RNA-binding protein HuR enhances p53 translation
in response to ultraviolet light irradiation
Krystyna Mazan-Mamczarz*, Stefanie Galba ´n*, Isabel Lo ´pez de Silanes*, Jennifer L. Martindale*, Ulus Atasoy†,
Jack D. Keene†, and Myriam Gorospe*‡
*Laboratory of Cellular and Molecular Biology, National Institute on Aging–Intramural Research Program, National Institutes of Health,
Baltimore, MD 21224; and†Duke University School of Medicine, Durham, NC 27710
Edited by Bert Vogelstein, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD, and approved May 19, 2003 (received for
review April 10, 2003)
Exposure to short-wavelength UV light (UVC) strongly induces p53
expression. In human RKO colorectal carcinoma cells, this increase
was not due to elevated p53 mRNA abundance, cytoplasmic export
of p53 mRNA, or UVC-triggered stabilization of the p53 protein.
Instead, p53 translation was potently enhanced after UVC irradi-
ation. The 3? UTR of p53 was found to be a target of the RNA-
binding protein HuR in a UVC-dependent manner in vitro and in
vivo. HuR-overexpressing RKO cells displayed elevated p53 levels,
whereas cells expressing reduced HuR showed markedly dimin-
ished p53 abundance and p53 translation. Our results demonstrate
a role for HuR in binding to the p53 mRNA and enhancing its
UV light ? embryonic lethal abnormal vision
of mitogenic and stressful stimuli such as DNA damage, hypoxia,
and nutrient deprivation. In turn, p53 induces the transcription
of many genes, including several that govern cell cycle arrest,
apoptosis, and DNA repair such as Gadd45 (growth arrest and
DNA damage-inducible 45), Bcl-2, PUMA (p53-up-regulated
mediator of apoptosis), p21WAF1, and mdm2 (1, 2). In human
cancer, p53 function has been found to be impaired either
through mutation (described in more than half of all malignan-
cies) or through aberrant regulatory events, underscoring its
critical function as tumor suppressor (3). Given the central role
that p53 plays in both normal cell division and tumor suppres-
sion, understanding the molecular pathways through which p53
expression and function are regulated has been the subject of
intense investigation over the past decade (3).
After exposure to mitogenic and stressful agents, adaptive
responses occur in the cell that culminate in the implementation
of altered gene expression patterns. Such gene expression
changes can be regulated at many levels, including transcription,
splicing, mRNA transport, and stability, as well as protein
translation and stability. p53 expression and function are exten-
sively regulated through mechanisms that are stress-, species-,
and cell type-specific (4). Although instances of transcriptional
regulation of p53 expression have been described (5), it is widely
accepted that p53 expression is primarily regulated through
modulation of the steady-state levels of p53 in the cell, its
subcellular localization, and its activity (1). In unstimulated cells,
p53 expression is typically maintained at very low levels, mainly
through continuous ubiquitin-mediated proteolysis, and can be
rapidly induced by blocking this degradation process (3, 6). In
addition, genotoxic agents have been shown to increase the
stability and activity of p53 protein through posttranslational
modification such as phosphorylation, acetylation, and altered
intracellular localization (4). In recent years, however, evidence
has emerged revealing that p53 mRNA stability and translation
underlying regulatory events remain largely unknown.
n mammalian cells, the expression and activity of the tumor
suppressor gene product p53 is induced in response to a variety
Here, we set out to examine the mechanisms governing the
elevation in p53 expression triggered by irradiation with short-
wavelength UV light (UVC) in human RKO colorectal carci-
noma cells. Our findings reveal that UVC irradiation potently
enhanced p53 translation. We further demonstrate that the 3?
UTR of p53 is a target of the RNA-binding protein HuR in a
UVC-dependent manner in vitro and in vivo. RKO cells display-
ing either elevated or reduced HuR levels showed either en-
hanced or diminished p53 expression, respectively, linked to
HuR in binding to the p53 mRNA and enhancing its translation.
Materials and Methods
Cell Culture, Treatment, and Plasmids. Human RKO colorectal
carcinoma cells (10) were maintained in minimum essential
medium (GIBCO?BRL) supplemented with 10% FBS and
antibiotics. For UVC treatment, medium was removed and
saved, cells were rinsed with PBS and irradiated, and medium
was restored. Unless otherwise specified, cells were irradiated
with 15 J?m2UVC and collected at the times indicated there-
after. S11 and zeo cells were generated after stable transfection
with pZeoSV2 (Invitrogen) and pZeoSV2-HuR, respectively.
Lactacystin was from Calbiochem. Plasmid pGL3-Luc-p53 3?
UTR was constructed by inserting the p53 3? UTR (positions
1421–2629), into the XbaI site of pGL3-promoter (Promega).
Northern and Western Blot Analyses. Total RNA was isolated from
intact cells. Nuclear and cytoplasmic RNA were prepared from
the pellet and supernatant, respectively, obtained after lysis of
cells in 10 mM Tris (pH 7.4), 1 mM KCl, 1 mM MgCl2, and 10%
Triton X-100 and brief centrifugation (420 ? g, 6 min, 4°C). All
RNA extractions were carried out by using STAT-60 (Tel-Test,
Friendswood, TX), and Northern blot analysis was performed as
described (11). For detection of p53 and 18S transcripts, oligo-
GACAGCATCAAATCATCCATTGCTTGGG and ACGGT-
ATCTGATCGTCTTCGAACC, respectively, were end-labeled
by using [?-32P]dATP and terminal transferase. Signals were
quantified with a PhosphorImager (Molecular Dynamics). Cal-
culation of p53 mRNA half-life was carried out by using acti-
nomycin D-based assays, as described (11).
For Western blot analysis, 20-?g aliquots of either total,
cytoplasmic, cytosolic, or polysome-bound protein were resolved
by electrophoresis in SDS-containing polyacrylamide gels, trans-
ferred, and hybridized by using monoclonal antibodies that
recognize p53 (DO-1, Santa Cruz Biotechnology), HuR (3A2,
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: ELAV, embryonic lethal abnormal vision; UVC, short-wavelength UV light;
REMSA, RNA electrophoretic mobility-shift assay; IP, immunoprecipitation; siRNA, small
interfering RNA; CR, coding region.
‡To whom correspondence should be addressed at: Box 12, Laboratory of Cellular and
Molecular Biology, National Institute on Aging–Intramural Research Program, National
Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224. E-mail: myriam-
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no. 14 www.pnas.org?cgi?doi?10.1073?pnas.1432104100
stabilized and translated (11, 14, 29). In this study, we report that
translation of the p53 mRNA, a target of HuR, was significantly
increased after DNA damage by low levels of UVC irradiation. It
is specifically stimulated after DNA damage so that transcription
can pause while DNA repair events proceed. This mechanism
would prevent the production of aberrant transcripts or proteins
synthesized mRNAs that encode important growth regulatory
proteins to be preserved, thereby permitting the protein products
to maintain homeostasis during a period of DNA repair. HuR and
other ELAV proteins would be ideal candidates for responding to
genotoxins and other damaging agents because they interact with
mRNAs encoding important growth regulatory factors and partic-
ipate in their production at the posttranscriptional level (22, 28). In
conclusion, such a broad posttranscriptional function for HuR and
other ELAV proteins will have particular significance after expo-
sure of mammalian cells to toxic agents: it will help ensure that key
as the cell assesses the damage and prepares to undergo either
growth arrest or apoptosis.
We thank X. Yang and W. Wang for helpful discussions.
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