoxyribonucleic acid-dependent ribonucleic acid polymerase in the mouse myeloma, MOPC 315.J. Biol. Chem. 1974; 249: 5889-5897Abstract Full Text PDF PubMed Google Scholar). Although Pol III is present mostly in the nucleus (20.Schwartz L.B. Sklar V.E. Jaehning J.A. Weinmann R. Roeder R.G. Isolation and partial characterization of the multiple forms of deoxyribonucleic acid-dependent ribonucleic acid polymerase in the mouse myeloma, MOPC 315.J. Biol. Chem. 1974; 249: 5889-5897Abstract Full Text PDF PubMed Google Scholar, 21.Jaehning J.A. Roeder R.G. Transcription of specific adenovirus genes in isolated nuclei by exogenous RNA polymerases.J. Biol. Chem. 1977; 252: 8753-8761Abstract Full Text PDF PubMed Google Scholar), cytosolic Pol III was proposed to play a role in the sensing of AT-rich DNA via the RIG-I (retinoic acid-inducible gene I) pathway (22.Chiu Y.H. Macmillan J.B. Chen Z.J. RNA polymerase III detects cytosolic DNA and induces type I interferons through the RIG-I pathway.Cell. 2009; 138: 576-591Abstract Full Text Full Text PDF PubMed Scopus (877) Google Scholar, 23.Ablasser A. Bauernfeind F. Hartmann G. Latz E. Fitzgerald K.A. Hornung V. RIG-I-dependent sensing of poly(dA:dT) through the induction of an RNA polymerase III-transcribed RNA intermediate.Nat. Immunol. 2009; 10: 1065-1072Crossref PubMed Scopus (673) Google Scholar24.Valentine R. Smith G.L. Inhibition of the RNA polymerase III-mediated dsDNA-sensing pathway of innate immunity by vaccinia virus protein E3.J. Gen. Virol. 2010; 91: 2221-2229Crossref PubMed Scopus (46) Google Scholar). Despite the regulation of Pol III by genes associated with tumorigenesis, little is known about the role of Pol III in the cellular function of transformed cells. Here, we identified the presence of cytosolic RNA:DNA hybrids in immortalized and transformed human tumor cells. Chemical inhibition of Pol III abrogated the presence of cytosolic RNA:DNA hybrids in cells. Cytosolic RNA:DNA hybrids were bound by microRNA (miRNA) machinery-associated proteins, such as DDX17 (DEAD (Asp-Glu-Ala-Asp) box polypeptide 17) and AGO2 (argonaute 2). We also identified Pol III-regulated intracellular miRNAs in A549 lung cancer cells. In summary, we demonstrate the constitutive presence of cytosolic RNA:DNA hybrids in a variety of cell lines, and this accumulation depends on Pol III, at least in A549 lung carcinoma cells. The human lung adenocarcinoma (A549), colorectal adenocarcinoma (LoVo and HT29), colorectal carcinoma (HCT116), acute monocytic leukemia (THP-1), human cervix carcinoma (HeLa), and normal lung tissue-derived (MRC-5) cell lines were purchased from American Type Culture Collection (Manassas, VA). Cells were grown in Dulbecco's modified Eagle's medium (Nacalai Tesque) supplemented with 10% fetal bovine serum (Nichirei Biosciences Inc., Tokyo, Japan), 1% penicillin/streptomycin (Nacalai Tesque), and 2% HEPES (Life Technologies). Cells were maintained with 5 μg/ml Plasmocin (InvivoGen) to prevent mycoplasma infection. Ara-C (cytarabine) was purchased from Wako Chemicals. Aphidicolin, Pol III inhibitor ML-60218, and leptomycin B were purchased from Calbiochem. ATM inhibitor KU60019 (Tocris Bioscience) and ATR inhibitor VE821 (Axon Medchem) were used at 10 μm. PicoGreen dsDNA reagent (Life Technologies) was used at 1:100 dilution. MitoTracker Red CM-H2XRos (Life Technologies) was dissolved in dimethyl sulfoxide (DMSO) and used at 500 nm. Fixed cells were treated with 0.5 units/ml RNase H (New England Biolabs) for 3 h at 37 °C. Cells were fixed in 4% paraformaldehyde (Nacalai Tesque) for 10 min and permeabilized in 0.2% Triton X-100 for 15 min. Nonspecific sites were blocked with 2% goat serum and 1% BSA in 0.2% Triton X-100 for 1 h. Transfected cells were stained with anti-cytochrome c oxidase subunit IV (COX IV) antibody (ab16056, Abcam), anti-POLR3G antibody (polymerase (RNA) III (3) (DNA-directed) polypeptide G; LS-C163858, LSBio), or anti-DDX17 antibody (19910-1-AP, Proteintech). The RNA:DNA hybrid-specific antibody S9.6 was a kind gift of Dr. D. Koshland (University of California, Berkeley) (25.Boguslawski S.J. Smith D.E. Michalak M.A. Mickelson K.E. Yehle C.O. Patterson W.L. Carrico R.J. Characterization of monoclonal antibody to DNA·RNA and its application to immunodetection of hybrids.J. Immunol. Methods. 1986; 89: 123-130Crossref PubMed Scopus (259) Google Scholar). The secondary polyclonal antibodies used were Alexa Fluor 488-conjugated goat anti-mouse IgG F(ab′)2 fragment (H+L) and Alexa Fluor 555-conjugated goat anti-rabbit IgG F(ab′)2 fragment (H+L) (Life Technologies). PicoGreen staining of DNA and MitoTracker staining of mitochondria were performed according to the manufacturer's instructions. Cells were stained with 2 μg/ml Hoechst for 10 min and mounted in mounting medium (Dako). Cell images were taken with a Leica TCS SP2 laser confocal scanning microscope and analyzed using Volocity (version 6.2.1) and Imaris. Micrographs show cells representative of total cell populations. A549 cells were transfected with POLR3G siRNA (Qiagen) using Lipofectamine RNAiMAX transfection reagent (Life Technologies) according to the manufacturer's instructions. AllStars negative control siRNA (Qiagen) was used as a control in transfection, and its sequence is proprietary. The POLR3G siRNA sequences used were 5′-AAGGCACACCACTCACTAATA-3′ (siPOLR3G_1) and 5′-TCAGAGTACTCAAGTGTACAA-3′ (siPOLR3G_2). Cells were lysed in cold radioimmune precipitation assay buffer (Nacalai Tesque), and lysates were electrophoresed in 4–12% NuPAGE Bis-Tris gel (Life Technologies) and then blotted onto PVDF membranes. Antibodies specific to DDX17 (sc-86409, Santa Cruz), AGO2 (C34C6, Cell Signaling Technology), and GAPDH (M171-3, MBL International) and horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology) were used to develop the blots with Immobilon Western chemiluminescent HRP substrate (Millipore). Digital images were acquired using ImageQuant LAS 500 (GE Healthcare). A549 cells (2 × 106) were seeded into 100-mm dishes and fixed in 1% paraformaldehyde for 10 min, followed by treatment with 125 mm glycine (Wako Chemicals) for 5 min. Cells were fractionated using a cell fractionation kit (MS861, MitoSciences). The cytosolic fraction was precleared by incubation with 5 μl of protein G-Sepharose beads (GE Healthcare) for 20 min at 4 °C on a rolling shaker. The cleared supernatant was incubated overnight at 4 °C on a rolling shaker with 10 μg/ml RNA:DNA hybrid-specific antibody and 10 μl of protein G-Sepharose beads. Immunoprecipitates were washed sequentially with radioimmune precipitation assay buffer, low salt buffer (20 mm Tris-HCl (pH 8.1), 150 mm NaCl, 0.1% SDS, 1% Triton X-100, and 2 mm EDTA), high salt buffer (20 mm Tris-HCl (pH 8.1), 600 mm NaCl, 0.1% SDS, 1% Triton X-100, and 2 mm EDTA), final wash buffer (20 mm Tris-HCl (pH 8.0), 0.1% SDS, 1% Triton X-100, and 1 mm EDTA), and Tris/EDTA buffer. Beads were resuspended in Tris/EDTA buffer with 1% SDS and incubated overnight at 65 °C to release protein complexes for subsequent gel electrophoresis. For mass spectrometry, similarly processed cell lysates were immunoprecipitated with RNA:DNA hybrid-specific antibody and silver-stained using a Silver Stain Plus kit (Bio-Rad) according to the manufacturer's instructions. Bands of interest were cut out and sent for mass spectrometry analysis at the Osaka University mass spectrometry facility. A549 cells were treated with 10 μm Pol III inhibitor for 24 h and subsequently treated with 10 μm Ara-C or DMSO for 15 h. DMSO-treated cells served as a control. Total RNA was extracted with TRIzol (Life Technologies) and labeled using a 3D-Gene miRNA labeling kit. The labeled RNA was hybridized to a human miRNA V19 microarray chip containing 2019 miRNA probes and analyzed on a ProScanArray microarray scanner (Toray Industries). miRNA profiles were provided as sample-wise median-normalized data by Toray Industries. Data were further normalized with an all-sample quantile normalization protocol using the corresponding Bioconductor package developed by Bolstad et al. (26.Bolstad B.M. Irizarry R.A. Astrand M. Speed T.P. A comparison of normalization methods for high density oligonucleotide array data based on variance and bias.Bioinformatics. 2003; 19: 185-193Crossref PubMed Scopus (6397) Google Scholar). Original miRNA profiles consisted of 2019 miRNA probes, of which only a small fraction showed significant expression in any of these experiments. After replacing the missing valued data (no expression observed) with the minimum of all observed expression values, miRNA probes that showed at least 3-fold differential expression between any pair of four experiments were used for further quantitative analysis. Identified miRNA sequences were used to obtain predicted gene targets, as acquired from the public domain resource DIANA-mirPath (27.Vlachos I.S. Kostoulas N. Vergoulis T. Georgakilas G. Reczko M. Maragkakis M. Paraskevopoulou M.D. Prionidis K. Dalamagas T. Hatzigeorgiou A.G. DIANA-mirPath v.2.0: investigating the combinatorial effect of microRNAs in pathways.Nucleic Acids Res. 2012; 40: W498-W504Crossref PubMed Scopus (442) Google Scholar). A p value threshold of 0.05 and a MicroT threshold of 0.8 were applied. After RNA extraction, cDNA was synthesized using a miScript II RT kit (Qiagen). miRNA levels were analyzed using assay kits for mature miR-4499 (Qiagen), precursor (includes detection for precursor and primary miR) miR-4499 (Qiagen), and TaqMan primary miR-4499 (Life Technologies) and quantified by quantitative PCR using iTaq Universal SYBR Green Supermix (Bio-Rad) or TaqMan Gene Expression Master Mix (Life Technologies). Precursor miR expression was determined using the equation in Ref. 28.Schmittgen T.D. Jiang J. Liu Q. Yang L. A high-throughput method to monitor the expression of microRNA precursors.Nucleic Acids Res. 2004; 32: e43Crossref PubMed Scopus (414) Google Scholar. Experiments were performed as described previously (9.Lam A.R. Le Bert N. Ho S.S. Shen Y.J. Tang M.L. Xiong G.M. Croxford J.L. Koo C.X. Ishii K.J. Akira S. Raulet D.H. Gasser S. RAE1 ligands for the NKG2D receptor are regulated by STING-dependent DNA sensor pathways in lymphoma.Cancer Res. 2014; 74: 2193-2203Crossref PubMed Scopus (108) Google Scholar). The following primers were used: XPO1-5′ (exportin-1), 5′-AGGTTGGAGAAGTGATGCCA-3′; XPO1-3′, 5′-GCACCAATCATGTACCCCAC-3′; KPNB1-5′ (karyopherin (importin) beta 1), 5′-GACCGACTACCCAGACAGAG-3′; KPNB1-3′, 5′-GACTCCTCCTAAGACGACGG-3′; NUP153-5′ (nucleoporin 153kDa), 5′-GCCCAAATCTTCCTCTGCAG-3′; NUP153-3′, 5′-GAAAGGAGCCACTGAAGCAC-3′; HPRT1-5′, 5′-CCCTGGCGTCGTGATTAGTG-3′; and HPRT1-3′, 5′-TCGAGCAAGACGTTCAGTCC-3′. For statistical analysis, Student's one-tailed t test (p < 0.05) was used unless stated otherwise after data were tested positive for normality by the Shapiro-Wilk test. For data that failed the normality test, a non-parametric Mann-Whitney Wilcoxon rank-sum test was used. A p value of <0.05 was considered statistically significant. We previously reported (9.Lam A.R. Le Bert N. Ho S.S. Shen Y.J. Tang M.L. Xiong G.M. Croxford J.L. Koo C.X. Ishii K.J. Akira S. Raulet D.H. Gasser S. RAE1 ligands for the NKG2D receptor are regulated by STING-dependent DNA sensor pathways in lymphoma.Cancer Res. 2014; 74: 2193-2203Crossref PubMed Scopus (108) Google Scholar) the presence of cytosolic ssDNA and dsDNA in cancer cells using specific antibodies and the vital dye PicoGreen, which detects dsDNA and RNA:DNA hybrids (29.Dragan A.I. Casas-Finet J.R. Bishop E.S. Strouse R.J. Schenerman M.A. Geddes C.D. Characterization of PicoGreen interaction with dsDNA and the origin of its fluorescence enhancement upon binding.Biophys. J. 2010; 99: 3010-3019Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 30.Haugland R.P. The Molecular Probes Handbook–A Guide to Fluorescent Probes and Labeling Technologies.10th Ed. Invitrogen, Carlsbad, CA2005Google Scholar). Here, we sought to test if RNA:DNA hybrids are present in the cytosol of human cancer cell lines. PicoGreen stained DNA in the cytosol of the human lung carcinoma cell line A549 and other cancer cell lines (Fig. 1, A and B, and Fig. 2, A and B). Three-dimensional surface rendering of confocal images showed that the majority of extranuclear DNA is present outside of mitochondria (Fig. 1B). To analyze if RNA:DNA hybrids contribute to the PicoGreen signals in the cytosol, A549 cells were treated with RNase H, which degrades RNA in RNA:DNA hybrids (31.Frank P. Albert S. Cazenave C. Toulmé J.J. Purification and characterization of human ribonuclease HII.Nucleic Acids Res. 1994; 22: 5247-5254Crossref PubMed Scopus (40) Google Scholar), prior to staining with PicoGreen. Pretreatment of cells with RNase H abrogated the cytosolic PicoGreen signals (Fig. 1, A–C). As expected, the staining of nuclear genomic DNA by PicoGreen was not changed.FIGURE 2.Presence of cytosolic RNA:DNA hybrids in human tumor cell lines. A, the human colorectal carcinoma cell lines LoVo, HCT116, and HT29; the human acute monocytic leukemia cell line THP-1; the human cervix carcinoma cell line HeLa; and the human normal lung tissue-derived cell line MRC-5 were stained for RNA:DNA hybrids (red) in the presence of Hoechst (blue). Scale bars = 10 μm. B, the colorectal adenocarcinoma cell lines LoVo, HCT116, and HT29 were stained with PicoGreen (upper panels). Bright-field images (differential interference contrast (DIC)) of cells are shown in the lower panels. C, three-dimensional isosurface rendering of confocal images of A549 cells stained for the presence of dsDNA (green) and RNA:DNA hybrids (red) in the presence of Hoechst (blue). Co-localization of dsDNA and RNA:DNA hybrids (yellow) was determined using Volocity computational image analysis.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To further investigate the presence of cytosolic RNA:DNA hybrids, we stained cells using the RNA:DNA hybrid-specific antibody S9.6 (25.Boguslawski S.J. Smith D.E. Michalak M.A. Mickelson K.E. Yehle C.O. Patterson W.L. Carrico R.J. Characterization of monoclonal antibody to DNA·RNA and its application to immunodetection of hybrids.J. Immunol. Methods. 1986; 89: 123-130Crossref PubMed Scopus (259) Google Scholar). In agreement with the PicoGreen results, S9.6 staining of tumor cells (A549, LoVo, HCT116, HT29, HeLa, and THP-1) and human fetal lung fibroblast cells (MRC-5) showed the presence of RNA:DNA hybrids in the cytosol and, to a lesser extent, in the nucleus (Fig. 1, D and E, and Fig. 2, A and B). As RNA:DNA hybrids can also form during replication of mitochondrial DNA (32.Xu B. Clayton D.A. RNA-DNA hybrid formation at the human mitochondrial heavy-strand origin ceases at replication start sites: an implication for RNA-DNA hybrids serving as primers.EMBO J. 1996; 15: 3135-3143Crossref PubMed Scopus (129) Google Scholar), we co-stained cells with the mitochondria-specific vital dye MitoTracker. Three-dimensional surface rendering of confocal images showed that the majority of RNA:DNA hybrids were localized outside of mitochondria (Fig. 1E). Pretreatment of cells with RNase H prior to S9.6 staining significantly reduced the cytosolic RNA:DNA hybrid staining (Fig. 1, D–F). Antibody S9.6 only partially co-stained with PicoGreen (Fig. 1G), suggesting that PicoGreen stains cytosolic dsDNA and RNA:DNA hybrids in A549 cells. Consistent with this possibility, staining of A549 cells using a dsDNA-specific antibody showed the presence of cytosolic dsDNA, which partially co-localized with the cytosolic PicoGreen staining (Fig. 2C). In summary, our data show that RNA:DNA hybrids are constitutively present in the cytosol of all tested cells. RNA:DNA hybrids can occur during DNA transcription (33.Shaw N.N. Arya D.P. Recognition of the unique structure of DNA:RNA hybrids.Biochimie. 2008; 90: 1026-1039Crossref PubMed Scopus (81) Google Scholar). In addition, cytosolic DNA is transcribed by Pol III in the cytosol and potentially in the nucleus into an RNA:DNA hybrid and dsRNA intermediate (22.Chiu Y.H. Macmillan J.B. Chen Z.J. RNA polymerase III detects cytosolic DNA and induces type I interferons through the RIG-I pathway.Cell. 2009; 138: 576-591Abstract Full Text Full Text PDF PubMed Scopus (877) Google Scholar, 23.Ablasser A. Bauernfeind F. Hartmann G. Latz E. Fitzgerald K.A. Hornung V. RIG-I-dependent sensing of poly(dA:dT) through the induction of an RNA polymerase III-transcribed RNA intermediate.Nat. Immunol. 2009; 10: 1065-1072Crossref PubMed Scopus (673) Google Scholar). To understand the mechanism by which cytosolic RNA:DNA hybrids are generated and regulated, A549 cells were treated with Pol III inhibitors prior to staining with S9.6 or PicoGreen (34.Wu L. Pan J. Thoroddsen V. Wysong D.R. Blackman R.K. Bulawa C.E. Gould A.E. Ocain T.D. Dick L.R. Errada P. Dorr P.K. Parkinson T. Wood T. Kornitzer D. Weissman Z. Willis I.M. McGovern K. Novel small-molecule inhibitors of RNA polymerase III.Eukaryot. Cell. 2003; 2: 256-264Crossref PubMed Scopus (61) Google Scholar). Treatment of A549 cells with the Pol III inhibitor ML-60218 decreased the cytosolic RNA:DNA hybrid staining at doses above the published half-maximal inhibitory concentration (IC50), but had no effect on nuclear PicoGreen staining and affected RNA:DNA hybrid staining at doses above IC50 (Fig. 3). Treatment of A549 cells with α-amanitin, a Pol II inhibitor, was toxic to cells compared with the Pol III inhibitor ML-60218. To investigate if the genetic inhibition of Pol III also reduced RNA:DNA hybrid levels, A549 cells were transfected with siRNA against POLR3G (siPOLR3G_1 and siPOLR3G_2), a subunit of the Pol III complex, or negative control siRNA. Knockdown of POLR3G protein expression resulted in the disappearance of cytosolic RNA:DNA hybrids by immunofluorescence staining, consistent with reduced POLR3G mRNA gene expression (Fig. 4). In contrast, negative control siRNA did not affect cytosolic RNA:DNA hybrid levels or POLR3G expression. This corroborated with previous results of Pol III chemical inhibition, which decreased the presence of RNA:DNA hybrids. In contrast to Pol II, which is localized exclusively in the nucleus, a fraction of Pol III is present in the cytosol. To investigate if cytosolic Pol III contributes to the generation of RNA:DNA hybrids in the cytosol, we co-stained A549 cells for POLR3G, a subunit of the Pol III complex, and RNA:DNA hybrids (35.Wang Z. Roeder R.G. Three human RNA polymerase III-specific subunits form a subcomplex with a selective function in specific transcription initiation.Genes Dev. 1997; 11: 1315-1326Crossref PubMed Scopus (129) Google Scholar). No significant co-staining of POLR3G and S9.6 or PicoGreen was observed in the cytosol of A549 cells (Fig. 5A). Furthermore, POLR3G was localized largely in the nucleus, suggesting that the presence of cytosolic RNA:DNA hybrids depends on nuclear Pol III activity. RNA:DNA hybrids can cause stalling of the replication fork and formation of dsDNA breaks (1.Aguilera A. García-Muse T. R loops: from transcription byproducts to threats to genome stability.Mol. Cell. 2012; 46: 115-124Abstract Full Text Full Text PDF PubMed Scopus (640) Google Scholar, 36.Chan Y.A. Hieter P. Stirling P.C. Mechanisms of genome instability induced by RNA-processing defects.Trends Genet. 2014; 30: 245-253Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 37.Hamperl S. Cimprich K.A. The contribution of co-transcriptional RNA:DNA hybrid structures to DNA damage and genome instability.DNA Repair. 2014; 19: 84-94Crossref PubMed Scopus (170) Google Scholar). To test if stalling of replication forks and the associated DNA dama" @default.
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• W2128741337 date "2015-03-01" @default.
• W2128741337 modified "2023-10-11" @default.
• W2128741337 title "RNA Polymerase III Regulates Cytosolic RNA:DNA Hybrids and Intracellular MicroRNA Expression" @default.
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• W2128741337 doi "https://doi.org/10.1074/jbc.m115.636365" @default.
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