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-
July 8, 2003 ?
vol. 100 ?
no. 14 www.pnas.org?cgi?doi?10.1073?pnas.1432104100
Santa Cruz Biotechnology), or ?-actin (Abcam, Cambridge,
U.K.). After incubation with appropriate secondary antibodies,
signals were detected by enhanced chemiluminescence.
Preparation of Protein Fractions. Cytoplasmic, nuclear, and whole-
cell fractions were prepared as described (11). For preparation
of cytosolic and polysomal fractions, cytoplasmic lysates were
layered onto a cushion of 30% sucrose in ice-cold buffer
containing 20 mM Hepes (pH 7.4), 50 mM KOAc, 5 mM
MgOAc, 1 mM DTT, 1 unit of RNasin per ?l, 1 ?g of leupeptin
per ml, 1 ?g of aprotinin per ml, and 0.5 mM phenylmethylsul-
supernatant (cytosolic fraction) was stored at ?80°C; the pellet
was resuspended in ice-cold buffer A containing 0.3 M NaCl,
incubated on ice for 1 h, and centrifuged at 10,000 ? g for 15 min
at 4°C; and the resulting supernatant (polysomal extract) was
stored at ?80°C.
Preparation of Synthetic RNA Transcripts. Total RNA from RKO
cells was reverse transcribed, and the cDNAs generated were
used as templates for PCR amplification of the coding region
and 3? UTR of p53. The 5? primers contained the T7 RNA
polymerase promoter sequence (T7): CCAAGCTTCTAATAC-
GACTCACTATAGGGAGA. To prepare the coding region
(encompassing positions 252 to 1439), oligonucleotides (T7)AT-
GGAGGAGCCGCAGTCAGATCCTAGC and AGAATGT-
CAGTCTGAGTCAGGC were used. To prepare the 3? UTR
template (encompassing positions 1421 to 2629), oligonucleo-
tides (T7)TGACTCAGACTGACATTCTCC and TGGCAGC-
AAAGTTTTATTGTAAAATAAGAGATCG were used.
PCR-amplified products were resolved on agarose gels, purified,
and used as templates for the synthesis of corresponding RNAs.
For RNA electrophoretic mobility-shift assay (REMSA), PCR-
amplified products were use for the synthesis of radiolabeled
transcripts, as described (11) and used at a specific activity of
100,000 cpm??l. For pull-down assays, PCR-amplified DNA was
used as template to transcribe biotinylated RNA by using T7
RNA polymerase in the presence of biotin-cytidine 5?-
triphosphate (CTP), and purified as described (12).
RNA Protein-Binding Assays: REMSA, REMSA Supershift, and Pull-
Down. REMSA analysis was carried out as described (11), by
using 10 ?g of cytoplasmic fractions. For REMSA supershift
analysis, cytoplasmic protein aliquots were incubated with an-
tibodies recognizing HuR, tristetraprolin (TTP), TIAR, TIA-1
(Santa Cruz Biotechnology), or AUF1 (a gift of G. Brewer,
University of Medicine and Dentistry of New Jersey, Piscat-
away), or with IgG1 (BD Life Sciences), for 30 min on ice before
addition to the binding reaction mixture; all subsequent steps
were as described above for REMSA. For biotin pull-down
assays, 6 ?g of biotinylated transcripts were incubated with 120
?g of cytoplasmic lysate for 30 min at room temperature.
Complexes were isolated with paramagnetic streptavidin-
conjugated Dynabeads (Dynal, Oslo), and pull-down material
was analyzed by Western blotting.
Immunoprecipitation (IP) of endogenous HuR-mRNA com-
plexes, used to assess the association of endogenous HuR with
endogenous p53 mRNA, was performed as described (13).
Twenty million RKO cells were collected per sample, and lysates
were used for IP for 4 h at room temperature in the presence of
excess (30 ?g) IP antibody [either mouse monoclonal anti-HuR
antibody 3A2 (Santa Cruz Biotechnology) or IgG1]. RNA in IP
material was used in RT-PCR reactions to detect the presence
of p53 mRNA; the p53-coding region was amplified by using the
primer pair described above, and the following amplification
conditions: 1 min at 94°C, 1 min at 55°C, and 1 min at 68°C, for
35 cycles. PCR products were visualized by ethidium bromide
staining of 1.5% agarose gels.
Analysis of Nascent p53. Newly translated p53 protein was mea-
sured by incubating 106cells with 1 mCi (1 Ci ? 37 GBq)
L-[35S]methionine and L-[35S]cysteine (Easy Tag EXPRESS,
NEN?Perkin–Elmer) per 60-mm plate for 20 min, whereupon
cells were lysed by using TSD lysis buffer (50 mM Tris, pH
7.5?1% SDS?5 mM DTT), and lysates were immunoprecipitated
by using either monoclonal anti-p53 antibody DO-1 (Santa Cruz
Biotechnology) or IgG1 for 1 h at 4°C. After extensive washes in
TNN buffer (50 mM Tris, pH 7.5?250 mM NaCl?5 mM EDTA?
0.5% Nonidet P-40), immunoprecipitated material was resolved
by 12% SDS?PAGE, transferred onto poly(vinylidene difluo-
ride) filters, and visualized by using a PhosphorImager (Molec-
Small Interfering RNA (siRNA) Transient Transfection. The siRNA
sequence targeting HuR (HuR4, AACACGCTGAACGGCTT-
GAGG) was derived from nucleotides 377–397 (GenBank ac-
cession no. BC003376). The sequence of the control (Ctrl)
siRNA, AAGTGTAGTAGATCACCAGGC, does not match
any known human gene. siRNA molecules were synthesized by
using a kit from Ambion (Austin, TX) and were transfected at
a concentration of 20 nM by using Olifectamine (Invitrogen).
Cells were treated with UVC 48 h after transfection and
collected for analysis 4 h later.
Results and Discussion
Here, we set out to examine the mechanisms underlying the
elevation in p53 expression triggered by UVC. Treatment of
human colorectal carcinoma RKO cells, which exhibit wild-type
p53 function (10), with the relatively low UVC doses of 15 or 30
J?m2, increased p53 expression, peaking by 6 h and remaining
elevated for up to 72 h (Fig. 1A). The rapid and robust increase
in p53 expression was not accompanied by a concomitant
elevation in p53 mRNA abundance (Fig. 1B), p53 mRNA
stability (Fig. 1C), or relative levels of cytoplasmic p53 mRNA
(Fig. 1D), indicating that the UVC-triggered induction of p53
was regulated by either translational or posttranslational events.
UVC was previously shown to increase p53 protein stability by
blocking its ubiquitin-mediated degradation through the pro-
teasome (6). To assess the extent to which p53 protein increase
examined whether inhibition of the proteasome alone could
recapitulate the effects of UVC on p53 induction in RKO cells.
Cells were incubated with lactacystin, a potent and highly
selective inhibitor of the proteasome, using doses that blocked
proteasome activity completely and had been found to elicit
maximal p53 induction (not shown). As shown, lactacystin
treatment alone did cause an increase in p53 levels, but com-
bined lactacystin and UVC treatments further induced p53
expression (Fig. 2A), indicating that inhibition of the proteasome
was not the sole mechanism leading to p53 induction by UVC.
We therefore investigated whether increased p53 translation
might also contribute to elevating p53 expression after UVC
irradiation by comparing the rate of new p53 synthesis between
untreated and UVC-treated cells. RKO cells were incubated in
the presence of L-[35S]methionine and L-[35S]cysteine for 20 min,
whereupon newly translated p53 was visualized by IP. The brief
incubation period was chosen to minimize the contribution of
p53 degradation in our analysis. As shown in Fig. 2B, newly
synthesized p53 was remarkably more abundant in UVC-
irradiated cells, demonstrating that enhanced p53 protein trans-
cells after UVC exposure.
Earlier studies examining p53 translational control had im-
plicated sequences in the 5? and 3? UTRs of the p53 transcript
(14–16). We thus examined whether the p53 mRNA was a target
of RNA-binding proteins in RKO cells, particularly if such
association(s) occurred in a UVC-dependent manner. Depicted
Mazan-Mamczarz et al. PNAS ?
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vol. 100 ?
no. 14 ?
in Fig. 3 (schematic) are transcripts spanning different sections
of the p53 mRNA that were synthesized in the presence of
radiolabeled UTP for use in REMSA or biotinylated CTP for
use in pull-down assays. REMSA analysis was carried out by
using lysates that were prepared from either untreated or
UVC-irradiated RKO cells. Transcripts corresponding to the
5? UTR and coding region (CR) revealed almost undetectable
binding to proteins present in cytoplasmic lysates, and complex
abundance did not change in a UVC-dependent manner (Fig.
3A, and data not shown). By contrast, transcripts encompassing
the p53 3? UTR were capable of extensive association with
cytoplasmic proteins, and the abundance of the complexes
increased when using lysates prepared from UVC-treated cells
(Fig. 3A). In control REMSA analyses, nuclear lysates revealed
abundant binding to all transcripts tested and exhibited no
UVC-dependent inducibility, similar to what was previously
demonstrated with other transcripts (refs. 11 and 12, and data
not shown). Evidence that the p53 3? UTR indeed possessed
translational enhancing properties came through the analysis of
chimeric transcripts. Plasmid vectors expressing either luciferase
mRNA (pGL3-Luc) or a chimeric mRNA comprising the lucif-
erase coding region linked to the p53 3? UTR [pGL3-Luc-
p53(3?UTR)] were transiently transfected into RKO cells. Ex-
pression levels of chimeric transcripts were unaffected by UVC
irradiation (not shown), and therefore assessment of luciferase
activity served as a measure of mRNA translation, as described
(17). The presence of the p53 3? UTR was found to significantly
enhance the translation of chimeric mRNAs after UVC irradi-
ation (Fig. 3B).
Given previous reports linking members of the ELAV RNA-
binding protein family to translational regulation (18), our
were prepared for Western blotting at the times indicated. (B) Northern blot
analysis of p53 expression 4 h after exposure to the indicated doses of UVC.
Relative p53 abundance was calculated by densitometry and is expressed as
fold increase relative to p53 levels in unirradiated cells. (C) Half-life of the p53
mRNA was calculated by addition of actinomycin D (ActD, 2 ?g?ml) to RKO
cells 4 h after either UVC irradiation (15 J?m2) or no treatment (untreated), as
described (11). Relative p53 mRNA abundance is expressed as fold increase
relative to p53 mRNA levels in unirradiated cells. (D) Total, cytoplasmic (Cy-
topl.), nuclear, and polysomal fractions were prepared for Northern blot
analysis 4 h after exposure to UVC (15 J?m2); 18S signals reveal even loading
Expression of p53 in RKO cells irradiated with UVC. (A) Cells were
prepared at the times indicated after treatment with lactacystin (5 ?M), UVC
(graph). (B) Newly translated p53 was measured by incubating cells with
L-[35S]methionine and L-[35S]cysteine for 20 min, followed by immunoprecipi-
tation by using either anti-p53 antibody or IgG1, resolving immunoprecipi-
tated samples by SDS?PAGE, and transferring for visualization of signals by
using a PhosphorImager.
(A) Western blot analysis of p53 expression in RKO whole-cell lysates
www.pnas.org?cgi?doi?10.1073?pnas.1432104100 Mazan-Mamczarz et al.
earlier reports of a UVC-dependent increase in HuR function in
RKO cells (11), and the existence of U-rich and AU-rich
elements, akin to other HuR-target sequences, in the p53
3? UTR (Fig. 3 Upper, gray shading), we directly examined
whether HuR, the only ELAV member shown to be expressed in
RKO cells (11), might be involved in the translational regulation
of p53. To directly investigate the potential association of HuR
with the p53 3? UTR in a UVC-dependent fashion, we carried
out three types of assays. First, we used a p53 3? UTR transcript
to perform REMSA supershift analysis with anti-HuR antibod-
ies. As shown in (Fig. 4A), HuR indeed forms a complex with the
p53 3? UTR, as revealed by the appearance of slower electro-
phoretic mobility bands (arrowhead) in anti-HuR antibody
incubations, but not in control antibody (IgG1) incubations.
Furthermore, the intensity of the supershift increased when
using lysates from UVC-treated cells. No supershifts were
observed when assessing the presence of additional AU-rich
element RNA-binding proteins implicated in regulating either
mRNA turnover or translation [AUF1, TTP, TIAR, and TIA-1
(19–21)]. Second, use of biotinylated p53 transcripts in pull-
down assays using streptavidin-coated beads revealed an asso-
ciation of the p53 3? UTR with HuR present in cytoplasmic and
polysomal preparations, in greater abundance when lysates were
prepared from UVC-treated cells. However, no such complexes
were found when using biotinylated p53(CR) or cytosolic lysates
(Fig. 4B). Finally, evidence for the in vivo association of endog-
enous p53 mRNA and HuR in the cell was obtained through
and AU-rich sequences (gray), as well as transcripts (CR and 3? UTR) used for
analysis. (A) REMSA (11) by using either CR or 3? UTR p53 radiolabeled
transcripts and cytoplasmic lysates prepared 4 and 6 h after treatment of RKO
cells with either 15 or 30 J?m2UVC. (B) Plasmids pGL3-Luc and pGL3-Luc-
p53(3?UTR) were used in transient transfections; 24 h after transfection, cells
were either irradiated (15 J?m2) or left untreated, and the relative luciferase
activity was calculated 6, 12, and 24 h later.
Binding of cytoplasmic proteins to the p53 3? UTR is linked to p53
supershift using either IgG1 or an anti-HuR antibody (11) (Left), or antibodies
recognizing the RNA-binding proteins shown (Right). f, free probe, digested
with RNase T1 but left without incubating with cytoplasmic lysates; arrow-
head, supershift of HuR-containing complexes. (B) Detection of HuR after
pull-down by using biotinylated p53 transcripts CR and 3? UTR (described in
or polysomal fractions of cells that were either left untreated or treated with
15 J?m2UVC, the presence of HuR and ?-actin was detected by Western
preserve the association of RNA-binding proteins with target mRNAs was
followed by RT-PCR analysis to detect endogenous p53 mRNA; PCR products
were resolved by electrophoresis in 1.5% agarose gels stained with ethidium
bromide. (D) Subcellular localization of HuR in cytoplasmic (Cytopl.), cytosolic
(Cytosol), and polysomal (Polysome) fractions prepared from cells that were
either left untreated or UVC-treated.
HuR binds to the p53 3? UTR in a UVC-dependent manner. (A) REMSA
Mazan-Mamczarz et al. PNAS ?
July 8, 2003 ?
vol. 100 ?
no. 14 ?
association with target mRNAs, by using a previously described
method (13). RT-PCR analysis revealed the presence of endog-
enous p53 mRNA in the material immunoprecipitated by using
anti-HuR antibodies, but not when nonspecific antibodies
(IgG1) were used (Fig. 4C). As earlier reported, whole-cell HuR
levels did not change with UVC irradiation, and HuR was
primarily nuclear at all times examined (11, 12). By contrast, its
cytoplasmic presence (Fig. 4D, Cytopl.) increased on UVC
irradiation, and was almost exclusively found in association with
the polysomal fraction (Fig. 4D, Polysome). These findings
demonstrate that HuR binds to the p53 3? UTR mRNA in a
UVC-dependent manner, and that HuR-p53 mRNA complexes
colocalize with the cell’s polysomes.
Direct analysis of HuR’s role in p53 expression was carried out
through experiments in which HuR expression levels in RKO
cells were modified. RKO cells stably overexpressing HuR (S11)
revealed heightened p53 levels, both basally and in response to
UVC (Fig. 5A). Conversely, reduction of HuR levels through
HuR-directed siRNA (HuR4; routinely reaching 20–30% of
control ‘‘Ctrl’’ siRNA transfections) was found to markedly
decrease p53 levels and to attenuate p53 induction after UVC
treatment (Fig. 5B). To directly assess whether HuR influenced
p53 translation, siRNA-transfected cells expressing lower HuR,
of L-[35S]methionine and L-[35S]cysteine for 20 min, and nascent
p53 was immunoprecipitated. As shown, p53 translation was
potently induced by UVC in control siRNA-transfected cells,
whereas cells expressing reduced HuR exhibited strikingly lower
p53 production (Fig. 5C). These data reveal that HuR enhances
p53 translation in a UVC-dependent manner.
Together, these findings demonstrate that HuR binds to the
p53 mRNA in vitro and in vivo. Despite HuR’s established role
in the stabilization of target mRNAs, HuR was not found to
influence p53 mRNA turnover in this stress paradigm (Fig. 1C)
but instead enhanced p53 translation. A role for HuR in
translational regulation has been previously proposed, based on
the effects of other ELAV proteins on translational control, and
the association of HuR with polysomes (18, 22, 23). Two recent
works link HuR to the translational regulation of p27 expression
through binding to the 5? UTR of the p27 mRNA, one demon-
strating an inhibitory role for HuR in p27 translation (24),
another consistent with an HuR-mediated enhancement in p27
translation (25). Our results support a model whereby HuR,
through its association with the p53 3? UTR, positively influ-
ences p53 translation. These findings recapitulate the enhanced
translation of neurofilament M previously reported for Hel-N1,
which also occurs via a 3? UTR-binding site, but does not involve
mRNA stabilization (18). The precise mechanisms mediating
HuR’s enhanced translation of p53 are unclear, but may be
linked to a mechanism of recruitment of mRNAs to active
polysomes. For example, neurofilament M (NF-M) and Glut-1
mRNAs shift their positions on polysome gradients from the
monosome peaks to the active polysome regions after transfec-
tion of HuB into hNT2 cells and 3T3L1 cells, respectively.
Although the observed increase in half-life of target mRNAs by
being mRNA stabilization (reviewed in refs. 22 and 28), those
results are consistent also with a role of HuR in translational
the apparent stabilization of HuR’s mRNA targets could be a
function of their physical sequestration during recruitment to
active polysomes via a mechanism intended to activate their
The relationship between mRNA turnover and translation
remains controversial. In some instances, translation of a given
mRNA has been shown to render it unstable (27), whereas other
studies support a link between mRNA stabilization and trans-
lation (23). In the present investigation, no differences in p53
mRNA abundance or stability were observed, and only an
HuR-mediated enhancement in p53 translation was apparent
(Fig. 5). To date, HuR has been shown to bind to many mRNAs
and promote their stabilization (for review, see ref. 28), but no
formal studies addressing potential links between HuR-
mediated stabilization and translation of target transcripts have
been reported. Should HuR prove to have a general function in
enhancing the translation of target mRNAs, it will lend support
to an emerging model whereby HuR binds to a given mRNA,
likely assists in its nuclear export, protects it from degradation
in the cytoplasm, and directs it to ribosomes, enhancing its
translation in concert with that of other HuR-bound mRNAs
It is striking that several RNA-binding proteins, including HuR,
respond to UVC, heat shock, and other perturbations by shifting
their location to the cytoplasm where their target mRNAs can be
levels. (A) Western blot analysis of HuR and p53 expression levels in RKO cells
that had been stably transfected with either an insert less plasmid (zeo) or an
HuR-expressing plasmid (S11). (B) RKO cells were transiently transfected with
either a control (Ctrl) siRNA or siRNA directed to HuR (HuR4), and exposed to
15 J?m2UVC 48 h later. HuR expression levels were analyzed by Western
blotting using whole-cell lysates that were prepared 4 h after UVC. (C) Newly
translated p53 was assessed as explained in the legend of Fig. 2B. Forty-eight
irradiated or left untreated; 4 h later, they were incubated with L-[35S]methi-
onine and L-[35S]cysteine for 20 min. Lysates were prepared and immunopre-
cipitated by using either anti-p53 antibody or IgG1, then processed as de-
scribed in Fig. 2B.
Modulation of HuR levels affects p53 translation and steady-state
www.pnas.org?cgi?doi?10.1073?pnas.1432104100Mazan-Mamczarz et al.
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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