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Gene
journal homepage: www.elsevier.com/locate/gene
a r t i c l e
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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.
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
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).
46
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.
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.
47
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.
48
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.).
49
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.
50
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.
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