Cell lines and cell culture reagents

HeLa human ovarian carcinoma (ATCC CCL-2), HEK293T human embryonic kidney (ATCC CRL-11268), HCT116 human colon carcinoma (ATCC CCL-247), HT1080 human fibrosarcoma (ATCC CCL-121), U2OS human osteosarcoma (ATCC HTB-96), WI38-VA13 human virus-transformed fibroblasts (ATCC CCL-75.1), Saos2 human osteosarcoma (ATCC HTB-85), WI38 lung fibroblast (ATCC CCL-75), and IMR90 lung fibroblast cells (ATCC CCL-186) were used in this study. Adar1^−/− MEF cells and isogenic control cells were established from Adar1^−/− mice¹⁸. Adar2^−/− MEF cells and isogenic control cells were established from Adar2^−/− mice⁴¹. HEK293T cells expressing FLAG-ADAR1p110-WT, FLAG-ADAR1p110-EAA, FLAG-ADAR2-WT, or FLAG-ADAR2-EAA were established by co-transfection of various p3XFLAG-CMV-10 plasmids (Sigma) with a puromycin resistance plasmid pPUR (Clontech)⁶⁹. These cell lines were free of mycoplasma contamination.

HeLa, HEK293T, HCT116, HT1080, Saos2, IMR90, Adar1^−/− MEF, Adar2^−/− MEF cells, and isogenic control MEF cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Gemini), penicillin (100 U/ml), and streptomycin (100 μg/ml) at 37 °C in a humidified atmosphere with 5% CO₂. U2OS, WI38-VA13, and WI38 cells were cultured in DMEM/F12 supplemented with 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 μg/ml).

HEK293T cells expressing FLAG-ADAR1p110-WT were treated with thymidine and nocodazole to synchronize in the M phase. The cells were cultured in T175 flask and treated with 2.5 mM of thymidine (Sigma) for 24 h. To release from the thymidine block, the cells were washed twice with phosphate-buffered saline (PBS) and culture medium. After incubation with a fresh medium for 3 h, the cells were treated with 0.1 μg/ml of nocodazole (Sigma) for 12 h.

Plasmid construction

An NheI restriction site was added to the multi-cloning site (MCS) of CSII-EF-MCS-IRES-puromycin-resistant gene (puro) by inserting the new MCS site oligonucleotide into NotI–BamHI-digested CSII-EF-MCS-IRES-puro vector^70,71. CSII-EF-FLAG-ADAR1p110-WT-IRES-puro, CSII-EF-FLAG-ADAR1p110-E912A-IRES-puro, or CSII-EF-FLAG-ADAR1p150-WT-IRES-puro used for protein overexpression in human cells was prepared by PCR cloning using p3XFLAG-CMV10-ADAR1p110-WT, p3XFLAG-CMV10-ADAR1p110-E912A, or p3XFLAG-CMV10-ADAR1p150-WT, respectively⁶⁹. The FLAG-ADAR1p110 PCR products were amplified using primers CSII-FLAG-p110-F and CSII-FLAG-p110-R. The PCR products were digested with NotI and BamHI and then inserted into CSII-EF-MCS-IRES-puro. The FLAG-ADAR1p150 PCR products were amplified using primers CSII-p150-F and CSII-p150-R. The PCR products were digested with NotI and NheI and then inserted into CSII-EF-MCS-IRES-puro. CSII-EF-FLAG-RNaseH2A was prepared by PCR cloning using pcDNA3.1-RNaseH2A⁷² The FLAG-RNaseH2A PCR products were amplified using primers CSII-FLAG-RNaseH2A-F and CSII-RNaseH2A-R. The PCR products were digested with NotI and BamHI and then inserted into CSII-EF-MCS-IRES-puro. CSII lentivirus vector was a kind gift from Hiroyuki Miyoshi and Toru Nakano.

pET28-FLAG-RNaseH2A used for recombinant protein purification was prepared by PCR cloning using a pET28-His-RNaseH2A plasmid. The PCR products were amplified using primers pET28-FLAG-RNaseH2A-F and FLAG-RNaseH2A-R, digested with XbaI and XhoI, and inserted into pET28 vector⁷³. pET28-His-RNASEH2A and pET15-His-RNaseH2B/2C were kind gifts from Marcin Nowotny. Oligo DNAs used for plasmid construction are listed in Supplementary Data 1.

Gene knockdown

Gene knockdown experiments were done by RNA interference methods using Lipofectamine RNAiMax (Life Technologies) or HiperFect at a final short interfering RNA (siRNA) concentration of 1, 2, or 5 nM. All siRNAs used in this study are listed in Supplementary Data 1.

Lentivirus infection

HEK293FT cells (5–6 × 10⁶) incubated in a 10 cm dish for a confluency of 80% were transfected with the following three plasmids using Lipofectamine 3000: 17 µg of CSII-EF-FLAG-ADAR1 or CSII-EF-FLAG-RNaseH2A plasmid, 10 µg of pCAG-HIVgp (GAG-POL DNA), and 10 µg of the vesicular stomatitis virus G (VSV-G) envelope plasmid pCMV-VSV-G. After 48 h incubation, the cell culture supernatant was filtered using a 0.45 μm filter and concentrated by Lenti-X concentrator (Clontech). The lentiviral pellet was resuspended in 2 ml of fresh culture medium containing 8 µg/ml of polybrene (Sigma) and added to 1 × 10⁵ cells cultured in a 6-well plate. Infected cells were then incubated with puromycin (1 μg/ml) for 48 h post infection for antibiotic selection. The extent of infection of CSII-EF-FLAG-ADAR1 or CSII-EF-FLAG-RNaseH2A was evaluated by western blotting analysis and immunostaining with anti-FLAG M2 antibody. ADAR1 rescue experiments required exogenous FLAG-ADAR1 expression in every cell and were carried out using early passage cells (≤passage 6).

Immunofluorescence staining

Transfection of siRNAs (siADAR1-1) into HeLa cells at 1 nM concentration was carried out as described above. After incubation for 24 h, the culture medium was replaced with a fresh medium containing CellLight Tubulin-GFP and BacMam 2.0 (Thermo Fisher Scientific). Nuclei were stained with SiR-DNA reagent (Cytoskeleton) at 0.25 μM for 6 h. Cells were cultured on Ibidi μDish 3.5 cm. After 72 h, cells were fixed with 4% paraformaldehyde and soaked in Dulbecco’s PBS. Microscopic images were obtained by using a Leica TCS SP5 DMI6000 CS Confocal Microscope and LAS X software (Leica), equipped with ultraviolet 405 diode, Argon, DPS3561, and HeNe594 lasers. Fluorescent images were captured with a 40× lens with a 512 × 512 frame. For multicolor experiments, the following wavelength settings were used: Tubulin-GFP (Ex 488 nm/Em 498–630 nm) and SiR-DNA reagent (Ex 647 nm/Em 657–800 nm). Nuclear morphological analysis was performed using 4′,6-diamidino-2-phenylindole (DAPI)-stained HeLa cells.

Immunoblot analysis

Cell lysates were prepared in Laemmli buffer containing benzonase nuclease (Sigma), complete EDTA-free proteinase inhibitor cocktail (Roche), and PhosStop phosphatase inhibitor cocktail (Roche) and size-fractionated by 4–20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were blotted to Immobilon-P nylon membrane (Millipore). Membranes were blocked with 10% Blocker BSA (bovine serum albumin) buffer (Thermo Fisher Scientific) and incubated with the primary antibodies overnight at 4 °C. After incubation with each appropriate secondary antibody, bands were detected with ECL (GE Healthcare) using X-ray films. Antibodies were diluted in 10% Blocker BSA buffer (Thermo Fisher Scientific). Antibodies used in this study are listed in Supplementary Data 4.

Dot blot analysis of genomic DNA

Cells were treated with siRNA for 72 h in a 10 cm dish, detached from the dish surface with TrypLE Express Enzyme, and harvested by centrifugation. After PBS wash, genomic DNA was purified using QIAGEN Blood & Cell Culture DNA Midi Kit. Briefly, the cell pellet was resuspended in buffer C1. After repeated buffer C1 wash and removal of the cell debris, the nuclear pellet was resuspended in buffer G2 (without RNase A) and treated with 2 mg of proteinase K at 50 °C for 60 min. The nuclear fraction was applied to a buffer QBT-treated QIAGEN Genomic-tip and washed twice with buffer QC. Genomic DNA was eluted with buffer QF and precipitated with 2-propanol. The DNA pellet was washed twice with 80% ethanol and air-dried. Genomic DNA was dissolved in TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA) and incubated overnight at 4 °C.

Genomic DNA was diluted in 100 μl of 6× saline sodium citrate (SSC) and spotted onto a Hybond N⁺ (GE Healthcare) using a Bio-Dot Apparatus (#1706545, Bio-Rad). The membrane was cross-linked with ultraviolet (UV) (0.24 J) and blocked with 5% non-fat dry milk (LabScientific) in PBS with 0.1% Tween-20 (PBST) for 1 h and then with SuperBlock buffer (Thermo Fisher Scientific) for 1 h at room temperature. The membrane was incubated with S9.6 antibody (Sigma) at 0.1 μg/ml in SuperBlock buffer overnight at 4 °C. After washing three times with PBST, the membrane was incubated with horseradish peroxidase-conjugated donkey anti-mouse IgG secondary antibody (Jackson Immuno Research) (0.04 μg/ml) at 0.1 μg/ml in SuperBlock buffer for 1 h at room temperature. After washing four times with PBST, dot signals were detected with ECL (GE Healthcare) using X-ray films. For the treatment with RNase H, 1 μg of genomic DNA was preincubated with 2 U of E. coli-RNase H (NEB) for 2 h at 37 °C. DNA:DNA, RNA:RNA, or RNA:DNA oligo duplex controls were annealed in buffer containing 10 mM Tris-HCl (pH 7.6) and 50 mM NaCl at 80 °C for 5 min, followed by slow cooling to room temperature. Oligonucleotides were used as controls are listed in Supplementary Data 1.

DNA:RNA hybrid immunoprecipitation

Fifty micrograms of genomic DNA prepared as described above was diluted in 250 μl of sonication buffer (10 mM Tris-HCl pH 8.5, 300 mM NaCl) and sonicated using Bioruptor (Diagenode) (20 cycles at high power, 30 s ON/60 s OFF) or Sonicator W-220 (Heat Systems Ultrasonics) (20 cycles at Lv3.5, 10 s ON/40 s OFF). During sonication, samples were kept cold very carefully. Sonicated genomic DNA was mixed with 0.02 pmol spike RNA:DNA oligonucleotide duplexes. A fraction of the genomic DNA sample (90%, 225 μl) was used for immunoprecipitation with the S9.6 antibody (Sigma), and the remaining fraction (10%, 25 μl) was used as the input control. Protein A beads (Dynabeads Protein A, Invitrogen) (100 μl) were blocked with 0.5% BSA in PBS containing 5 mM EDTA overnight at 4 °C. After washing twice with DRIP buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% NP-40, 0.1% sodium deoxycholate), 20 μg of S9.6 antibody (Sigma) or control mouse IgG (sc-2025, Santa Cruz) were applied to the blocked Dynabeads in DRIP buffer overnight at 4 °C. After washing twice with DRIP buffer, the beads were resuspended in 100 μl of DRIP buffer containing 500 U of RNasin Plus inhibitor (Promega). Sonicated DNA was diluted to half concentration in a buffer containing 100 mM Tris-HCl pH 7.4, 10 mM EDTA, 2% NP-40, and 0.2% sodium deoxycholate. Then, 100 μl of the beads were added to DNA solution and incubated overnight at 4 °C with rotation. The beads were washed by the following steps: (1) twice with DRIP buffer; (2) twice with DRIP high buffer (50 mM Tris-HCl, pH 7.4, 500 mM NaCl, 5 mM EDTA, 1% NP-40, 0.1% sodium deoxycholate); (3) twice with DRIP Li buffer (50 mM Tris-HCl, pH 8.0, 250 mM LiCl, 1 mM EDTA, 0.5% NP-40, 0.5% sodium deoxycholate); (4) once with DRIP TE NaCl+ buffer (100 mM Tris-HCl pH8.0, 10 mM EDTA, 50 mM NaCl); and (5) once with DRIP TE buffer (100 mM Tris-HCl pH8.0, 10 mM EDTA). After removal of DRIP TE buffer using a magnetic stand, RNA:DNA hybrids were eluted from the beads in 200 μl of DRIP elution Buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% sodium dodecyl sulfate (SDS)) by shaking at 65 °C for 30 min at 1400 r.p.m. (Benchmark Scientific, MultiTherm shaker H5000-H). The beads were removed from the supernatant by another round of separation with a magnetic stand and centrifugation. The supernatant was treated with 80 μg of proteinase K (Roche) in the presence of 160 U of RNasin Plus inhibitor for 30 min at 42 °C. RNA:DNA hybrids were purified using QIAquick Nucleotide Removal Kit (Qiagen) and eluted in 200 μl of buffer containing 5 mM Tris-HCl pH 8.5. For RNase H treatment, 50 μg of sonicated genomic DNA was preincubated with 25 U of E.coli-RNase H (NEB) overnight at 37 °C. Recovery of RNA:DNA hybrids and RNase H treatment were evaluated by quantitative PCR analysis of spike RNA:DNA duplex. Oligonucleotides used as spike RNA:DNA duplexes are listed in Supplementary Data 1.

qPCR analysis of DRIP products

Two microliters of DRIP products was used for qPCR with Power SYBR Green PCR Master Mix (Thermo Fisher Scientific) and QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems, QuantStudio Real-Time PCR software). Primers used are listed in Supplementary Data 1.

Dot blot analysis of DRIP products

DRIP products or whole genomic DNA (10 μl) were mixed with 15 μl of 0.13 N NaOH/3.3 mM EDTA solution and incubated at 90 °C for 10 min. The denatured DRIP products were diluted in 100 μl of 6× SSC and spotted onto a Hybond N+ using Bio-Dot Apparatus. The membrane was cross-linked with UV (0.24 J) and pre-hybridized with ULTRAhyb Ultrasensitive Hybridization Buffer (Invitrogen) overnight at 42 °C. 5′-³²P-labeled DNA or LNA-oligonucleotide probe was added to the hybridization buffer and incubated overnight at 42 °C. The membrane was washed three times with 2× SSC/0.1% SDS solution for 15 min at 42 °C or 50 °C. A fraction of DRIP samples (5%) was spotted as an input control onto the membrane. Hybridized probe signals were detected using Typhoon RGB Imager (GE Healthcare, Amersham Typhoon Control software). Oligonucleotides used as probe and washing temperature are also listed in Supplementary Data 1. Consensus α-satellite, Alu, and LINE1 probes were hybridized and washed at 42 °C. Using these less stringent hybridization and washing conditions, these probes target variations known to exist within sub-family members of each repetitive element. In particular, the Alu consensus probe is 44 nucleotides, so it can recognize all Alu subfamilies, except Alu that is missing the 3′ region. Therefore, consensus Alu and LINE1 probes recognize ~11% and 18% of the human genome, respectively.

RNA strand analysis of DRIP products

Fifty micrograms of genomic DNA prepared as described above was diluted in 250 μl of sonication buffer and sonicated using Sonicator W-220 (20 cycles at Lv3.5, 10 s ON/40 s OFF). During sonication, samples were kept cold very carefully. A fraction of the genomic DNA sample (90%, 225 μl) was used for immunoprecipitation with the S9.6 antibody (Sigma or Kerafast), and the remaining fraction (10%, 25 μl) was used as the input control. Protein A beads (Dynabeads Protein A, Invitrogen) (100 μl) were blocked with 0.5% BSA in PBS containing 5 mM EDTA overnight at 4 °C. After washing twice with DRIP buffer, 20 μg of S9.6 antibody or control mouse IgG (sc-2025, Santa Cruz) was applied to the blocked Dynabeads in DRIP buffer overnight at 4 °C. After washing twice with DRIP buffer, the beads were resuspended in 100 μl of DRIP buffer containing 500 U of RNasin Plus inhibitor (Promega). Sonicated DNA was diluted to half concentration in a buffer containing 100 mM Tris-HCl pH 7.4, 10 mM EDTA, 2% NP-40, and 0.2% sodium deoxycholate. Then, 100 μl of the beads was added to the DNA solution and incubated overnight at 4 °C with rotation. The beads were washed by the following steps: (1) twice with DRIP buffer; (2) twice with DRIP High buffer; (3) twice with DRIP Li buffer; (4) one with DRIP TE NaCl+ buffer; and (5) twice with 100 mM Tris-HCl pH 8.0 buffer. After removal of 100 mM Tris-HCl pH 8.0 buffer using a magnetic stand, the beads were treated with 10 U of TURBO DNase (Thermo Fisher Scientific) at 37 °C for 1 h in 100 μl of TURBO DNase buffer containing 160 U of RNasin Plus inhibitor. After adding 100 μl of double concentration DRIP elution buffer (100 mM Tris-HCl, pH 8.0, 20 mM EDTA, 2% SDS), RNAs were eluted from the beads by shaking at 65 °C for 30 min at 1400 r.p.m. (Benchmark Scientific, MultiTherm shaker H5000-H). The beads were removed from the supernatant by another round of separation with a magnetic stand and centrifugation. The supernatant was treated with 80 μg of proteinase K (Roche) for 30 min at 42 °C. RNAs were purified using QIAquick Nucleotide Removal Kit (Qiagen) and eluted in 100 μl of buffer containing 5 mM Tris-HCl pH 7.6. For RNase H treatment, 50 μg of sonicated genomic DNA was preincubated with 25 U of E. coli-RNase H (NEB) overnight at 37 °C. The input control was treated with RNase I and TURBO DNase and was purified using QIAquick Nucleotide Removal Kit.

DRIP products were incubated at 80 °C for 10 min. The denatured DRIP products were diluted in 100 μl of 6× SSC and spotted onto a Hybond N+ using Bio-Dot Apparatus. The membrane was cross-linked with UV (0.24 J) and pre-hybridized with ULTRAhyb Ultrasensitive Hybridization Buffer (Invitrogen) overnight at 42 °C. 5′-³²P-labeled LNA-oligonucleotide probe was added to hybridization buffer and incubated overnight at 42 °C. The membrane was washed three times with 2× SSC/0.1% SDS solution for 15 min at 42 °C or 55 °C. Hybridized probe signals were detected using a Typhoon RGB Imager (GE Healthcare, Amersham Typhoon Control software). A fraction of DRIP samples (5%) was spotted onto the membrane. Oligonucleotides used as probes and washing temperatures are listed in Supplementary Data 1.

Preparation of duplex substrates

Sense or antisense oligonucleotides of telomere sequences were purchased from IDT and Dharmacon. The 5′ ends of RNA and DNA strands to be analyzed were biotinylated. Sense and antisense oligonucleotides were annealed in annealing buffer (10 mM Tris-HCl pH 7.5, 50 mM NaCl) to prepare perfectly matched or mismatched dsRNAs or RNA:DNA hybrids, which were used as substrates for in vitro editing assay.

Preparation of recombinant ADAR1 proteins

All procedures were carried out at 4 °C. HAT-ADAR1p110-WT-, FLAG-ADAR1p110-WT-, or HA-ADAR1p110-EAA-expressing Sf9 cells were prepared with baculovirus⁶⁹. The cells were washed with PBS and resuspended in Tris+ buffer (250 mM Tris pH 7.8, 1 mM dithiothreitol (DTT), 0.6 mM phenylmethylsulfonyl fluoride (PMSF), proteinase inhibitor cocktail). The cells were sonicated and debris was removed by centrifugation. The supernatant (cell extract) was diluted with an equal volume of 2× TGK buffer (100 mM Tris-HCl pH 7.8, 200 mM NaCl, 40% glycerol, 1 mM DTT, 0.6 mM PMSF, proteinase inhibitor cocktail) and stored at −80 °C.

HAT-ADAR1p110-WT was purified using TALON Metal Affinity Resin (Clontech). The resin was prewashed with STD300 buffer (50 mM Tris-HCl pH 7.0, 300 mM NaCl, 20% glycerol, 1 mM β-mercaptoethanol, 0.05% NP-40). After buffer exchange to STD300 using Zeba^TM 7 K molecular weight cut-off (MWCO) spin desalting column (Thermo Fisher), the cell extract was loaded onto the resin. After washing with STD300 buffer, the resin was treated with 80 kU of micrococcal nuclease (NEB) for 30 min in STD300 buffer containing 1 mM CaCl₂ at room temperature and washed with STD300 buffer containing 0.5 mM EDTA and 0.5 mM EGTA and then washed with STD300 buffer containing 7.5 mM imidazole. HAT-ADAR1p110-WT recombinant protein was eluted with STD300 buffer containing 150 mM imidazole and proteinase inhibitor cocktail. Imidazole was removed by using Zeba^TM 7 K MWCO spin desalting column.

FLAG-ADAR1p110-WT or HA-ADAR1p110-EAA was purified using anti-FLAG M2 agarose (Sigma) or anti-HA agarose (Thermo Fisher Scientific), respectively. The agarose was washed with STD150 buffer (50 mM Tris-HCl pH 7.0, 150 mM NaCl, 20% glycerol, 1 mM β-mercaptoethanol, 0.05% NP-40). After buffer exchange to STD150 using Zeba^TM 7 K MWCO spin desalting column, the cell extract was loaded onto the agarose. After washing with STD150 buffer, the agarose was treated with 80 kU of micrococcal nuclease (NEB) for 30 min in STD150 buffer containing 2 mM CaCl₂ at room temperature and washed with STD150 buffer containing 3 mM EDTA and 3 mM EGTA, STD150 buffer, STD500 buffer (50 mM Tris-HCl pH 7.0, 500 mM NaCl, 20% glycerol, 1 mM β-mercaptoethanol, 0.05% NP-40), and again STD150 buffer. FLAG-ADAR1p110-WT or HA-ADAR1p110-EAA recombinant protein was eluted with STD150 buffer containing protease inhibitor cocktail and 0.1 mg/ml FLAG peptide or HA peptide, respectively.

All recombinant proteins purified were stored in STD150 buffer containing 1 mM DTT, instead of 1 mM β-mercaptoethanol, at −80 °C

In vitro editing assay

The in vitro editing reaction mixture, containing 5 nM of telomere RNA:RNA duplex substrates and 75 nM of HAT-ADAR1p110-WT, FLAG-ADAR1p110-WT, or HA-ADAR1p110-EAA protein, was incubated at 37 °C for 2 h in in vitro editing buffer I (20 mM HEPES-KOH pH 7.5, 100 mM NaCl, 0.01% NP-40, 5% glycerol, 1 mM DTT). For editing of RNA:DNA hybrid substrates, in vitro editing buffer II (20 mM HEPES-KOH pH 7.5, 20 mM NaCl, 0.01% NP-40, 5% glycerol, 1 mM DTT) was used. Edited RNA or DNA strands were purified using Dynabeads MyOne Streptavidin C1 (Thermo Fisher Scientific). To remove opposite RNA or DNA strands, RNase H (NEB) or TURBO DNase (Thermo Fisher Scientific) was used, respectively. For sequencing of edited substrates, reverse transcription-PCR was carried out for RNA strands, while PCR was carried out for DNA strands. Each reaction used specific primer sets (Supplementary Data 1). RT reactions were carried out using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific), and PCR reactions were performed using Platinum Taq DNA polymerase (Thermo Fisher Scientific). PCR products were sequenced using a specific sequencing primer, and the ratio of A and G peaks in the chromatograms were analyzed by CodonCode Aligner (CodonCode Corporation).

Protein co-IP

Cells expressing FLAG-ADAR1p110-WT, FLAG-ADAR1p110-EAA, FLAG-ADAR2-WT, FLAG-ADAR2-EAA, or FLAG-RNaseH2A were fixed with 0.3% formaldehyde in PBS containing 1 mM DTT at room temperature for 10 min. After washing twice with PBS, the cells were suspended in co-IP buffer (50 mM Tris-HCl pH 7.6, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, proteinase inhibitor cocktail, PhosStop, RNasin Plus inhibitor) and sonicated. The debris was removed by centrifugation and the supernatant was incubated overnight at 4 °C with 50 μl of anti-FLAG M2 magnetic beads (Sigma), prewashed and blocked with 20% BSA blocker in co-IP buffer. The beads were washed three times with co-IP buffer and NP-40 wash buffer (50 mM Tris-HCl pH 7.6, 150 mM NaCl, 1 mM EDTA, 1% NP-40). Laemmli buffer containing proteinase inhibitor cocktail and PhosStop phosphatase inhibitor cocktail was added to the beads and boiled at 98 °C for 10 min. Interacting proteins with ADAR1p110-WT, ADAR2, or RNaseH2A were detected by immunoblot analysis as described above; 2.5% extracts of co-IP products were used as input controls.

Preparation of RNase H2 complex and RNA:DNA hybrid cleavage assay

To prepare recombinant human RNase H2A/2B/2C triple complex, pET28-FLAG-RNaseH2A and pET15-His-RNaseH2B/2C vectors were co-transformed into E. coli BL21 cells cultured in LB medium containing 100 μg/ml ampicillin and 20 μg/ml kanamycin. Protein induction was started at an optical density of 0.6 with 0.4 mM isopropyl β-d-1-thiogalactopyranoside and incubated overnight at 30 °C. After harvesting cells by centrifugation, cells were suspended in 40 mM HEPES pH 7.0, 75 mM NaCl, 5% glycerol, proteinase inhibitor cocktail, and 1 mg/ml lysozyme and then sonicated. The debris was removed by centrifugation twice at 12,000 × g. The supernatant was diluted with an equal volume of dilution buffer (40 mM NaH₂PO₄ pH 7.0, 500 mM NaCl, 5% glycerol) and mixed with Ni-NTA agarose (Qiagen) at 4 °C. The agarose was washed with STD300 buffer containing 5 mM imidazole. Recombinant RNase H2A/2B/2C complex was eluted with 20 ml of STD300 buffer containing 150 mM imidazole and proteinase inhibitor cocktail. Anti-FLAG M2 affinity gel (Sigma) was washed with STD150 buffer and mixed with an eluted fraction of Ni-NTA agarose at 4 °C. After washing with STD150 buffer, the resin was treated with 80 kU of micrococcal nuclease (NEB) for 30 min in STD150 buffer containing 2 mM CaCl₂ at room temperature and washed with STD500 buffer and then with STD150 buffer. FLAG-RNase H2A/His-RNase H2B/2C complexes were eluted with 0.1 mg/ml FLAG peptide in STD150 buffer containing proteinase inhibitor cocktail.

5′-³²P-labeled oligonucleotide RNAs were annealed with complementary DNAs as described above (Supplementary Data 1). Cleavage assays of RNA:DNA hybrid by RNase H2A/2B/2C complex were done in a 50 μl reaction mixture containing 50 mM Tris-HCl pH 8.5, 75 mM KCl, 3 mM MgCl₂, 10 mM DTT, 2 nM RNase H2A/2B/2C complex, 1 nM RNA:DNA substrate, and RNasin plus inhibitor. Cleavage assays by recombinant human RNase H1 (ab153634, Abcam) were done in a 50 μl reaction mixture containing 25 mM Tris-HCl pH 7.5, 50 mM KCl, 5 mM MgCl₂, 1 mM DTT, 10 μg/ml BSA, 5 nM RNase H1, 1 nM RNA:DNA substrate, and RNasin plus inhibitor. Reaction mixtures were incubated at 37 °C and 7.5 μl aliquots were taken after 0, 5, 10, 30, and 60 min. At each time point, to stop the reaction, gel loading buffer (80% formamide, 20% glycerol, 0.025% bromophenol) was added to the aliquots. After heating at 80 °C for 10 min, samples were analyzed by 10% Urea-PAGE. 5′-³²P-labeled RNA signals were detected using a Typhoon RGB Imager (GE Healthcare, Amersham Typhoon Control software).

Telomere FISH analysis

Exponentially growing cells were treated with colcemid (60 ng/ml) for 1 h and harvested. Then, cells were subsequently swollen in a hypotonic 0.075 M KCl solution for 20 min at room temperature and then fixed in a freshly prepared 3:1 mix of methanol:acetic acid four times. After fixation, cells were dropped onto glass microscope slides and allowed to dry for 2 days at room temperature. The slides were immersed in PBS at 37 °C for 30 min, fixed in 4% formaldehyde in PBS for 2 min, and washed three times with PBS for 5 min. The slides were then treated with 1 mg/ml pepsin solution (pH 2.0) at 37 °C for 2–5 min. After washing with PBS for 10 s, the slides were fixed in 4% formaldehyde in PBS and washed three times with PBS for 5 min. Then, 10 μl of hybridization mixture containing 70% formamide, 1% (w/v) blocking reagent (Roche) in a maleic acid buffer (pH 7.0), and 3 ng of fluorescence-labeled telomeric peptide nucleic acid (PNA) probe FITC-(CCCTAA)₄ were applied to each slide and mounted under a coverslip. The slides were heated on an aluminum heat block at 80 °C for 3 min and hybridized with PNA probe for 5 h in 37 °C. After hybridization, the slides were washed twice in 70% formamide/10 mM Tris (pH 7.2) for 15 min, followed by washing three times with 50 mM Tris/150 mM NaCl (pH 7.5)/0.05% Tween-20. Finally, DNA was counterstained with Vectashield with DAPI (Vector Lab). The chromosome samples were observed using a fluorescence microscope and digital images were recorded using a CCD camera and LAS X software (Leica).

Immuno-FISH assay for MEF cells

Cells were seeded onto coverslips and cultured overnight. The adhered cells were washed twice with cold PBS for 5 min and fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. Cells were then washed three times with PBS for 5 min each and permeabilized with ice-cold 0.5% NP-40 in PBS for 10 min on ice. Cells were washed three times with PBS for 5 min each and incubated with anti-phosphorylated histone γH2AX antibody (Millipore), followed by Alexa 488 secondary antibody (Molecular Probes) with 30% Blocker BSA (bovine serum albumin) buffer (Thermo Fisher Scientific). After staining, labeled protein was cross-linked with 4% paraformaldehyde in PBS for 20 min at room temperature. The samples were washed two times with PBS for 5 min and then dehydrated in 70, 90, and 100% ethanol for 3 min each and air-dried. Hybridization mixtures (10 μl) containing 3 ng of fluorescence-labeled telomeric PNA probe were applied to the slide and mounted under a coverslip. The slides were heated for 3 min on a hot plate at 80 °C. After hybridization with a telomeric PNA probe for 5 h, the cells were washed three times with 70% formamide/10 mM Tris (pH 6.8) for 15 min, followed by a 5-min wash with 0.05 M Tris/0.15 M NaCl (pH 7.5)/0.05% Tween-20 and a 5-min wash with PBS. Mounting and microscopic analysis was performed as for the telomere FISH analysis.

Immuno-FISH assay for HeLa cells

Cells were seeded onto collagen-coated Ibidi 3-well chambers and transfected with siControl or siADAR1-1. The adhered cells were washed twice with PBS and fixed with 4% paraformaldehyde (Thermo Fisher Scientific) in PBS at room temperature for 10 min. After washing twice with PBS, the cells were permeabilized with 0.25% Tween-20 in PBS. After washing twice with PBS, the cells were treated with 20 μg of RNase A and 500 U of RNase I in PBS at 37 °C for 1 h. After washing three times with PBS, the cells were blocked with 30% Blocker BSA buffer (Thermo Fisher Scientific) for 30 min and incubated with anti-γH2AX antibody (Abcam), followed by Alexa 488 or 647 secondary antibody (Thermo Fisher Scientific) with 30% Blocker BSA buffer (Thermo Fisher Scientific). After staining, labeled protein was cross-linked with 4% paraformaldehyde in PBS at room temperature for 10 min. After washing twice with PBS, the cells were dehydrated in 70%, 85%, and 100% ethanol for 2 min each and air-dried. Fifty microliters of hybridization mixture containing 500 nM TelC-Cy3 PNA probe (PNA bio), 20 mM Tris-HCl pH 7.4, 60% formamide, and 0.5% blocking reagent (Roche) was applied to the chamber. The chamber was heated for 3 min on a heat block at 80 °C. After hybridization with TelC-Cy3 PNA probe at room temperature for 5 h, the cells were washed three times with PBS at 45 °C for 10 min. Finally, DNA was counterstained with DAPI. The slides were mounted with ProLong Gold (Thermo Fisher Scientific). Microscopic images were obtained by using a Leica TCS SP5 DMI6000 CS Confocal Microscope (Leica). Fluorescent images were captured with a ×63.0 lens by LAS X software (Leica). Ectopic expression of FLAG-ADAR1p110 was evaluated by immunostaining with anti-FLAG M2 antibody.

Chromosome orientation-FISH

Cells were cultured in a medium containing a 3:1 ratio of 5′-bromo-2′-deoxyuridine (BrdU, Sigma):5′-bromo-2′-deoxycytidine (BrdC, Sigma) at a total final concentration of 10 μM during the final 24 h. Colcemid addition led to the accumulation of mitotic cells. Cultures were trypsinized and then treated with hypotonic KCl, fixed, and dropped onto microscope slides. Prior to hybridization of the single-stranded telomere probe (as above for FISH), slides were treated with 0.5 mg/ml RNase A (Sigma) for 10 min at 37 °C and then stained with 0.5 μg/ml Hoechst 33258 (Sigma) in 2× SSC for 15 min at room temperature. Slides were then exposed to 365 nm UV light for 25 min. The BrdU/BrdC-substituted DNA strands were digested with 3 U/μl of exonuclease III (Promega) in a buffer supplied by the manufacturer (50 mM Tris-HCl, 5 mM MgCl₂, and 5 mM dithiothreitol, pH 8.0) for 10 min at room temperature. An additional denaturation was performed in 70% formamide, 2× SSC at 70 °C for 1 min, followed by dehydration in a cold ethanol series (70, 85, and 100%). The CO-FISH procedure results in the original parental strands serving as single-stranded chromosomal target DNA for hybridization of single-stranded probes.

Time-lapse imaging

HeLa cells were treated with CellLight Tubulin-GFP and BacMam 2.0 (Thermo Fisher Scientific) for 12 h before siRNA transfection. Transfection of siRNAs into HeLa cells at 1 nM concentration was carried out as described above. Nuclei were visualized by staining of DNA with SiR-DNA reagent (Cytoskeleton) (0.25 μM) for 6 h. Cells were cultured in CellView 3.5 cm glass-bottomed dishes (Greiner). Time-lapse images were obtained using a Leica TCS SP5 DMI6000 CS Confocal Microscope between 48 and 72 h post transfection.

Statistics and reproducibility

All experiments were performed at least twice or more independent times with similar results. Image quantitation was done using Image J or ImageQuant software (GE Healthcare). Data were analyzed using Microsoft Excel (Microsoft Corporation) and were presented as means ± SD or SEM. Two-tailed t tests were conducted where the minimum level of significance was P < 0.05.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.