Gene 559 (2015) 4451

Contents lists available at ScienceDirect

Gene
journal homepage: www.elsevier.com/locate/gene

DNA damage stress induces the expression of Ribosomal Protein S27a

gene in a p53-dependent manner
Nagisa Nosrati, Neetu Rohit Kapoor, Vijay Kumar
Virology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, India

a r t i c l e

i n f o

Article history:
Received 30 August 2014
Received in revised form 28 December 2014
Accepted 8 January 2015
Available online 12 January 2015
Keywords:
DNA damage stress
RPS27a
RNA interference
p21Waf1
p53
Ribosomal RNA

a b s t r a c t
The small ribosomal protein RPS27a is known to play a role in the activation of cellular checkpoints via p53 which
links ribosome biogenesis to cell cycle progression. Here, we show that RPS27a gene is a direct transcriptional target of p53 and is overexpressed in response to DNA damage. Elevated RPS27a level was associated with increased
expression of p53 and its target p21Waf1 gene. The RPS27a activity was specically inhibited in the presence of a
dominant negative mutant of p53. Down-regulation of ectopically expressed RPS27a by RNA interference blocked
the activation of p21waf1 in response to DNA damage. Thus, RPS27a appears to be a novel stress sensor in the cell
which amplies p53 response to arrest cell cycle.
2015 Elsevier B.V. All rights reserved.

1. Introduction
Ribosomal proteins (RPs) are ubiquitous and abundant RNA-binding
proteins that are essential for ribosome biogenesis. RPs are now fast
emerging as novel regulators of cell growth linking aberrant ribosome
biogenesis to cell cycle arrest (Warner and McIntosh, 2009). The extraribosomal functions of RPs include regulation of p53 and apoptosis by diverse mechanisms including inhibition of Mdm2 activity, and enhanced
translation and translocation of p53 inside the cell (Warner and
McIntosh, 2009; Wool, 1996). RPs such as L5, L11, L23, L26 and S7 are
known to act as inhibitors of Mdm2 activity in response to nucleolar stress
leading to p53 stabilization (Dai and Lu, 2004; Lohrum et al., 2003; Zhang
et al., 2003, 2010; Bhat et al., 2004; Dai et al., 2004; Jin et al., 2004; Takagi
et al., 2005; Or-Rosenfeld et al., 2008; Chen et al., 2007; Zhu et al., 2009).
In turn, p53 responds to diverse cellular insults by activating CDK inhibitors such as p21Waf1, which participate in cell cycle arrest, premature senescence and apoptotic cell death (Beckerman and Prives, 2010; Berkers
et al., 2013).
Under normal conditions, the intracellular levels of p53 are regulated via Mdm2-mediated ubiquitination and degradation processes.
Abbreviations: ARF, alternative reading frame; ARPP P0, acidic ribosomal phosphoprotein P0; ChIP, chromatin immunoprecipitation; DDR, DNA damage response; IHH, immortalized human hepatocyte; rDNA, ribosomal DNA; rRNA, ribosomal RNA; RPs, ribosomal
proteins; RPS27a, ribosomal protein S27a; RT-PCR, Reverse transcriptasePCR; RT-qPCR,
real time quantitative PCR; UBCEP80 gene, ubiquitin carboxyl extension protein 80 gene.
Corresponding author at: Virology Group, International Centre for Genetic
Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi 110067, India.
E-mail address: vijay@icgeb.res.in (V. Kumar).

http://dx.doi.org/10.1016/j.gene.2015.01.014
0378-1119/ 2015 Elsevier B.V. All rights reserved.

However, under stress conditions Mdm2 activity may be sequestered

leading to p53 stabilization (Ozaki and Nakagawara, 2011; Carvajal
and Manfredi, 2013). An intricate relationship also exists between the
accumulation of p53 and nucleolar/ribosomal proteins (Teng et al.,
2013). For example, the small ribosomal protein RPS3 can regulate ribosome biogenesis in response to DNA damage (Kim et al., 2013) while
RPS27a can activate cellular checkpoints via p53 to inhibit cell cycle
(Xiong et al., 2011; Sun et al., 2011). Although RPS27a emerged as a crucial regulator of p53 under nucleolar stress, its molecular underpinning
which links DNA damage to cell cycle arrest remains elusive. Hence we
sought to understand the mechanism by which RPS27a is able to
activate p53 and impose cell cycle arrest in response to DNA damage.
We show that the human RPS27a gene is both an inducer and a target
of p53. p53 rapidly bound to specic sites on the RPS27a promoter
and stimulated transcription under DNA damage condition. The
RPS27a gene expression was dependent on p53 as this could be abrogated in the presence of a dominant negative mutant of p53 (p53DD). Further, RPS27a behaved as an effector that facilitated the p53 activation
signal to its downstream target p21waf1 linking the nucleolar stress
and cellular genotoxic response to cell cycle regulation.
2. Materials and methods
2.1. Chemicals and antibodies
All chemicals were procured from Calbiochem, UK. Protein-A or A/G
sepharose and protease inhibitor cocktail (PIC) were from Amersham Biotech, UK. Luciferase assay system was from Promega, USA. Dulbecco's

N. Nosrati et al. / Gene 559 (2015) 4451

modied Eagle's medium was obtained from GIBCO, USA. SYBR Green
was from (Sigma-Aldrich, St Louis, MO, USA). The working concentrations of genetoxic agents and other chemicals were as follows:
Etoposide (50 M), methylmethanesulfone (MMS, 0.02%, w/v), paclitaxel (5 M), cycloheximide (CHX, 100 g/ml) and tumor necrosis factor
(TNF, 50 ng/ml). Ultra Violet (UV) treatment of cells was given using a
germicidal lamp (254 nm).
Antibody for RPS27a was from Novus Biologicals (Colorado, USA).
Antibodies for Myc-A14, p21Waf1, pp53, p53, pMDM2, MDM2, pERK,
ERK, pAKT, AKT, pJNK, JNK, cyclin E, E2F1 and GAPDH were purchased
from Santa Cruz Biotechnologies (California, USA).

45

2.6. RNA isolation and quantitative RT-PCR assay

Total RNA was isolated from cells using TRIzol reagent as per the
supplier's instructions (Invitrogen). Reverse transcriptase-PCR (RTPCR) was performed with M-MuLV reverse transcriptase (Fermentas)
according to the manufacturer's guidelines. RT-qPCR was done using
specic primers (Table S1) as described previously (Fatima et al.,
2012). Results were analyzed by using the comparative Ct method
(Schmittgen and Livak, 2008). Actin or ARPP (acidic ribosomal phosphoprotein P0) mRNA were used as internal control in these samples.
2.7. Cell synchronization and cell cycle analysis

2.2. Cloning, expression vectors and reporter constructs

The development of expression vector for the human RPS27a is described elsewhere (Fatima et al., 2012). pIRES2-EGFP and pEGFP-N3
were procured from Clontech, (USA). siRNA for RPS27a (RPS27asiRNA) was from Santa Cruz Biotechnologies (California, USA).
The expression vectors for p53 and p21Waf1 luciferase promoter reporter construct (WWP-LUC) were kindly provided by Dr. Bert Vogelstein
(The Johns Hopkins Oncology Center, Baltimore, USA). The p53 dominant
negative (p53DD) expression vector was kind gift from Dr. Ghanshyam
Swarup (Center for Cellular and Molecular Biology, Hyderabad, India).
The full-length RPS27a reporter construct (FL-RPS27a) was developed by PCR amplifying a 1.6 kb promoter region using the human genomic DNA as template using specic primers (Table S1). The DNA
fragment was cloned into the pGL3 basic luciferase vector between
SmaI and HindIII sites (Promega). Deletion mutants of RPS27a promoter
were derived from the FL-RPS27a reporter construct using a combination of restriction enzymes (for 2-RPS27a, NheI/SpeI and 3-RPS27a,
PstI). All the RPS27a reporter constructs were veried by restriction
digestion and DNA sequencing.

2.3. Cell culture and transfection

IHH cells were synchronized by serum starvation. Cells were rst

serum starved for 72 h followed by incubation with complete medium
for indicated time points and harvested for further analysis.
Cell cycle analysis was performed as described previously (Mukherji
et al., 2007; Pandey and Kumar, 2012). Briey, cells were washed with
PBS (1), xed in 70% ethanol, and stained with propidium iodide (50
mg/ml). The cell suspension was analyzed by uorescence-activated
cell sorting (FACS) using FACS Calibur (BD Bioscience, San Jose, CA,
USA). The percentages of cell cycle distribution were determined
manually.
2.8. Chromatin immunoprecipitation (ChIP) assay
ChIP assay was carried out as described earlier (Pandey and Kumar,
2012). Briey, IHH cells were cross-linked with formaldehyde (1%),
lysed, and sonicated over ice (7 pulses at 30% amplitude) and centrifuged
at 13,000 rpm for 10 min to obtain the supernatant. Samples were precleared for 12 h with protein A-Sepharose beads and incubated overnight with specic antibodies. The immune complexes were pulled
down using protein A-Sepharose beads. After a series of washing steps,
the beads were extracted in 500 l of elution buffer (0.1 M NaHCO3, 1%
SDS) and analyzed by SYBR green PCR using RPS27a ChIP primers
(Table S1). Data were normalized with input DNA and expressed as fold
enrichment over mock. Results were analyzed by using the comparative
Ct method (Schmittgen and Livak, 2008).

The immortalized human hepatocyte (IHH) cells were maintained in

complete medium comprising DMEM with antibiotics (Penicillin/Streptomycin) and 10% fetal bovine serum (Hyclone, USA) at 37 C in a humidied incubator with 5% CO2. The immortalized human hepatocyte (IHH)
cell line was kindly provided by F. Danniel (Schippers et al., 1997).
Cells were transiently transfected with Lipofectamine (Invitrogen, USA)
according to the manufacturer's instructions. In general, cells were
transfected at a density of 0.6 106 or 1.5 106 (~60%) in 60 or
100 mm culture dishes with 2 g or 5 g respectively, of indicated plasmid
DNA or siRNA. pIRES2-EGFP and EGFP-N3 were used as a vector and
transfection controls in parallel set of experiments. Typically, a 6070%
cell transfection level of cells was accomplished in these experiments.

The TFSEARCH program owing to the TRANSFAC database was used

to predict transcription factor binding sites on RPS27a promoter sequence (http://www.cbrc.jp/research/db/TFSEARCH.html).

2.4. Western blot analysis

3. Results

Cell lysates were prepared in cell lysis buffer (Promega, USA). Protein concentration was determined in the cleared lysates using
Bradford's reagent. Equal amounts of protein were re-suspended in
4 SDS dye, boiled and resolved on 1015% SDS-PAGE followed by
Western blotting (Mukherji et al., 2007). The protein bands were visualized using enhanced chemiluminescence (ECL) from Santa Cruz Biotechnologies (California, USA).

3.1. RPS27a gene is expressed in response to DNA damage

2.5. Luciferase assay

Luciferase assay was performed according to the manufacturer's instructions. The relative luciferase activity was measured after normalizing each sample with protein amount and transfection efciency.

2.9. Bioinformatic analysis

2.10. Statistical analysis

Data are expressed as mean S.E. Statistical signicance was calculated using Student's t test. P values b 0.05 were considered signicant.

RPs such as RPL11, RPL5 and RPL23 exhibit anti-cancer properties

owing to their ability to activate p53 and impose cell cycle arrest or
cell death (Lindstrom, 2009). Since RPS27a is reported to interfere
with the p53-Mdm2 axis under ribosomal stress (Sun et al., 2011), we
wondered if RPS27a could also function as a mediator of p53-stress
response. Therefore, the level of RPS27a transcript was measured by
RT-qPCR after treating cells with some common DNA damaging agents
such as Etoposide, MMS and UV light that are known to induce cell
cycle arrest via p53. There was a marked increase (p b 0.001) in the expression of RPS27a gene in the presence of these agents (Fig. 1A). Note
that, the well-known pro-apoptotic agent TNF also induced RPS27a
expression. Interestingly, the expression of RPS27a gene was inhibited

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N. Nosrati et al. / Gene 559 (2015) 4451

Fig. 1. Expression of RPS27a in response to genotoxic stress. (A) IHH cells were treated either with Etoposide or TNF for 24 h, or with MMS or UV light for 30 min and the levels of RPS27a
transcripts were measured by RT-qPCR. (B) Cells were transiently transfected with control or expression vectors for p53 and/or p53DD and treated with Etoposide for 24 h after transfection. Total RNA (2.5 g) was used to measure the levels of RPS27a transcripts as above. Results are shown as pooled data of three independent experiments (mean S.D.). Statistically
signicant at *, p b 0.001.

in the presence of a dominant negative mutant of p53 (p53DD). Further,

enforced expression of p53 alone caused a marked increase in the expression of RPS27a gene (p b 0.001) which could be specically
inhibited in the presence of p53DD (Fig. 1B). Besides, the RPS27a

protein level was also stabilized under the DNA damage stress conditions which could have important bearing on the cell fate (Fig. S1). Together, these results suggested that RPS27a could be an important
player in the DNA damage response (DDR) pathway.

Fig. 2. Transcriptional regulation of RPS27a promoter by p53. (A) Schematic representation of the RPS27a luciferase reporter constructs: The full length RPS27a promoter reporter (FLRPS27a) is shown on the top along with its two luciferase reporter deletion mutant constructs 2 and 3. p53 binding site in the human RPS27a promoter along with the consensus binding sequence are also shown. (B) IHH cells were transiently transfected with FL-RPS27a or its deletion mutants 2- and 3-RPS27a, along with either control or p53 expression vectors and
harvested after 48 h. Total cell lysates were used to measure luciferase activity after normalizing with control extracts. The occupancy of p53 on RPS27a gene promoter was measured by
ChIP-qPCR both in asynchronously growing cells (C) or cells treated with Etoposide for indicated time periods (D). (E) Asynchronized IHH cells were transiently transfected with control or
expression vectors for p53 and/or p53DD and treated with Etoposide for 24 h after transfection to analyze the occupancy of H3K9ac on RPS27a promoter. Result represents pooled data
from three independent experiments (mean S.D.). Statistical signicance: *, p b 0.001 vs. control/mock.

N. Nosrati et al. / Gene 559 (2015) 4451

3.2. RPS27a gene promoter is responsive to p53

Since RPS27a gene appeared to be a transcriptional target of p53, we
next performed the TFSEARCH analysis of the promoter region of the
human RPS27a gene. The bioinformatics analysis predicted a canonical
p53 binding site ( 213 to 203 bp) in the RPS27a gene promoter
sequence (Fig. 2A). To validate the TFSEARCH predictions, we next investigated the regulation of RPS27a gene promoter using the luciferase
reporter constructs of full length (FL-RPS27a) and two deletion mutants
of RPS27a promoter (2- and 3-RPS27a) (Fig. 2A). As shown in Fig. 2B,
p53 could induce the FL-RPS27a reporter activity by nearly 2.5 fold (p b
0.001). No change in the luciferase activity was observed with 3RPS27a, which lacked the p53 binding site. However, a higher reporter
activity with 2-RPS27a could be attributed to the removal of some inhibitory sequences in the promoter region.
To further validate the role of p53 in the RPS27a gene activation, the
occupancy of p53 on the RPS27a promoter was analyzed by ChIP-qPCR
using primers anking the p53-binding region (Table S1). There was a
marked (~8 fold) increase in the recruitment of p53 on the RPS27a promoter as compared to mock (Fig. 2C). Interestingly, a rapid and dramatic increase in promoter occupancy of p53 (~ 500 fold in 15 min) was
observed under the conditions of genotoxic stress with Etoposide
(Fig. 2D).

47

As histone acetylation is a mark of active or euchromatin where

transcriptional activation takes place via chromatin remodeling, we
sought to decode the histone modication marks near the RPS27a promoter. Our ChIP studies (Fig. 2E) showed a signicant enrichment of
H3K9ac on the RPS27a promoter. Interestingly, the H3K9ac mark coincided with the recruitment of p53 which was specically inhibited in
the presence of p53DD (Fig. 2E). Taken together, these results strongly
suggested that p53 could be an important transcriptional regulator of
RPS27a gene during the DNA damage response.
3.3. Interference with RPS27a expression down-regulates p21Waf1 gene
expression
After establishing the involvement of p53 in the RPS27a gene expression, we next analyzed the effect of p53-RPS27a axis on the regulation of
p21Waf1 gene which is one of the main effectors of p53 (Macleod et al.,
1995; He et al., 2005). The p21Waf1 luciferase reporter gene activity was
measured after enforced expression of p53, RPS27a and RPS27a-siRNA.
As shown in Fig. 3A, both p53 and RPS27a stimulated the reporter gene
activity, which could be reversed in the presence of p53DD or by RNA interference against RPS27a expression. Further, analysis of the endogenous
p21Waf1 transcripts by RT-qPCR corroborated the luciferase assay ndings
and indicated the expression of p21Waf1 gene just as p53 (Fig. 3B). To

Fig. 3. Regulation of p21Waf1 promoter in the presence of RPS27a and p53. (A) IHH cells were transiently transfected with WWP-LUC along with combinations of RPS27a and p53 expression vectors. In addition, a set of cells were also transfected with RPS27a-siRNA. Cells were harvested after 48 h and total cell lysates were used to measure luciferase activity. (B) Cells were
transfected with indicated expression plasmids and the total RNA was used to measure the levels of p21Waf1 mRNA by RT-qPCR. IHH cells were transiently transfected with control or
RPS27a expression plasmid (2 g) alone or with an increasing concentration of RPS27a-siRNA (0.25, 0.5, 0.75 and 1 g) and p21Waf1 mRNA (C) or protein (D) levels were measured by
RT-qPCR and Western blotting. GAPDH served as internal control. Result represents pooled data from three independent experiments (mean S.D.). Statistical signicance was tested
at a probability level of *, p b 0.001 vs. control.

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N. Nosrati et al. / Gene 559 (2015) 4451

delineate the molecular mechanism of inhibition of the p21Waf1 promoter

by RPS27a-siRNA, we titrated the p21Waf1 promoter activation using
increasing amounts of RPS27a-siRNA. As expected, there was a dose
dependent inhibition in the levels of both p21Waf1 mRNA (Fig. 3C) as
well as protein (Fig. 3D). Further, a similar expression prole of RPS27a
and p53 mRNAs was also observed in the presence of RPS27a-siRNA
(Fig. S2). Thus, these results suggested the existence of a new link between the ribosomal protein and the p53-related p21Waf1 pathway.
3.4. RPS27a overexpression supports anti-proliferative and pro-apoptotic
signaling

genotoxic stress. Ironically, no major difference in the levels of different

rRNA forms (viz., 45S, 18S, 28S, 5.8S and 5S) was observed (Fig. 4).
However, analysis of the levels of different proliferative and apoptotic
markers in these cells indicated down-regulation of some key proliferation markers such as pERK, pAKT and Cyclin E. On the contrary, there
was a marginal increase in the levels of pro-apoptotic markers such as
phosphorylated p53 and Mdm2 (Fig. S3). Thus, these results highlighted
the role of RPS27a as a molecular sensor that seems to monitor the proliferative potential of healthy cells and divert them to undergo apoptosis
in response to DNA damage.
4. Discussion

As RPS27a is known to be involved in ribosome biogenesis and now

emerged as an important effector of DNA damage, we next investigated
the effect of RPS27a overexpression on rRNA transcription under

Previous studies have shown that RPs can regulate p53 stability, cell
cycle progression and apoptosis through a variety of mechanisms

Fig. 4. Expression of rRNA genes in the presence of RPS27a. IHH cells were transiently transfected either with control, RPS27a or p53 expression vectors (2 g each) and harvested after
48 h. One set of cells was also treated with Etoposide (50 M) for 24 h. Total RNA isolated from these cells and used for measuring the levels of 45S rRNA (A), 5.8S rRNA (B) and 5S rRNA
(C) transcripts by RT-qPCR. ARPP mRNA level was used as internal control. Result represents pooled data from three independent observations (mean S.D.).

N. Nosrati et al. / Gene 559 (2015) 4451

including the inhibition of Mdm2 activity and enhancing the translation

and intracellular localization of p53 (Dai and Lu, 2004; Lohrum et al.,
2003; Zhang et al., 2003, 2010; Bhat et al., 2004; Dai et al., 2004; Jin
et al., 2004; Takagi et al., 2005; Or-Rosenfeld et al., 2008; Chen et al.,
2007; Zhu et al., 2009). Under stress conditions the small ribosomal
protein RPS27a is reported to play an essential role in the activation of
cellular checkpoints via p53 which links ribosome biogenesis to cell
cycle progression (Xiong et al., 2011; Sun et al., 2011). RPS27a is a fusion
protein encoded by the ubiquitin carboxyl extension protein 80 gene
(UBCEP80 gene). The N-terminal domain of UBCEP80 gene codes for
mono ubiquitin whereas the C-terminal region codes for a carboxyl extension protein, which eventually becomes a part of the mature 40S ribosomal subunit (Redman and Rechsteiner, 1989; Monia et al., 1989).
Interestingly, RPS27a is also overexpressed in renal, breast and colon
carcinomas (Kanayama et al., 1991; Adams et al., 1992; Wong et al.,
1993). Recently, we have also reported a dramatic increase in the
expression of RPS27a gene in an oncomouse model of hepatocellular
carcinoma (Fatima et al., 2012).
In this study, we aimed at linking DNA damage-induced p53 activation to RPS27a gene expression. We observed that RPS27a gene was
induced following DNA damage which was specically blocked by regulating p53 activity in the cell (Fig. 1A). We identied p53 as a direct
transcriptional activator of RPS27a gene using reporter assay, qRT-PCR
and ChIP analyses (Figs. 1B, 2B and C). Note worthily, our data also indicated a rapid recruitment of p53 (peaking at 15 min post-treatment) to
the RPS27a gene promoter in response to DNA damage (Fig. 2D). Further, our epigenetic studies conrmed an increase in the acetylation
(H3K9ac) mark on the RPS27a promoter upon Etoposide treatment or
p53 overexpression (Fig. 2E). Notably, the RPS27a gene expression
was dependent on p53 as this could be abrogated in the presence of

49

p53DD. Interestingly, the half-life of RPS27a protein also increased

from 45 min to more than 2 h after Etoposide treatment to ensure the
availability of RPS27a under stress conditions (Fig. S1). Led by these observations, we postulate that the p53-RPS27a axis could be an early
stress response system in the cell.
The elevated expression and stability of RPS27a under stress conditions seemed to play a signicant role in transducing p53 signal to its
downstream effectors such as CDK2 inhibitor, p21Waf1 perhaps to
delay/inhibit the G1/S phase transition (Berkers et al., 2013; Ozaki and
Nakagawara, 2011; Carvajal and Manfredi, 2013). Accordingly, RPS27a
overexpression also augmented the expression of p21waf1 via p53 leading to a dramatic rise in both p21 mRNA as well as its reporter activity
(Fig. 3A, B). Also a dose dependent inhibition was observed in the
p21waf1 mRNA and protein levels in the presence of RPS27a-siRNA
(Figs. 3C, D and S2). It was interesting to note that the activators of
DDR (e.g., etoposide) as well as ARF tumor suppressor pathways
(e.g., UV irradiation) could induce RPS27a expression albeit both pathways are considered to function independent of each other (Velimezi
et al., 2013). As both DDR and ARF pathways act by activating tumor
suppressor p53, the enhanced RPS27a expression could be another important indicator of stress response. Note however that DDR machinery
has a dominant role in early response to limited oncogenic stress
whereas ARF is unable to trigger cell cycle arrest or cell death. It appears
that RPS27a is able to alarm p53 through positive feedback loop to activate its own expression. Subsequently, the binding of RPS27a to MDM2
would free more p53 for the activation of p21Waf1 that in turn could impose cell cycle arrest and cell death. Therefore, it would be interesting to
investigate if these pathways are engaged concurrently or sequentially
during oncogenic insult, and that signal threshold had a role in the regulation of these pathways (Evangelou et al., 2013). Interestingly, when

Fig. 5. Schematic network of auto feedback regulatory loop involving p53 and RPS27a under DNA damage conditions. Ribosome biogenesis is an energy-consuming step, and thus is regulated at multiple levels to conserve the wasteful expenditure. RPs and other nucleolar proteins play extra ribosomal roles to regulate ribosome biogenesis. RPS27a is also one such molecule that regulates p53 stability under nucleolar stress and links ribosome biogenesis to cell cycle progression. Based on our studies, the model depicts dual role played by RPS27a, both as
an inducer of p53 under nucleolar stress as well as target of p53 under DNA damage conditions. Data provides evidence for link between DNA damage and nucleolar stress as over expression of RPS27a under DNA damage will halt ribosome biogenesis by starting up feedback loop in which p53 is activated. Our data also highlights that RPS27a is a crucial molecular sensor
that must be maintained in optimal amounts for normal cell cycle progression. Both over expression or knock down of RPS27a lead to activation of cell cycle inhibitors such as p21Waf1.

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N. Nosrati et al. / Gene 559 (2015) 4451

the expression of exogenous RPS27a was knocked down by RNA interference, the levels of mRNA for both p21waf1 and p53 were reduced.
However, the silencing of endogenous RPS27a led to an increase in the
expression of both p53 and p21waf1 (Fig. 3C, S2B). We speculate that
apart from transducing the p53-mediated DNA damage response,
RPS27a could also be involved in the maintenance of cellular homeostasis. Thus, depletion of endogenous RPS27a may impose additional stress
which would further induce p53 and p21waf1 expression. Thus, RPS27a
appears to be a crucial molecule sensor whose levels must be maintained in a correct stoichiometric ratio failing which could have deleterious consequences on cells.
The dependence of rRNA transcription on RPS27a is already known
(O'Donohue et al., 2010). However, we did not observe any signicant
change in the levels of rRNA transcripts under the experimental conditions used here (Fig. 4). Nevertheless, as DNA damage response is reported to regulate rRNA metabolism (Antoniali et al., 2014), we did
observe a marginal decline in the 5.8S, 18S and 28S rRNA forms
upon etoposide treatment. Note that no change in the levels of prerRNA (45S rRNA) was observed under these conditions (Fig. 4).
These results suggested the differential effect of genotoxic stress on
the stability of processed rRNA forms as compared to pre-rRNA.
Under these conditions, the activation of apoptotic signaling as evident from increase in the phosphorylation of p53 (Ser-392) and
MDM2 (Thr-218) is testimony of the loss of interaction between
p53 and MDM2. This might contribute towards p53 stabilization
and induction of apoptotic signaling pathway (Fig. S3) (Ozaki et al.,
2013). Therefore, the elevated levels of RPS27a in healthy hepatocytes may impart a role in the active action of an auto-feedback regulatory loop of p53 where its induction following DNA damage could
activate RPS27a and strengthen p53 signaling.

5. Conclusion
The present study highlights the signicant role of RPS27a as signal
transmitter between DNA damage response and cell cycle progression/
ribosome biogenesis. Since synthesis of ribosomes is a highly energy
consuming process, it must be tightly and quickly regulated to conserve
cellular energy under unfavorable conditions. Thus, RPS27a appears to
be a novel stress sensor in the cell, which rapidly accumulates under
stress conditions and act as an amplier of p53 response to arrest cells
in the G1 phase (Fig. 5). Perhaps RPS27a could be used as a molecular
marker in cancer management.

Acknowledgments
This work was supported by grant no. 37(1398)/10/EMR-II of the
Council of Scientic and Industrial Research (CSIR), New Delhi. The authors thank Dr. Bert Vogelstein (The Johns Hopkins Oncology Center,
Baltimore, USA) for p53 expression vector and p21Waf1 luciferase promoter reporter construct (WWP-LUC) and Dr. Ghanshyam Swarup
(Center for Cellular and Molecular Biology, Hyderabad, India) for
p53DD. The IHH cell line was kindly provided by F. Danniel, Institut National de la Sant et de la Recherche Mdicale Unite 481, Universite Paris
7, Paris, France. Technical assistance by R. Kumar and T. Choedon is
gratefully acknowledged. Nagisa Nosrati received a pre-doctoral fellowship from ICGEB, Trieste while Neetu Rohit Kapoor is a recipient of fast
track fellowship for young investigators from Department of science
and technology, New Delhi, India.

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.gene.2015.01.014.

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