nature cell biology
Articlehttps://doi.org/10.1038/s41556-024-01427-6

Maintenance of R-loop structures byphosphorylated hTERT preserves
genome integrity
Received: 18 July 2023
Accepted: 23 April 2024
Published online: xx xx xxxx
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Mitsuhiro Machitani 1,10, Akira Nomura 1,2,10, Taro Yamashita 3,
Mami Yasukawa 1, Saori Ueki1, Ken-Ichi Fujita1, Toshihide Ueno 4,
Akio Yamashita 5, Yoshikazu Tanzawa2, Masahiko Watanabe 2,
Toshiyasu Taniguchi6, Noriko Saitoh 7, Shuichi Kaneko 3, Yukinari Kato
Hiroyuki Mano 4 & Kenkichi Masutomi 1,

8,9

As aberrant accumulation of RNA–DNA hybrids (R-loops) causes DNA damage
and genome instability, cells express regulators of R-loop structures. Here
we report that RNA-dependent RNA polymerase (RdRP) activity of humantelomerase reverse transcriptase (hTERT) regulates R-loop formation. We
found that the phosphorylated form of hTERT (p-hTERT) exhibits RdRP activity
in nuclear speckles both in telomerase-positive cells and telomerase-negative
cells with alternative lengthening of telomeres (ALT) activity. The p-hTERT
did not associate with telomerase RNA component in nuclear speckles but,instead, with TERRA RNAs to resolve R-loops. Targeting of the TERT genein ALT cells ablated RdRP activity and impaired tumour growth. Using agenome-scale CRISPR loss-of-function screen, we identified Fanconi anaemia/
BRCA genes as synthetic lethal partners of hTERT RdRP. Inactivation of RdRP
and Fanconi anaemia/BRCA genes caused accumulation of R-loop structures
and DNA damage. These findings indicate that RdRP activity of p-hTERT guards
against genome instability by removing R-loop structures.
Cells counteract the induction of genome instability through DNA
repair facilitated by DNA damage response (DDR), which recruits various DDR effectors to DNA lesions for DNA repair (reviewed in ref. 1).
Such genome instability also associates with abnormal genome structures. An R-loop is a three-stranded nucleic acid structure containing RNA–DNA hybrids, formed along with transcriptional elongation
due to annealing of the nascent RNA strand with the template DNA
strand. R-loops enrich in specific regions such as transcription initiation and termination sites, DNA damage sites and telomere regions2.

Accumulation of aberrant R-loops induces destabilization of genome
structure and inhibition of transcript maturation, causing DNA damage and cell death. To prevent such adverse effects by accumulation ofR-loop structures, cells express regulators. For example, RNase H family
proteins digest the RNA strand of R-loops3,4, and the helicase senataxin(SETX)5 and DEAH box helicase 9 (DHX9)6 unwind R-loop structures.
Telomere structures also maintain genome stability by protecting
the ends of chromosomes (reviewed in ref. 7). Most cancers maintaintelomeres relying on upregulation of telomerase, which consists of a

Division of Cancer Stem Cell, National Cancer Center Research Institute, Tokyo, Japan. 2Department of Orthopaedic Surgery, Surgical Science, Tokai
University School of Medicine, Isehara, Japan. 3Department of Gastroenterology, Kanazawa University Graduate School of Medical Sciences, Kanazawa,
Japan. 4Division of Cellular Signaling, National Cancer Center Research Institute, Tokyo, Japan. 5Department of Investigative Medicine, University of the
Ryukyus Graduate School of Medicine, Nakagami, Japan. 6Department of Molecular Life Science, Tokai University School of Medicine, Isehara, Japan.
7
Division of Cancer Biology, The Cancer Institute of JFCR, Tokyo, Japan. 8Department of Antibody Drug Development, Tohoku University Graduate Schoolof Medicine, Sendai, Japan. 9Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, Sendai, Japan. 10These authors
contributed equally: Mitsuhiro Machitani, Akira Nomura.
e-mail: kmasutom@ncc.go.jp
1

Nature Cell Biology

Article
catalytic component hTERT8–10 and a template RNA of human telomerase RNA component (hTERC)11. Although telomerase plays an essential
role in maintaining telomeres, previous studies have reported thathTERT has non-canonical functions beyond telomere maintenance12,13.
We reported previously that hTERT has RdRP activity, which functions
to transcribe an antisense RNA from a template RNA14,15. Phosphorylation of hTERT at threonine 249 by the serine/threonine kinase CDK1 isa key mechanism to regulate RdRP activity16. The synthesis of antisense
RNAs by the RdRP activity of hTERT negatively regulates various target
RNA levels such as transcripts of tumour suppressor genes, eventuallyleading to tumour formation14–16. In addition, we detected high levels of
p-hTERT expression in aggressive cancers with poor prognosis including lung, pancreatic and liver cancers17.
Here we found a functional role of the RdRP reaction by hTERT
in R-loop regulation and maintenance of genome stability. We foundthat p-hTERT localizes in nuclear speckles (NSs) but not telomeres. It
associates with telomeric repeat-containing (TERRA) noncoding RNAstranscribed from subtelomeric regions and dissolves R-loop structures,indicating that RdRP activity of hTERT functions to guard against
genome instability through removal of R-loop structures.

https://doi.org/10.1038/s41556-024-01427-6

with an anti-hTERT mAb (clone 10E9-2) followed by immunoblotting (IB). Consistent with the IIF images, we observed endogenous
hTERT (Fig. 1g) and p-hTERT (Fig. 1h) in U2OS, VA-13 and Saos-2 cells
synchronized in the mitotic phase. Another mAb specific for p-hTERT(clone TpMab-35) also detected p-hTERT signals in mitotic U2OS
cells (Extended Data Fig. 1h,i). These observations suggest that synchronization in the mitotic phase facilitated detection of p-hTERT
in ALT cells. In addition, knockdown of hTERT eliminated the hTERT(Fig. 1i) and p-hTERT (Fig. 1j) signals, indicating that these signals
are specific for hTERT and p-hTERT. Moreover, we confirmed thattreatment with the CDK1 inhibitor RO-3306 ablated p-hTERT signals(Fig. 1k). These findings indicate that p-hTERT is present even in
ALT cells lacking telomerase activity.

p-hTERT in ALT cells exhibits RdRP activity

To examine p-hTERT expression, we performed indirect immunofluorescence (IIF) staining using previously described anti-p-hTERT
mAbs (TpMab-1 and TpMab-3)16,17 and observed the specific signalof p-hTERT in the nuclei of several immortal cell lines (Fig. 1a,b). We
included a subset of cancer cells that retain telomere length by atelomerase-independent mechanism based on homologous recombination, called ALT. We found that p-hTERT was also detected in ALT cells(U2OS, VA-13 and Saos-2 cells), although these cells lack telomerase
activity, in agreement with previous reports (Fig. 1a,b). When we suppressed hTERT expression using siRNAs specific for hTERT (siTERT#1
and #2), the p-hTERT signals were diminished (Fig. 1c). To confirm thephosphorylation specificity of these signals, we treated HeLa and U2OS
cells with an inhibitor of CDK1 that catalyses phosphorylation of hTERT
at threonine 249 (ref. 16). Treatment with CDK1 inhibitor RO-3306
diminished the specific signal of p-hTERT in these cells (Fig. 1d). In addition, we generated another mAb specific for p-hTERT (clone TpMab-35)
and observed identical signals of p-hTERT in all cell lines (Extended
Data Fig. 1a–c). To further confirm the expression of hTERT in ALT cellsby semi-quantitative and quantitative RT-PCR (qRT-PCR) analyses, we
optimized conditions to identify hTERT expression (Extended Data
Fig. 1d–g) using primers specific for exons 15 and 16, and detected
hTERT mRNA in U2OS, VA-13 and Saos-2 cells (approximately one-third
versus that in HeLa cells; Fig. 1e).
Next, we examined p-hTERT expression in mitotic ALT cells,
because hTERT is enriched during mitosis in telomerase-positive
cells15,18. When we synchronized ALT cells in mitotic phase, we foundthat mitotic U2OS cells expressed high levels of p-hTERT spreading throughout the whole cell (Fig. 1f). We further investigated theexpression of p-hTERT in ALT cells using lysates of mitotic cells synchronized by treatment with nocodazole. Because it is difficult to
detect endogenous hTERT, we performed immunoprecipitation (IP)In agreement with previous reports19,20, we confirmed lower but detectable levels of hTERC in addition to hTERT mRNA in ALT cells (Fig. 1e),
which is essential for telomerase activity. To assess telomerase and
RdRP activities of hTERT in ALT cells, we isolated hTERT immune complex with hTERT-specific antibodies from ALT cells, and subsequently
performed a direct telomerase assay and RdRP assay. We confirmed that
IP with the hTERT-specific antibodies recovered endogenous hTERT
proteins (Extended Data Fig. 2a). Although the hTERT immune complex
isolated from telomerase-positive 293T cell lysate with the anti-hTERT
antibodies (10E9-2 and abx120550; refs. 21,22) exhibited detectablelevel of telomerase activities, the p-hTERT immune complex isolatedwith the p-hTERT-specific antibody (clone TpMab-3) exhibited no telomerase activity (Fig. 2a). In addition, we found that p-hTERT does not
associate with hTERC (Fig. 2b), suggesting that p-hTERT does not form
a complex with hTERC and thus lacks telomerase activity.
Consistent with several studies23–25, we ascertained that ALT cells
exhibited no telomerase activity (Fig. 2a and Extended Data Fig. 2b).
We found that hTERT in ALT cells does not associate with hTERC (Fig. 2c
and Extended Data Fig. 2c), even if ALT cells express both hTERT and
hTERC (Fig. 1e). These results suggest that hTERT does not form a complex with hTERC in ALT cells, explaining the previously reported lackof telomerase activity in ALT cells. By contrast, we found that p-hTERT
isolated from these ALT cells exhibited RdRP activity (Fig. 2d,e). In
addition, knockdown of hTERT (Fig. 2f) and inhibition of hTERT phosphorylation with the CDK1 inhibitor RO-3306 (Fig. 2g) decreased the
RdRP activity of hTERT in mitotic U2OS cells. We previously verified
the RdRP activity with a recombinant hTERT (rhTERT) protein partiallypurified from E. coli14. Consistent with the previous finding, we confirmed that not only hTERT immunoprecipitated from cells but alsorhTERT expressed by a baculovirus expression system exhibits RdRP
activity (Extended Data Fig. 2d). Because the recombinant protein
partially purified from a non-mammalian system does not associate with other mammalian factors, these observations suggest thathTERT itself, but not a protein that associates with hTERT, possesses
RdRP activity. Together, these findings indicate that p-hTERT exhibits
RdRP activity in a hTERC-independent manner instead of telomerase
activity in ALT cells.
Since various hTERT alternative splicing (AS) variants (such as α, β,γ, Δ2 and Δ4-13 variants) have been identified even in ALT cells26–28, we

Fig. 1 | Expression of phosphorylated hTERT proteins in ALT cells. a,b, IIF
imaging of p-hTERT. Telomerase-positive cell lines (293T, HeLa and Huh-7 cells)and ALT cell lines (U2OS, VA-13 and Saos-2 cells) were immunostained with
anti-p-hTERT mouse mAbs (TpMab-3 (a) and TpMab-1 (b)). Scale bars, 10 μm.
c,d, IIF imaging of p-hTERT (TpMab-3) in HeLa and U2OS cells treated withhTERT siRNAs (c) or a CDK1 inhibitor (RO-3306; d). Scale bars, 10 μm.
e, hTERT mRNA and hTERC expression levels in telomerase-positive HeLa and
ALT (U2OS, VA-13 and Saos-2) cells, assessed by quantitative RT-PCR analysis
using hTERT_ex15-16 primer pairs (n = 3 independent experiments per group).

Data are presented as mean ± s.d. f, Detection of p-hTERT (TpMab-3) signals in
HeLa and U2OS cells synchronized in mitosis with nocodazole. Scale bars,
10 μm. g,h, Detection of endogenous hTERT (2E4-2; g) and p-hTERT (TpMab-3;h) by IP and the subsequent IB from the cells with nocodazole treatment.
i–k, IP-IB of endogenous hTERT and p-hTERT proteins in U2OS cells with hTERT
knockdown or CDK1 inhibitor (RO-3306) treatment. The cells were treated with
nocodazole. Experiments were repeated three times (a,b,d,f–k) or twice (c) withsimilar results. Numerical source data and unprocessed blots are available assource data.

Results

ALT cells express a phosphorylated form of hTERTNature Cell Biology

Articlehttps://doi.org/10.1038/s41556-024-01427-6

aTelomerasepositive cells
Huh-7

HeLa

U2OS

VA-13

Telomerasepositive cells
293T

Saos-2HeLa

ALT cells
Huh-7

U2OS

VA-13

Saos-2

Merge

Merge

p-hTERT(TpMab-1)

p-hTERT(TpMab-3)

HoechstHoechst293Tb

ALT cells

c

U2OS

HeLa
siTERT#1

siTERT#2

siCtrlsiTERT#1

siTERT#2

HeLa

RO-3306 0 µM

5 µM

U2OS
10 µM

0 µM

5 µM

10 µM

IP: hTERT(10E9-2)

0.001
102

HeLa

p-hTERT

IB: hTERT(2E4-2)

76

VA-13 Saos-2
Merge

HeLa

52

150

IP: hTERT(10E9-2)

102

IB: p-hTERT(TpMab-3)

7652

Nocodazole(mitotic phase)

U2OS

Control

Nature Cell Biology

225
150
102
76

IP:
p-hTERT(TpMab-3)

j

(kDa)
IB:
hTERT
(2E4-2)

k

IP:
hTERT
(10E9-2)

Nocodazole
Control (mitotic phase)

(kDa)
225

225
150
102
76

IP: hTERT(10E9-2)
IB: p-hTERT(TpMab-3)

siCtrlsiTERT#1
siTERT#2

(kDa)

IP:
hTERT
(10E9-2)

siCtrlsiTERT#1
siTERT#2

isiCtrlsiTERT#1
siTERT#2

Nocodazole(mitotic phase)

HeLa

Control

HoechstU2OS

Saos-2225

150

0.0001

VA-13

Nocodazole(mitotic phase)

(kDa)

225

0.01

f

U2OS

Nocodazole(mitotic phase)Control
Nocodazole(mitotic phase)Control

HeLa

Control

Saos-2

Telomerase
positive

ALT

Nocodazole(mitotic phase)(kDa)

Control

0.1

Control1

Relative expression

VA-13

U2OS

10

Nocodazole(mitotic phase)Control
Nocodazole(mitotic phase)ControlhTERC

Nocodazole(mitotic phase)

hTERT

h

Telomerase
positive

ALT

Nocodazole(mitotic phase)geControl

Merge

Merge

p-hTERT(TpMab-3)

p-hTERT(TpMab-3)

HoechstHoechstsiCtrldIB:p-hTERT(TpMab-3)

150
102
76

–

+

–

+ RO-3306

Articlehttps://doi.org/10.1038/s41556-024-01427-6

ab

Direct telomerase assay

HeLa

ALT

10 µg

p-hTERT (TpMab-3)

hTERT (10E9-2)

hTERT (abx120550)

p-hTERT (TpMab-3)

hTERT (10E9-2)

hTERT (abx120550)

IP

Saos-2

VA-13

p-hTERT (TpMab-3)

hTERT (10E9-2)

hTERT (abx120550)(nt)

hTERT (10E9-2)

IP

U2OS
p-hTERT (TpMab-3)

hTERT (abx120550)

293T

hTERT
(10E9-2)

293T

p-hTERT(TpMab-3)
10 µg

hTERT
(10E9-2)

40 µg

10 µg

p-hTERT(TpMab-3)
10 µg

40 µg

hTERC

70
60

d

50

RdRP assay
ALT

p-hTERT (TpMab-3)

hTERT (10E9-2)

IgG

hTERT (10E9-2)

60
50
40

RdRP
product

30

20

hTERC

e

Saos-2

70

Saos-2hTERT (10E9-2)

p-hTERT (TpMab-3)

VA-13

hTERT (10E9-2)

U2OS

hTERT (10E9-2)

hTERT (10E9-2)

IP

p-hTERT (TpMab-3)

HeLap-hTERT (TpMab-3)

c

VA-13

IgG

(nt)

U2OS
hTERT (10E9-2)

IP

20

IgG

30

hTERT (10E9-2)

HeLa

IgG

40

RdRP assay
Saos-2fp-hTERT (TpMab-3)g

RdRP assay

70

RdRP assay
siTERT#2

siTERT#1(nt)

siCtrlU2OS

None

hTERT (10E9-2)

p-hTERT (TpMab-3)

VA-13

hTERT (10E9-2)(nt)

p-hTERT (TpMab-3)

IP

hTERT (10E9-2)

U2OS

U2OS
RO-3306 –
(nt)

70
60

50

50

50

40

40

40

20

RdRP
product

30

20

HeLa
–

+

70

60

30

+

60

RdRP
product

30

RdRP
product

20

Fig. 2 | Enzymatic activity of hTERT in ALT cells. a, Telomerase activity of hTERT
and p-hTERT immunoprecipitated with anti-hTERT (abx120550 and 10E9-2) andanti-p-hTERT (TpMab-3) antibodies from the indicated cells. The 293T cells are
positive controls. b,c, Association of hTERC with hTERT and p-hTERT isolatedwith the 10E9-2 and the TpMab-3 antibodies from HeLa, 293T (b) and ALT cells(U2OS, VA-13, and Saos-2 cells; c). d, IP-RdRP assay using hTERT protein prepared

identically to those in a from cells treated with nocodazole. Mouse IgG is an
isotype control for the immunoprecipitation. e, IP-RdRP assay using hTERT
and p-hTERT proteins from cells treated with nocodazole. f,g, RdRP activity of
mitotic U2OS cells with hTERT knockdown (f) or CDK1 inhibition (RO-3306;
g). Experiments were repeated three times with similar results. Source
unprocessed blots are available as source data.

examined whether the AS variants of hTERT exhibit RdRP activity using
recombinant hTERT AS variant proteins. Although we detected α, β, γand Δ4-13 variants as well as the full-length hTERT mRNA in U2OS cells(Extended Data Fig. 3a,b), recombinant AS variant proteins exhibited
no RdRP activity (Extended Data Fig. 3c,d). These results indicate thatthe full-length hTERT is essential for the RdRP activity.

Nature Cell Biology

Articlehttps://doi.org/10.1038/s41556-024-01427-6

aHoechstp-hTERT

SRRM2

b

Merge

Huh-7

Whole cell

Triton X-100
4 °C, 30 min

MNase digestion
4 °C, 30 min

VA-13

ALT cells

U2OS

Cytoplasm/nucleus
fractionation

Saos-2

NaCl extraction
0.5 M, on ice, 5 min × 3

Sup
Nucleoplasm/chromatin

Whole cell

Nucleoplasm/chromatin
NS-enrichedfraction

Nucleus
NS-enriched fraction
12

38

12

12

2
0

U2AF1

150

150

102

102

76

76

30

52

52

20

Nature Cell Biology

G
epr

225

Nucleoplasm/chromatin
NS-enrichedfraction

(nt)

225

Fig. 3 | p-hTERT localizes in NSs in ALT cells. a, Super-resolution imaging of
p-hTERT and SRRM2 in Huh-7, U2OS, VA-13 and Saos-2 cells. Scale bars, 10 μm.
b, Strategy for biochemical purification of NS-enriched fraction. c, Detection of NS
marker proteins such as phospho-SR proteins (clone 1H4), SF3B1 and U2AF1 in the
NS-enriched fraction. H2A is a marker for chromatin proteins. d, Determination of
hTERC levels in NSs by quantitative RT-PCR analysis. MALAT1 long noncoding RNA

AP

AT
AL

RdRP assay

Nucleus

Nucleoplasm/chromatin
NS-enrichedfraction

Cytoplasm

Whole

(kDa)

M

f

U2OS
Nucleoplasm/chromatin
NS-enrichedfraction

Nucleus

Cytoplasm

(kDa)

Whole

HeLa

Nucleuse

Histone
H2A

C

38

SF3B1

ER

150

4

hT

150

6

TB

31

8

AC

31

10

e-

38

38

PhosphoSR proteins
(1H4)

pr

52

1

52

Relative copy numbers

76

76

DH

Cytoplasm

(kDa)

Whole

Nucleoplasm/chromatin
NS-enrichedfraction

Nucleus

Cytoplasm

Whole

(kDa)

d

U2OS

HeLa

Nucleusc

PelletNS-enriched fraction60

IP: hTERT(10E9-2)
IB: p-hTERT(TpMab-3)

50
40

RdRP
product

and precursor mRNAs (pre-GAPDH and pre-ACTB) are marker RNAs localized in NSs.
Data are presented as mean ± s.d. (n = 3 independent experiments per group).
e, Detection of hTERT proteins immunoprecipitated from the indicated biochemical
fractions. f, IP-RdRP assay using hTERT proteins prepared identically to those in e.
Experiments were repeated four times (e), three times (c,f), and twice (a) with similar
results. Numerical source data and unprocessed blots are available as source data.

Article
p-hTERT localizes in nuclear speckles in ALT cells
To study a functional role of p-hTERT in ALT cells, we initially examined localization of p-hTERT in ALT cells. The hTERT signal, which
includes both unphosphorylated and phosphorylated hTERT, exists
throughout the nucleus, whereas p-hTERT localizes in the nuclear
structures (Extended Data Fig. 4a). The formation of ALT-associatedpromyelocytic leukaemia (PML) bodies (APBs) around telomeres isessential for the maintenance of telomeres in ALT cells29. However, thesignal of p-hTERT did not colocalize with that of APB markers PML andTRF2 in U2OS cells (Extended Data Fig. 4b,c), indicating that p-hTERT
exists outside telomeres. We focused on the shape of p-hTERT signals in immunofluorescence imaging. Emerging evidence has shownthat RNA and intrinsically disordered proteins undergo liquid–liquid
phase separation (LLPS) to form liquid droplets inside the cells30,31.
Since p-hTERT forms foci like liquid droplets (Fig. 1a and Extended
Data Fig. 4a), we hypothesized that p-hTERT undergoes LLPS and subsequently localizes in subnuclear structures. Proteins, potentiallyinducing LLPS, harbour intrinsically disordered regions (IDRs) on theirprotein domains32. Putative analyses of IDRs by several web interfaces
predicted the existence of an IDR on hTERT protein (Extended Data
Fig. 4d–g). To confirm the involvement of LLPS, we treated HeLa andU2OS cells with 1,6-hexanediol (1,6-HD) which disrupts hydrophobic
interactions and inhibits LLPS. Treatment with 1,6-HD rapidly disassembled the dot structure of p-hTERT (Extended Data Fig. 4h), suggesting that p-hTERT localizes in subnuclear structures formed by LLPS.
The 1,6-HD treatment also allowed us to efficiently detect endogenous
hTERT (Extended Data Fig. 4i) and its RdRP activity (Extended Data
Fig. 4j). In addition, we lysed cells in high salt to inhibit weak electrostatic interactions, since the electrostatic interactions are essential for
LLPS. Each treatment with 1 M NaCl and 1,6-HD increased the hTERT
protein amounts isolated from U2OS and HeLa cells (Extended Data
Fig. 4k). Combination of treatments with 1 M NaCl and 1,6-HD further
enriched the yield of hTERT proteins recovery (Extended Data Fig. 4k).
These observations suggest that p-hTERT does not co-localize withtelomeres but is present in another subnuclear structure through
LLPS in both telomerase-positive and ALT cells.
Next, we assessed where p-hTERT is localized. We focused on NSs
that contain RNA regulators such as precursor mRNA (pre-mRNA) splicing factors, transcription factors and 3′-end RNA processing factors33,34,
because p-hTERT regulates the expression of specific genes by synthesizing antisense RNAs15,16. Multicolour immunofluorescence imaging
using a super-resolution microscopy demonstrated that the signal of
p-hTERT completely merged with an NS marker SRRM2 in ALT cells(Fig. 3a). We then confirmed that the p-hTERT antibody (TpMab-3)
does not cross-react to phospho-Ser-Arg-rich (SR) proteins contained
abundantly in NSs. While the treatment with hTERT siRNAs eliminatedthe p-hTERT signal, it did not affect the signal of phospho-SR proteins(Extended Data Fig. 5a). Furthermore, we treated cells with a CLK1
inhibitor KH-CB19 to inhibit phosphorylation of SR proteins, because
CLK1 is a specific kinase for SR proteins35. The treatment with the CLK1
inhibitor inhibited the phosphorylation of SR proteins, but not that ofhTERT (Extended Data Fig. 5b).
To further confirm the localization of p-hTERT in NSs, we biochemically isolated an NS-enriched fraction (Fig. 3b and Methods).
We confirmed that the NS-enriched fraction contained a series ofFig. 4 | Involvement of RdRP in R-loop resolution on TERRA sequences.
a,b, Detection of TERRA and ARRET RNAs associated with p-hTERT
immunoprecipitated from NS-enriched fraction, analysed by RNA dot blot(a) and quantitative RT-PCR analyses (b; n = 3 independent experiments pergroup). Data are presented as mean ± s.d. c, FISH for TERRA RNAs in U2OS cells.
An NS marker SRRM2 is co-stained. Treatment with RNase A was included as acontrol. Scale bars, 10 μm. d, RdRP activity of p-hTERT purified from NSs against
a synthetic TERRA RNA template. e,f, Dot blot analyses of dsRNAs ( J2; e) andR-loop structures (S9.6; f) using RNAs and gDNAs associated with p-hTERT in NSs.

Nature Cell Biology

https://doi.org/10.1038/s41556-024-01427-6

phospho-SR proteins, SF3B1, and U2AF1 that are maker proteins localized in NSs (Fig. 3c). We also observed the enrichment of pre-mRNAs
and MALAT1 long noncoding RNA in the NS-enriched fraction, whichwere localized in NSs36. By contrast, we found that hTERC is less abundant in the NS-enriched fraction than in nuclear fraction (Fig. 3d).
IP-IB assay using an anti-p-hTERT antibody demonstrated that thispurification process concentrated p-hTERT into the NS-enrichedfraction (Fig. 3e). In addition, hTERT immunoprecipitated from the
NS-enriched fraction exhibited RdRP activity (Fig. 3f). These results
indicate that p-hTERT localizes outside telomeres but in NSs in both
telomerase-positive and ALT cells.

RdRP regulates R-loop structure on TERRA sequences
To investigate the function of p-hTERT in NSs, we isolated hTERT complex by IP from NSs purified biochemically and conducted RIP-seq
analysis for RNAs associated with hTERT in NSs (Extended Data Fig. 6a).
Interestingly, the RIP-seq suggested that hTERT interacts with TERRA
RNAs37 transcribed from subtelomeric regions (Extended Data Fig. 6b).
We also verified the association of hTERT and p-hTERT with TERRA
RNAs by RNA dot blot analysis (Fig. 4a) and qRT-PCR analysis (Fig. 4b).
hTERT knockdown cells had higher levels of TERRA RNA expression(Extended Data Fig. 6c). Since we studied the NS fraction in these analyses, these results suggest that TERRA RNAs exist in NSs. In concordancewith these findings, previous reports revealed that not all TERRA RNAs
localize at telomeres and some TERRA RNAs exist outside telomeres,
although TERRA RNAs are enriched at telomeres38,39. To further investigate the localization of TERRA RNAs into NSs, we performed FISH
(fluorescence in situ hybridization) analyses for TERRA RNAs and verified that TERRA RNAs localized in NSs (Fig. 4c).
To examine whether p-hTERT exhibits RdRP activity with TERRA
RNAs, we used TERRA RNAs as a template for IP-RdRP assay and confirmed that hTERT (Extended Data Fig. 6d) and p-hTERT (Fig. 4d) synthesized RdRP products using TERRA RNAs as a template. To determine
whether TERRA RNAs are preferentially used for RdRP activity, we
performed a titration assay using a mixture of template TERRA RNAs
and artificial RNAs without TERRA sequences and found that TERRA
RNAs exhibited robust RdRP activity (Extended Data Fig. 6e). Moreover,hTERT and p-hTERT purified from NSs interact with double-stranded
RNAs (dsRNAs) detected with a dsRNA-specific antibody (Fig. 4e).
These observations revealed that hTERT RdRP synthesizes antisense
RNAs of TERRA RNAs and produces dsRNAs. Since TERRA RNAs havebeen shown to associate with telomeric repeat sequences in genomic
DNA and form RNA–DNA hybrid (R-loop) structures38,40, we speculated
that hTERT might also regulate R-loop formation especially on TERRA
sequences. An analysis of hTERT and p-hTERT immunoprecipitatedfrom NSs identified R-loop signals associated with hTERT and p-hTERT(Fig. 4f). To clarify a function of p-hTERT in R-loop regulation, we incubated telomeric repeat RNA–DNA hybrids with hTERT (Extended Data
Fig. 6f,g) and p-hTERT (Fig. 4g) immune complex and observed degradation of telomeric repeat RNA–DNA hybrids, suggesting that p-hTERT
dissolves R-loop structures. We obtained these data using hTERT and
p-hTERT purified from NSs fractions, indicating that the R-loop regulation by RdRP occurs in NSs. Given these results, we propose a model for
regulation of R-loop formation via RdRP reaction (Fig. 4h). Following
RNA transcription, nascent RNA transcript and DNA template hybridize
Treatment with dsRNA-specific RNase III and RNA–DNA hybrid-specific RNase
H was included as controls. g, In vitro R-loop degradation assay using p-hTERT
complex immunoprecipitated from the NS-enriched fraction and 5′-32P-labelled
TERRA RNA–DNA hybrids. The reaction products were treated with RNase III
to remove dsRNAs generated by RdRP activity of p-hTERT. RNase H is a positive
control to digest RNA–DNA hybrids. h, Model of resolution of R-loop structuresby p-hTERT. Experiments were repeated three times (a,e,f) and twice (c,d,g) withsimilar results. Numerical source data and unprocessed blots are available assource data.

Articlehttps://doi.org/10.1038/s41556-024-01427-6

IP

hTERT
IgG (10E9-2)––+

Input (1%)

Input (1%)

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hTERT
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p-hTERT(TpMab-3)

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NS-enrichedfraction

U2OS

p-hTERT(TpMab-3)

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p-hTERT(TpMab-3)

(nt)

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Input (1%)

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p-hTERT(TpMab-3)

p-hTERT(TpMab-3)

p-hTERT(TpMab-3)

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BufferIncubation time 2 h
(nt)

2h

0.5 h

1h

2h

2h

UUAGGGAATCCCRNA 50DNA

Pol IIUUAGGGTERRA sequences
Digestion by
RNase H

Synthesis ofantisense RNA
P

60

P

Genomic
DNA
RNA 5’

70

32

R-looph

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p-hTERT
AAUCCCUUAGGG

5’

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Pol IIUUAGGG5’TERRA sequences

40

5’Nature Cell Biology

Prevention ofR-loop formation

5’

Digestion of
RNA strand

Article

293T

U2OS (PD 6)
Clone #1 (PD 6)
Clone #2 (PD 6)
U2OS (PD 77)
Clone #1 (PD 72)
Clone #2 (PD 71)

Clone #2

Clone #1

U2OS (WT)

Clone #2

Clone #1c

Nocodazole(mitotic phase)Control
U2OS (WT)

Clone #2

Clone #1

Clone #2

U2OS (WT)

Clone #1

(kDa)b

Nocodazole(mitotic phase)Control

U2OS (WT)aU2OS (PD 109)
Clone #1 (PD 102)
Clone #2 (PD 100)

https://doi.org/10.1038/s41556-024-01427-6

225

(nt)
70
60
50

150

hTERT
IP: hTERT(10E9-2)
IB: hTERT(2E4-2)

102
76

(kb)
5
4

40

3

RdRP
product

30

1

20

d

2

7

5

U2OS

4

Clone #1

3

Clone #2

2
1

n.d.

)

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N
P
RI
(D

R1

ne

g

Clone #1

EG

M
AL
C

f

C

PC
RI
(D

3

13
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q-

-2
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P

RR
TE
q-

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1q

Clone #1

Clone #2
10 mm

Clone #2

35

U2OS

Clone #2

Tumour volume (mm3)

20

300

P = 0.0001

P = 0.0003

Cell numbers (× 104)

25

15
10
5
0

Clone #1

350

30

250
200
150
100

P = 0.0002

e

)

A

A
RR
TE

RR
TE
q13

10

q-

TE

RR

A

A

0

P = 0.0003

Relative expression

6

50
0
0

2

4

6

8

Day

0

20

40

60

80

Day

Fig. 5 | CRISPR-Cas9 editing of TERT gene locus in U2OS cells. a, Expression ofhTERT protein (2E4-2) in hTERT-CRISPR clones with nocodazole treatment. b, IPRdRP assay using hTERT-CRISPR clones treated as in a. c, Evaluation of telomere
lengths in hTERT-CRISPR clones by Southern blotting analysis for telomere
restriction fragments. PD, population doublings. d, Determination of TERRA RNA
levels by quantitative RT-PCR analysis of DRIP products. CALM3 and ERG1 wereanalysed as positive and negative controls, respectively, for DRIP analysis. n.d.,
not detected. Data are presented as mean ± s.d. (n = 3 independent experiments

per group). e, Cell proliferation assay using hTERT-CRISPR clones. Data arepresented as mean ± s.d. (n = 3 independent experiments per group). f, Tumours
generated from hTERT-CRISPR clones in NOD/SCID mice (top panel). The volumeof each tumour is shown as mean ± s.e.m. (n = 8 mice per group; bottom panel).
One-way ANOVA with Dunnett correction for multiple comparisons between
control and other groups (e,f). Experiments were repeated three times (a,b) or
twice (c) with similar results. Numerical source data and unprocessed blots areavailable as source data.

and form R-loop structures, where p-hTERT binds to TERRA sequencesof the nascent RNA transcripts and synthesizes antisense RNAs through
RdRP activity. The RdRP reaction converts RNA–DNA hybrids of R-loops
to dsRNAs, preventing R-loop formation. Since an accumulation ofR-loop formation has anti-proliferative effects, by resolving R-loops
hTERT may allow the proliferation of ALT cells.

TERT gene editing impairs the proliferation of ALT cells

Nature Cell Biology

To further assess the biological significances of p-hTERT in ALT cells,
we used the CRISPR-Cas9 system targeting the TERT gene. We failed todevelop hTERT knockout clones with homozygous mutations, probably
because the loss of TERT gene is lethal. However, we established two
clones with heterozygous mutations in the TERT gene. We found by

Article
Sanger sequencing analysis that hTERT-CRISPR clone #1 has an allele
with 16 bp deletion (853–868 nt in exon 2) in the TERT locus in addition
to wild-type allele (Extended Data Fig. 7a), and that hTERT-CRISPR clone#2 has two mutant alleles with 16 bp deletion (848–863 nt or 853–868 nt
in exon 2) in the TERT locus (Extended Data Fig. 7b). These 16 bp deletion
mutations result in frameshifting and causes a premature termination
codon (PTC; 1,033 nt from the transcription start site). The PTC in thecoding region of hTERT mRNA would be recognized and degraded by
nonsense-mediated mRNA decay41. Indeed, hTERT expression bothin hTERT-CRISPR clones #1 and #2 was lower than that in wild-type
U2OS cells at protein (Fig. 5a) and mRNA (Extended Data Fig. 7c) levels. Furthermore, we observed that CRISPR-Cas9 editing of the TERT
locus ablated the RdRP activity dependent on hTERT (Fig. 5b). Genome
edition of the TERT gene did not affect telomere lengths in U2OS cells,
confirming that hTERT is not involved in telomere maintenance in
ALT cells (Fig. 5c). To investigate R-loop structures in hTERT-CRISPR
clones, we performed DRIP (DNA–RNA immunoprecipitation) assay
with an RNA–DNA hybrid-specific S9.6 antibody and determined TERRARNA levels contained in R-loop structures. This analysis demonstrated
that TERRA RNAs are more abundant in DRIP samples of hTERT-CRISPR
clones than those of wild-type U2OS cells, suggesting that R-loops
with TERRA RNAs accumulate in hTERT-CRISPR clones (Fig. 5d). We
next performed RNA-seq and clustering of expression profiles on thesubset of genes expressed differentially in hTERT-CRISPR clones. The
subsequent gene ontology (GO) analysis showed enrichment of genes
involved in cell cycle process and DNA replication within genes downregulated in hTERT-CRISPR clones (Extended Data Fig. 7d). We thenmonitored the proliferation of these cells. The TERT gene mutationin hTERT-CRISPR clones #1 and #2 negatively affected cell proliferation (Fig. 5e). To further evaluate the influence of TERT gene editing in
ALT cells in vivo, we implanted wild-type U2OS cell and hTERT-CRISPR
clones (clone#1 and #2) subcutaneously into non-obese diabetic/severe
combined immunodeficiency (NOD/SCID) mice. Consistent with theresults of the cell proliferation assay, the volume of tumours generatedusing hTERT-CRISPR clones (clone #1 and #2) was significantly smaller
than that of wild-type U2OS control (Fig. 5f).

Synthetic lethality between inhibition of RdRP and FANC/
BRCA
Next, we performed genome-wide CRISPR screening using celllines that harbour the mutant hTERT-CRISPR allele. We infected thehTERT-CRISPR clone with the Brunello human CRISPR/Cas9 lentivirallibrary42, which contains 76,441 single-guide RNAs (sgRNAs) targeting 19,114 genes. After puromycin selection, we collected a portionof the cells as a sample for day 0, and we then collected the rest ofcells after 21 days of culture (Fig. 6a). We conducted next-generation
sequencing to determine sgRNAs contained in each cell populationand plotted the gene essentiality score calculated with the MAGeCK
algorithm43 (Fig. 6b). We focused on gene groups with lower essentiality scores (< –0.5) for the hTERT-CRISPR clone but not for wild-type
U2OS cells, implying that these genes are specifically essential for thehTERT-CRISPR clone (Fig. 6b). The subsequent GO analysis and pathway
Fig. 6 | Synthetic lethality between inhibition of RdRP and Fanconi anaemia/
BRCA. a, Scheme of genome-wide CRISPR screening using an RdRP-mutant
hTERT-CRISPR clone. U2OS cells and hTERT-CRISPR clone #2 were transducedwith lentivirus gRNA library and selected with puromycin for 48 h, and a portionof the cells was collected as a baseline (day 0). The remainder was harvested
after 21 days of culture. b, The MLE beta scores for each gene generated using the
MAGeCK algorithm, which shows gene essentiality in the cells. The red squareindicates RdRP synthetic lethal genes. c,d, GO analysis (c) and KEGG pathway
analysis (d) for RdRP synthetic lethal genes. The P values were calculated using
the clusterProfiler package. e, The MLE beta essentiality scores for FANC/
BRCA genes. f, Colony formation assay using hTERT-CRISPR clones transfectedwith siRNAs specific for FANC/BRCA genes (n = 3 independent experiments

Nature Cell Biology

https://doi.org/10.1038/s41556-024-01427-6

analysis of these genes showed the enrichment of genes involved in DNA
repair and Fanconi anaemia (FANC) pathway (Fig. 6c,d). These genes
included BRCA1, BRCA2 and FANC genes (Fig. 6e), which function in
DNA repair processes in concert with BRCA1 and BRCA244,45. Deletionof the FANC/BRCA genes only exhibited an effect in the absence ofhTERT expression (Fig. 6e). To assess synthetic lethality by inhibition ofhTERT and the FANC/BRCA pathway, we induced knockdown of genes
involved in the FANC/BRCA pathway in hTERT-CRISPR clones (Extended
Data Fig. 8a). Suppression of FANC/BRCA gene expression with thesesiRNAs significantly decreased the viability of hTERT-CRISPR clones
more greatly than that of U2OS cells (Fig. 6f and Extended Data Fig. 8b).
The CRISPR screening did not define PARP1 as an essential gene for thehTERT-CRISPR clone (Extended Data Fig. 8c), although PARP1 also plays
an important role in DNA repair46. Indeed, PARP knockdown did nothave an effect on the viability of hTERT-CRISPR clones (Extended Data
Fig. 8d,e). We then overexpressed wild-type or T249A-mutant hTERT in
hTERT-CRISPR clones to validate the significance of RdRP activity in thesynthetic lethality (Extended Data Fig. 8f). T249A-mutant, in which thethreonine 249 of hTERT is replaced with alanine, is not phosphorylated
and thus lacks the RdRP activity16. Although overexpression of wild-type
hTERT rescued the synthetic lethal phenotype of hTERT-CRISPR clones
with FANC/BRCA knockdown, overexpression of the T249A-mutant had
no effect on the viability of hTERT-CRISPR clones (Fig. 6g and Extended
Data Fig. 8g). These results suggest that the RdRP activity, but not otherfunctions, is involved in the synthetic lethal pathway.
Next, we induced hTERT knockdown in telomerase-positive
Capan-1 cells (Extended Data Fig. 9a,b), which carry a BRCA2 mutation, and Capan-1 clones reverted from BRCA2 mutation, in which
BRCA2 gene expression is restored47. The Capan-1 clones with reversionof BRCA2 gene mutation exhibited a significantly higher survival rate
after treatment with siTERT#1 and #2 than that in control Capan-1 cells(Fig. 6h and Extended Data Fig. 9c). We also utilized FANCF-deficient
TOV-21G cells and TOV-21G cells corrected for FANCF (designatedTOV-21G FANCF)48. TOV-21G FANCF cells were more resistant to hTERT
knockdown (Fig. 6i and Extended Data Fig. 9d) and to a CDK1 inhibitor
RO-3306, which blocks the RdRP activity, as well as to a PARP inhibitor, olaparib, used as a positive control (Fig. 6j,k and Extended Data
Fig. 9e,f). By contrast, when we treated these cells with a telomerase
inhibitor, 6-thio-dG49, both TOV-21G and TOV-21G FANCF cells were notresistant to the telomerase inhibitor (Fig. 6l and Extended Data Fig. 9g),
implying that inhibition of telomerase activity did not induce the synthetic lethality. Collectively, these observations reveal that inhibitionof RdRP and the FANC/BRCA pathway is synthetic lethal.

Inhibition of RdRP and FANC/BRCA accumulates R-loops
Biochemical experiments demonstrated that p-hTERT regulates a formation of R-loop structures (Fig. 4). Notably, previous reports have also
shown that FANC/BRCA genes, including BRCA1, BRCA2 and FANCD2,
dissolve R-loop structures and prevent excessive R-loop accumulation50–52. To investigate cooperative control in R-loop formation by
hTERT and FANC/BRCA pathway, we induced double-knockdown ofhTERT and FANC/BRCA gene and analysed R-loop levels in the cells.
per group). g, Survival of hTERT-CRISPR clones with FANC/BRCA knockdown
when overexpressing a wild-type hTERT or an RdRP-deficient T249A mutant(n = 3 independent experiments per group). h,i, Survival rates following hTERT
knockdown in Capan-1 (h) and TOV-21G (i) cell clones corrected for BRCA2 andFANCF deficiencies, respectively (n = 3 independent experiments per group).
j–l, Sensitivity of TOV-21G FANCF cells to a CDK1 inhibitor (RO-3306; j), a PARP
inhibitor (olaparib; k) and a telomerase inhibitor (6-thio-dG; l) (n = 3 independent
experiments per group). Data are presented as mean ± s.d. (f–l). Two-tailed
unpaired t-test (i–k) and one-way ANOVA method with Dunnett correction for
multiple comparisons between control and other groups (f,h). Numerical source
data are available as source data.

Articlehttps://doi.org/10.1038/s41556-024-01427-6

R-loop signals accumulated after single-knockdown of hTERT (Fig. 7a,leftmost lane). In addition, slight accumulation of R-loop signals wasobserved in single-knockdown of FANC/BRCA genes (Fig. 7a, rightfour lanes, first line). Strikingly, they were particularly enriched in thecells with double-knockdown of hTERT and FANC/BRCA genes (Fig. 7a,right four lanes, second and third lines). In addition, we immunoprecipitated R-loop structures from the cells by DRIP53 and determinedbsgRNA library
infection

Gene essentiality
scores(MLE beta score)sgRNA counts at day 21
sgRNA counts at day 0

1

MLE beta score(RdRP-mutant cells)

Wild-type cells (U2OS)

RdRP-mutant cells (clone#2)0–1

Synthetic lethal

–2
–2

d

GO analysis

Pathway analysis

mRNA methylation

Fanconi anaemia pathway

Mitochondrial translation

Nucleocytoplasmic transport

DNA repair

RNA polymerase

Protein monoubiquitination

Huntington disease
0.1

0.001

0.00001

1

0.1

0.01

P value

–0.5
–0.7

1.0
0.8
0.6
0.4
0.2

0.8
0.6
0.4
0.2

1.4
1.2

0.6
0.4
0.2

0.4
0.2
0

Nature Cell Biology

DMSO

RO-3306

TOV-21G FANCF
P = 0.0034

1.0
0.8
0.6
0.4
0.2
0

DMSO

F
N
FA

si

N

C

C

E

#2
D2

si

FA

si

FA

#1
C
N

N

TOV-21G
FANCF
P = 0.0064

0.8
0.6

P = 0.0161

0.4
0.2

C

si

si

si

TOV-21G
1.2

OlapariblRelative survival rate

0.6

Relative survival rate

1.0
0.8

k

TOV-21G

tr
l
TE
RT
#
si
TE 1
RT
#2

tr
l
TE
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#1
si
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#2

si

C
FA
N
si

P = 0.0017

1.2
1.0

0

C

D2

A2
BR

C
BR

C

A1

l
tr
C
si

si

si

TOV-21G FANCF

C

A2
C

si

C2-2

0

TOV-21G

FA

BR
si

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0.8

0

1.2

D2

#2

#1
A2
C

BR
si

C
BR
si

1.0

Capan-1

Relative survival rate

Clone#2+T249A

P = 0.0005
P = 0.0014

Clone#2+hTERT

1.0

P = 0.0010
P = 0.0002

i

Clone#2

1.2

A1

A1
C

BR

h

U2OS

Relative survival rate

Relative survival rate

g 1.4

Relative survival rate

#1

l
tr
C
si

si

–1.1

#2

0

–0.9

j

Clone#2

P = 0.0001
P = 0.0001

–0.3

1.2

P = 0.0002
P = 0.0033

Relative survival rate

MLE beta score

Clone#2

Clone#1

P = 0.0012
P = 0.0058

I
C

F

N

C

FA

E
C

N
FA

D2
FA

N

C

B

N

C

C

N

FA

A2

FA

A1
C

BR

BR

U2OS

U2OS

1.4

P = 0.0005
P = 0.0006

f

–0.1

0.001

P valuee
0.1

0MLE beta score(wild-type cells)

P = 0.0001
P = 0.0001

c

Day21

P = 0.0005

Day0

–1

P = 0.0003

a

TERRA RNA levels associated with R-loops by a strand-specific reverse
transcription and the subsequent qPCR (Extended Data Fig. 9h,i). Consistent with the dot blot analysis, this DRIP analysis found that TERRA
RNAs associated with R-loop structures accumulated in the cells withdouble-knockdown of hTERT and FANC/BRCA genes (Fig. 7b). Theseobservations demonstrated that inhibition of hTERT and the FANC/
BRCA pathway leads to accumulation of R-loop structures. We were

TOV-21G

TOV-21G FANCF

1.2
1.0
0.8
0.6
0.4
0.2
0

DMSO

6-thio-dG

Articleb9

siFANCF

siBRCA2

siBRCA1

siCtrlasiFANCD2https://doi.org/10.1038/s41556-024-01427-6

7

Fold enrichment

siCtrlsiTERT#1
IB: S9.6(R-loop)

siCtrlsiTERT#1

6
5
4
3

siTERT#1

siCtrlsiTERT#1

siBRCA2 siFANCD2 siFANCF

siCtrlsiBRCA1

siFANCF

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siCtrlsiTERT

RNaseH +siTERT#2

siBRCA2 siFANCD2 siFANCF

siCtrlsiBRCA1

siBRCA2 siFANCD2 siFANCF

Merge γH2AX Hoechst

siBRCA1

siBRCA1

siCtrlRNaseH –csiFANCFsiFANCD2

siBRCA1

siTERT

siBRCA2

siCtrlsiFANCF

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IB:
dsDNA

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1
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siCtrl2siTERT#2

siBRCA1+ RNaseH

siTERT#2+ RNaseH

10q-TERRA
13q-TERRA
16q-TERRA
1q-2q-10q-13q-TERRA

8

30
25
20
15

1.4

10

U2OS
1

P = 0.0028
P = 0.0006

P = 0.0031

0

2

4

6

8

U2OS

1.2

Clone#1

1.0
0.8

Clone#2
P = 0.0001

P = 0.0019

0.4
0.2
0
0.001

0.1

Clone
#1
Clone
#2

C
N

C

si

FA

N
FA
U2OS
Clone#2
1.2

Relative survival rate

U2OS

Clone#1
P = 0.0003
P = 0.0031

1.0

BRCA2

h

p-hTERT

Inhibition ofhTERT expression
orInhibitors against
RdRP activity

Genetic mutation
R-loop0.6

Genomic DNA

0.4
0.2

RNA 5’DMSO

CPTFanconi anaemia
factor

BRCA1

P

0.8

0

10

CPT (uM)

F

D2

A2
si

BR
si

C
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si

C

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l
tr
C
si

CPT
DMSO (2 nM)

P = 0.0001

0.6

IR (Gy)0gClone#2

0.1

0.01

5

Clone#1

P = 0.0170

Relative survival rate

35

f
10

Relative survival rate

P = 0.0111
P = 0.0192

40

P = 0.0004
P = 0.0040

γH2AX-positive cells (%)

45

esiTERT#2
P = 0.0218
P = 0.0168

siTERT#1
P = 0.0009
P = 0.0010

siCtrl

P = 0.0011
P = 0.0009d

Pol IIUUAGGGTERRA sequences
Accumulation of R-loop structures
Excessive DNA damage and cell deathFig. 7 | Genome instability triggered by suppression of hTERT. a,b, Analysesof R-loop structures in U2OS cells co-transfected with siRNAs specific for hTERT
and FANC/BRCA genes. R-loop structures were detected by dot blot analysis (a).
TERRA RNA levels in R-loops were determined by strand-specific quantitative
RT-PCR analysis of DRIP products (b; n = 3 independent experiments per group).
RNase H, a control to digest RNA–DNA hybrids; dsDNA, a loading control.
c,d, IIF imaging of γH2AX in U2OS cells with double-knockdown of hTERT and
FANC/BRCA genes (n = 4 independent experiments per group). Scale bars, 50 μm.

Nature Cell Biology

e–g, Cell viability assays using hTERT-CRISPR clones treated with IR (e) and CPT(f,g; n = 3 independent experiments per group). Cell viability was determined bycolony formation assay (e,g) and MTT assay (f). h, Model of synthetic lethality
between inhibition of RdRP activity and the FANC/BRCA pathway. Data arepresented as mean ± s.d. (b,d–g). One-way ANOVA with Dunnett correction for
multiple comparisons between control and other groups (d–g). Experiments
were repeated three times (a) or twice (c) with similar results. Numerical source
data and unprocessed blots are available as source data.

Articleunable to detect interaction of RdRP with BRCA2, suggesting that RdRP
and the FANC/BRCA pathway function redundantly (Extended Data
Fig. 10a,b). Excessive R-loop structures cause DNA damage and subsequent genome instability2. Consistently, double-knockdown of hTERT
and FANC/BRCA genes efficiently induced γH2AX, which is a marker
associated with DNA lesions (Fig. 7c,d). Therefore, we finally examined the effect of hTERT on vulnerability to DNA damage. In results,
hTERT-CRISPR clones were more sensitive to DNA damage inducedby ionizing radiation (IR) and camptothecin (CPT; Fig. 7e–g). Taken
together, hTERT RdRP dissolves R-loop structures on TERRA sequencesand guarantees genomic stability, like the FANC/BRCA proteins (Fig. 7h
and Extended Data Fig. 10b).

Discussion

Expression and activity of p-hTERT in ALT cells
The discovery of ALT activity in immortalized cell lines without telomerase activity was first reported in 1995. The authors revealed thatthese ALT cells completely lack telomerase activity23 and that the ALT
mechanism occurs in human tumours24. The subsequent studies haveshown that most cancer cells with ALT activity lack telomerase activity
and maintain the telomere without any contribution of hTERT23,24,54–56.
This series of studies resulted in emphasizing the mutually exclusive
properties of telomerase and ALT activities, and hence, the expressionof hTERT in ALT cells has scarcely been investigated. However, several
reports verified that hTERT is functional in ALT cells54–56. For example,
ectopic expression of a mutant hTERT in ALT cells showed that hTERT
contributes to tumourigenesis by a telomerase-independent mannerin ALT cells25. In this study, we have focused on the role of hTERT in
ALT cells lacking telomerase activity (Figs. 1 and 3) and provided evidence showing that p-hTERT exhibits RdRP activity but not telomerase
activity (Fig. 2). Genome editing of the TERT gene in ALT cells had inhibitory effects on proliferation of ALT cells (Fig. 5). These observations
reveal that p-hTERT in ALT cells exhibits RdRP activity and contributes
to cancer progression in a telomerase-independent manner. We previously conducted genome editing of TERT in telomerase-positive
293T cells and demonstrated that the genome editing of TERT in
293T cells also inhibited tumour formation16. Taken together, theseresults suggest that the RdRP activity of hTERT contributes to cancer
progression in various cells including telomerase-positive and ALT cells.
One question is why ALT cells do not show telomerase activity.
We confirmed expression of both minimum essential components fortelomerase activity, hTERT and hTERC, in ALT cells (Fig. 1). Despite theexpression of hTERT and hTERC, ALT cells did not exhibit telomerase
activity (Fig. 2a). We noticed that the signal of p-hTERT completely
merged to NS markers, indicating that p-hTERT mostly, if not completely, localizes in NSs of ALT cells (Fig. 3a). Furthermore, we observedrare hTERC signal in NSs (Fig. 3d), probably because hTERC localizes
partly in another subnuclear structure, Cajal body, with hTERT, whichis essential for the maturation of telomerase enzyme complex57. These
results imply that p-hTERT and hTERC exist in separate compartments.
We previously reported that phosphorylation of hTERT promotes
RdRP activity but dispensable for telomerase activity16. In addition,
we confirmed that p-hTERT does not exhibit telomerase activity evenin telomerase-positive 293T cells (Fig. 2a), and that p-hTERT does not
associate with hTERC, even if hTERC is present (Fig. 2b). We also foundthat hTERT does not form a complex with hTERC in ALT cells (Fig. 2c).
Alternatively, hTERT binds to TERRA RNAs in ALT cells (Fig. 4a,b). Previous reports demonstrated that ALT cells express high levels of TERRA
RNAs38,58 and that TERRA RNAs associate with hTERC and inhibit telomerase activity39. These data suggest that TERRA RNAs might inhibit theformation of telomerase complex in ALT cells.

Detection of endogenous hTERT proteins
While we detected hTERT proteins in mitotic cells, hTERT was difficult
to detect in cells without nocodazole treatment (Fig. 1g,h). Consistent
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https://doi.org/10.1038/s41556-024-01427-6

with our data, several groups have reported that it is difficult to detect
endogenous hTERT in interphase59,60 and that hTERT is enriched in
mitotic phase61. In the present study, we revealed that endogenous
hTERT localizes in NSs in interphase (Fig. 3a), and found that treatment
to disassemble the NS structure with 1,6-HD promotes to solubilize
hTERT proteins even in interphase (Extended Data Fig. 4i). We consider
that the manipulation of cells in mitosis, where nuclear membrane
disappears and nuclear structures disassemble, is equivalent to thetreatment with 1,6-HD (Fig. 1f). These results suggest that the disassembly of NS structures would enable us to solubilize and efficiently
detect hTERT proteins.

Regulation of R-loop formation by p-hTERT
In terms of the physiological significance of hTERT, we demonstratethat hTERT regulates R-loop structure through RdRP (Fig. 4). To prevent the deleterious effects of accumulation of R-loop structures,
cells express regulators of R-loop structures, including RNase H
family protein3,4 and helicases such as SETX5 and DHX96. A previous
ChIP-seq analysis revealed the predominant association of RNase H1
with R-loops in gene promoter regions62. Since it is not clear in thisprevious study whether RNase H1 regulates TERRA RNAs, we analysed
their ChIP-seq data and found no binding signal of RNase H1 in subtelomeric regions. In the present study, we report that hTERT associateswith and dissolves R-loop structures on TERRA sequences (Fig. 4),
suggesting that hTERT regulates R-loop structures by different modeof action from RNase H1.
We propose a model for regulation of R-loop formation via RdRP
reaction (Fig. 4h). In this model, the RdRP reaction targeting TERRA
sequences strips RNA transcripts from the RNA–DNA hybrids to dissolve R-loop structures. Consistent with our model, a previous report
indicated that R-loop formation in transcription termination regions
facilitates synthesis of antisense RNAs that hybridize with the sense
RNAs, subsequently generating dsRNAs63. Although this previous
report did not provide any data on what kind of polymerases catalyse
the synthesis of antisense RNAs, we here discovered that p-hTERT is asynthesizer of antisense RNAs in genomic regions with R-loops. Ourdata support and strengthen the model that generation of dsRNAs
through synthesizing antisense RNAs regulates R-loop formation.

Gene dependency of ALT cells
A previous report analysed the gene dependency of ALT cells, indicating that ALT cells depend on genes involved in DNA replication andreplication stress response64. In particular, this analysis demonstrated
that FANCM is the most essential gene in ALT cells, probably because
FANCM suppresses telomeric replication stress in ALT cells65. Consistent
with this previous finding, the CRISPR screening in this study identified FANCM as a gene with high dependency in wild-type U2OS cells,
and revealed that FANCM was also essential for hTERT-CRISPR clones.
These observations imply that FANCM is an essential gene in ALT cells,
regardless of hTERT status.

Inhibition of RdRP activity and FANC/BRCA pathway
Several laboratories have reported that the FANC/BRCA family members remove R-loop structures, thereby preventing genome instability50–52. In particular, association of BRCA1 with TERRA RNAs prevented
the formation of R-loops mediated by TERRA RNAs52. In the current
study, we performed a genome-wide CRISPR/Cas9 loss of function
screen and revealed that concurrent inhibition of hTERT and the FANC/
BRCA genes significantly decreased cell viability (Fig. 6). hTERT did notassociate with BRCA2, suggesting that RdRP and FANC/BRCA pathway
function redundantly (Extended Data Fig. 10). The FANC/BRCA genes
maintain genomic DNA homeostasis, suppressing cancer progression,and cells lacking the FANC/BRCA pathway are more cancer prone66.
Therefore, inhibition of the RdRP activity of hTERT may be an effective
therapeutic strategy for such tumours lacking the FANC/BRCA pathway.

Article
In summary, our results indicate that p-hTERT is universally
expressed in not only telomerase-positive cells but also ALT cells. We
further found the localization of p-hTERT into NSs and affirmed thatp-hTERT regulates R-loop formation through RdRP activity, which
contributes to cancer cell proliferation. These observations implicatethat p-hTERT maintains genome stability throughout the genome fromtelomere to telomere by taking advantage of the two distinct enzymatic
activities (Extended Data Fig. 10c). These results provide important
insights for a deeper understanding of the mechanisms underlying thetelomerase-independent contribution of hTERT to tumours.

Inclusion and ethics statement
All collaborators of this study have fulfilled all authorship criteria
required by Nature Portfolio journals and have been listed as authors, as
their participation was essential for this study throughout the research
process. Findings in this study were determined with these local collaborators. The collaborators agreed on their roles and responsibilities ahead of the research. This research was not severely restrictedor prohibited in the setting of the researchers. This research does not
result in stigmatization, incrimination, discrimination or otherwise
personal risk to participants.

Online content

Any methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information,
acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at https://doi.org/10.1038/s41556-024-01427-6.

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Article

Methods

Our research complies with the relevant ethical regulations. All experiments using mice and recombinant DNA were performed in accordancewith the study protocols approved by the Ethics Committee and the
Recombinant DNA Experiment Committee of National Cancer Center(2015-187, B60M7-20 and B60M8-22) and the Animal Care and Use
Committee of Kanazawa University (2017-050) and Tohoku University(2019NiA-001).

Cell culture and reagents
The human osteosarcoma cell line U2OS (300364, CLS) was cultured
in DMEM/F-12 with 5% heat-inactivated fetal bovine serum (IFS). The
SV40 transformed human fibroblast cell line VA-13 (RCB0251, RIKEN
BRC), the human osteosarcoma cell line Saos-2 (RCB0428, RIKEN BRC),
the human cervical carcinoma cell line HeLa (CVCL_0030, ATCC), thehuman hepatocellular carcinoma cell line Huh-7 ( JCRB0403, JCRB Cell
Bank), the human ovarian cancer cell line TOV-21G (CVCL_3613, ATCC),
the FANCF transduced human ovarian cancer cell line TOV-21G FANCF48,
the BRCA2-mutated human pancreatic cancer cell line Capan-1 (HTB79, ATCC), the BRCA2-restored Capan-1 clones (C2-1 and C2-2)47, andthe SV40 transformed human embryonic kidney cell line HEK-293T(CVCL_0063, ATCC) were cultured in DMEM with 10% IFS. To synchronize cells to mitotic phase, the cells were treated with medium containing 0.1 μg ml–1 nocodazole overnight. A CDK1 inhibitor RO-3306, CPT
and nocodazole were purchased from Merck. A telomerase inhibitor 6-thio-dG and a CLK1 inhibitor KH-CB19 were purchased from
Selleck Chemicals.

Antibodies
Anti-hTERT mouse mAbs (clones 10E9-2 and 2E4-2)15,18 and ananti-p-hTERT mouse mAb (clone TpMab-3)17 were previously generated,
and the specificity was evaluated. An anti-p-hTERT mouse monoclonal
antibody (mAb; clone TpMab-35) was newly generated in this study.
An anti-hTERT mouse mAb (clone 2E4-2, 1:200), anti-p-hTERT mouse
mAbs (clones TpMab-3 and TpMab-35, 1:200), an anti-phospho-SR
proteins mouse mAb (1H4, Merck, MABE50, 1:800), an anti-SF3B1
mAb (clone 16, MBL, D221-3, 1:1,000), anti-U2AF1 polyclonal antibodies (pAbs; MBL, RN085PW, 1:1,000), anti-histone H2A rabbit pAbs
(Cell Signaling Technology, 2578S, 1:1,000), an anti-BRCA2 mousemAb (Ab-1, Merck, OP95, 1:250), an anti-dsRNA mouse mAb ( J2, SCICONS, 10010200, 1:200), an anti-RNA–DNA hybrid mouse mAb (S9.6,
Merck, MABE1095, 1:5,000) and an anti-dsDNA mouse mAb (HYB33101, Santa Cruz Biotechnology, sc-58749, 1:5,000) were used for IB. An
anti-hTERT mAb (clone 10E9-2, MBL, M216-3, 1:100), an anti-p-hTERT
mouse mAb (clone TpMab-3, 1:50), anti-hTERT sheep pAbs (abx120550,
Abbexa, 1:50) and an anti-RNA–DNA hybrid mouse mAb (S9.6, Merck,
MABE1095, 1:100), were used for IP. Anti-p-hTERT mouse mAbs (clones
TpMab-3 and TpMab-35, 1:100), an anti-SC-35 mAb (against SRRM2)(SC-35, Merck, S4045, 1:2,000), an anti-γH2AX rabbit mAb (20E3, Cell
Signaling Technology, 9718, 1:500), an anti-PML rabbit mAb (EPR16792,
abcam, ab179466, 1:500) and anti-TRF2 goat pAbs (Novus Biologicals,
NB100-56694, 1:250) were used for IIF staining.
TpMab-35 was established as described previously17. Briefly, female
BALB/cAJcl mice (6 weeks old) were purchased from CLEA Japan. They
were maintained in specific pathogen-free (SPF) rooms at 20 °C, 50%
humidity and 11 h/13 h light/dark cycle condition. The Animal Careand Use Committee of Tohoku University approved all animal experiments. To develop mAbs against p-hTERT, 100 μg of keyhole limpet
hemocyanin (KLH)-conjugated hTERT phosphopeptide 244CEPERpTPVGQG254 (Eurofins Genomics) was immunized intraperitoneally (i.p.)with Imject Alum (Thermo Fisher Scientific) into one BALB/c mouse.
The procedure included three additional immunizations of 100 μg,
followed by a final booster intraperitoneal injection of 100 μg twodays before its spleen cells were harvested. The harvested spleen cells
were subsequently fused with P3U1 cells, using polyethylene glycol
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1500 (PEG1500; Roche Diagnostics). Then, hybridomas were grownin an RPMI-1640 medium, supplemented with 10% heat-inactivated
FBS, 100 units ml–1 penicillin, 100 μg ml–1 streptomycin, 0.25 μg ml–1
amphotericin B, and hypoxanthine/aminopterin/thymidine (HAT) for
the selection (Thermo Fisher Scientific). Cultured supernatants were
finally screened using enzyme-linked immunosorbent assay (ELISA) for
the detection of hTERT phosphopeptide and nonphosphopeptide16.
The clone TpMab-35, which is specific for the p-hTERT peptide, wasfinally established.

Indirect immunofluorescence staining
Cells were fixed with 4% formaldehyde in PBS, permeabilized with 0.2%
TritonX-100 in PBS, and blocked with 2% bovine serum albumin in PBS.
The cells were incubated with 10 μg ml–1 of anti-phospho-hTERT mouse
mAbs (clones TpMab-3 and TpMab-35), 10 μg ml–1 of an anti-hTERT
mouse mAb (clone 2E4-2), 1:2,000 dilution of an anti-SC-35 mAb(against SRRM2; SC-35, Merck), 1:200 dilution of an anti-phospho-SR
proteins mouse mAb (1H4, Merck), 1:500 dilution of an anti-γH2AX
rabbit mAb (20E3, Cell Signaling Technology), 1:500 dilution of ananti-PML rabbit mAb (EPR16792, abcam) and/or 1:250 dilution ofanti-TRF2 goat pAbs (Novus Biologicals), followed by incubation in
the presence of Alexa488- or Alexa568-labelled secondary antibody(1:1,000; Thermo Fisher Scientific). The nuclei were stained with Hoechst 33342 (NucBlue; Thermo Fisher Scientific).

TERRA RNA-FISH
The PNA probe against TERRA RNAs (TelC-FITC probe) was obtained
from Panagene. Cells were fixed with 4% formaldehyde in PBS andpermeabilized with 0.5% TritonX-100 in PBS. For a control sample,
the cells were preincubated with 100 μg ml–1 of RNase A for 20 min
at 37 °C. After blocking with 2% BSA (Merck) in PBS, the cells wereincubated with the primary antibody, followed by an incubation in thepresence of an Alexa568-labelled secondary antibody (1:1,000; Thermo
Fisher Scientific). The telomeric PNA probe was diluted at 1:100 in 60%
formamide and boiled for 10 min at 85 °C. Cells were refixed with 4%
formaldehyde in PBS and then hybridized with a hybridization solution
containing the FITC-labelled telomeric PNA probe at room temperaturefor 2 h. The nuclei were stained with Hoechst 33342 (NucBlue; Thermo
Fisher Scientific).

IP-IB of hTERT
Cells were lysed in Lysis Buffer A (0.5% NP-40, 20 mM Tris-HCl (pH7.4) and 150 mM NaCl). After sonication for 10 seconds, lysates were
centrifuged at 21,000g at 4 °C for 15 min and collect the supernatant.
Lysate from 1 × 107 cells (Fig. 1) or 1 mg of lysate (Figs. 3 and 5) werepre-absorbed with 40 μl of Pierce Protein A Plus Agarose (Thermo
Fisher Scientific) for 30 min at 4 °C. Pre-absorbed lysate was mixed with
10 μg of an anti-hTERT mAb (clone 10E9-2) and 40 μl of Pierce Protein
A Plus Agarose, and incubated overnight at 4 °C. Immune complexes
were washed three times with Lysis Buffer A and eluted in 2× SDS loading buffer (2% β-mercaptoethanol, 20% glycerol, 4% SDS and 100 mM
Tris-HCl (pH 6.8)) with heating at 95 °C for 5 min, and then subjected
to SDS-PAGE in 8% polyacrylamide gels. After electrophoresis under
reducing conditions, bands of protein were transferred to nitrocellulose membranes (Amersham; Cytiva). After blocking with 5% skimmed
milk prepared in TBS-T (Tween-20, 0.1%) or Immobilon Signal Enhancer(Merck), the membrane was incubated with an anti-hTERT mousemAb (clone 2E4-2) or anti-p-hTERT mouse mAbs (clones TpMab-3 andTpMab-35), followed by incubation in the presence of Mouse TrueBlot
ULTRA Anti-Mouse Ig horseradish peroxidase (HRP; Rockland).

IP-RdRP assay
hTERT and p-hTERT proteins were immunoprecipitated from humancell lines as described for the IP-IB assay with an anti-hTERT mAb (clone
10E9-2) and an anti-p-hTERT mAb (clone TpMab-3), respectively. The

Articlebead suspension with immune complexes was washed four times
with 1× acetate buffer (10 mM HEPES-KOH (pH 7.8), 100 mM potassium acetate, and 4 mM MgCl2) containing 10% glycerol, 0.1% Triton-X
and 0.06× Complete EDTA-free (Roche Diagnostics), and once with
AGC solution (1× acetate buffer containing 10% glycerol and 0.02%
CHAPS) containing 2 mM CaCl2. The bead suspension was treated with
0.25 units per μl Micrococcal Nuclease (MNase; Takara Bio) at 25 °C for
15 min. Immunoprecipitants were subsequently washed twice with AGC
solution containing 3 mM EGTA and once with 1× acetate buffer containing 0.02% CHAPS. Reaction mixture (40 μl) was prepared by combining 20 μl of the bead suspension with 6 μl of [α-32P] UTP (3,000 Ci
per mmol) and 25 ng μl–1 (final concentration) of RNA template, andincubated at 32 °C for 2 h. The following RNA templates were used:
DN3AS (5ʹ-GGGAUCAUGUGGGUCCUAUUACAUUUUAAACCCA-3ʹ, usedin Figs. 2, 3 and 5 and Extended Data Fig. 4j) and TERRA (5ʹ-(GUUAGG)8-3,
used in Fig. 4). These RNAs have hydroxyl groups at both the 5ʹ and3ʹ ends. The final concentrations of ribonucleotides were 1 mM ATP,0.2 mM GTP, 10 μM UTP and 0.2 mM CTP. The resulting products weretreated with Proteinase K to stop the reaction, purified several times
with phenol/chloroform until the white interface disappeared andprecipitated using ethanol. The RdRP products were treated with
RNase I (2 U, Promega) at 37 °C for 2 h to digest single-stranded RNAs
completely, followed by Proteinase K treatment, phenol/chloroform
purification and ethanol precipitation. The products were electrophoresed in a 15% polyacrylamide gel containing 7 M urea, and detectedby autoradiography.

Direct telomerase assay
For the direct telomerase assay, we modified the original methods21,22. TERT protein was immunoprecipitated from human cell lines
as described for the IP-IB assay with incubation on ice for 30 min
without sonication. Immune complexes were washed three times
with Lysis buffer A, and then suspended in 30 μl of TRAP lysis buffer(10 mM Tris-HCl (pH 7.5), 1 mM MgCl2, 1 mM EGTA, 0.5% CHAPS, 10%
glycerol, 100 μM Pefabloc SC, and 0.035% 2-mercaptoethanol). The
direct telomerase assay was carried out with 10 μl of the suspension
and 40 μl of reaction mixture (2.5 μl of [α-32P] dGTP (6000 Ci/mmol),
50 mM Tris-HCl (pH 8.0), 50 mM KCl, 1 mM MgCl2, 1.25 mM Spermidine, 5 mM 2-mercaptoethanol, 2.5 mM dTTP, 2.5 mM dATP, 25 μM
dGTP and 2.5 μM a5 primer (5ʹ-TTAGGGTTAGGGTTAGCGTTA-3ʹ)) by
incubation at 37°C for 2 h. The products were purified with phenol/
chloroform and precipitated using ethanol. The products were electrophoresed in a 10% polyacrylamide gel containing 7 M urea and detectedby autoradiography.

Telomere length measurement
To measure telomere length by Southern blotting analysis,
hTERT-CRISPR clones were cultured and their population doublings
were calculated. Genomic DNAs (gDNAs) were isolated with the GenElute Mammalian genomic DNA miniprep kit (Merck) according to the
manufacturer’s instructions. The isolated gDNAs were then digested
with HinfI and AfaI, electrophoresed in a 0.8% agarose gel and hybridized
with a 32P-labelled telomeric (CCCTAA)3 probe as described previously67.

Semi-quantitative and quantitative reverse transcription PCR
Total RNA was isolated from cells using TRIzol (Thermo Fisher Scientific) and treated with RQ1 DNase (Promega). cDNA was synthesizedusing 500 ng of total RNA with oligo (dT)12-18 primer (Thermo Fisher
Scientific), random hexamers (Thermo Fisher Scientific) and one ofreverse transcriptases, PrimeScript reverse transcriptase (Takara Bio),
Superscript IV reverse transcriptase (Thermo Fisher Scientific) or
Superscript IV VILO master mix (Thermo Fisher Scientific), followedby PCR analyses. Semi-quantitative PCR analysis was performed using
Ex-Taq DNA polymerase (TaKaRa Bio). qRT-PCR analysis was performed
using Fast SYBR Green Master Mix (Thermo Fisher Scientific) andNature Cell Biology

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QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific). Alldata of qRT-PCR analysis were normalized by the data of GAPDH mRNAlevels. For determination of RNA levels in NSs, the data of qRT-PCR analysis were normalized by the amount of RNAs (Fig. 3d). The sequences ofthe primers used in this study are described in Supplementary Table 1.

Transfection of siRNA
Cells were transfected with siRNAs using lipofectamine 2000 (Thermo
Fisher Scientific) according to the manufacturer’s instructions. To
suppress hTERT expression efficiently, we treated the cells with siRNAs
specific for hTERT twice for IIF analysis. The sequences of siRNAs wereas follows: siTERT#1, GUGUCUGUGCCCGGGAGAATT; siTERT#2, GCAUUGGAAUCAGACAGCATT. MISSION siRNA Universal Negative Control#1 (Merck) was used as a negative control. The following Silencer SelectsiRNAs (Thermo Fisher Scientific) were used: siBRCA1 (s457, s458),
siBRCA2 (s2083, s2085), siFANCD2 (s4988, 4989), siFANCE (s4992),
siFANCF (s5015), siPARP1 (s1098, s1099).

Genome editing using CRISPR-Cas9
Recombinant Cas9 protein (TrueCut Cas9 Protein v2; Thermo Fisher
Scientific) and TERT-specific gRNA (TrueGuide synthetic guide RNA,
Thermo Fisher Scientific, CRISPR1117444_SGM) were premixed
and transfected into U2OS cells using lipofectamine CRISPRMAX
Cas9 transfection reagent (Thermo Fisher Scientific) according to
the manufacturer’s instructions. After 4 days’ incubation, a single
cell was cloned. The transfection and clonal selection process wererepeated once more. Genomic DNA was extracted with the GenElute Mammalian genomic DNA miniprep kit (Merck) according to the
manufacturer’s instructions. Presence of mutation in single-cellclones was confirmed by PCR amplification and Sanger sequencing using primers 5′-TTGAATTCGAACCATAGCGTCAGGGAGG-3′ and5′-TTGAATTCGAACCATAGCGTCAGGGAGG-3′.

RNA-seq and data analysis
RNA sequence library preparation, sequencing, mapping, gene expression and GO enrichment analysis were performed by DNAFORM. Qualities of total RNA were assessed by Bioanalyzer (Agilent) to ensure that
RIN (RNA integrity number) is over 7.0. Sequencing cDNA libraries(RNA-seq libraries) were prepared using SMARTer stranded Total RNA
Sample Prep Kit-HI Mammalian (Takara Bio) according to the manufacturer’s instruction. RNA-seq libraries were sequenced using paired end
reads on a NextSeq 500 instrument (Illumina). Obtained raw reads were
trimmed and quality-filtered using the Trim Galore! (version 0.6.4_dev),
Trimmomatic (version 0.39) and cutadapt (version 2.10) software.
Trimmed reads were then mapped to the human GRCh38 genome
using STAR (version 2.7.9a)68. Reads on annotated genes were counted
using featureCounts (version 2.0.1)69. FPKM values were calculated
from mapped reads by normalizing to total counts and transcript. Differentially expressed genes were detected using the DESeq2 package(version 1.20.0). The list of differentially expressed genes was used forclustering analysis with MBCluster.Seq package70 and GO enrichment
analysis with clusterProfiler package71.

Recombinant human hTERT
rhTERT and AS variant proteins were produced by Fujifilm Wako Pure
Chemical using a baculovirus expression system. The TERT and AS
variant genes were synthesized and inserted into a transfer vector.
The transfer vector was cotransfected with baculovirus DNA into Sf9
cells. After incubation, the supernatant containing the recombinantbaculovirus was harvested and then infected into expressSf+ cells forlarger scale expression of recombinant proteins. The cell pellet was
harvested and lysed in lysis buffer (0.5% NP-40, 20 mM Tris-HCl (pH7.4), and 150 mM NaCl). After sonication for 10 seconds, lysates were
centrifuged at 21,000g at 4 °C for 15 min and collect the supernatant.
The recombinant hTERT and AS variant proteins were purified with anArticleanti-hTERT mAb (clone 10E9-2) and Pierce Protein A Plus Agarose fromthe supernatant and used for the IP-IB and IP-RdRP assays.

Generation of stable cell lines
Lentiviruses were created using the lentiviral vectors pLV-puro,
pLV-puro-hTERT or pLV-puro-T249A, constructed by Vectorbuilder.
T249A mutant, in which the threonine 249 of hTERT is replaced withalanine, are not phosphorylated and thus lack the RdRP activity16. After
infection, polyclonal cell populations were purified by selection withpuromycin (2 µg ml–1) for 2 days.

Genome-wide CRISPR screening
Human Brunello CRISPR knockout pooled library lentiviral prep was
obtained from Addgene (73179-LV)42. U2OS cells or the hTERT-CRISPR
clone #2 (108 cells) were infected with the pooled lentivirus library at a
multiplicity of infection of ∼0.4 and >500-fold coverage of the library.
Forty-eight hours postinfection, cells were selected with puromycin(2 μg ml–1). After 48 h selection, 4 × 107 cells were collected as a control sample (day 0 timepoint), and the others were seeded to start thescreen. Every 3 days, 4 × 107 cells were passaged (~500-fold coverageof the library). After 21 days of culture, 4 × 107 cells were harvested,
and gDNAs were isolated from the cell pellets using the Blood and Cell
Culture DNA Maxi Kit (Qiagen). sgRNA sequences were amplified from200 μg of gDNA by 20 parallel PCR reactions (10 μg of gDNA per tube)
using Q5 High-Fidelity DNA Polymerase (New England Biolabs). The
resulting amplicons were mixed and purified by gel extraction using
Wizard SV Gel and PCR Clean-Up System (Promega). Purified PCR
products were subjected to high-throughput sequencing analysis with
Illumina HiSeq 2500 platform (Illumina). The sequences of the primers
used in this study are described in Supplementary Table 1. Sequencing
data were analysed to obtain the read counts for each sgRNA and to
generate a maximum-likelihood estimation (MLE) of gene essentiality
score (β score) for each gene using the MAGeCK algorithm43. Genes witha β score < −0.5 in hTERT-CRISPR groups, but with β score > −0.3 in U2OS
groups were picked up as potential synthetic lethal genes. To predict
the biological functions and pathways associated with the putative
genes, GO analysis and KEGG pathway analysis were performed using
the DAVID online tool (https://david.ncifcrf.gov/).

Cell viability assay
For the colony formation assay, cells were seeded into 6-well plates
at a density of 500 cells per well and subjected to transfection with
the indicated siRNAs, treatment with chemical reagents or IR. Afterapproximately 12 days, the cells were fixed with 4% formaldehyde
in PBS, washed with PBS, and stained with 0.2% crystal violet in 20%
methanol for 15 min at room temperature. After cells were washed with
PBS twice, colony numbers were counted.
For MTT assay, cells were seeded into 96-well plates at a densityof 10,000 cells per well and treated with chemical reagents. After 72 h
incubation, cell viabilities were determined using Cell Proliferation Kit
I MTT (Merck) according to the manufacturer’s instructions.

Purification of nuclear speckles
To separate cells into cytoplasmic fraction and nuclear pellet, ~80–90%
confluent cells in 150 mm dishes were lysed with 4 ml of HLB/NP40
buffer (10 mM Tris-HCl (pH 7.5), 10 mM NaCl, 0.5% NP40 and 2.5 mM
MgCl2) and incubated for 5 min on ice. After the incubation, 1 ml of
HLB/NP40/Sucrose buffer (10 mM Tris-HCl (pH 7.5), 10 mM NaCl, 0.5%
NP40, 2.5 mM MgCl2 and 10% sucrose) was added under the lysate
layer, followed isolation of nuclear pellet by centrifugation at 400g at
4 °C for 5 min. Isolated nuclear pellet was treated with 1% Triton X-100
in TM5 buffer (10 mM Tris–HCl (pH 7.4) and 5 mM MgCl2) for 5 min on
ice and the supernatant was removed by centrifugation at 700g at
4 °C for 5 min. The pellet was treated with 50 units of MNase (TakaraBio) at 4 °C for 30 min. The pellet was washed twice with TM5 buffer
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containing 3 mM EGTA to inactivate MNase activity and 500 mM NaCl
to remove nucleoplasmic extracts and chromatin. After centrifugation
at 800g for 5 min, the pellet was treated with 500 mM NaCl as above.
The final pellet was lysed in 800 μl of Lysis Buffer A. After sonicationfor 10 seconds, lysates were centrifuged at 21,000g at 4 °C for 15 min
and the supernatant was collected as an NS-enriched fraction.

R-loop analysis by dot blot
HeLa cells were transfected with siRNAs. After 72 h incubation, gDNAs
were isolated from the cells using the Blood and Cell Culture DNA
Midi Kit (Qiagen). gDNA (2 μg) was dotted onto a Hybond N+ (Amersham) using a Bio-Dot Apparatus (Bio-Rad). The membrane was thencross-linked with ultraviolet (UV; 240 mJ cm–2). After blocking with 5%
skimmed milk prepared in TBS-T (Tween-20, 0.1%) and subsequently
with SuperBlock buffer (Thermo Fisher Scientific), the membrane wasincubated with an anti-RNA–DNA hybrid mouse mAb (S9.6, Merck) at
0.2 μg ml–1 in SuperBlock buffer (Thermo Fisher Scientific) overnight at
4 °C, followed by incubation in the presence of HRP-labelled anti-mouse
IgG secondary antibody ( Jackson Immunoresearch Laboratories).

Dot blot analysis of hTERT IP products
hTERT protein was immunoprecipitated from 1 mg of NS-enrichedfraction as described for the IP-IB assay with an anti-hTERT mAb (clone
10E9-2) or an anti-p-hTERT mAb (clone TpMab-3). The bead suspension
with immune complexes was washed four times with Lysis Buffer A
and RNA co-immunoprecipitated with the p-hTERT immune complex
was isolated using TRIzol (Thermo Fisher Scientific). The isolated
RNA was treated with RQ1 RNase-Free DNase (Promega) and dotted
onto a Hybond N+ (Amersham) using a Bio-Dot Apparatus (Bio-Rad).
The membrane was then cross-linked with UV (120 mJ cm–2) and thenprobed with 32P-labelled synthetic oligonucleotides that were complementary to the sequence of TERRA RNA or ARRET RNA (TERRA RNA:
5′-(CCCTAA)4-3′; ARRET RNA: 5′-(TTAGGG)4-3′).
For detection of dsRNAs, RNA co-immunoprecipitated with hTERT
or p-hTERT was treated with DNase (Promega) and dotted onto themembrane as above. The membrane was then cross-linked with UV(240 mJ cm–2). After blocking with 5% skimmed milk prepared in TBS-T(Tween-20, 0.1%) and subsequently with SuperBlock buffer (Thermo
Fisher Scientific), the membrane was incubated with anti-dsRNA
mouse mAb ( J2, SCICONS) in SuperBlock buffer (Thermo Fisher Scientific) overnight at 4 °C, followed by incubation in the presence of
HRP-labelled anti-mouse IgG secondary antibody ( Jackson Immunoresearch Laboratories). For prepare a control sample treated withdsRNA-specific RNase III, RNA was preincubated with 1 unit of RNase
III (Thermo Fisher Scientific Inc.) for 2 h at 37 °C.

R-loop degradation assay
TERRA RNA oligonucleotide (5′-(GUUAGG)8-3′) and the complementary oligonucleotide DNA (5′-(CCTAAC)8-3′) were synthesized by Integrated DNA Technologies. To prepare TERRA RNA:DNA oligo duplex,
5′-32P-labelled TERRA RNA and the complementary oligonucleotide
DNA were annealed in annealing buffer (10 mM Tris-HCl (pH 7.5), 50 mM
NaCl and 1 mM EDTA) at 80 °C for 5 min and slowly cooled to room
temperature. hTERT immune complex was immunoprecipitated from
1 mg of NS-enriched fraction as described for the IP-IB assay with ananti-hTERT mAb (clone 10E9-2) or an anti-p-hTERT mAb (clone TpMab3). The hTERT immune complex was washed three times and incubatedwith TERRA RNA:DNA hybrids in a buffer containing 50 mM NaCl,
10 mM Tris-HCl (pH 7.9), 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 0.2 mM
GTP, 0.2 mM UTP, and 0.2 mM CTP, and RNasin plus inhibitor for 2 h
at 37 °C. The reaction samples were treated with Proteinase K to stop
the reaction. After phenol/chloroform purification and ethanol precipitation, the reaction products were treated with 1 unit of RNase III(Thermo Fisher Scientific) at 37 °C for 1 h to digest dsRNAs generated by
p-hTERT immune complex. The reaction products were subsequently

Articleelectrophoresed in a 10% polyacrylamide gel containing 7 M urea, anddetected by autoradiography.

DNA–RNA hybrid immunoprecipitation (DRIP)
HeLa cells were transfected with siRNAs. After 72 h incubation, gDNAs
were isolated from the cells using the Blood and Cell Culture DNA Midi
Kit (Qiagen). gDNA (20 μg) were sonicated using Bioruptor (CosmoBio)
at high power with 20 cycles (30 s on and 50 s off) in sonication buffer
containing 10 mM Tris-HCl (pH 8.5) and 300 mM NaCl. The sonicatedgDNAs were mixed with 5 μg of an anti-RNA–DNA hybrid mouse mAb(S9.6, Merck) and 40 μl of Pierce Protein A Plus Agarose and incubatedovernight at 4 °C in DRIP buffer containing 50 mM Tris-HCl (pH 7.4),
150 mM NaCl, 5 mM EDTA and 1% NP-40. Immune complexes were
washed with DRIP buffer, DRIP high buffer (50 mM Tris-HCl, pH 7.4,
500 mM NaCl, 5 mM EDTA, 1% NP-40), DRIP Li buffer (50 mM Tris-HCl,
pH 8.0, 250 mM LiCl, 1 mM EDTA, 0.5% NP-40), and DRIP TE buffer(100 mM Tris-HCl pH 8.0, 10 mM EDTA), and then eluted in elution
buffer containing Proteinase K (Takara Bio), 10 mM EDTA and 0.5% SDS
by shaking at 37 °C for 30 min. After phenol/chloroform purificationand ethanol precipitation, the DRIP products were treated with RQ1
DNase (Promega) to purify the RNA strand and subjected to qRT-PCR
analysis with strand-specific primers. The sequences of the primers
used in this study are described in Supplementary Table 1.

Xenotransplantation
Six-week-old male NOD/SCID mice (NOD/NCrCRL-Prkdcscid) were purchased from Charles River Laboratories and used as recipients forxenotransplantation. They were maintained in SPF rooms at 20 °C,
50% humidity, and 12 h/12 h light/dark cycle condition. 1 × 105 cells
were suspended in a mixture of serum-free medium and Matrigel (BDBiosciences; 1:1 volume). The mixture was injected subcutaneously
through a 26-gauge needle into the right dorsal areas of anaesthetized
NOD/SCID mice. We monitored tumour formation and tumour size
every two or three days, and dissected out the tumours 10 weeks later.
The study protocol was approved by the Kanazawa University Animal
Care and Use Committee, and all procedures were performed in accordance with the guidelines and regulations of Kanazawa University.

Statistical analysis
Statistical significance was determined using two-tailed unpaired
t-test or one-way ANOVA method with Dunnett correction for multiple
comparisons. Data are presented as the mean ± s.d. or s.e.m.

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

Data availability

All sequencing datasets have been deposited at the Gene Expression
Omnibus with accession codes GSE226966 and GSE226994. All the data
supporting the findings of this study are available within the article andfrom the corresponding author upon reasonable request. Source data
are provided with this paper.

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68. Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner.
Bioinformatics 29, 15–21 (2013).
69. Liao, Y., Smyth, G. K. & Shi, W. featureCounts: an efficient general
purpose program for assigning sequence reads to genomic
features. Bioinformatics 30, 923–930 (2014).
70. Si, Y., Liu, P., Li, P. & Brutnell, T. P. Model-based clustering forRNA-seq data. Bioinformatics 30, 197–205 (2014).
71. Yu, G., Wang, L. G., Han, Y. & He, Q. Y. clusterProfiler: an R package
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Acknowledgements

This work was supported in part by the Japan Society for the
Promotion of Science KAKENHI (22K15550 to M.M., 20J00036 and
20H03438 to A.Y.), the Japan Science and Technology Agency(JPMJMS2022 to A.Y.) and several grants from the Japan Agency
for Medical Research and Development: the P-CREATE grant(JP21cm0106584 and JP23ama221523 to M.M. and JP21cm0106115
to K.M.), the Practical Research for Innovation Cancer Control(JP20ck0106403 to K.M.), Basis for Supporting Innovative Drug
Discovery and Life Science Research (BINDS; JP22ama121008 to Y.K.)
and the Translational Research programme (JP24ym0126804 to M.M.).
T.T. was supported by the Uehara Memorial Foundation and Tokai
University Tokuda Memorial Cancer/Genome Basic Research Grant.
This work was supported by the Center for Promotion of Translational
Research, National Cancer Center Japan.

Author contributions

M.M., A.N., M.Y., S.U. and K.-I.F. performed biochemical and cellularexperiments. T.Y. and S.K. performed animal experiments. T.U. and
H.M. conducted bioinformatics analyses of CRISPR screening. Y.K.
generated a phospho-specific monoclonal antibody against hTERT. T.T.
provided FANC/BRCA mutant cell clones. N.S. supported purificationof NSs. M.M., A.N., A.Y., Y.T., M.W. and K.M. designed the experiments
and discussed the interpretation of the results. M.M., A.N. and K.M.
wrote the paper.

Competing interests

The authors declare no competing interests.

Additional information

Extended data is available for this paper athttps://doi.org/10.1038/s41556-024-01427-6.
Supplementary information The online version
contains supplementary material available athttps://doi.org/10.1038/s41556-024-01427-6.
Correspondence and requests for materials should be addressed to
Kenkichi Masutomi.
Peer review information Nature Cell Biology thanks the anonymous
reviewers for their contribution to the peer review of this work. Peerreviewer reports are available.
Reprints and permissions information is available atwww.nature.com/reprints.

Article

Extended Data Fig. 1 | See next page for caption.

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Article
Extended Data Fig. 1 | Detection of phosphorylated hTERT proteins andhTERT mRNAs in ALT cells. a, IIF imaging of p-hTERT. Telomerase-positive celllines (293T, HeLa, and Huh-7 cells) and ALT cell lines (U2OS, VA-13, and Saos-2
cells) were immunostained with an anti-p-hTERT mouse mAb (TpMab-35). Scalebar, 10 μm. b, c, IIF imaging of p-hTERT (TpMab-35) in HeLa and U2OS cells after
treatment with hTERT siRNAs (b) and a CDK1 inhibitor (RO-3306) (c). Scale bar, 10
μm. d, e, Comparison between reverse transcriptases for RT-PCR analysis(n = 4 independent experiments per group). cDNA was synthesized from total
RNAs with PrimeScript (PS), Superscript IV (SSIV), or Superscript IV VILO.

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The relative expressions are noted below the panel (for b). f, Schematic diagramof hTERT mRNA. Arrows depict locations of primers used for quantitative
RT-PCR analysis. g, Determination of hTERT mRNA levels by quantitative RT-PCR
analysis using LT5/6 primer pairs or new primer pairs targeting the exon 15 and 16
junction of hTERT mRNA (n = 3 independent experiments per group).
h, i, Detection of p-hTERT (TpMab-35) in U2OS and HeLa cells synchronized inmitosis with nocodazole. Data are presented as mean ± SD (for e, g). Experiments
were repeated three times (for a, c) and twice (for b, d, h, i) with similar results.
Source numerical data and unprocessed blots are available in source data.

Article

Extended Data Fig. 2 | Telomerase and RdRP activity in ALT cells. a, Detectionof endogenous hTERT immunoprecipitated from the indicated cells with
nocodazole treatment. b, Direct telomerase assay using hTERT proteins
immunoprecipitated with an anti-hTERT mouse mAb (10E9-2) from the indicatedmitotic cells treated with nocodazole. 293T cells are positive controls.
c, quantitative RT-PCR analysis for hTERC associated with hTERT and p-hTERTNature Cell Biology

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isolated with the 10E9-2 and the TpMab-3 antibodies from HeLa, U2OS, VA-13,
and Saos-2 cells (n = 3 independent experiments per group). Data are presented
as mean ± SD. d, RdRP activity of endogenous hTERT immunoprecipitatedfrom HeLa cells and a recombinant hTERT (rhTERT) protein. Experiments wererepeated three times (for a, b, d) with similar results. Source numerical data andunprocessed blots are available in source data.

Article

Extended Data Fig. 3 | See next page for caption.

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Article
Extended Data Fig. 3 | RdRP activity of hTERT alternative splicing variants.
a, Schematic diagram of hTERT alternative splicing (AS) variants. Arrows depict
locations of primers used for quantitative RT-PCR analysis. b, Determinationof hTERT AS variant levels by quantitative RT-PCR analysis (n = 3 independent
experiments per group). Data are presented as mean ± SD. N.D., not detected.

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c, Detection of recombinant proteins of AS variants purified by a baculovirus
expression system. * indicates migration of full-length hTERT or AS variants. d, IPRdRP assay using recombinant AS variant proteins. Experiments were repeatedthree times (for c, d) with similar results. Source numerical data and unprocessedblots are available in source data.

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Extended Data Fig. 4 | See next page for caption.

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Article
Extended Data Fig. 4 | hTERT undergoing liquid-liquid phase separation in
ALT cells. a, IIF imaging of hTERT (2E4-2) and p-hTERT (TpMab-3) in HeLa andU2OS cells. Scale bar, 10 μm. b, c, Multicolor immunofluorescence imagingof p-hTERT and PML (b) or TRF2 (c) in U2OS cells. TEN, telomerase essential
N-terminal domain; RBD, RNA-binding domain; RT, reverse transcriptase
domain; CTE, C-terminal extension. Scale bar, 10 μm. d-g, Prediction of disorderregions on hTERT protein using the IUPred2A web interface (d) and the PONDR
predictor VL-XT (e), VL3-BS (f), or VSL2 (g). The bold lines show putative disorderNature Cell Biology

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regions. h, IIF imaging of p-hTERT in HeLa, Huh-7, and U2OS cells disrupting LLPS
with 10% 1,6-HD for 5 min. Scale bar, 10 μm. i, j, Detection of endogenous hTERT
proteins (i) and RdRP activity (j) in the cells inhibiting LLPS with 1,6-HD. The 10E92 and 2E4-2 clones are antibodies for immunoprecipitation and immunoblottingof hTERT, respectively. k, Detection of endogenous hTERT protein underconditions for inhibiting LLPS (1,6-HD treatment and lysis with 150 or 1000 mM
NaCl). Experiments were repeated three times (for a, h-k) and twice (for b, c) withsimilar results. Source unprocessed blots are available in source data.

Articlehttps://doi.org/10.1038/s41556-024-01427-6

Extended Data Fig. 5 | Specificity of an anti-p-hTERT antibody (TpMab-3). a, b, IIF imaging of p-hTERT (TpMab-3) and phospho-SR proteins (1H4) in HeLa cells
treated with siRNAs specific for hTERT (a) and a CLK1 inhibitor (KH-CB19) (b). Experiments were repeated twice with similar results. Scale bar, 10 μm.

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Extended Data Fig. 6 | See next page for caption.

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Article
Extended Data Fig. 6 | Association between hTERT and TERRA RNAs and
RdRP activity of p-hTERT against TERRA RNAs. a, Scheme of hTERT RIP-seq,
which analyzed RNAs co-immunoprecipitated with hTERT using an anti-hTERT
antibody (10E9-2) from nuclear speckle (NS)-enriched fraction. b, RIP-seq
data of hTERT purified from nuclear speckles. Arrows show reads mapped tosubtelomeric regions. c, Quantitative RT-PCR analysis of TERRA RNAs fromdifferent subtelomeric regions in hTERT knockdown HeLa and U2OS cells (n = 4
independent experiments per group). Data are presented as mean ± SD. One-way
ANOVA method with Dunnett correction for multiple comparisons between
control (siCtrl) and other groups. d, RdRP activity of hTERT purified from nuclear

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speckles against a synthetic TERRA RNA template. e, IP-RdRP assay using hTERT
proteins immunoprecipitated with an anti-hTERT antibody (10E9-2) from the
HeLa cells. A mixture of two RNA templates, control DN3AS and 5ʹ-(CCUAAC)83ʹ for TERRA, was used for the IP-RdRP assay. f, g, In vitro R-loop degradation
assay using hTERT (f) and p-hTERT (g) complex immunoprecipitated from the
NS-enriched fraction and 5′-32P-labeled TERRA RNA:DNA hybrids. RNase H is apositive control to digest RNA:DNA hybrids. Experiments were repeated threetimes (for d, f) and twice (for e, g) with similar results. Source numerical data andunprocessed blots are available in source data.

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Extended Data Fig. 7 | See next page for caption.

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Extended Data Fig. 7 | Establishment of hTERT-CRISPR clones. a, b, Sanger
sequencing for the hTERT-CRISPR clones #1 (a) and #2 (b). Black lines and red
arrowheads indicate the gRNA sequence and deletion regions, respectively.
c, Determination of hTERT mRNA levels in hTERT-CRISPR clones by quantitative
RT-PCR analysis (n = 3 independent experiments per group). Data are presented

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as mean ± SD. One-way ANOVA method with Dunnett correction for multiple
comparisons between control (U2OS) and other groups. d, Clustering and GO
analyses of genes differentially expressed in hTERT-CRISPR clones. The p-values
were calculated by MBCluster.Seq package. Source numerical data are available
in source data.

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Extended Data Fig. 8 | See next page for caption.

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Extended Data Fig. 8 | Effects of FANC/BRCA knockdown and PARP1
inhibition in hTERT-CRISPR clones. a, Knockdown efficiencies of siRNAs
specific for FANC/BRCA genes in hTERT-CRISPR clones (n = 3 independent
experiments per group). b, The representative images of colony formation
assay using hTERT-CRISPR clones transfected with siRNAs specific for FANC/
BRCA genes. c, The MLE beta essentiality scores for FANC/BRCA and PARP1 genes
generated from MAGeCK algorithm. d, e, The representative images (d) andcolony counting (e) of colony formation assay using hTERT-CRISPR clones with

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FANC/BRCA and PARP1 knockdowns (n = 3 independent experiments per group).
f, g, Detection of hTERT proteins (f) and the representative images of colony
formation assay (g) using hTERT-CRISPR clones overexpressing a wild-type
hTERT or a T249A mutant. Data are presented as mean ± SD (for a, e). One-way
ANOVA method with Dunnett correction for multiple comparisons between
control and other groups (e). Experiments were repeated twice with similar
results (for f). Source numerical data and unprocessed blots are available insource data.

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Extended Data Fig. 9 | See next page for caption.

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Extended Data Fig. 9 | Effects of hTERT knockdown in cells lacking FANC/
BRCA genes. a, Knockdown efficiencies of siRNAs specific for hTERT in Capan-1
and TOV-21G cell clones corrected for BRCA2 and FANCF deficiencies (n = 4
independent experiments per group). b, Direct telomerase assay using hTERT
proteins immunoprecipitated from the indicated cells. c-g, The representative
images of colony formation assay using these revertant clones. h, Schematic
diagram of a strand-specific RT-PCR. DRIP RNAs were subjected to quantitative

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RT-PCR (qRT-PCR) analysis with strand-specific primers with a tag sequence.
i, qRT-PCR analysis of DRIP RNAs using tagged primers specific for sense orantisense strand TERRA RNAs (n = 3 independent experiments per group).
Strand-specific qPCR primers only recognize each strand-specific cDNA. Data arepresented as mean ± SD (for a, i). Experiments were repeated twice with similar
results (for b). Source numerical data and unprocessed blots are available insource data.

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Extended Data Fig. 10 | See next page for caption.

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Extended Data Fig. 10 | Maintenance of R-loop structures by p-hTERT
and FANC/BRCA proteins. a, Immunoblotting for BRCA2 in proteins coimmunoprecipitated with hTERT from the indicated biochemical fractions.
Experiments were repeated three times with similar results. b, Model ofmaintenance of R-loop structures by p-hTERT and FANC/BRCA proteins. c, Modelof different roles of hTERT in regulation of genome stability. hTERT elongatestelomeres as the catalytic enzyme of telomerase. The 3′ telomeric overhangs
invades into the telomere duplex and forms a small loop at the chromosome

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ends, called T-loop structure, which protects the end of linear chromosomesfrom degradation, fusion, and recombination. On the other hand, hTERT binds to
TERRA sequences of the nascent RNA transcripts and synthesizes antisense RNAs
through RdRP activity. The RdRP reaction converts RNA:DNA hybrids of R-loops
to dsRNAs, preventing R-loop formation. hTERT guarantees genome stabilityby the two enzymatic activities. Source unprocessed blots are available insource data.