RNA-splicing factor SART3 regulates translesion DNA synthesis
Nucleic Acids Research
RNA-splicing factor SART3 regulates translesion DNA synthesis
Min Huang 1
Bo Zhou 1
Juanjuan Gong 0
Lingyu Xing 1
Xiaolu Ma 1
Fengli Wang 0
Wei Wu 1
Hongyan Shen 1
Chenyi Sun 1
Xuefei Zhu 0
Yeran Yang 1
Yazhou Sun 1
Yang Liu 1
Tie-Shan Tang 0
Caixia Guo 1
0 State Key Laboratory of Membrane Biology, Institute of Zoology, University of Chinese Academy of Sciences, Chinese Academy of Sciences , Beijing 100101 , China
1 CAS Key Laboratory of Genomics and Precision Medicine, Beijing Institute of Genomics, University of Chinese Academy of Sciences, Chinese Academy of Sciences , Beijing 100101 , China
Translesion DNA synthesis (TLS) is one mode of DNA damage tolerance that uses specialized DNA polymerases to replicate damaged DNA. DNA polymerase (Pol ) is well known to facilitate TLS across ultraviolet (UV) irradiation and mutations in POLH are implicated in skin carcinogenesis. However, the basis for recruitment of Pol to stalled replication forks is not completely understood. In this study, we used an affinity purification approach to isolate a Pol -containing complex and have identified SART3, a pre-mRNA splicing factor, as a critical regulator to modulate the recruitment of Pol and its partner RAD18 after UV exposure. We show that SART3 interacts with Pol and RAD18 via its C-terminus. Moreover, SART3 can form homodimers to promote the Pol /RAD18 interaction and PCNA monoubiquitination, a key event in TLS. Depletion of SART3 also impairs UV-induced single-stranded DNA (ssDNA) generation and RPA focus formation, resulting in an impaired Pol recruitment and a higher mutation frequency and hypersensitivity after UV treatment. Notably, we found that several SART3 missense mutations in cancer samples lessen its stimulatory effect on PCNA monoubiquitination. Collectively, our findings establish SART3 as a novel Pol /RAD18 association regulator that protects cells from UV-induced DNA damage, which functions in a RNA bindingindependent fashion.
Mammalian cells are under endogenous and exogenous
attacks every day, causing a variety of genomic lesions. These
DNA lesions can often result in replication forks stalling,
which if not resolved in time lead to replication fork collapse
and even double strand breaks (DSBs), one of the most
deleterious DNA damages. In order to cope with this
situation, damage tolerance pathways are evolved for the cellular
). Translesion DNA synthesis (TLS) is one
major mode of DNA damage tolerance, which utilizes
specialized polymerases that lack 3 -5 exonucleolytic proofreading
activity and replicate DNA with low fidelity and
processivity. The best-characterized TLS polymerases are Y-family
polymerases, including Pol , Pol , Pol and REV1 (
Among them, Pol can correctly bypass ultraviolet (UV)–
induced cis–syn thymine–thymine cyclobutane–pyrimidine
dimers (CPDs). Loss of functional Pol has been known to
cause the variant form of the skin cancer-prone syndrome,
Xeroderma Pigmentosum (XPV) (
It is well known that the TLS pathway can be efficiently
triggered by replication stress, which usually generates
stretches of single-stranded DNA (ssDNA) through
uncoupling of replicative polymerase and helicase activities. The
ssDNAs are rapidly coated by replication protein A (RPA),
which recruits the ubiquitin E3 ligase RAD18 to stalled
replication forks to promote monoubiquitination of
Proliferating cell nuclear antigen (PCNA) at Lys164 (
Monoubiquitinated PCNA (PCNA-mUb) then facilitates
optimal TLS through its enhanced binding with Y family
). Compelling evidence reveals that
PCNAmUb, the key event in TLS, is tightly regulated by
several DDR factors, including USP1, MSH2, BRCA1, Pol ,
REV1 and Parkin (
). In contrast to USP1, which can
deubiquitinate PCNA-mUb (
), MSH2, BRCA1 and
Parkin have been shown to facilitate UV-induced
PCNAmUb through promoting ssDNA generation and RPA
focus formation (
). And Pol and REV1, two TLS
polymerases, can promote RAD18 recruitment after UV
). Pol has also been shown to bridge
RAD18 and PCNA to promote efficient PCNA-mUb
formation after DNA damage. Notably, this Pol scaffolding
function is independent of its DNA polymerase activity but
relies on the Pol /RAD18 association (17). Interestingly,
RAD18 is also required to guide Pol to stalled
replication sites through Pol /RAD18 physical interaction (
Although RAD18 phosphorylation mediated by Cdc7 or
JNK enhances the Pol /RAD18 association (
Pol /RAD18 interaction is regulated remains largely
SART3 (Squamous Cell Carcinoma Antigen Recognized
By T-Cells 3) is a nuclear RNA-binding protein (RBP),
which contains half-a-tetracopeptide repeats (HAT) in the
N-terminus and two RNA recognition motifs (RRM1 and
RRM2) near the C-terminus. Being a U4/U6 recycling
factor, SART3 can assist pre-mRNA splicing, thereby
regulating gene expression (
). In line with this, SART3 is
indispensable for embryonic development, whose deficiency is
reported to be embryonic lethal (
). Moreover, SART3
also expresses at high levels in the nucleus of malignant
tumor cell lines and majority of cancer tissues (27). Here, we
identified SART3 to be a novel partner of Pol , whose
depletion decreases ssDNA generation, RPA focus formation
as well as the chromatin binding of RAD18 in the
presence of UVC treatment. Consistently, knockdown SART3
impairs Pol focus formation and CPD lesion bypass
after UVC exposure, leading to UV hypersensitivity.
Furthermore, we found that SART3 can form homodimers
and associate with RAD18. And SART3 can promote the
Pol /RAD18 interaction though its coiled-coil domain to
facilitate PCNA-mUb formation. Lastly, several missense
mutations of SART3 identified in tumor samples fail to
augment PCNA-mUb levels and activate TLS pathway.
Collectively, we define an RNA binding-independent function for
SART3 in TLS by facilitating RAD18 /Pol interaction and
RAD18 chromatin accumulation to promote PCNA-mUb
formation, providing insights into how SART3 promotes
genome integrity and contributes to cancer development.
MATERIALS AND METHODS
Plasmids and reagents
SART3 cDNA was a gift from Dr. Jiahuai Han (Xiamen
University). SFB (S-Flag–Streptavidin binding
peptide)and Myc-tagged RAD18 plasmids were gifts from Dr Jun
Huang (Zhejiang University). Full-length and truncations
of SART3 were PCR amplified and cloned into
pEGFPC3 (Clontech) or pCMV5-Flag to generate GFP- or
Flagtagged fusion proteins. Full-length and truncations of Pol
were amplified and cloned into p2xFlag-CMV-14 (Sigma)
or pEGFP-C3 vector.
Anti-Flag M2 agarose affinity gel, mouse monoclonal
antibody against Flag and BrdU for labeling ssDNA
were purchased from Sigma (St. Louis, MO, USA).
Antibody against BrdU was from BD science. Antibodies
against RAD18 for western blotting, SART3 and RPA32
were from Abcam. Anti-RAD18 antibody for
immunofluorescence was purchased from Bethyl Laboratories.
Antibodies against CPD was from Cosmo Bio Co (Tokyo,
Japan). Monoclonal antibodies against PCNA (PC10) and
GFP (FL) were from Santa Cruz Biotechnology. Antibody
against USP1 was from Cell Signaling Technology. Alexa
Fluor-555-labeled goat anti-mouse-IgG was from
Cell culture and reagents
Human U2OS and 293T cells were obtained from the
American Type Culture Collection (Rockville, MD, USA).
SV40transformed MRC5 (Pol normal) and XP30RO (Pol
deficient) cells were kindly provided by Dr Alan Lehmann.
RAD18 knockout 293T cell lines were prepared through
TALEN as described (
). All cells were treated with
mycoplasma removal agent (MPbio) and cultured in DMEM
medium supplemented with 10% fetal bovine serum (FBS)
at 37◦C in the presence of 5% CO2 if not specified. For
transient transfection experiments, cells were transfected with
indicated constructs using VigoFect (Vigorous
Biotechnology Beijing Co., Ltd, China) following the manufacturer’s
protocol. Forty-eight hours later, transfected cells were
collected for further experiments.
For RNAi experiments cells were transfected with
siRNAs purchased from GenePharma (Shanghai, China)
using RNAiMAX (Invitrogen) according to manufacturer’s
instruction, and analyzed 72 h later. The gene-specific
target sequences were as follows: SART3 (3 UTR-1) (GGAG
ACAGGAAATGCCTTA), SART3 (3 UTR-2) (GATG
TGGTGTCCTGAGATA), SART3 (CDS) (GCUGAGAA
GAAAGCGUUAA) and POLH (CAGCCAAATGCCCA
TTCGCAA). The negative control (siNC) sequence (UU
CUCCGAACGUGUCACGU) was also obtained from
GenePharma. Unless otherwise specified, SART3-3
UTR2 was used as the representative siRNA against SART3 in
all experiments. Western blots were used to validate
knockdown efficiency of theses siRNAs. For focus formation
assay, cells were further transfected with GFP-Pol 48 h later
after siRNA transfection.
Co-immunoprecipitation and western blotting
HEK293T cells were transfected with Flag-SART3 and
GFP-Pol or Flag-Pol and GFP-SART3. Forty-eight
hours later, the cells were harvested and lysed with HEPES
buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1mM
EDTA, 1 mM EGTA, 10% glycerol, 1% Triton X-100, 25
mM NaF, 10 M ZnCl2). The whole cell lysates were
immunoprecipitated with anti-Flag M2 agarose in the
presence or absence of RNase A, ethidium bromide (EB), as
indicated. For mapping the regions within SART3
responsible for its interaction with Pol and RAD18, GFP-tagged
wild-type (WT) and a series of truncated SART3 were
co-transfected with Flag-Pol or SFB-RAD18 (SFB:
SFlag-Streptavidin binding peptide) in HEK293T cells for
co-immunoprecipitation experiments. The
immunoprecipitated products were separated by SDS-PAGE and detected
by immunoblotting with indicated antibodies. For isolation
of chromatin-fractions, U2OS cells were treated with UVC
(15 J/m2), and the triton-insoluble fractions were harvested
as previously described (
Mutation frequencies were measured using the supF
shuttle vector system as described previously (
), which is used
to measure TLS activity in mammalian cells. HEK293T
cells were transfected with siNC or SART3 siRNAs twice.
Forty-eight hours after the first transfection, cells were
transfected with UVC-irradiated (400 J/m2 UVC) pSP189
reporter plasmid. 48 h later, pSP189 plasmid from 293T
cells was extracted by using a DNA miniprep kit (Tiangen,
China) and the purified plasmid was digested with Dpn1
followed by transformation into the MBM7070 bacterial
strain. The transformed MBM7070 cells were cultured on
Luria-Bertani plates with 200 M IPTG, 100 g/ml X-gal
and 100 g/ml ampicillin. The mutation frequency in the
supF coding region was calculated by the ratio of white
(mutant) and blue (WT) colonies. The pSP189 plasmid and
MBM7070 strain were gifts from Dr M. Seidman (
Micronucleus assay was performed as described (
Briefly, U2OS cells transfected with siNC or siSART3 were
irradiated with UVC (3 J/m2) followed by incubating with
6 g/ml Cytochalasin B in complete medium for 48 h. The
cells were trypsinized and washed with PBS once, further
treated with 0.075 M KCl for 20 min. Cells were centrifuged
to remove most hypotonic buffer and stained with 0.01%
acridine orange. Cells with two nuclei were counted to
calculate the micronuclei rate.
IF was performed as described (
). Briefly, cells were
seeded on cover glasses and irradiated with UVC. The cells
were permeabilized with 0.5% Triton X-100 for 5–30 min
before being fixed in 4% paraformaldehyde. The samples
were then blocked with 5% donkey serum (for RAD18
staining) or 5% FBS and 1% goat serum (for RPA32
staining) for 30 min. The cells were next incubated with indicated
antibodies for 45 min followed by incubation with Alexa
Fluor 568 goat anti-mouse (Invitrogen, Molecular Probes)
for 45 min. The cells were later counterstained with DAPI
and images were acquired with a Leica DM5000 (Leica)
equipped with HCX PL S-APO 63 × 1.3 oil CS immersion
objective (Leica) and processed with Adobe Photoshop 7.0.
For quantitative analysis of UV-induced Pol focus
formation, U2OS cells transfected with GFP-Pol were treated
with 15 J/m2 UVC and fixed with 4% paraformaldehyde 10
h later as previously described. GFP-Pol -expressing cells
with more than 30 Pol foci were counted as GFP-Pol foci
positive cells. More than 200 cells were analyzed for each
Detection of un-replicated CPDs in UV-treated cells
Detection of CPDs in single-stranded DNA templates was
performed as previously described (
). Briefly, cells
cultured on coverslips were treated with 0 or 10 J/m2 UVC.
Four hours later, cells were permeabilized with 1%
Triton X-100 in PBS for 2 min, followed by fixation with 2%
formaldehyde in 0.5% Triton X-100/PBS for 15 min at room
temperature. Un-bypassed CPDs in non-denatured DNA
were detected by using primary mouse monoclonal
antibodies against it (TDM2, CosmoBio). Cells were
subsequently incubated with Alexa Flour 568 goat anti-mouse
(Invitrogen, Molecular Probes) and stained with DAPI. To
check the UV-induced CPDs, cells were further treated with
2 M HCl for 10 min to denature DNA after fixation. Images
were obtained using a fluorescence microscope.
Immunofluorescent detection of ssDNA
Cells were seeded on glass coverslips to reach a confluence
of about 50% the next day. BrdU (Sigma) (10 M) was
added to the cells and incubated for 48 h. Cells were then
irradiated with 20 J/m2 UVC. Two hours later, the cells were
permeabilized with solution buffer (20 mM HEPES pH 7.4,
50 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 0.5%
Triton X-100) for 5 min at room temperature, followed by
fixation in 4% paraformaldehyde for 10 min. SsDNAs were
identified in non-denatured DNA with primary
antibodies against BrdU (BD). To check the BrdU incorporation
efficiency, cells were further treated with 2 M HCl for 10
min after fixation. Images were analyzed by fluorescent
SART3 interacts with Pol and regulates Pol focus formation after UV radiation
To determine the mechanism of regulation of Pol in
response to UV irradiation, we transfected 2xFlag-Pol in
HEK293T and performed immunopurification as described
). The protein samples at the indicated region on a
SDS-PAGE gel were digested and analyzed with liquid
chromatography-tandem mass spectrometry
(Supplementary Figure S1) (
). The analysis identified several
potential Pol -interacting proteins, including SART3, which
is a factor implicated in pre-mRNA splicing and gene
expression. The association of GFP-Pol with Flag-SART3
was confirmed by co-immunoprecipitation (co-IP) (Figure
1A). Given that the C-terminus of SART3 has two
RNArecognition motifs (RRM1 and RRM2), which can
recognize and bind RNA, we examined whether RNA is
required for the interaction between SART3 and Pol . Cell
lysates were treated with RNase A prior to co-IP. The
result showed that RNase A treatment had no apparent
effect on the association between Flag-SART3 and GFP-Pol
(Figure 1B). We further observed that the binding between
Flag-Pol and GFP-SART3 was not affected after
treatment of cell lysates with ethidium bromide (EtBr), which is
known to disrupt protein–DNA interactions (Figure 1C).
These results exclude the possibility that the association
between SART3 and Pol is mediated via DNA or RNA.
Additionally, we found that Flag-Pol could also precipitate
endogenous SART3 (Figure 1D). Intriguingly, their
association manifested a dynamic change after UV treatment,
namely, the level of interaction between the two proteins
increased at 4 h and decreased to background at 8 h after UV
treatment (Figure 1D).
Given that Pol plays an important role in UV-induced
TLS, to uncover the significance of SART3 binding to Pol
in vivo, we examined whether SART3 depletion impairs
Pol focus formation after UV irradiation. SART3 in U2OS
cells was depleted by siRNA directed to its 3 UTR. The
knockdown cells were transfected with GFP-Pol , followed
by UVC (15 J/m2) treatment and incubated for 10 h. We
noted that the proportion of GFP-Pol foci-positive cells
in the SART3-depleted group (39.3%) was significantly
decreased compared to non-targeting siRNA (siNC)-treated
control (55.2%) (Figure 1E). To exclude the possibility that
the reduced Pol focus formation results from siRNA
offtarget effect, we also co-transfected Flag-SART3 and
GFPPol into the SART3-depleted U2OS cells and checked its
Pol focus formation. We found that expression of
FlagSART3 (57.3%) could rescue the reduced Pol focus
formation in SART3-depleted cells to the siNC-treated
control level (55.2%) (Figure 1E and F). This result indicates
that SART3 is required for optimal Pol association with
stalled replication forks following UV exposure.
SART3 depletion impairs UV-induced PCNA-mUb and
RAD18 focus formation
It is well-established that PCNA-mUb plays an
important role in the recruitment of Y-family TLS polymerases
to stalled replication factories after replication stress. We
then determined whether the reduced Pol focus
formation caused by SART3 depletion is due to an impaired
formation of PCNA-mUb after UV irradiation. To test that,
we first tried to establish SART3 knockout cells using the
CRISPR/Cas9 method, but were unsuccessful, hinting that
SART3 might be important for cell survival. We then
generated U2OS stable cells expressing either GFP-SART3
or GFP vector (GFP) by lentivirus infection (Figure 2A).
The expression levels of exogenous and endogenous SART3
were comparable in the U2OS stable cells expressing
GFPSART3. The cells were transfected with two independent
siSART3 oligos targeting SART3 3 UTR regions followed
by UVC treatment. The triton-insoluble fractions were
collected and analyzed by immunoblotting. We noticed that
depletion of endogenous SART3 in GFP stably expressing
cells led to a dramatically decrease in PCNA-mUb at 4 h
after UV irradiation, compared to siNC control (Figure 2B).
Similarly, SART3 ablation with two different siRNA
oligos targeting its 3 UTR or CDS regions, attenuated the
UV-inducible level of PCNA-mUb (Supplementary Figure
S2A). In contrast, depletion of endogenous SART3 in
GFPSART3 stably expressing cells manifested no obvious effect
on PCNA-mUb, supporting that the reduced PCNA-mUb
observed in SART3-depleted cells results from knockdown
of SART3 (Figure 2C). Moreover, U2OS cells stably
overexpressing GFP-SART3 showed more efficient formation of
PCNA-mUb than GFP-expressing cells (Figure 2D).
Given that the level of PCNA-mUb is negatively
regulated by the USP1 deubiquitinase (
), we compared the
levels of USP1 in SART3-depleted and control U2OS cells
under the presence and absence of UV radiation. SART3
depletion did not cause appreciable alterations in USP1
expression (Figure 2C), excluding the possibility that the
compromised PCNA-mUb formation in SART3-depleted cells
is caused by upregulation of USP1.
The level of PCNA-mUb is also positively regulated by
the RAD18-RAD6 ubiquitin ligase complex. We first
compared the level of RAD18 in the control and
SART3depleted cells and found that SART3 depletion had no
obvious effect on RAD18 expression (Figure 2C).
Considering that PCNA-mUb occurs in a chromatin context, we
further analyzed whether SART3 regulates the chromatin
association of RAD18. Interestingly, the result showed
that depletion of SART3 reduced the level of
chromatinassociated RAD18 (Figure 2C), while expressing
siRNAresistant GFP-SART3 could rescue this reduction. We also
wanted to know whether SART3 is required for RAD18
focus formation after UV irradiation. IF results showed that
depletion of SART3 in GFP but not GFP-SART3
expressing cells led to a dramatic decrease in the RAD18 focus
formation at 4 h after UV treatment (Figure 2E-F). These
results indicated that the reduced formation of PCNA-mUb
observed in SART3-depleted cells after UV treatment
results from impaired RAD18 accumulation at stalled
To rule out the possibility that SART3 depletion reduces
Pol focus formation through downregulation of Pol , we
knocked down the endogenous SART3 in U2OS cells
stably expressing GFP-SART3 or GFP and examined the
protein level of Pol with or without UVC treatment
(Figure 2G). No apparent reduction in Pol level was noted
after SART3 depletion. In addition, SART3 ablation
attenuated UV-inducible PCNA-mUb in MRC5 cells as well
as in XP30RO (Pol -deficient) cells (Supplementary
Figure S2B), suggesting that SART3 regulates formation of
PCNA-mUb in a Pol -independent pathway.
Collectively, these results indicate that SART3 regulates
UV-induced Pol focus formation through RAD18 and
SART3 regulates UV-induced RPA focus formation and single-stranded DNA generation
As RAD18 is recruited to chromatin through its direct
interaction with RPA-coated ssDNA (
), we then tested
whether SART3 depletion impairs the chromatin
association of RPA. Endogenous SART3 in GFP or GFP-SART3
expressing cells was depleted and RPA focus formation
as well as the extents of RPA binding to chromatin
after UV exposure were compared. IF results showed that
UV-induced RPA32 focus formation was dramatically
decreased in SART3-depleted cells (13.3%) relative to siNC
control (23.3%) (Figure 3A and B). Expression of
siRNAresistant GFP-SART3 was able to rescue the impaired RPA
focus formation (28.0%). Consistently, we found that
depletion of SART3 in GFP but not GFP-SART3 expressing
cells reduced the level of RPA32 on chromatin (Figure 3C),
while it had no effect on RPA32 expression (Figure 3C).
We also pulsed labeled SART3-depleted U2OS cells with
EdU (10 M) for 1 h followed by reaction with
Alexa488azide. After checking the proportion of EdU positive cells
in siNC and siSART3, we found that SART3 depletion also
impaired DNA replication (Supplementary Figure S3A).
RPA32 is known to be recruited to stalled replication
forks through its avid affinity with ssDNA. We then checked
whether the compromised RPA32 chromatin loading in
SART3 knockdown cells results from impaired ssDNA
generation after UV exposure. The cells were labeled with a
thymidine analogue 5-bromo-2 -deoxyuridine (BrdU) for
48 h prior to UVC exposure. BrdU corresponding to
ssDNA but not dsDNA was monitored by
immunofluorescence with an anti-BrdU antibody without
denaturation of DNA. The result revealed that, at 2 h post-UV,
SART3-depleted cells exhibited a significant reduction in
UV-induced ssDNA formation (Figure 3D and E). The
percentage of BrdU-positive cells in SART3-depleted cells
(24.3%) was remarkably reduced, compared with that in
siNC controls (34.5%). Moreover, exogenous expression of
SART3 could rescue the ssDNA formation defect (34.5%)
(Figure 3D and E). We also performed a denatured
immunofluorescence to show that the entire nuclear DNA was
more or less evenly labeled with BrdU (Supplementary
Figure S3B). These results indicate that SART3 facilitates
efficient ssDNA generation after exposure to UV.
SART3 is required for optimal cellular response after UV treatment
To further define the role of SART3 in UVC-induced TLS
pathway, we determined the ability of SART3-depleted
cells to bypass UV-induced CPD lesions as previously
reported. Analogous to Pol -depleted cells (27.4%), SART3
knockdown cells (21.9%) exhibited an obviously increased
CPD signal in ssDNA compared to siNC-treated control
cells (10.9%) (Figure 4A and B). In line with the result
that SART3 is required for optimal Pol focus formation
after UV treatment, expressing siSART3-resistant
GFPSART3 in SART3 knockdown cells reversed the CPD
signal in ssDNA (13.1%) close to siNC level (Figure 4A and
B and Supplementary Figure S3C). Notably, equivalent
amounts of CPDs were detected in denatured DNA in these
cells (Supplementary Figure S3C). These results reveal that
SART3 is required for gap filling opposite genomic CPD
Previous studies have shown that cells depleted of Pol
exhibit an elevated mutation frequency (
). Given that
depletion of SART3 abrogates Pol recruitment after UVC
exposure, we speculated that SART3 depletion may also
increase UV-induced genome mutagenesis. To test that, a
mutagenesis assay based on a UV–irradiated shuttle vector
pSP189 that carries a mutant supF suppressor tRNA33 was
performed (29). As expected, the normalized mutation
frequencies in SART3 depleted cells were elevated (1.87-fold
increase for siUTR-1, 1.79-fold increase for siUTR-2, as
compared to siNC control) (Figure 4C). We also noticed
that depletion of SART3 in U2OS cells sensitized the cells to
UV killing, although the extent was less than that of
depletion of Pol in GFP-expressed U2OS cells (Figure 4D and
E). Moreover, the SART3 depletion-derived UV
hypersensitivity could be rescued by exogenously expressed
siRNAresistant SART3 (Figure 4D). To further uncover the
biological significance of SART3 in the cellular response to
UV irradiation, we also performed a micronucleus (MN)
assay. We found that knockdown of SART3 significantly
increased the proportion of cells with unperturbed MN
(13.4% vs 7.03%) or MN after UV exposure (21.4%
versus 10.4%), which was completely rescued by exogenously
expressed siRNA-resistant SART3 (Figure 4F). These data
suggest that SART3 plays an important role in preventing
genomic instability in response to UV irradiation.
SART3 interacts with RAD18 and Pol through its RRMs
To identify the domains in SART3 responsible for its
interaction with Pol , Flag-Pol and a series of GFP-SART3
truncations (Figure 5A) were transiently co-transfected into
293T cells followed by co-IP. We found that peptides
containing RRMs (RNA recognition motifs) of SART3
associated with Pol (Figure 5B), indicating that the HAT domain
does not appear to be the major interactor with Poln. We
also examined the interactions between SFB-SART3 and
GFP-Pol truncations (Figure 5C). We found that SART3
bound to the N terminus of Pol , which is distinct from the
Pol -RAD18 binding region (Figure 5D). Similar result was
obtained through a GST-SART3 pull down assay
(Supplementary Figure S4A).
Interestingly, we found that SART3 also associated with
RAD18 through a co-IP assay (Figure 5E), which was
further confirmed by showing that GST-SART3 bound to
endogenous RAD18 but not GST protein alone
(Supplementary Figure S4B). To map the domain in SART3 required
for its interaction with RAD18, we co-transfected
GFPSART3 truncations and SFB-RAD18 into 293T followed
by co-IP. Intriguingly, the RRMs in SART3 also bound with
SFB-RAD18 (Figure 5F), suggesting that SART3 binds
both RAD18 and Pol through the same domain.
SART3 promotes RAD18/Pol association through its ho
Since RAD18 can associate with Pol (
), it was
necessary to rule out the possibility that the interaction
between SART3 and Pol is mediated by RAD18. To do this,
we transfected Flag-Pol into WT or RAD18 knockout
293T cells (
) followed by co-IP experiments (Figure 6A).
The result showed that the association between Flag-Pol
and endogenous SART3 were comparable in these two cell
lines, indicating that RAD18 is not necessary for the
interaction. Meanwhile, Flag-Pol precipitated endogenous
RAD18 as well (Figure 6A). We then determined whether
Pol mediates the interaction between RAD18 and SART3
by transfecting SFB-RAD18 into MRC5 and XP30RO
(Pol -deficient) cells followed by a co-IP assay. We found
that SFB-RAD18 still associated with SART3 in the
absence of Pol (Figure 6B). To further support this result,
we generated a GFP-RAD18 mutant lacking the binding
domain for Pol (GFP-RAD18- PID). We found that
although GFP-RAD18- PID failed to interact with
FlagPol (Supplementary Figure S4C), it still bound to
FlagSART3 (Supplementary Figure S4D, lane 5). These results
suggest that SART3 interacts with both RAD18 and Pol
Given that SART3 interacts with Pol and RAD18
through the same RRMs and the coiled-coil domain is one
of the principal subunit oligomerization motifs in proteins
), we wondered whether SART3 can interact with
itself through its coiled-coil domain, then promote Pol and
RAD18 association through forming a complex. To test
this hypothesis, we first co-transfected 293T cells with
GFPSART3 and Flag-SART3 followed by immunoprecipitation
with anti-Flag antibodies. The result showed that
GFPSART3 but not GFP-SART3- CC (deletion of the
coiledcoil domain) could be efficiently co-precipitated (Figure
6C). To further confirm that, 293T cells transfected with
Flag-SART3 were treated with different concentrations of
a crosslinking reagent. We found that Flag-SART3 could
form homodimers in vivo, which was not affected by UVC
exposure (Figure 6D). These data indicate that SART3 can
form homodimer mediated via its coiled-coil domain.
We then determined whether SART3 enhances the
association between Pol and RAD18. We found that depletion of
SART3 in 293T cells significantly impaired the interaction
between Flag-Pol and endogenous RAD18 (Figure 6E).
In addition, overexpression of GFP-SART3, which could
form homodimers, enhanced the interaction between
FlagPol and RAD18 (Figure 6F). In contrast, overexpression
of GFP-SART3- CC showed no stimulatory effect
(Figure 6F), although it still localized in the nucleus
(Supplementary Figure S4E). Considering that Pol /RAD18
interaction plays a key role in targeting RAD18 to PCNA
and promoting formation of PCNA-mUb at stalled
replication forks (
), we isolated the chromatin fractions to
examine whether SART3 homodimerization is vital for
PCNAmUb as well. As shown in Figure 6G, the overexpression of
GFP-SART3, instead of GFP or GFP-SART3- CC,
promotes formation of PCNA-mUb. Notably,
CC manifested a dramatically reduced chromatin
association (Figure 6G).
Cancer-associated SART3 mutants affect TLS
Given that TLS is important for genome stability and
cancer progression, we wondered whether SART3
mutants identified in patients affect the TLS process.
SART3V591M (Valine591 is mutated to Methionine) is a SART3
missense mutation found in a Chinese pedigree with
disseminated superficial actinic porokeratosis (DSAP) (
When complemented into SART3-depleted cells, both WT
and SART3-V591M were able to restore Pol focus
formation and PCNA-mUb formation in SART3 depleted cells
(Supplementary Figure S5A-C), suggesting that
SART3V591M does not lose its ability to activate TLS. Through
exploring the cBioPortal database (http://www.cbioportal.
), several missense mutations with
repeated incidence in SART3 coiled-coil and RRM domains
were identified in multiple cancers, including melanoma,
cervical squamous cell carcinoma and breast cancer
(Figure 7A, Table 1). We first constructed several GFP-SART3
mutants in which the target amino acids were mutated into
alanine (K614A, R742A and R836A) and transfected them
into U2OS cells. In contrast to WT and SART3-V591M,
K614A and R836A mutants were less efficient in
promoting PCNA-mUb formation (Figure 7B), while R742A
mutation did not obviously compromise SART3 ability to
stimulate PCNA-mUb formation (Figure 7B). Then we
generated several mutants as are the cases in patients, including
K614N, K614R and R836W. We found that they displayed
lower efficiency in promoting PCNA-mUb formation than
that of WT (Figure 7C). We found that the mutations at
K614 (K614N, K614R and K614A) did not impair SART3
homodimerization (Supplementary Figure S6A). However,
K614N, K614R and R836W mutations significantly
compromised SART3 ability to stimulate RAD18/Pol
association (Supplementary Figure S6B). We further found that
K614N, K614R and R836W mutations also impaired the
ability of SART3 binding to chromatin after UV
irradiation (Supplementary Figure S6C), indicating that SART3
chromatin binding is likely a prerequisite for its effect on
RAD18/Pol association. We also noticed that mutation
of R580I did not impair SART3 homodimerization as well
as its chromatin association (Supplementary Figures S6D
and S6E). Taken together, the ability to promote
PCNAmUb formation after UV was impaired in several SART3
missense mutants of coiled-coil and RRMs domains,
hinting that the dysfunction of SART3 mutants in
promoting RAD18/Pol association and activating TLS may
contribute to genome instability and cancer progression.
Notably, taking all the SART3 coding mutations into
comparison, we found that the mutation burden of cancer
patients with SART3 mutants was higher than those with WT
SART3 (Supplementary Figure S7).
Pol /RAD18 physical interaction plays very important
roles in TLS regulation. On one hand, RAD18 can guide
Pol to stalled replication sites (
), and on the other hand,
Pol has been shown to bridge RAD18 and PCNA to
promote efficient PCNA-mUb formation after DNA damage
). Therefore, it seems that RAD18 and Pol play
mutually dependent roles in TLS pathway activation through
their association. Given their interaction confers cellular
resistance to UV killing and regulates genome mutagenesis
). It is necessary to explore how Pol /RAD18
interaction is regulated in vivo.
SART3 was previously regarded as a pre-mRNA
splicing factor, which can promote the formation of the U4/U6
di-snRNP to regulate pre-mRNA splicing and gene
). In this study, we have provided several
lines of evidence to show that SART3 is a critical
regulator of Pol /RAD18 interaction and PCNA-mUb
formation in response to UV damage. First, SART3 physically
interacts with Pol and RAD18. Second, depletion of SART3
significantly impairs UV-induced Pol and RAD18 focus
formation. Third, SART3 depletion remarkably decreases
PCNA-mUb and RPA focus formation after UV
irradiation. Fourth, cells depleted of SART3 displays a defective
TLS efficiency, an increased genome instability and
hypersensitivity in response UV treatment. Finally, SART3
promotes Pol /RAD18 association through its dimerization.
Notably, the novel role of SART3 in TLS is independent
of its RNA binding activity. Hence, our data suggest that
SART3 not only promotes the generation of ssDNAs at
stalled replication forks to facilitate RPA focus formation,
but also promotes Pol /RAD18 association, which
synergistically enhances PCNA-mUb and Pol focus formation
after UV irradiation, modulating TLS process (Figure 7D).
The TLS pathway is known to be efficiently triggered by
replication stress, which leads to uncoupling of replicative
polymerase and helicase activities, and generating stretches
of ssDNA (
). However, how ssDNA generation is
regulated remains largely unclear. Here, we found that SART3
regulates ssDNA generation after UV irradiation.
Considering that SART3 can function as a histone chaperone to
associate with USP15 for the H2B deubiquitination (
while the absence of H2BK123 ubiquitination (H2Bub1) in
yeast leads to an ‘open’ chromatin structure (
), it is
plausible that SART3 might modulate ssDNA generation by
regulating chromatin dynamics. In support of it,
H2Bub1deficiency cells accumulate unrepaired DNA lesions and/or
replication intermediates enriched with RPA foci indicative
of ssDNA gaps (
We found that SART3 could form homodimers in vivo,
which is consistent with a recent report (
). In addition,
we noticed that SART3 interacts with both RAD18 and
Pol . Interestingly, we found that depletion of SART3
attenuates the association of RAD18 with Pol , indicating
that, through dimerization, SART3 is required for enhanced
RAD18/Pol interaction. Moreover, based on the fact that
K614N and K614R mutations compromised SART3
abilities in chromatin binding and in promoting RAD18/Pol
association but not in SART3 dimerization, it is likely that
SART3 chromatin binding is a prerequisite for its
stimulatory effect on RAD18/Pol association. This mode of
regulation of SART3 on RAD18/Pol interaction is
distinct from that of Cdc7, which was reported to function via
phosphorylation of RAD18 (
). Moreover, unlike SART3,
Cdc7 depletion has no effect on PCNA-mUb formation.
SART3 has been implicated as a candidate gene in DSAP,
which is an uncommon autosomal dominant chronic
disorder of keratinization and develops on sun-exposed areas of
). We found that V591M mutation does not affect
UV-induced PCNA-mUb and Pol focus formation,
suggesting that the potential DSAP-causing mutation does not
function through regulation of TLS to contribute to this
disease. We also found that several SART3 missense mutations
in SART3 coiled-coil and RRMs with repeated incidence
detected in cancer patients impair their ability to promote
UV-induced PCNA-mUb formation. Notably, the mutation
burden of cancer patients harboring SART3 coding
mutations was higher than patients with WT SART3, hinting
that other Pol -independent error-prone DDR pathways
might be responsible for it.
In conclusion, our work identified a novel role of SART3
in TLS regulation. Interestingly, SART3 was found
upregulated in cisplatin resistant ovarian cancer cell lines
compared with the parental cell lines (P = 0.007 and 0.04,
respectively) in two datasets (GSE45553 and GSE43694).
Given that Pol can protect tumor cells from cisplatin
), the function of SART3 in these tumor cells
might also contribute to cisplatin chemoresistance.
Additional studies of the functions of SART3 in DDR should
yield a greater insight into its role in governing genome
Supplementary Data are available at NAR Online.
The authors thank Drs Jiahuai Han, Alan Lehmann,
Jianguo Ji and Jun Huang for reagents.
National Nature Science Foundation of China ;
CAS Strategic Priority Research Program [XDA16010107];
Natural Science Foundation of Beijing ;
National Key Research and Development Program of China
[2017YFC1001001]; National Nature Science
Foundation of China [91754204, 31570816, 31471331, 31670822,
31701227 and 31470784], CAS Strategic Priority Research
Program [XDB14030300]. State Key Laboratory of
Membrane Biology and CAS Key Laboratory of Genomic and
Precision Medicine. Funding for open access charge:
National Nature Science Foundation of China [81630078,
Conflict of interest statement. None declared.
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