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https://doi.org/10.1038/s41467-021-21921-x

OPEN

ADAR1 RNA editing enzyme regulates R-loop
formation and genome stability at telomeres incancer cells
1234567890():,;

Yusuke Shiromoto

1,4, Masayuki Sakurai1,2,4, Moeko Minakuchi1,4, Kentaro Ariyoshi1,3 & Kazuko Nishikura1 ✉ADAR1 is involved in adenosine-to-inosine RNA editing. The cytoplasmic ADAR1p150 edits
3’UTR double-stranded RNAs and thereby suppresses induction of interferons. Loss of thisADAR1p150 function underlies the embryonic lethality of Adar1 null mice, pathogenesis of thesevere autoimmune disease Aicardi-Goutières syndrome, and the resistance developed incancers to immune checkpoint blockade. In contrast, the biological functions of the nuclearlocalized ADAR1p110 remain largely unknown. Here, we report that ADAR1p110 regulates Rloop formation and genome stability at telomeres in cancer cells carrying non-canonicalvariants of telomeric repeats. ADAR1p110 edits the A-C mismatches within RNA:DNA
hybrids formed between canonical and non-canonical variant repeats. Editing of A-C mismatches to I:C matched pairs facilitates resolution of telomeric R-loops by RNase H2. This
ADAR1p110-dependent control of telomeric R-loops is required for continued proliferation of
telomerase-reactivated cancer cells, revealing the pro-oncogenic nature of ADAR1p110 and
identifying ADAR1 as a promising therapeutic target of telomerase positive cancers.

1 The Wistar Institute, Philadelphia, PA, USA. 2Present address: Research Institute for Biomedical Sciences, Tokyo University of Science, Chiba, Japan.
3

Present address: Integrated Center for Science and Humanities, Fukushima Medical University, Fukushima, Japan. 4These authors contributed equally:
Yusuke Shiromoto, Masayuki Sakurai, Moeko Minakuchi. ✉email: kazuko@wistar.org
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denosine deaminase acting on RNA (ADAR) is the
enzyme involved in adenosine-to-inosine RNA editing(A-to-I RNA editing), and three ADAR gene family
members (ADAR1, ADAR2, and ADAR3) have been identiﬁed invertebrates1–5. ADARs share common domain structures, such as
multiple dsRNA-binding domains (dsRBDs) and a separate catalytic domain6,7. Both ADAR1 (ADAR, DRADA) and ADAR2(ADARB1) are catalytically active enzymes, whereas no catalytic
activity of ADAR3 (ADARB2) has been shown so far1–4. A-to-I
editing occurs most frequently in noncoding regions that containrepetitive elements Alu and LINE8,9, and many millions of editing
sites have been identiﬁed in the human transcriptome of theserepetitive sequences9–11.
Two ADAR1 isoforms, p150 and p110, are generated by the useof separate promoters and alternate splicing12. ADAR1p150 ismostly in the cytoplasm, whereas ADAR1p110 mainly localizes in
the nucleus13. The cytoplasmic ADAR1p150 regulates the dsRNAsensing mechanism mediated by melanoma-differentiationassociated protein 5 (MDA5), mitochondrial antiviral signaling
protein (MAVS), and interferon signaling (MDA5-MAVS-IFN
signaling)14–16. The cytoplasmic ADAR1p150 edits 3′-untranslated
region (3′-UTR) dsRNAs primarily comprising inverted Alu
repeats and thereby suppresses activation of MDA5-MAVS-IFN
signaling14–16. This ADAR1p150 function in the regulation of the
MDA5-MAVS-IFN pathway underlies the embryonic lethality of
Adar1-null mice17,18 and also the pathogenesis of AicardiGoutières syndrome (AGS; AGS1–7 subgroups known), a severe
human autoimmune disease against endogenous nucleic acids14–16.
Mutations of seven genes, including RNaseH2A (AGS4), RNaseH2B (AGS2), RNaseH2C (AGS3), and ADAR1 (AGS6), havebeen identiﬁed in association with AGS, and ten AGS6 mutationsof ADAR1 have been reported so far19. Finally, this ADAR1p150mediated suppression of IFN signaling also represses tumor
responsiveness to immune checkpoint blockade20, revealing the
pro-oncogenic ADAR1p150 function. Analysis of The Cancer
Genome Atlas database revealed elevated ADAR1 expression andA-to-I editing levels in almost all types of cancers21,22, indicatingthat this pro-oncogenic ADAR1p150 function helps cancer cells
suppress inﬂammatory responses and thus avoid hostimmunosurveillance20. In contrast to the recent advance in theknowledge of ADAR1p150 functions, the biological functions of
the nuclear-localized ADAR1p110, other than its involvement inediting of intronic Alu dsRNAs23, have remained mostly unknown.
Newly transcribed RNA usually dissociates from its template
DNA strand immediately after transcription, but occasionally it
forms a stable RNA:DNA hybrid, which consequently leaves thesense DNA in a single-stranded form. This structure, called an Rloop, often spans 100–2000 bp and causes abortive transcription
and instability of the genome24,25. Several mechanisms are known
to suppress the formation of R-loops, for example, degradation of
RNA strands of RNA:DNA hybrids by RNase H126 and RNase
H227,28 and unwinding of RNA:DNA hybrids by helicases such as
DExH-box helicase 9 (DHX9)29 and senataxin (SETX)30. Humandiseases including amyotrophic lateral sclerosis type 4, ataxiaocular apraxia type 2, and AGS are caused by the accumulation ofR-loops due to deﬁciency in one of those suppression
mechanisms31,32. Telomere end regions consisting of repetitive
sequences are important for protection of these regions fromrecombination and degradation33,34. However, telomeric repeat
regions are also known to be naturally prone to the formation ofR-loops24,35, which in turn causes telomere instability and perhaps underlies carcinogenesis of certain cancers31,36. The canonical hexameric repeat sequence of the telomeric G-strand (sense
strand) is TTAGGG33,37. Interestingly, detection of widespread
telomeric variant repeats such as TCAGGG and TTGGGG has
been reported in cancer cells38–40. Mutations/variations
2(nucleotides) of telomeric canonical repeat DNA sequence
TTAGGG (antisense sequence CCCTAA) detected in cancer cells
such as TTGGGG (CCCCAA) and TCAGGG (CCCTGA) areemphasized by underlining. Their RNA sequence versions are
UUGGGG (CCCCAA) and UCAGGG (CCCUGA). In addition,adenosine residues to be edited by ADAR1 were also emphasized
by underlining: TTAGGG (RNA sequence UUAGGG).
In this study, we found an important role for the ADAR1p110
isoform in resolution speciﬁcally of the R-loops formed at telomeric repeat regions. ADAR1p110 edits both the RNA and the
DNA strands of telomeric repeat RNA:DNA hybrids containing
mismatched base pairs formed between canonical and variant
repeats. ADAR1p110-mediated editing of A–C-mismatched basepairs, which converts them to I:C-matched base pairs, is requiredfor degradation of the RNA strands of telomeric repeat RNA:DNA hybrids by RNase H2. We found that RNase H2 is incapable of resolving mismatch-containing telomeric RNA:DNA
hybrids by itself. This newly found ADAR1p110 role in suppression of telomeric R-loops seems to be essential for the continued proliferation of telomerase-reactivated cancer cells withaccumulated variant telomeric repeats, revealing yet another prooncogenic function of ADAR1.
Results
Depletion of ADAR1 results in telomere DNA damage, telomere abnormalities, and mitotic arrest. We recently made several observations that indicated the involvement of ADAR1 in themaintenance of telomere stability and mitosis. First, signiﬁcantly
increased telomere abnormality, such as telomere fusions, wasdetected with Adar1-null mouse embryonic ﬁbroblast (MEF) cells
derived from Adar1-null mouse embryos18 (Supplementary
Fig. 1a, b). In contrast, no signiﬁcant telomere abnormality was
detected with Adar2-null MEF cells (Supplementary Fig. 1c)
derived from Adar2-null mouse embryos41. Chromosome orientation ﬂuorescence in situ hybridization (CO-FISH) analysis
revealed signiﬁcantly increased involvement of leading strands intelomere fusions detected in Adar1-null MEF cells (Supplementary
Fig. 1d), indicating that ADAR1 is involved in the mechanism thatmaintains the integrity of the telomere leading strands. Detectionof signiﬁcantly increased telomere dysfunction-induced foci (TIF)
revealed by telomere FISH and γH2AX immunostaining suggested
accumulation of DNA damage, if not exclusively, mainly at telomeres in Adar1-null MEF cells (Supplementary Fig. 1e), whichmust be closely related to the increased telomere fusions (Supplementary Fig. 1b, c). Second, time-lapse imaging of HeLa cells
undergoing ADAR1 gene knockdown revealed many aberrantly
shaped nuclei (Fig. 1a and Supplementary Fig. 2a) that appeared to
be arrested during mitosis, and these aberrant cells eventually died,most likely by apoptosis (Supplementary Movies 1 and 2). Close
examination revealed the presence of increased bridged nuclei,
micronuclei, and multinuclei in ADAR1-depleted cells (Fig. 1b andSupplementary Fig. 2a). Signiﬁcantly increased TIFs were detectedalso in ADAR1-depleted HeLa cells (Fig. 1c). Ectopic expression of
ADAR1p110-WT but not ADAR1-E912A, a mutant without catalytic activity6 (Supplementary Fig. 2b), suppressed induction of
TIFs in ADAR1-depleted cells (Supplementary Fig. 3), demonstrating the importance of ADAR1-mediated A-to-I editing
activity in the maintenance of telomere stability. Western blotting
analysis revealed signiﬁcantly upregulated DNA damage markers
such as phosphorylated DNA-dependent protein kinase catalytic
subunit (DNA-PKcs), γH2AX, and phosphorylated RPA32 in
ADAR1-depleted HeLa cells (Fig. 1d). In addition, elevated levelsof CCNB1 and histone H3 phosphorylated at serine 10 as well as
decreased expression of phosphorylated CDC2 indicated that
ADAR1-depleted cells were arrested during mitosis (Fig. 1d).

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siControlasiADAR1-1 (magnified)60

Frequency (%)

bsiADAR1-1

40

20

HeLa siControl
HeLa siADAR1-1

**

**
**

0

BridgeMicronuclei

Multinucleic

HeLa siControl
HeLa siADAR1-1

siControl

TIF Positive Cell (%)

25

siADAR1-1

telFISH

JH2AX

telFISH
JH2AX

d ADAR1p150
ADAR1p110

e

GAPDH

3’

Phos-T2609
DNA-PKcs

RPA32

5’

Whole

15
10
5
0

telFISH
JH2AX(Magnified)
RPA

ss

DN

RPA

RP
A

A

R-loop

A
RN
NA
DNA
eD
:RNA
plat
hybr
m
e
id
T
RNase
ADAR1
H2A
RNase RNase
RNase H1 DHX9 SETX
H2C H2B

RNA Pol II

5’

Whole
Phos-T21

telFISH
JH2AX
DAPI

**
20

DNA damage
marker?

J-H2AX
CCNB1
Phos-CDC2

Mitotic marker

siC
on
tro
siA
l
DA
R
1-1
siA
DA
R1
-2

Phos-H3(S10)These results together suggest that ADAR1 deﬁciency causes
aberrant mitotic arrest with extensive DNA damage at telomeres.
Accumulation of R-loops speciﬁcally at telomeric repeat
regions in ADAR1-depleted cells. One of the DNA damage
markers increased in ADAR1-depleted cells was replication
protein A 32 kDa subunit (RPA32 or RPA2) (Fig. 1d). Phosphorylated RPA32 binds to single-stranded DNAs (ssDNAs) and,thus, is an effective marker for R-loops42 (Fig. 1e). We reasoned
that ADAR1 depletion might result in the accumulation of Rloops, which could cause DNA damage, telomere abnormalities,

and mitotic arrest43–45. Dot blot analysis using the S9.6 antibody
speciﬁc to RNA:DNA hybrids46 (Fig. 2a) revealed that ADAR1
depletion indeed resulted in signiﬁcantly increased the formationof RNA:DNA hybrids (Fig. 2b and Supplementary Fig. 2a).
Treatment with Escherichia coli-RNase H, which digests RNA
strands of RNA:DNA hybrids, eliminated dot blot signals, conﬁrming speciﬁc detection of RNA:DNA hybrids (Fig. 2b). ADAR2
depletion had almost no effect on the formation of RNA:DNA
hybrids (Fig. 2c and Supplementary Fig. 2a), indicating that the
R-loop regulatory function is speciﬁc to ADAR1.
RNase H126 and H228 degrade RNA strands of RNA:DNA
hybrids, while DHX929 and SETX30 unwind RNA:DNA hybrids:

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Fig. 1 ADAR1 depletion resulted in abnormalities of the nucleus and upregulation of DNA damage pathway and cell cycle marker genes. a HeLa cells
were ﬁrst transfected with siControl or siADAR1 (siADAR1-1) for 72 h and then treated with CellLight Tubulin-GFP. Nuclei were visualized by staining of
DNA with SiR-DNA reagent. Representative images taken from real-time videos (Supplementary Movies S1 and 2) are presented. Scale bar, 50 μm. b The
frequency of abnormalities of the nucleus (nucleoplasmic bridge, micronuclei, and multinuclei) was estimated by examining at least 200 individual HeLa
cells treated with siControl or siADAR1-1 RNAs. Values are mean ± standard error (n = 3, biologically independent samples) with signiﬁcant differences by
two-tailed Student’s t test indicated, **P < 0.01. Scale bar, 5 μm. c Telomere DNA damages in ADAR1-depleted cells. Telomere FISH and immunostainingfor γH2AX revealed signiﬁcantly increased telomere dysfunction-induced foci (TIF, indicated by yellow arrowheads), suggesting the causative relevance ofthe telomeric repeat DNA damage to chromosome abnormality detected in ADAR1-depleted HeLa cells. At least 200 individual HeLa cells treated withsiControl or siADAR1-1 RNAs were examined. HeLa cells with one or more TIFs were counted as TIF-positive cells. Values are mean ± SD (n = 3,
biologically independent samples) with signiﬁcant differences by two-tailed Student’s t test indicated, **P < 0.01. Scale bar, 10 μm. b, c All individual
experimental data values and exact P values are presented in Source Data ﬁle. d Western blotting analysis was done using total cell extracts from HeLa
cells treated with siControl or two separate siADAR1 (siADAR1-1 and -2) RNAs for 72 h. Protein molecular weight markers are presented in Source Dataﬁle. e The R-loop structure consisting of an RNA:DNA hybrid formed between the RNA strand newly transcribed by RNA polymerase II and the template
DNA strand with the single-stranded antisense DNA bound with ssDNA-binding protein RPA as well as major regulators are schematically shown.

a

0.1

0.5

Genomic DNA
0.5 Pgb 2 Pg0.5 pmolNo

siControl
No

Control oligo

DNA:DNA
0.02

RNA:RNA
0.1 pmolsiADAR1-1

Buffer only

siADAR1-2

RNase H 1U

RNA:DNAc

Genomic DNA(0.25 Pg) 0

siControl siADAR1-1

Relative Amount of RNA:DNA Hybrid
1

2

3

4

siControl*****

siADAR1-1*
n.s.

siADAR1-2
siADAR2-1
siADAR2-2

d

Genomic DNA(1 Pg) 0

Relative Amount of RNA:DNA Hybrid
1

2

3

4

siControl**
n.s.

siADAR1-1***

siRNase H1****

siRNase H2A
siRNase H1 & H2A
siDHX9siSETXe

Genomic DNA(0.25 Pg) 0
siControl

Relative Amount of RNA:DNA Hybrid
1

2

3

4

Vector Only**n.s.

Vector Only*

F-ADAR1p110-WT**

F-ADAR1p110-E912A

n.s.

*

siADAR1-1

F-ADAR1p150-WT

Fig. 2 A-to-I editing activity of ADAR1p110, not ADAR1p150 or ADAR2, is required for suppression of R-loops. a–e Dot blot analysis for RNA:DNA
hybrids was conducted using control oligos (a) or genomic DNA (b–e). a The S9.6 antibody recognized speciﬁcally RNA:DNA but not DNA:DNA or RNA:RNA oligo duplex controls. b, c Increased RNA:DNA hybrids were detected only in ADAR1-depleted but not in ADAR2-depleted HeLa cells. b The S9.6
antibody signals were abolished by E. coli-RNase H treatment, conﬁrming speciﬁc detection of RNA:DNA hybrids. d Comparison of RNA:DNA hybrid levels
between depletion of ADAR1 versus depletion of known R-loop regulators. e Increased RNA:DNA hybrid formation resulting from depletion of endogenous
ADAR1 was rescued by infection of ADAR1p110-WT (wild type) but not by infection of ADAR1p110-E912A deamination defective mutant or ADAR1p150WT. c–e Data are mean ± SD (n = 3, biological replicates); signiﬁcant differences were identiﬁed by two-tailed Student’s t tests: *P < 0.05, **P < 0.01,***P < 0.001, n.s., not signiﬁcant. All individual experimental data values and exact P values are presented in Source Data ﬁle.

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they all are capable of resolving already formed R-loop structures(Fig. 1e). Depletion of these R-loop regulators, either by single
knockdown or combination knockdown of RNase H1 and RNase
H2, except for RNase H1 single knockdown (see “Discussion”),
resulted in signiﬁcant accumulation of RNA:DNA hybrids in
HeLa cells (Fig. 2d and Supplementary Fig. 2c). Importantly, theamount of RNA:DNA hybrid formed in ADAR1-depleted cells
was equivalent to or even more than those formed upon depletionof these known regulators (Fig. 2d). Using various ADAR1expressing lentivirus systems, we conducted rescue experiments:
repression of RNA:DNA hybrids formed in ADAR1-depleted
HeLa cells. As expected, ectopic expression of FLAG-tagged
ADAR1p110-WT (wild type) efﬁciently suppressed the formationof RNA:DNA hybrids (Fig. 2e). In contrast, the ADAR1p110E912A could not rescue despite being expressed at a higher level
than that of endogenous ADAR1p110 (Fig. 2e and Supplementary
Fig. 2b). Furthermore, ADAR1p150-WT did not suppress theformation of RNA:DNA hybrids (Fig. 2e and Supplementary
Fig. 2b). These results demonstrated that A-to-I editing activity of
ADAR1p110, but not ADAR1p150, is required for suppression ofR-loops.
We next investigated where in the genome ADAR1p110
controls the formation of R-loops. It has been reported thatcertain genome loci such as actively transcribed genes andmitochondrial genes are particularly prone to the formation of Rloops47–49. Accordingly, we conducted quantitative PCR (qPCR)
analysis of DNA:RNA immunoprecipitation (DRIP) products
pulled down with the S9.6 antibody. The results revealed that
ADAR1 depletion had no effects on the formation of R-loops at
the known transcription start sites of NEAT1, JUN, PMS2, and
CLSPN genes, an intronic site of the β-actin gene, and an exonicsite of the mitochondrial gene CYTB47–49 (Fig. 3a). In addition toactively transcribed genes, centromeric and telomeric repeat
regions have been reported to be prone to the formation of Rloops24,25,35. The repetitive elements of retrotransposons such as
Alu and LINE are known to be the most frequent targets for
ADAR19,50. To this end, DRIP products were next tested for dot
blot hybridization analysis using four different probes carrying
telomeric repeat, α-satellite centromeric repeat, Alu, and LINE1
sequences (Supplementary Fig. 4a, b and Supplementary Data 1).
We found that ADAR1 depletion resulted in the accumulation of
RNA:DNA hybrids speciﬁcally at telomeric repeat regions, but
not at α-satellite centromeric repeats nor Alu and LINE1 repeats(Fig. 3b, c), which account for nearly 30% of the humangenome51. Accumulation of R-loops at telomeres could explain
the increased TIFs, various telomere abnormalities observed in
Adar1-null MEF cells (Supplementary Fig. 1), and mitotic arrest
detected in ADAR1-depleted HeLa cells (Fig. 1c, d). We conclude
that telomere repeats are, if not exclusive, the major targets of
ADAR1.
ADAR1p110 cannot edit completely complementary telomericrepeat RNA:DNA hybrids. ADAR1 was originally identiﬁed asan A-to-I editing enzyme speciﬁc to dsRNAs52,53. However,recent studies by Beal and his colleagues54 revealed that a catalytic
domain fragment of ADAR1 can edit DNA strands of RNA:DNA
hybrid substrates in vitro. However, it was not known whether
the full-length ADAR1 also had such RNA:DNA editing activity
and, if so, whether that RNA:DNA hybrid editing activity issigniﬁcant in vivo. Interestingly, the adenosine within the hexameric TTAGGG (UUAGGG) sequence of canonical telomericrepeats (Fig. 4a) is the most favored A-to-I editing sites of
ADAR1: it prefers 5′ nearest-neighbor U and 3′ nearest-neighbor
G55. Together with our results (Fig. 3b, c), we reasoned that
ADAR1p110 might edit RNA:DNA hybrids carrying telomeric

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repeat sequences. Editing of A:U or A:T base pairs to I:U or I:T
wobble base pairs within telomeric repeat RNA:DNA hybrids
would reduce their thermodynamic stability, which in turn would
facilitate their dissociation or unwinding by R-loop regulatory
helicases such as DHX9 and SETX (Fig. 1e). Accordingly, we
prepared telomeric repeat duplex substrates (RNA:RNA and
RNA:DNA hybrid) consisting of perfectly complementary sense
and antisense strands (Fig. 4b, c) and tested them for in vitro
editing with ADAR1p110, the ADAR1 isoform relevant to R-loop
regulation (Fig. 2e). As expected, ADAR1p110 very efﬁciently
edited six adenosine residues of the G-strand RNA (Fig. 4b, top),
and also, with lower efﬁciency, C-strand adenosines (Fig. 4b,bottom) of the completely complementary telomeric repeat
dsRNA. In contrast, ADAR1p110 editing of the completely
matched telomeric repeat RNA:DNA hybrids (Fig. 4c, top andbottom for G-strand RNA and C-strand DNA, respectively) wasvery inefﬁcient, only at the background level, that is, <5% (Supplementary Data 2), revealing a clear difference in activity toward
telomeric repeat dsRNAs versus RNA:DNA hybrids.
Widespread detection of telomeric variant repeats in ALT and
non-ALT cancer cells. Variant telomeric repeats such as
TCAGGG and TTGGGG are detected in the telomeres of cancer
cells including HeLa cells. In some cases, the proportion of variant repeats almost equals that of canonical repeats38,39. Cancer
cells maintain telomere length either by the non-canonical telomere extension mechanism, known as the alternative lengtheningof telomeres (ALT), in ALT-positive cancer cells (ALT cells) or byreactivating telomerase in ALT-negative cancer cells such as HeLa
cells (non-ALT cells)33,37. Telomeric variant repeats are ampliﬁed
by homologous recombination between telomeric repeats in
ALT cells, and also by a currently unknown mechanism in nonALT cells39. Using telomeric canonical TTAGGG and variant
TCAGGG or TTGGGG repeat-speciﬁc probes capable of distinguishing a single-nucleotide mismatch (Supplementary Fig. 4a,b), we detected indeed signiﬁcant amounts of telomeric variant
TCAGGG and TTGGGG repeats in four non-ALT cell lines,
including HeLa cells, as well as three ALT cell lines, whereas theamount of telomeric variant repeats was much less in two primary ﬁbroblast cell lines examined (Fig. 5a, b).
Accumulation of RNA:DNA hybrids containing telomericvariant repeats in ADAR1-depleted cells. Although ADAR1
does edit A:U base pairs of completely matched dsRNAs, A–Cmismatched base pairs present in naturally occurring dsRNAs
such as inverted Alu dsRNAs are, in fact, the favored ADAR1
target sites9,50. Detection of TCAGGG and TTGGGG variant
repeats surrounded by TTAGGG canonical repeats within astretch of telomeric sequence has been reported in HeLa cells38.
We hypothesized that RNA:DNA hybrids containing A–C-mismatched base pairs could arise in two ways: ﬁrst, from slippedbinding of telomeric repeat-containing RNAs (TERRAs) derived
from a stretch of TCAGGG variant repeats (UCAGGG) to the Cstrand of canonical TTAGGG repeats (CCCTAA) (Fig. 6a); second, binding of TERRA RNAs derived from canonical TTAGGG
repeats (UUAGGG) to the C-strand of TTGGGG variant repeats(CCCCAA) (Fig. 6b). In particular, TTGGGG variant repeats(both DNA and RNA strands) are prone to the formation of a
G-quadruplex structure, which then causes frequent formation ofR-loops56. G-quadruplex formation of the G-strand TERRAs
carrying this particular variant (Fig. 6b, UUGGGG repeats
highlighted in orange) is expected to leave its C-strand DNA
single stranded, which in turn may cause more frequent slippedhybridization with the canonical repeat G-strand TERRAs(Fig. 6b). Second, there would be an alternative type of pairing,

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Transcription Start Site

a

NEAT1

3

n.s.

PMS2-TSS (% Input)

2

1

n.s.

CLSPN

n.s.

IgG

S9.6

1.2

0.8

0.4

0

0

CLSPN-TSS (% Input)

JUN

2.5

JUN-TSS (% Input)

PMS2
1.6

n.s.

2.0
1.5
1.0
0.5

1.6
1.2
0.8
0.4

0

0

IgG

S9.6

RNase H
& S9.6Intronic Site

Mitochondrial Gene
siControl
siADAR1-1
30

n.s.

CYTB

2.0

CYTB (% Input)

E-actin Intron 3 (% Input)

siControl
siADAR1-1

E-actin
1.5
1.0
0.5
0

n.s.

20

10

0

IgG

b

S9.6

RNase H
& S9.6

IgG

Telomere

Satellite

Alu

LINE

Input
IgG

siA

siA

on

tro

l

DA
R1
DA -1
R1
-2
siC
on
tro
siA
l
DA
siA R1-1
DA
R1
-2
siC
on
tro
siA
l
DA
siA R1-1
DA
R1
-2
siC
on
tro
siA
l
DA
siA R1-1
DA
R1-2

S9.6 IP

siC

S9.6

RNase H
& S9.6c

Repetitive Oligo Probe Used

RNase H
& S9.6 IP

RNase H
& S9.6

Telomeric TTAGGG Repeat(% Input)

NEAT1-TSS (% Input)

siControl
siADAR1-1

siControl
siADAR1-1

2.5

Telomere

**

IgG

S9.6

2.0
1.5
1.0
0.5
0

RNase H
& S9.6

Fig. 3 Accumulation of R-loops at telomeres in ADAR1-depleted cells. a ADAR1 depletion had no effects on already known sites prone to the formation ofR-loops. Six sites were examined by qPCR analysis of DRIP products. PCR primers used are listed in Supplementary Data 1. Data are mean ± SD (n = 3,
biological replicates); signiﬁcant differences were identiﬁed by two-tailed Student’s t tests: n.s., not signiﬁcant. b DRIP products were subjected to genomic
DNA dot blot hybridization analysis with a probe containing the G-rich-telomere canonical repeat (TTAGGG), or a probe for α-satellite repeat, Alu, or LINE1
consensus sequence (Supplementary Data 1). ADAR1 depletion resulted in increased the formation of RNA:DNA hybrids speciﬁcally at telomeric repeats,
which was abolished by E. coli-RNase H treatment prior to DRIP. c Signiﬁcance of the increased R-loop formation at telomeric repeats was conﬁrmed byconducting three independent dot blot hybridization analyses of DRIP products. Data are mean ± SD (n = 3, biological replicates); signiﬁcant differenceswere identiﬁed by two-tailed Student’s t tests: **P < 0.01, n.s., not signiﬁcant. a, c All individual experimental data values and exact P values are presented in
Source Data ﬁle.

namely in trans formation of RNA:DNA hybrids, which has beenpreviously implicated for telomeric repeat sequences25,57. Thus,in trans hybridization of telomeric repeat RNAs transcribed fromone loci either with canonical or variant repeats to C-strand
DNAs containing variant or canonical repeats from the other loci,respectively, could also result in telomeric repeat RNA:DNA
hybrids containing A–C-mismatched base pairs (Supplementary
Fig. 5a, b). G-strand TERRA RNAs containing variant TCAGGG
6(UCAGGG) and canonical TTAGGG (UUAGGG) repeat
sequences are expected to accumulate in C–A (Fig. 6a and Supplementary Fig. 5a) and A–C (Fig. 6b and Supplementary Fig. 5b)
mismatch-containing RNA:DNA hybrids, respectively. Similarly,
C-strand DNAs containing canonical TTAGGG (CCCTAA) andvariant TTGGGG (CCCCAA) repeat sequences are anticipated to
be present in C–A (Fig. 6a and Supplementary Fig. 5a) and A–C(Fig. 6b and Supplementary Fig. 5b) mismatch-containing RNA:NATURE COMMUNICATIONS | (2021)12:1654 | https://doi.org/10.1038/s41467-021-21921-x | www.nature.com/naturecommunications

NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-21921-x

ARTICLE

Fig. 4 ADAR1p110 cannot edit completely matched RNA:DNA hybrids carrying telomeric repeat sequences. a Canonical telomeric repeat sequences of
G-strand RNA (red) and C-strand DNA (blue) are shown. Six adenosines of the G-strand RNA and twelve adenosines of the C-strand DNA are indicated bynumbers 1–6 and 1–12, respectively. b In vitro editing assay for completely matched telomeric repeat dsRNA was conducted using ADAR1p110-WT
recombinant protein. c In vitro editing assay for completely matched telomeric repeat RNA:DNA hybrids using ADAR1p110-WT recombinant protein. No
signiﬁcant levels of editing for matched RNA:DNA hybrids were detected. b, c PCR products (RT-PCR-ampliﬁed RNA strands and PCR-ampliﬁed DNA
strands) were subjected to Sanger sequencing. The editing frequency was estimated as the % ratio of the guanosine (black) peak over the sum ofguanosine and adenosine (green) peaks of the sequencing chromatograms. Editing frequency estimated for three independent experiments (n = 3,
technical replicates) is presented in Supplemental information (Supplementary Data 2 and Source Data ﬁle).

DNA hybrids, respectively. That is exactly what we observed by
dot blot analysis of DRIP products using canonical and variant
repeat-speciﬁc LNA-oligonucleotide probes (Supplementary
Fig. 4c, d): ADAR1 depletion resulted in the accumulation of both
RNA and DNA strands of RNA:DNA hybrids consisting of
canonical and variant telomere repeats (Fig. 6c, d). These results
strongly suggest that ADAR1p110 regulates telomeric RNA:DNA
hybrids containing variant repeats and mismatched base pairs.
ADAR1p110 edits A–C-mismatched base pairs of telomeric
RNA:DNA hybrids formed between canonical and variant
repeats. To this end, we prepared additional telomeric repeat
duplex substrates containing A–C-mismatched base pairs andtested them for in vitro editing with ADAR1p110. ADAR1p110
again edited very efﬁciently six adenosine residues of telomericrepeat dsRNA containing A–C mismatches, as expected (Fig. 7a,
top). In contrast, ADAR1p110 editing of C-strand adenosines of
A–C-mismatched dsRNA was less efﬁcient, perhaps due to thefact that two adenosines are not in the most favored UAG
sequence context (Fig. 7a, bottom)55. Most importantly,
ADAR1p110 edited adenosine residues of both the RNA and the
DNA strands of RNA:DNA hybrids, provided that they were atmismatched A–C base pairs (Fig. 7b, top and 7c, bottom). The
level of editing of the opposite strands of these RNA:DNA
hybrids, that is, C-strand DNA and G-strand RNA, respectively,
was insigniﬁcant (Fig. 7b, bottom and 7c, top). As RNA:DNA
hybrid editing activity was lost completely with ADAR1p110EAA, a dsRNA-binding defective mutant58 (Supplementary
Fig. 6), binding of ADAR1p110 to RNA:DNA hybrids must be
mediated via its dsRBDs, rather than other domains such as ZDNA-binding domain (Zβ), and is essential for editing of RNA:DNA hybrids. Previous screening has identiﬁed ADAR1 as one ofmany RNA:DNA hybrid-binding proteins, although its biologicalsigniﬁcance was not addressed47.

Editing of A–C mismatches in telomeric repeat RNA:DNA
hybrids facilitates their resolution by RNase H2. In order toobtain insight into the mechanism by which ADAR1p110mediated editing of telomeric repeat RNA:DNA hybrids contribute to resolution of telomeric R-loops, we looked for candidate cofactors of ADAR1p110 required for removal of RNA
strands from telomeric RNA:DNA hybrids. We conducted
immunoprecipitation experiments using HEK293T cells stably
expressing FLAG-ADAR1p110-WT. We found that FLAGADAR1p110-WT co-immunoprecipitated with RNase H2A andH2C subunits but not with RNase H1 (Fig. 8a, upper panels),
revealing the close association of ADAR1p110 with RNase
H2 subunits. Similar experiments with FLAG-ADAR2 revealed
no association of ADAR2 with RNase H1 or RNase H2 subunits(Supplementary Fig. 7). The ADAR1p110 interaction with RNase
H2 subunits was lost when FLAG-ADAR1p110-EAA dsRNAbinding defective mutant was used as the bait (Fig. 8a, upper
panels). Finally, we conducted a reciprocal pull-down experimentusing FLAG-tagged RNase H2A as the bait. FLAG-RNase H2Apulled down endogenous ADAR1p110 (Fig. 8a, lower panels).
Furthermore, both FLAG-ADAR1p110-WT and FLAG-RNase
H2A pulled down TRF2, a telomere-binding protein and amember of the Shelterin complex33,34. These results together
indicate that ADAR1p110 interacts with RNase H2, but not with
RNase H1, on the telomeric RNA:DNA hybrids, and possibly
collaborates in dissociating telomeric RNA:DNA hybrids containing mismatched A–C base pairs.
Accordingly, we prepared telomeric repeat RNA:DNA hybrids
containing different numbers of A–C-mismatched base pairs aswell as I:C-matched RNA:DNA hybrids (mimicking A-to-I-edited
hybrids), which were then subjected to in vitro assay using
puriﬁed human recombinant RNase H1 and RNase H2A/2B/2C
complex proteins. To our surprise, RNase H1 digested RNA
strands of telomeric repeat RNA:DNA hybrids regardless of thenumber of A–C-mismatched base pairs (Fig. 8b, upper panels and

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a0.2

Non-ALT

ALT

Primary

Probe (TCAGGG)3
Probe (TTGGGG)3
Oligo
Oligo
(CCCCAA)4(CCCTGA)4
5 fmol5 fmol
0.2 1
5 fmol
0.2 1

Probe (TTAGGG)3
Oligo(CCCTAA)4
1

HeLa

HeLa

HeLa

HEK293T

HEK293T

HEK293T

HCT116

HCT116

HCT116

HT1080HT1080HT1080

U2OS

U2OS

U2OS

Saos2

Saos2

Saos2

WI38-VA13

WI38-VA13

WI38-VA13

WI38

WI38

WI38

IMR90
Genome
DNA

b

IMR90

IMR90
4

20 100 ng

4

20 100 ng

4

20 100 ng

Telomeric Repeat (f mol/Pg Genomic DNA)
0

0.1

0.2

0.3

HeLa
HEK293T
Non-ALT
HCT116
HT1080
U2OS
Saos2
ALT
WI38-VA13
WI38
Primary
IMR90

0.4

0.5

Canonical TTAGGG

0

0.005

0.010

0.015

0.020

HeLa
HEK293T
Non-ALT
HCT116
HT1080
U2OS
Saos2
ALT
WI38-VA13
WI38
Primary
IMR90

0.205

0.210 0.215

Variant TCAGGG

0

0.02

0.04

0.06

HeLa
HEK293T
Non-ALT
HCT116
HT1080
U2OS
Saos2
ALT
WI38-VA13
WI38
Primary
IMR90

0.08

0.10

0.12

0.14

Variant TTGGGG

Fig. 5 Detection of telomeric variant repeats in ALT-positive and non-ALT cancer cell lines. a Dot blot hybridization analysis for genomic DNA samples
was conducted using three separate telomeric repeat probes capable of distinguishing a single-nucleotide mismatch (Supplementary Fig. 4a, b). In addition
to canonical TTAGGG signals, varying amounts of TCAGGG and TTGGGG variant repeat signals were detected in both ALT and non-ALT cancer cell lines,
but not in primary human ﬁbroblast cells. b Quantitation of canonical and variant telomeric repeats was done by comparing dot blot signals of genomic
DNA and canonical and variant repeat-speciﬁc control oligos. Three independent dot blot hybridization analyses were performed. Data are mean ± SD (n =3, biological replicates). All individual experimental data values are presented in Source Data ﬁle.

8c, left). In contrast, we found that digestion by RNase H2 is very
sensitive to the presence of A–C-mismatched base pairs. The
RNA strand of the RNA:DNA hybrids containing six
A–C-mismatched base pairs was almost completely resistant todigestion by RNase H2 (Fig. 8b, lower panels and 8c, right). As
8

A–C-mismatched base pairs are replaced with matched I:Cbase pairs, the RNA:DNA hybrids became more permissive to
RNase H2-mediated digestion (Fig. 8c, right): more efﬁcient
digestion for hybrids with more I:C-matched base pairs (Fig. 8d
and Supplementary Fig. 8).

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a

b

IgG

S9.6

RNase H
& S9.6TTAGGG Repeat (% Input)

l

R1

0

tro

RNase H
& S9.6 IP-1

S9.6 IP
10

DA

R1

IgG

20

siA

siC

on

tro

l

-1

RNase H
& S9.6 IP

Input

30

siControl
siADAR1-1

Canonical UUAGGG

IgG

S9.6

***

20

10

0

DA

S9.6 IP

**

on

IgG

30

Canonical Probe
LNA-(CCCTAA) 3

siC

Input

Variant UCAGGG

siControl
siADAR1-1

siA

Variant Probe
LNA-(CCCTGA) 3

TCAGGG Repeat (% Input)

c DRIP G-Strand RNA Analysis

RNase H
& S9.6

Input

IgG

0.8

IgG

S9.6

RNase H
& S9.6

DA

0

R1

tro

RNase H
& S9.6 IP

l

0.4

-1

S9.6 IPTTGGGG Repeat (% Input)***

Variant Probe
LNA-(TTGGGG)3

on

DA

R1

l
siA

siC

on

tro

RNase H
& S9.6 IP-1

S9.6 IP

Canonical CCCTAA

siA

IgG

1.2

siControl
siADAR1-1

siC

Input

TTAGGG Repeat (% Input)

d DRIP C-Strand DNA Analysis
Canonical Probe
LNA-(TTAGGG)35

siControl
siADAR1-1

Variant CCCCAA

*

4
3
2
1
0

IgG

S9.6

RNase H
& S9.6

Fig. 6 Increased telomeric RNA:DNA hybrids containing variant repeats in ADAR1-depleted cells. Formation of telomeric repeat RNA:DNA hybrids
containing A–C mismatches by in cis slipped hybridization (a, b). a TERRA RNAs transcribed from the region containing four TCAGGG (green) variant
repeats surrounded by TTAGGG (gray) canonical repeats form an RNA:DNA hybrid containing four C–A mismatches by in cis slipped hybridization to theC-strand DNA containing canonical TTAGGG (CCCTAA) repeats. b TERRA RNAs transcribed from the region containing four TTGGGG (orange) variant
repeats surrounded by TTAGGG (gray) canonical repeats form an RNA:DNA hybrid containing four A–C mismatches by in cis slipped hybridization to theC-strand DNA containing TTGGGG (CCCCAA) variant repeats. c, d Detection of increased RNA:DNA hybrids containing TCAGGG and TTGGGG variant
repeats in ADAR1-depleted HeLa cells. c DRIP products were examined for G-strand RNAs of UCAGGG variant and UUAGGG canonical repeats by dot blot
analysis using high-afﬁnity LNA-oligonucleotide probes capable of distinguishing a single-nucleotide mismatch (Supplementary Fig. 4c). d Similarly, DRIP
products were examined for C-strand DNAs of TTAGGG (CCCTAA) canonical and TTGGGG (CCCCAA) variant repeats using LNA-oligonucleotide probes
capable of distinguishing a single-nucleotide mismatch (Supplementary Fig. 4d). c, d Dot blot signals were abolished by E. coli-RNase H treatment prior to
DRIP. The signiﬁcance of the increase in RNA:DNA hybrids containing telomeric canonical and variant repeats (RNA and DNA strands) was conﬁrmed byconducting three independent dot blot hybridization analysis of DRIP products. Data are mean ± SD (n = 3, biological replicates); signiﬁcant differenceswere identiﬁed by two-tailed Student’s t tests: *P < 0.05, **P < 0.01, ***P < 0.001. All individual experimental data values and exact P values are presented
in Source Data ﬁle.

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Fig. 7 ADAR1p110 edits both RNA and DNA strands of telomeric repeat RNA:DNA hybrids containing A–C mismatches. a–c In vitro editing assay fortelomeric repeat dsRNA (a) and RNA:DNA hybrids (b, c) containing A–C or C–A mismatches was conducted using ADAR1p110-WT recombinant protein.
PCR products (RT-PCR-ampliﬁed RNA strands and PCR-ampliﬁed DNA strands) were subjected to Sanger sequencing. Editing frequency estimated forthree independent experiments (n = 3, technical replicates) is presented in Supplemental information (Supplementary Data 2 and Source Data ﬁle).

ADAR1 depletion leads to the accumulation of RNA:DNA
hybrids only in non-ALT cancer cells. Our FLAG-ADAR1p110
immunoprecipitation experiments revealed the association of
ADAR1p110 with RNase H2 subunits, but not with RNase H1(Fig. 8a). Efﬁcient resolution of telomeric RNA:DNA hybrids by
RNase H1 has been reported speciﬁcally with ALT activity
positive cancer cell lines (ALT cells)45. Interestingly, the same
study also reported that RNase H1 had no or very little effects on
resolution of telomeric RNA:DNA hybrids in non-ALT celllines45, indicating different mechanisms used for regulation of
telomeric R-loops in ALT versus non-ALT cancer cells. Accordingly, we investigated the effects of ADAR1 depletion on accumulation of RNA:DNA hybrids in both ALT and non-ALT celllines as well as primary ﬁbroblast cells. ADAR1 depletion resulted
in increased RNA:DNA hybrids in all non-ALT cell lines examined, but had almost no effects in ALT cell lines nor in primaryﬁbroblast cells (Fig. 9a and Supplementary Fig. 2d). Furthermore,
dot blot analysis of DRIP products using a telomeric repeat proberevealed that ADAR1 depletion resulted in the formation of
telomeric repeat RNA:DNA hybrids speciﬁcally in non-ALT
cancer cell lines, for example, HeLa (Fig. 3c), HEK293T, andHCT116 (Fig. 9b and Supplementary Fig. 9). The reported
association of RNase H1 with the strong telomeric repeat R-loops
formed only in ALT cells45, perhaps highlights the capability of
RNase H1 to resolve telomeric RNA:DNA hybrids even in thepresence of mismatched base pairs (Fig. 8b, c).
We found that ADAR1p110 and RNase H2A expression levels
are much higher in non-ALT cancer cells as compared to those in
ALT cancer cells and primary ﬁbroblast cells (Fig. 10a).
Furthermore, the interaction of ADAR1p110 with RNase H2A
10

was detected only in non-ALT cancer cells, but not in ALT cancer
cells and primary ﬁbroblast cells (Fig. 10b). Interestingly, ADAR1
depletion resulted in the upregulation of M-phase-speciﬁc marker
genes (Fig. 1d), perhaps indicating an M-phase-speciﬁc
ADAR1p110 function in suppression of telomeric R-loops.
Accordingly, we analyzed the interaction of ADAR1p110 and
RNase H2 by F-ADAR1p110 co-immunoprecipitation (co-IP)
experiments in cells synchronized to the M phase. First, we
noticed that RNase H2A expression levels increased signiﬁcantly,>2-fold, at the M phase (Fig. 10c, compare unsynchronized andM-phase extract lanes), while endogenous ADARp110 levels
unchanged (Fig. 10c, compare unsynchronized and M-phaseextract vector-only lanes). Most importantly, we found that theADAR1p110–RNase H2A interaction increased >3-fold in Mphase synchronized cells as compared to unsynchronized cells(Fig. 10c, FLAG-IP lanes). These results perhaps indicate a special
need in non-ALT cancer cells for two-step removal of RNA
strands of telomeric RNA:DNA hybrids accumulated speciﬁcallyaround late G2 to M phase: ﬁrst correction of mismatched basepairs by the nuclear ADAR1p110 and then for degradation of
RNA strands by RNase H2 (Fig. 8d and Supplementary Fig. 8).
Taken together, our results indicate a differential role played by
RNase H1 and RNase H2 in resolution of R-loops accumulated in
ALT and non-ALT cancer cells, respectively, due to their different
activity in degrading mismatch-containing telomeric RNA:DNA
hybrids (Supplementary Fig. 10).
Discussion
Several regulators of telomeric R-loops such as 5′ to 3′ exonuclease, Rat1p59, ﬂap endonuclease 160, and RNase H1 andNATURE COMMUNICATIONS | (2021)12:1654 | https://doi.org/10.1038/s41467-021-21921-x | www.nature.com/naturecommunications

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F-A

On

R1

tor
Ve
c

F-A

DA

ly

a

p1
10
DA
-W
R1
Ve
T
p1
cto
r O 10-E
nly
AA
F-A
DA
R1
F-A
p1
10
DA
-W
R1
T
p1
10
-EA
A

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Bait

0 5 10 30 60b

0 5 10 30 60

0 5 10 30 60 min

50 (nt)40
30

RNase H2A
RNase H2C

RNase H1

Prey

RNase H1

20

TRF2
Extract

10FLAG-IP

tor
On
F-R
ly
Na
se
Ve
H2
cto
A
rO
nly
F-R
Na
se
H2
A

ADAR1p110
50 (nt)
40
30

Ve
c

RNase H2

20

Bait
ADAR1p110

10

Prey

TRF2

32 P

Extract

FLAG-IPA A A A I
P I

A A A A A A

32

P I I I I I I

32

C C C C C CC C C C C CC C C C C C

RNase H2A

RNase H2A/2B/2C reaction

RNase H1 reaction

1

0.8

0.6

0
0

10

20

30

40

50

0.6

***
***

6A-C
4A-C&2I:C
2A-C&4I:C
6I:C

0.2

0.8

*

0.4

Relative uncleaved RNA strand

1

n.s.

Relative Uncleaved RNA Strand

c

0.4
6A-C
4A-C&2I:C
2A-C&4I:C
6I:C

0.2

0

60 min

Time

0

10

20

30

40

50

60 minTimedA-to-I editing by ADAR1p110

I

I

I

I

I

I

C

C

C

C

C

C

Removal of RNA strand by RNase H2&Resolution of telomeric R-loops

Fig. 8 Editing of A–C mismatches in telomeric repeat RNA:DNA hybrids facilitates RNase H2 cleavage of RNA strands. a F-ADAR1p110-WT
recombinant protein was copuriﬁed with endogenous RNase H2 subunits H2A and H2C, but not with RNase H1. No RNase H2 association detected with adsRNA-binding defective mutant F-ADAR1p110-EAA (upper panels). Similarly, FLAG-RNase H2A pulled down endogenous ADAR1p110 (lower panels).
Note that both FLAG-AFAR1p110 and FLAG-RNase H2A pulled down endogenous TRF2, a telomere-binding protein and a member of the Shelterincomplex, supporting our hypothesis that ADAR1 and RNase H2 collaborate together to resolve telomeric R-loops. Protein molecular weight markers arepresented in Source Data ﬁle. b In vitro assay for digestion of 5′-32P-labeled RNA strands of telomeric repeat RNA:DNA hybrids by RNase H1 or RNase
H2A/2B/2C complexes. RNase H2 could not degrade the RNA strands of telomeric repeat RNA:DNA hybrids containing six A–C mismatches, but begandigesting RNA strands of RNA:DNA hybrids containing four A–C mismatches. Replacement of all A–C mismatches with I:C-matched base pairs resulted in
efﬁcient digestion of RNA strands by RNase H2. RNase H1 degraded RNA strands regardless of the number of A–C mismatches. c Time course of digestionof RNA strands of telomeric repeat RNA:DNA hybrids with varying numbers of A–C mismatches by RNase H1 or RNase H2. Data are mean ± SD (n = 4,
technical replicates); signiﬁcant differences were identiﬁed by two-tailed Student’s t tests: *P < 0.05, ***P < 0.001, n.s., not signiﬁcant. All individual
experimental data values and exact P values are presented in Supplementary Data 3. d A-to-I editing of A–C mismatches to I:C base pairs in RNA:DNA
hybrids facilitates digestion of RNA strands by RNase H2.

H245,59,61 have been reported. In this study, we identiﬁed
ADAR1p110 as a major regulator of telomeric R-loops speciﬁcally in cancer cells. Variant telomeric repeats such as
TCAGGG and TTGGGG repeats are widespread in both ALT
and non-ALT cancer cell lines38,39 (Fig. 5). Because of thesevariant repeats, cancer cells encounter a unique problem insolving telomeric RNA:DNA hybrids containing mismatchedbase pairs. Hybridization of TERRA molecules carrying the Gstrand UCAGGG variant sequences to the C-strand DNA carrying the canonical CCCTAA (antisense of TTAGGG) repeats

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Relative RNA:DNA Hybrid Formed(siADAR1-1/siControl)
1
2
3
4

a

0
siControl
HeLa**

siADAR1-1
siControl

HEK293T***

siADAR1-1
siControl

Non-ALT
HCT116*

siADAR1-1
siControl
HT1080**siADAR1-1
siControl
U2OS**

siADAR1-1
siControl

n.s.

Saos2

ALT

siADAR1-1
siControln.s.

WI38-VA13
siADAR1-1
siControl
WI38
Primary
Fibroblastn.s.

siADAR1-1
siControl
n.s.

IMR90
siADAR1-1

siControl
siADAR1-1
1.6

siControl
siADAR1-1

HEK293T

2.0

HCT116

**

*

1.2

1.5

0.8

1.0

0.4

0.5

0

0

IgG

S9.6

IgG

S9.6

Non-ALT

Telomeric TTAGGG Repeat(% Input)

Telomeric TTAGGG Repeat(% Input)

b

2.0

U2OS

5

n.s.

Saos2

n.s.

4

1.5

3
1.0
2
0.5

1
0

0

IgG

IgG

S9.6

S9.6

ALT

Telomeric TTAGGG Repeat(% Input)siControl
siADAR1-1
1.6

WI38

n.s.

2.5

IMR90
n.s.

2.0

1.2

1.5
0.8
1.0
0.4

0.5
0

0

IgG

S9.6

IgG

S9.6

Primary Fibroblast

Fig. 9 ADAR1 regulates accumulation of telomeric R-loops only in non-ALT cells. a Detection of increased RNA:DNA hybrids only in non-ALT cells.
Genomic DNA samples (0.25 µg) collected from various cells treated with siControl or siADAR1-1 RNAs for 72 h were examined by dot blot assay using
the S9.6 antibody. Data are mean ± SD (n = 3, biological replicates); signiﬁcant differences were identiﬁed by two-tailed Student’s t tests: *P < 0.05, **P <0.01, ***P < 0.001, n.s., not signiﬁcant. b DRIP products from select cell lines were further subjected to dot blot analysis for the C-strand telomeric repeat
DNA using the canonical telomeric repeat (TTAGGG) speciﬁc probe (Supplementary Fig. 4a). Telomeric repeat RNA:DNA hybrids were detected only in
HEK293T and HCT116 non-ALT cancer cell lines. Data are mean ± SD (n = 3, biological replicates); signiﬁcant differences were identiﬁed by two-tailed
Student’s t tests: *P < 0.05, **P < 0.01, n.s., not signiﬁcant. a, b All individual experimental data values and exact P values are presented in Source Data ﬁle.

(Fig. 6a and Supplementary Fig. 5a) or hybridization of TERRA molecules carrying the G-strand canonical UUAGGG
sequence to the C-strand DNA carrying the CCCCAA (antisense of TTGGGG) variant sequence (Fig. 6b and Supplementary Fig. 5b), either by slipped hybridization or in trans12hybridization, would result in the formation of telomeric repeat
RNA:DNA hybrids containing C–A or A–C mismatches,
respectively. The possibility of R-loop formation induced bymismatches between nascent RNA and DNA sequences has
been previously pointed out62.

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Non-ALTa ADAR1p150

ALT

Primary

ADAR1p110
RNase H2A
RNase H1

T
T1
16
HT
10
80
U2
OS
Sa
os
2
WI
38
-VA
13
WI
38
IM
R9
0

93

b

F-A

Ve
cto
r

On
ly

DA
R1
Ve
p1
10
cto
-W
rO
T
n
l
F-A
y
DA
R1
p1
10
-W
T

HC

HE

K2

He

La

E-actin

Bait

HeLa

RNase H2A

Non-ALTBait

HEK293T

RNase H2A
Bait

U2OS

RNase H2A

ALT

Bait
WI38-VA13
RNase H2A
Bait
WI38

RNase H2A

Primary

Bait

IMR90

RNase H2A

DA

On

F-A

Ve
c

tor

c

FLAG-IP

ly
R1
Ve
p1
10
cto
rO
n
ly
F-A
DA
R
1p
Ve
1
cto
r O 10
n
ly
F-A
DA
R1
Ve
p1
cto
10
rO
nly
F-A
DA
R1
p1
10

Extract

Bait
RNase H2A

Prey

ADAR1p110

Unsyn

Phos-H3(S10)M

FLAG-IP

E-actin
Unsyn

M

Extract

We found that ADAR1p110 could edit efﬁciently A–C-mismatched adenosines of both RNA and DNA strands within
telomeric RNA:DNA hybrids and convert them to I:C-matchedWatson–Crick base pairs. Interestingly, telomeric variant repeats
have been reported to expand during ALT-mediated inter- and/or
intra-telomeric recombination in ALT cells, and by a currently

ARTICLE

Fig. 10 M-phase-speciﬁc interaction of ADAR1p110 and RNase H2 in nonALT cancer cells. a Elevated ADAR1 and RNase H2A expression levels
detected in non-ALT cells. Western blotting analysis was performed using
total cell extract proteins and speciﬁc antibodies (Supplementary Data 4).
b Interaction between ADAR1p110 and RNase H2 detected only in non-ALT
cancer cells. FLAG-ADAR1p110-WT recombinant protein was copuriﬁed withendogenous RNase H2 subunit H2A only from non-ALT cell lines. c Elevated
RNase H2A expression levels and also increased interaction of ADAR1p110
with RNase H2A detected speciﬁcally at M phase. FLAG-ADAR1p110-WT
pull-down experiments were conducted using HEK293T cells synchronized
at M phase using the thymidine-nocodazole double block system. a–c Protein
molecular weight markers are presented in Source Data ﬁle.

unknown mechanism in non-ALT cells38,39. An interesting possibility arises upon A-to-I editing of these mismatched A–C basepairs to I:C-matched base pairs: replication of A-to-I-edited Cstrand DNA could generate more variant telomeric TCAGGG
repeats (Supplementary Fig. 8, bottom), perhaps explaining thereported expansion of this variant repeat in cancer cells38,39. Most
importantly, we found that A-to-I editing of A–C mismatcheswithin RNA:DNA hybrids is critical for efﬁcient digestion of RNA
strands by RNase H2, and consequent resolution of telomeric Rloops (Fig. 8d and Supplementary Fig. 8), because RNase H2,
unlike RNase H1, is incapable of digesting RNA strands ofmismatch-containing RNA:DNA hybrids. Our ﬁndings on association of ADAR1p110 with RNase H2, but not with RNase H1 inliving cells (Fig. 8a), together with elevated expression of RNase
H2A subunit (Fig. 10a), especially at the M phase (Fig. 10c),further conﬁrms collaboration between ADAR1p110 and RNase
H2 in resolving speciﬁcally telomeric RNA:DNA hybrids containing A–C mismatches, perhaps at late G2 to M phase, in nonALT cancer cells (Supplementary Fig. 10).
We currently do not know the exact reason why a speciﬁc
requirement of ADAR1p110 for suppression of telomeric R-loops
is restricted to non-ALT cells (Fig. 9a, b and Supplementary
Fig. 10), as widespread presence of telomeric variant repeats has
been detected in both ALT and non-ALT cancer cells (Fig. 5). The
cell cycle-dependent function of RNase H2, speciﬁcally at G2–M
phase, in resolution of R-loops, has been reported also in yeast63.
In contrast, RNase H1 has been reported to act through the entire
cell cycle in yeast63. We conﬁrmed elevated expression of RNase
H2 and its interaction with ADAR1p110 speciﬁcally at the Mphase in non-ALT cancer cells (Fig. 10c). Most interestingly, anassociation of RNase H1 with telomeric RNA:DNA hybrids atstrong R-loop-forming loci has been reported only in ALT cancer
cells with overly elevated TERRA expression levels45 (Supplementary Fig. 10, left). Association of RNase H1 to telomeric Rloops may also be further facilitated by more frequent binding of
RPA32 to the single-stranded G-strand DNA speciﬁcally in
ALT cells64. RPA32 has been shown to enhance the association of
RNase H1 and R-loops42 (Supplementary Fig. 10, left). Knockdown of RNase H1 in HeLa cells (non-ALT cells) indeed did notlead to a signiﬁcant increase of R-loops (Fig. 2d), in agreementwith the previous report: RNase H1-dependent resolution of
telomeric R-loops occurs only in ALT cells45 (Supplementary
Fig. 10, left). TERRA expression levels seem to be another factor
that differentiates non-ALT cells from ALT cells. In non-ALT
HeLa cells, expression of TERRA is regulated in a cell cycledependent manner: lowest in S and early G2, but increasing
toward the end of G2 and M phase65. Furthermore, TERRA
expression levels remain low in non-ALT cells such as HeLa and
HEK293T, and thus unable to support strong R-loop-forming lociand recruit RNase H145 (Supplementary Fig. 10, right). Instead,
RNase H2 together with ADAR1p110 is recruited to such not so

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strong R-loop-forming loci during G2–M phase, and RNase H2and ADAR1p110 cooperatively resolve telomeric repeat RNA:DNA hybrids containing mismatched base pairs (Supplementary
Fig. 10, right). Clearly, many issues remain to be resolved withregard to why and how two mechanisms are differentiallyemployed for resolution of telomeric R-loops in ALT and nonALT cancer cells.
Telomere abnormalities such as telomere losses and telomere
leading-strand-mediated fusions, most likely caused by unresolved telomeric R-loops, were detected in ADAR1-depleted cells(Supplementary Fig. 1). Although we have no direct evidence tospecify the exact timing, telomeric RNA:DNA hybrids may form
and accumulate from the late S phase through M phase in
ADAR1-depleted HeLa cells. Unresolved telomeric RNA:DNA
hybrids as well as prolonged exposure of single-stranded Gstrands most likely lead to double-stranded DNA breaks andeventually to telomere losses and fusions. Apparently, the telomerase activated in non-ALT cells does not mend the shortened
telomere ends. Non-homologous end joining and DNA-PKcs
participate in telomere end-capping, exclusively at telomeres
generated by leading-strand synthesis in non-ALT cancer
cells66,67. This end-capping process does not seem to function
efﬁciently in ADAR1-depleted cells. It has been reported thatunresolved and persistent R-loops interfere with the DNA
double-strand break (DSB) repair mechanism68. Although factors
involved in DSB repair, such as DNA-PKcs and γH2AX, areactivated in ADAR1-depleted cells (Fig. 1d), the DSB repair
mechanism may be hindered due to the presence of unresolved
RNA:DNA hybrids, which could also contribute to the telomere
abnormalities detected. Additional and speciﬁc studies will be
required to address these issues.
Recent elegant studies by Nicholas Haining and hiscolleagues20 revealed the possibility that ADAR1 inhibitors could
restore MDA5-MAVS-IFN signaling and inﬂammatory responses
in tumors and resurrect their response to therapy utilizing
immune checkpoint blockade. However, our studies presented
here suggest another possibility: elimination of ADAR1 and/or
suppression of its A-to-I editing activity would lead to theaccumulation of telomeric repeat R-loops and consequent genome instability and apoptosis particularly in non-ALT and
telomerase-positive cancers, which are, in fact, 70–80% of all
types of cancers45. We predict that ADAR1 inhibitors would bevery effective therapeutics for cancer treatment because they willinterfere with two completely different pro-oncogenic ADAR1
functions: suppression of MDA5-MAVS-IFN signaling by
the cytoplasmic ADAR1p150 and maintenance of telomere stability in telomerase-reactivated cancer cells by the nuclear
ADAR1p110.
Methods
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 humanvirus-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 andisogenic 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-ADAR2WT, 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
14

DMEM/F12 supplemented with 10% fetal bovine serum, penicillin (100 U/ml), andstreptomycin (100 μg/ml).
HEK293T cells expressing FLAG-ADAR1p110-WT were treated withthymidine 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 releasefrom the thymidine block, the cells were washed twice with phosphate-bufferedsaline (PBS) and culture medium. After incubation with a fresh medium for 3 h, thecells 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-purovector70,71. CSII-EF-FLAG-ADAR1p110-WT-IRES-puro, CSII-EF-FLAGADAR1p110-E912A-IRES-puro, or CSII-EF-FLAG-ADAR1p150-WT-IRES-puroused 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-FLAGp110-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 weredigested with NotI and NheI and then inserted into CSII-EF-MCS-IRES-puro.
CSII-EF-FLAG-RNaseH2A was prepared by PCR cloning using pcDNA3.1RNaseH2A72 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 FLAGRNaseH2A-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 inthis 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-FLAGRNaseH2A plasmid, 10 µg of pCAG-HIVgp (GAG-POL DNA), and 10 µg of thevesicular 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 andconcentrated 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 thenincubated 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 stainedwith 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 andsoaked in Dulbecco’s PBS. Microscopic images were obtained by using a Leica TCS
SP5 DMI6000 CS Confocal Microscope and LAS X software (Leica), equipped withultraviolet 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 sizefractionated 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 detectedwith ECL (GE Healthcare) using X-ray ﬁlms. Antibodies were diluted in 10%

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Blocker BSA buffer (Thermo Fisher Scientiﬁc). Antibodies used in this study arelisted 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, andharvested 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 treatedwith 2 mg of proteinase K at 50 °C for 60 min. The nuclear fraction was applied to abuffer 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 incubatedovernight at 4 °C.
Genomic DNA was diluted in 100 μl of 6× saline sodium citrate (SSC) andspotted 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) andblocked 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-conjugateddonkey 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. Afterwashing 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 waspreincubated 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 (sc2025, Santa Cruz) were applied to the blocked Dynabeads in DRIP buffer overnightat 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). SonicatedDNA 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 withrotation. 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 TrisHCl 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 roundof 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 waspreincubated 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.

ARTICLEDot 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 andincubated at 90 °C for 10 min. The denatured DRIP products were diluted in 100 μlof 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 andincubated 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 weredetected 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 andwashing 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). Duringsonication, 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 blockedwith 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 μlof DRIP buffer containing 500 U of RNasin Plus inhibitor (Promega). SonicatedDNA 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 withrotation. 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 DRIPTE NaCl+ buffer; and (5) twice with 100 mM Tris-HCl pH 8.0 buffer. Afterremoval of 100 mM Tris-HCl pH 8.0 buffer using a magnetic stand, the beads weretreated 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. Afteradding 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 witha 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 waspreincubated 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 prehybridized with ULTRAhyb Ultrasensitive Hybridization Buffer (Invitrogen)overnight at 42 °C. 5′-32P-labeled LNA-oligonucleotide probe was added tohybridization buffer and incubated overnight at 42 °C. The membrane was washedthree 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 (GEHealthcare, Amersham Typhoon Control software). A fraction of DRIP samples(5%) was spotted onto the membrane. Oligonucleotides used as probes andwashing 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 andDNA 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, whichwere 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-ADAR1p110EAA-expressing Sf9 cells were prepared with baculovirus69. The cells were washedwith 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

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NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-21921-x(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). Afterbuffer exchange to STD300 using ZebaTM 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 wasremoved by using ZebaTM 7 K MWCO spin desalting column.
FLAG-ADAR1p110-WT or HA-ADAR1p110-EAA was puriﬁed using antiFLAG 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). Afterbuffer exchange to STD150 using ZebaTM 7 K MWCO spin desalting column, thecell extract was loaded onto the agarose. After washing with STD150 buffer, theagarose 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 βmercaptoethanol, 0.05% NP-40), and again STD150 buffer. FLAG-ADAR1p110WT 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 oftelomere 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 weresequenced using a speciﬁc sequencing primer, and the ratio of A and G peaks in thechromatograms were analyzed by CodonCode Aligner (CodonCode Corporation).
Protein co-IP. Cells expressing FLAG-ADAR1p110-WT, FLAG-ADAR1p110EAA, FLAG-ADAR2-WT, FLAG-ADAR2-EAA, or FLAG-RNaseH2A were ﬁxed
with 0.3% formaldehyde in PBS containing 1 mM DTT at room temperature for10 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 thesupernatant 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-FLAGRNaseH2A 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 andthen 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
16

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 andthen 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 donein 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 thereaction, 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 ahypotonic 0.075 M KCl solution for 20 min at room temperature and then ﬁxed ina 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 acoverslip. The slides were heated on an aluminum heat block at 80 °C for 3 min andhybridized 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 bywashing 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 andﬁxed 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 icecold 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 twicewith 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

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NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-21921-x

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 FLAGADAR1p110 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 thenstained 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 singlestranded 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 asdescribed 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 glassbottomed 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 ormore 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 signiﬁcancewas P < 0.05.
Reporting summary. Further information on research design is available in the Nature
Research Reporting Summary linked to this article.

Data availability
Data supporting the ﬁndings of this study are available from the corresponding author
upon request. Source data are provided with this paper.

Received: 28 May 2020; Accepted: 19 February 2021;

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Acknowledgements
We thank Marcin Nowotny for RNase H2A, H2B, and H2C template plasmids, andRoger A. Greenberg and John M. Murray for a critical reading of the manuscript. We also
thank the Protein Expression and Genomics Shared Facilities of The Wistar Institute Cancer Center, which are supported by the National Cancer Institute (P30
CA010815), for the services provided. The work was supported by grants from the
National Institutes of Health (GM040536, CA175058, and GM130716), the Ellison
Medical Foundation (AG-55-2281-09), the Macula Vision Research Foundation, andEmerson Collective to K.N. Y.S., and M.M. were supported in part by a fellowship fromthe Japan Society for the Promotion of Science (JSPS 2017-19) and the Uehara Memorial
Foundation, respectively.

Author contributions
Y.S., M.S., and K.N. designed the study. Y.S., M.S., M.M., and K.A. performed experiments. Y.S., M.S., and K.N. wrote the manuscript. All authors contributed to the interpretation of the data and commented on the manuscript.

Competing interests
The authors declare no competing interests.

Additional information
Supplementary information The online version contains supplementary material
available at https://doi.org/10.1038/s41467-021-21921-x.
Correspondence and requests for materials should be addressed to K.N.
Peer review information Nature Communications thanks Heather Hundley and otheranonymous, reviewers for their contributions to the peer review of this work.
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NATURE COMMUNICATIONS | (2021)12:1654 | https://doi.org/10.1038/s41467-021-21921-x | www.nature.com/naturecommunications