Molecular Cell

Review
Translational Regulation of Gene Expression
during Conditions of Cell Stress
Keith A. Spriggs,2 Martin Bushell,1 and Anne E. Willis1,*
1Medical

Research Council Toxicology Unit, Lancaster Road, Leicester LE1 9HN, UK

for Biomolecular Sciences, School of Pharmacy, University of Nottingham, University Park, Nottingham NG7 2RD, UK
*Correspondence: aew5@le.ac.uk
DOI 10.1016/j.molcel.2010.09.028
2Centre

A number of stresses, including nutrient stress, temperature shock, DNA damage, and hypoxia, can lead to
changes in gene expression patterns caused by a general shutdown and reprogramming of protein
synthesis. Each of these stress conditions results in selective recruitment of ribosomes to mRNAs whose
protein products are required for responding to stress. This recruitment is regulated by elements within
the 50 and 30 untranslated regions of mRNAs, including internal ribosome entry segments, upstream open
reading frames, and microRNA target sites. These elements can act singly or in combination and are themselves regulated by trans-acting factors. Translational reprogramming can result in increased life span, and
conversely, deregulation of these translation pathways is associated with disease including cancer and
diabetes.
Introduction
All living organisms must respond to, and defend against, environmental stresses. These include temperature shock (Richter
et al., 2010), oxygen shock (hypoxia or oxidative stress)
(Majmundar et al., 2010), nutrient deprivation (Sengupta et al.,
2010), and DNA damage (Ciccia and Elledge, 2010). The ability
to respond rapidly to changes in the cellular environment is
essential for survival, and numerous strategies for dealing with
cell stress have evolved (Figure 1).
Since most cellular processes are catalyzed by proteins, it is
vital for a cell to effect rapid changes in protein levels in any
stress response. There are many levels at which the cellular
concentrations of proteins can be regulated, including chromatin
remodeling, transcription of genomic DNA into pre-messenger
RNA (pre-mRNA), splicing (Weake and Workman, 2010), export
(Keene, 2010), mRNA localization and sequestration (Keene,
2010), translation (Sonenberg and Hinnebusch, 2009), posttranslational modification of proteins, and protein degradation
(Andreou and Tavernarakis, 2009). This review will focus on the
translational control of gene expression in response to cellular
stress and specifically on regulated changes in the synthesis of
subsets of proteins from a pool of mRNAs. This level of control
is particularly important in stress responses, as it allows a rapid
change in the complement of proteins synthesized in a cell
without any lag while new mRNA is transcribed and processed
(Sonenberg and Hinnebusch, 2009), together with reprogramming of protein synthesis to elicit a response that is relevant to
the type of stress induced.
Eukaryotic Translational Control
Eukaryotic protein synthesis involves three stages, and the ratelimiting step is usually translation initiation (Sonenberg and
Hinnebusch, 2009). A typical eukaryotic mRNA comprises
a coding sequence which directs protein synthesis, flanked by
50 and 30 untranslated regions (UTRs). In the canonical model
of cap-dependent translation initiation, the smaller ribosome
228 Molecular Cell 40, October 22, 2010 2010 Elsevier Inc.

subunit is recruited to a modified base at the extreme 50 end of

the mRNA (the m7G cap). This process is coordinated by
a complex of eukaryotic initiation factors (eIFs) called eIF4F,
composed of (1) eIF4G, a large scaffold protein which facilitates
interactions between proteins and the small ribosomal subunit;
(2) eIF4E, which recognizes the 50 -terminal mRNA m7G cap
structure; and (3) eIF4A, an RNA helicase which processes
otherwise inhibitory RNA secondary structures (Figure 1). The
interaction of poly(A)-binding protein (PABP) with eIF4G and
eIF4B circularizes the mRNA and stabilizes the complex (Sonenberg and Hinnebusch, 2009; Bushell et al., 2001). Ternary
complex is recruited which is comprised of eIF2, methionyltRNAi, and GTP (Sonenberg and Hinnebusch, 2009). The ribosome loaded with these factors scans along the 50 UTR until it
recognizes an initiation codon, AUG, which prompts hydrolysis
of eIF2-GTP, uncoupling of tRNAi from eIF2, and release of initiation factors. Recruitment of the large ribosomal subunit then
occurs, and translation elongation begins (Jackson et al., 2010).
The complex nature of translation initiation in eukaryotes
offers a number of targets for regulation, many of which rely on
the modulation of phosphorylation states of eIFs by stressrelated kinases and phosphatases (Figure 1). One of the most
commonly used mechanisms for inhibiting global translation is
by phosphorylation of the initiation factor eIF2 (Hinnebusch
et al., 2007). In order to be recycled, eIF2 is recharged with
GTP by the guanine nucleotide exchange factor (GEF) eIF2B.
However, when eIF2 is phosphorylated on serine 51 of its
a subunit, it becomes a competitive inhibitor of eIF2B, preventing eIF2 recycling and reducing translation initiation rates by
lowering ternary complex concentration (Hinnebusch et al.,
2007). In mammalian cells, four proteins control the formation
of ternary complex: the GCN2, PERK, HRI, and PKR kinases
(Figure 1). These are activated by different external stimuli, and
the particular kinase that is activated is generally dependent
upon the specific stress induced (Figure 1; Hinnebusch et al.,
2007).

Molecular Cell

Review

Figure 2. RNA Elements within the 50 and 30 UTRs of an mRNA

Regulate Translation

Figure 1. Exposure to Cell Stresses Inhibits Translation

Many different types of cell stress cause a global inhibition of protein synthesis
via signaling cascades that lead to the reversible change in phosphorylation
status of proteins involved in translation initiation. A common consequence
of stress is the inhibition of mTOR, leading to a reduction in 4EBP phosphorylation, and consequent inhibition of the eIF4E-eIF4G interaction; or a reduction
in the activities of eIF4B, and indirectly, eIF4A. A parallel phosphorylation of the
a subunit of eIF2 by stress-related kinases (PKR, GCN2, PERK, and HRI)
causes it to become a competitive inhibitor of ternary complex recycling,
leading to a reduction in initiation codon recognition.

A second mechanism for nonspecifically reducing levels of

protein synthesis involves interfering with m7G cap recognition,
thereby preventing recruitment of the translational machinery
to the mRNA (Raught and Gingras, 2007). Ordinarily, the m7G
cap is recognized by eIF4E as part of the eIF4F complex;
however, there are several eIF4E-binding proteins (4EBPs)
which compete with eIF4G for a binding site on eIF4E and
prevent eIF4F complex formation (Marcotrigiano et al., 1999).
The strength of binding of 4EBPs to eIF4E is controlled by phosphorylation: hypophosphorylated 4EBPs bind strongly, whereas
phosphorylated 4EBPs bind less well (Figure 1). Recognition
of the m7G cap can also be inhibited by the eIF4E homolog
4E-HP that competes with eIF4E for m7G cap binding, but which
cannot form eIF4F complexes and hence promote translation
initiation (Tee et al., 2004).
mRNAs encoding some stress response proteins must be able
to evade global repression of translation, and several mechanisms have evolved to achieve this (Figure 2).
One such mechanism is to bypass cap-dependent recruitment
of the mRNA. An estimated 10% of mRNAs contain internal
ribosome entry sites (IRESs; Mitchell et al., 2005; Figure 2) in their
50 UTRs and therefore have the potential to be translated by
internal ribosome entry, a process which allows recruitment of
the translation machinery downstream of the 50 m7G cap at
a position within the 50 UTR (Spriggs et al., 2008). Originally identified in viruses, IRESs are typically structured RNA elements that

Superimposed onto the global inhibition of protein synthesis are multiple

mechanisms for regulating the expression of individual mRNAs. IRESs in the
50 UTRs of many stress-response mRNAs allow the synthesis of proteins
when cap-dependent translation is compromised. MicroRNA binding sites,
generally in the 30 UTRs of RNAs, specifically reduce the expression levels of
targeted mRNAs.

facilitate the binding of the mRNA to 40S subunits, usually

involving a subset of cofactors known as ITAFs (IRES transacting factors). Changes in the abundance or activity of these
ITAFs allow precise control of IRES mediated translation, and
this mode of initiation is frequently used during stress conditions
when cap-dependent translation is compromised (Powley et al.,
2009; Spriggs et al., 2005, 2009). There has been some disagreement in the translation field as to the existence of cellular IRESs,
since some mRNAs that were reported to harbor IRES elements
were subsequently shown to contain cryptic promoters or splice
sites. However, providing that all the controls are rigorously performed (see the supplemental section of Cobbold et al., 2008), it
has been possible to identify IRES activity in cellular mRNAs. It is
also important to use a variety of constructs to test for apparent
IRES activity, in particular monocistronic vectors containing
upstream hairpins to block scanning, in addition to dicistronic
vectors.
The presence of AUG codons in the 50 UTR is inhibitory in the
canonical model of cap-dependent translation since the scanning preinitiation complex will identify these as start codons
and initiate synthesis of an incorrect peptide (Figure 3). However,
when eIF2 is phosphorylated and global translation compromised, the presence of upstream open reading frames (uORFs;
in-frame start and stop codons in the 50 UTR) can promote the
increased expression of certain stress-related mRNAs. UORFs
are present in 50% of protein coding transcripts (Calvo et al.,
2009). Although first identified in yeast (Hinnebusch, 2005), the
prototypical mammalian example of this type of regulation is
ATF4, a stress response gene encoding a transcription factor
with many downstream targets (Ron and Harding, 2007). There
are two major uORFs in the mRNA of ATF4, with the second
overlapping the authentic ATF4 coding sequence albeit in
a different reading frame (Figure 3). Under normal conditions,
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Molecular Cell

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Figure 3. Upstream Open Reading Frames
Regulate Translation during Cell Stress
Under normal conditions, two upstream open
reading frames (uORFs) in the 50 UTR of the ATF4
mRNA are recognized by scanning preinitiation
complexes. Since the second uORF overlaps
with the authentic ATF4 coding sequence, but in
a different frame, expression of functional ATF4
is repressed (upper figure). However, under conditions of cell stress in which eIF2 a phosphorylation
leads to a reduction in ternary complex concentration, the second uORF is less likely to be recognized, allowing translation of functional ATF4
from the authentic open reading frame (lower
figure).

when eIF2 is not phosphorylated and ternary complex is not

limiting, the scanning preinitiation complex recognizes the first
uORF and translates a short peptide, and the 60S ribosome
dissociates upon reaching the stop codon marking the end of
the uORF. The 40S ribosomal subunit that remains associated
with the mRNA is then able to recruit ternary complex and initiate
translation of the second uORF. Because the second uORF
overlaps with the authentic coding sequence, this prevents
translation of the ATF4 coding sequence. However, in conditions
of reduced ternary complex availability, initiation of the second
uORF is less likely, as there is less chance of the scanning ribosomal subunit recruiting the ternary complex required for start
codon recognition (Figure 3). By this mechanism, a reduction in
active translation factor (eIF2) results in the increased expression
of mRNAs containing the correct configuration of uORFs (Ron
and Harding, 2007).
Other RNA elements such as iron responsive elements (IREs)
can recruit regulatory proteins to inhibit ribosome scanning or
modulate mRNA longevity (Leipuviene and Theil, 2007; Mukhopadhyay et al., 2009). Another notable example of regulatory
mRNA elements is the IFN-g-activated inhibitor of translation
(GAIT) element to which the GAIT complex binds. The GAIT
complex functions by simultaneously binding to the GAIT
element and to eIF4G, inhibiting eIF4G interactions with the
translation initiation factor eIF3 and consequently blocking
translation initiation (Kapasi et al., 2007).
MicroRNAs (miRNAs) provide a mechanism for the exquisite
posttranscriptional regulation of gene expression during cell
stress, which is important in coordinating this response (Babar
et al., 2008) (Leung and Sharp, 2010). miRNAs are short (22 nt)
noncoding RNAs that bind with imperfect complementarity to
their target mRNAs, usually at sites within the 30 UTR, to destabilize mRNA and inhibit translation by mechanisms that are not
fully understood (Figure 2). Up to 60% of mRNAs are regulated
by at least one of the thousand or more miRNAs in the human
genome, allowing a complex superimposition of regulation by
families of miRNAs.
Translational Control during Nutrient Stress
Nutrient Depletion
When eukaryotic cells are deprived of nutrients (e.g., following
depletion of glucose or essential amino acids), there is a coordinated cellular response (Sengupta et al., 2010). A decrease in
230 Molecular Cell 40, October 22, 2010 2010 Elsevier Inc.

global protein synthesis rates is mediated by two distinct

signaling pathways, the target of rapamycin (TOR) pathway
and the general amino acid control pathway (GAAC), in addition
to the increased synthesis of defined subsets of proteins.
The TOR pathway is the major nutrient-sensing pathway in
mammalian cells, the central component of which is the TOR
kinase (Wullschleger et al., 2006) that is found in two different
multiprotein complexes, termed TORC1 and TORC2 (Wullschleger et al., 2006). The downstream positive mediators of
mTORC1 (composed of mammalian TOR, raptor, and GbL)
include 4E-BP and S6K (Um et al., 2006) (Figure 1). Following
nutrient depletion, particularly that caused by starving the cells
of leucine, there is inhibition of mTORC1, which leads to inhibition of translation initiation and elongation (Ma and Blenis,
2009). A reduction in eIF4F complex formation that is, in part,
mediated by a dephosphorylation of the 4EBPs allows them to
bind to and sequester eIF4E (Wang and Proud, 2009). There is
also a rapid dephosphorylation of S6K1 (Ma and Blenis, 2009).
In agreement with a role for S6K in the regulation of protein
synthesis and cell growth, it has been shown that S6K-deficient
mice are smaller and have metabolisms that are similar to
calorie-restricted animals, although the exact mechanisms by
which this occurs are not well understood (Um et al., 2006).
The effects of extreme nutrient deprivation on whole organisms have been studied in several model invertebrate systems;
in each case, reduced or altered protein synthesis as a result
of calorie restriction is associated with enhanced life span
(Kaeberlein and Kennedy, 2008, 2009; Syntichaki et al., 2007).
Additional data suggest that in mammals mTOR signaling
directly influences aging and 600-day-old mice (comparable to
a human of 60 years of age) treated with rapamycin display
significant increases in longevity (Harrison et al., 2009), with
the same effect also observed for Drosophila (Bjedov et al.,
2010). The precise mechanisms that increase life span following
dietary restriction have yet to be fully described. It is known
that under starvation conditions TORC1 promotes autophagy,
which will supply starving cells essential bioenergetic components (Lum et al., 2005), and that the increased removal of
damaged molecules and organelles via this route could be beneficial for the prevention of aging (Mizushima et al., 2008).
However, the data also suggest that a reduction of TOR signaling
allows the reprogramming of protein synthesis to permit the
translation of mRNAs that promote longevity (Guertin et al.,

Molecular Cell

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2006). Translational profiling was performed to identify which
mRNAs were translationally regulated in Drosophila subjected
to dietary restriction (Zid et al., 2009). This technique couples
sucrose density gradient separation of actively translating ribosomes (polysomes) with cDNA microarray and allows a translational profile to be obtained under a defined set of conditions
(Spriggs et al., 2008). The data showed a selective upregulation
of translation of mRNAs whose products have a role in mitochondrial ATP generation, oxidative phosphorylation, and protein
folding, including those in complexes I and IV of the electron
transport chain (Zid et al., 2009). It was suggested that the
shorter, less structured 50 UTRs of these mRNAs permitted their
translation under conditions of low eIF4F complex formation (Zid
et al., 2009). Additional studies also suggest that an induction of
oxidative stress and mitochondrial respiration may be a general
mechanism by which life span is increased as a result of calorie
restriction. For example, in Caenorhabditis elegans, life span is
enhanced following glucose depletion by activating these stress
pathways (Schulz et al., 2007), and under nutrient depletion in
mammals there is an increase in mitochondrial biogenesis (Nisoli
et al., 2005). The translational reprogramming following nutrient
depletion would have the net effect of inducing the cellular level
of reactive oxygen species (ROS) and inducing oxidative stress,
a major cause of molecular damage that is associated with aging
(Muller et al., 2007). A key question to address, therefore, is how
does activation of these pathways increase life span? It has been
shown recently that in fission yeast calorie restriction resulting in
the production of ROS induces the mammalian ortholog of the
p38 kinase, Sty1, and it is the activation of this stress pathway
that is responsible for life span extension (Zuin et al., 2010).
This raises the possibility that p38 kinase, in addition to regulating senescence (Coulthard et al., 2009), may also control
aging (Zuin et al., 2010).
The general amino acid control pathway (GAAC) also contributes to stress adaptation following nutrient depletion. In this
pathway amino acid starvation results in the accumulation of
uncharged tRNAs which activate GCN2, which in turn phosphorylates eIF2 on the a subunit at serine 51, lowering its activity and
causing a reduction in overall protein synthesis rates. These
changes allow the cell to reprogram translation to alleviate
the nutritional stress. One protein central to this response is
GCN4, whose mRNA contains four uORFs (Hinnebusch, 2005).
In the fed state, these act to repress translation of the
authentic ORF, since the scanning ribosome selects each of
the four AUGs in the GCN4 leader before the start codon for
the protein coding ORF, and downstream reinitiation following
translation of a uORF is inefficient (Hinnebusch, 2005). However,
when the levels of ternary complex are low following nutrient
depletion, the leader of GCN4 provides additional distance/
time in which this complex can be recruited. Derepressing
GCN4 permits the transcription of more than 30 amino acid
biosynthetic genes (Hinnebusch, 2005). Polysome profiling of
the global response to amino acid starvation in yeast (Smirnova
et al., 2005) identified changes in the translational state of over
600 mRNAs, and several stress pathways were shown to be
translationally coregulated. It was shown that mRNAs encoding
amino acid permeases, nitrogenous compound permeases,
plasma membrane proteases, and others involved in protein

degradation were translationally activated, suggesting that these

could form part of the early amino acid scavenging pathways
(Smirnova et al., 2005).
In mammalian cells, amino acid starvation stimulates transport
system A for neutral amino acids (McGivan and Pastoranglada,
1994). mRNAs that encode proteins in this system are translationally regulated during amino acid starvation, and two
members of this pathway, the cat-1 Arg/Lys transporter (CAT-1)
and the sodium-coupled neutral amino acid transporter (SNAT2),
contain IRESs (Fernandez et al., 2005; Gaccioli et al., 2006;
Yaman et al., 2003).
The CAT-1 50 UTR contains a uORF that is activated under
conditions when ternary complex is low and whose translation
is essential for the translation of CAT-1 protein as it allows the
transient structural remodeling of the mRNA leader into an active
IRES (Yaman et al., 2003). Ribosome stalling on the uORF is
essential to the formation of an active IRES (Fernandez et al.,
2005). Other types of posttranscriptional regulation also control
CAT-1 synthesis; the 30 UTR of CAT-1 contains a target site for
miR122 that under normal-fed conditions represses the translation of CAT-1 and localizes this mRNA to processing bodies
(Bhattacharyya et al., 2006). However, upon nutrient deprivation,
this mRNA, aided by HuR, is liberated and becomes polysomally
associated (Bhattacharyya et al., 2006). The relative contributions of the IRES and the miRNA to the translation of CAT-1
are still under debate.
Both mRNA and protein levels of SNAT2 were induced
following amino acid starvation, and it was concluded that eIF2
a phosphorylation increased gene transcription, and IRES-mediated translation was required for the increased synthesis of this
protein under these conditions (Gaccioli et al., 2006).
Finally, although it would appear that two separate signaling
pathways are required for nutrient control, in yeast the data suggest that there is significant crosstalk between these systems
(Cherkasova and Hinnebusch, 2003). Inhibition of mTOR by
rapamycin induces dephosphorylation of GCN2 at serine 577
(which stimulates its tRNA binding function), thereby stimulating
the phosphorylation eIF2 a and GCN4 translation in starved cells.
In contrast, in nonstarved cells addition of rapamycin reduced
Ser 577 phosphorylation and decreased the tRNA-binding function of GCN2. These rapamycin-induced events were shown to
require dephosphorylation of the TAP42-regulated phosphatases SIT4/PP2A (Cherkasova and Hinnebusch, 2003).
Nutrient Overexposure
Under fed conditions, signaling through TORC1 phosphorylates
4EBPs, allowing their release from eIF4E, and stimulates eIF4F
complex formation. Similarly, under fed conditions, S6K phosphorylates several proteins that control overall translation rates,
including components of the small ribosomal subunit rpS6,
eukaryotic elongation factor 2 kinase (eEF2K; thus inactivating
this kinase, which negatively regulates eIF2), and eIF4B (which
activates overall translation rates by regulating the activity of
eIF4A) (Wang and Proud, 2009).
During the development of obesity, stress responses are
induced in metabolic tissues, leading to chronic low-grade
inflammation that results in the disruption of systemic metabolic
homeostasis (Hotamisligil, 2006). Using a genetic model of
obesity (ob/ob mice which are insulin resistant as a result of
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leptin deficiency) and in mice fed a high-fat diet, it has been
shown that there is an increase in the levels of mRNA, protein,
and, importantly, degree of phosphorylation of PKR (Figure 1)
in white adipose tissue and liver (Nakamura et al., 2010). PKR
was required for JNK activation in response to lipid exposure,
suggesting that signaling through this pathway is an important
component of this response. It was proposed that PKR may
form part of the adaptive response to prevent further accumulation of energy in the overfed state (Nakamura et al., 2010). PKR is
normally activated by double-stranded RNA, and interestingly
the RNA-binding domain of PKR was also shown to be required
for its activation in response to lipids. Consequently, endogenous RNA species produced during metabolic stress could be
required for the activation of this pathway. Moreover, PKR
modifies insulin signaling and is associated with apoptosis that
is induced by double-stranded RNA, suggesting that activation
of this pathway by viral infection may contribute to the development of diabetes (Nakamura et al., 2010).
Temperature Shock
Cold Shock
Exposure of cultured mammalian cells to mild cold stress (32 C)
results in changes in transcription rates, the cell cycle, the cytoskeleton, and metabolic processes. Many of these changes are
mediated at the level of translation, a major point at which the
cold-shock response is regulated (Lleonart, 2010; Al-Fageeh
and Smales, 2006; Roobol et al., 2009). The general decrease
in protein synthesis is mediated by an increase in eIF2a
phosphorylation (Underhill et al., 2007) and a decrease in the
synthesis of eIF3i, a subunit of initiation factor 3 (Roobol et al.,
2009). Upon recovery from temperature shock these changes
are reversed (Roobol et al., 2009). The data suggest that eIF3i
has a central role in regulating the cold shock response and
the subsequent recovery from cold shock (Roobol et al., 2009).
Under conditions of cold shock it would be expected, therefore,
that mRNAs that are less dependent on ternary complex and
eIF3i for their synthesis would be preferentially translated, for
example those that contain uORFs or IRESs.
The cold-shock proteins cold inducible RNA binding protein
(CIRP) and RNA binding protein 3 (RBM3) have different kinetics
of induction, reflecting their differential roles in protection/
recovery from cold shock (Al-Fageeh and Smales, 2009; Roobol
et al., 2009; Underhill et al., 2007). CIRP is regulated both at
the level of transcription and translation, and there are three
major transcripts of CIRP that are differentially regulated. At
37 C the main transcript does not contain the full-length
50 UTR, whereas in cells exposed to a 32 C cold shock, two transcripts are generated, one of which (the longest) contains an
IRES. The presence of the IRES aids the translation of CIRP
during cold shock, although the ITAFs that are required for its
translation have yet to be defined (Al-Fageeh and Smales,
2009). The mechanism by which the translation of RBM3 occurs
during cold shock is unknown at present. Both CIRP and RBM3
contain a glycine-rich domain (RGG motif), and CIRP has been
shown to bind to poly (U) tracts in the 50 and 30 UTRs of mRNAs
and affects their stability and translation (Wilusz et al., 1988).
For example, it has been shown that CIRP interacts with the
30 UTR of ATR and that overexpression of CIRP leads to
232 Molecular Cell 40, October 22, 2010 2010 Elsevier Inc.

increased ATR protein levels in response to stress, suggesting

that CIRP may directly affect the translation of this mRNA
(Yang et al., 2010).
Heat Shock
Following heat shock in mammalian cells there is a highly
coordinated response comprised of the activation of a set of
transcription units that encode heat shock proteins (HSPs) and
an increase in the synthesis of the corresponding proteins
(Morimoto, 1998), at the same time as a general decrease in
the global rates of protein synthesis. This overall reprogramming
of gene expression permits the selective synthesis of Hsps which
act as molecular chaperones (e.g., HSP 100, 90, 70, 60, 40, and
27 [Morimoto, 1998]) that facilitate protein refolding and increase
cell survival following temperature shock (Westerheide and
Morimoto, 2005). Inhibition of eIF4F complex formation during
heat shock is mediated, in part, by a reduction in the phosphorylation of eIF4E, an increase in the binding of hypophosphorylated 4EBP to eIF4E (Vries et al., 1997; Wang et al., 1998), and
a decrease in the phosphorylation of eIF2a (Coldwell et al.,
2001). However, none of these events are sufficient to cause
the complete inhibition of translation that is observed (Feigenblum and Schneider, 1996; Vries et al., 1997; Wang et al.,
1998). A major contribution to translational inhibition is mediated
by the binding of Hsp27 to eIF4G, which allows its dissociation
from the eIF4F complex and PABP into heat shock granules
(Cuesta et al., 2000; Ma et al., 2009). These granules form
when translation initiation is impaired following cell stress, and
the data suggest that they contain mRNAs that are stalled in
the process of translation initiation (Buchan and Parker, 2009).
Untranslated mRNAs are sorted or processed in stress granules
for reinitiation, degradation, or packaging into mRNPs (Buchan
and Parker, 2009).
Hsp mRNAs are translated during heat shock because they
have low requirements for eIF4F, or they are translated by alternative mechanisms requiring sequence elements in their 50 UTRs
(Yueh and Schneider, 2000). For example, translation of Hsp70
mRNA during heat shock is facilitated by complementarity of
this mRNA with 18 s rRNA (Yueh and Schneider, 2000). Other
mRNAs that encode proteins synthesized during heat shock
contain IRESs. For example, BIP and BAG-1 50 UTRs contain
IRESs, the latter of which is required for the increased synthesis
of the p36 isoform of BAG-1 during heat shock (Coldwell et al.,
2001). Some of the ITAFs for BAG-1 have been identified, namely
PTB and PCBP1 (Pickering et al., 2003, 2004); however, it is not
known if these proteins themselves are induced during heat
shock.
Cells that have previously been exposed to heat shock gain
thermotolerance, and although protein synthesis is still rapidly
inhibited following a temperature rise, they recover much faster
than naive heat-shocked cells due to the presence of Hsps
that remain after the initial insult. It is possible to recapitulate
this effect during recovery from heat shock by overexpressing
individual Hsps. For example, overexpression of Hsp70 protected both cap-dependent translation and translation mediated
by the FGF-2 IRES, whereas overexpression of Hsp27 only protected cap-dependent translation. This is likely to reflect the
different factor requirements of these two modes of translation
initiation (Doerwald et al., 2003).

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Translation Control following DNA Damage
In response to DNA damage, a series of signaling networks,
collectively referred to as the DNA damage response (DDR),
result in cell-cycle arrest or apoptosis, which prevents damaged
genetic material being inherited. The decision to undertake
a program of repair or cell death is dictated by both the severity
and the nature of the DNA damage experienced (Jackson and
Bartek, 2009). Different types of DNA damage are sensed by
parallel signaling pathways with members of the phosphatidylinositol-3-kinase-like (PI3K) family, ATM, ATR, and DNA-PKcs
at their core. These upstream kinases then, in turn, activate the
downstream checkpoint kinases, Chk1, Chk2, and MK2, which
activate negative regulators of the cell cycle and inhibit positive
regulators of the cell cycle such as p53 and CDC25A, respectively (Reinhardt and Yaffe, 2009). Translational reprogramming
following DNA damage permits the preferential synthesis of
a specific subset of mRNAs whose protein products are required
for the response to DNA damage (Powley et al., 2009). If the DNA
damage exceeds the capacity of the DNA repair machinery, an
apoptotic program is initiated, leading to a number of changes
to the translation apparatus (Clemens et al., 2000).
The exact mechanism by which global translational repression
is achieved depends on the type of DNA damage induced.
Ionizing radiation and the toposiomerase II inhibitor etoposide
cause double-strand breaks leading to repression of translation
and a decrease in mTOR activity. Decreased mTOR activity
attenuates phosphorylation of 4EBP1, rpS6, and presumably
eIF4B (which has been shown to be downstream; Sonenberg
and Hinnebusch, 2009). Decreased phosphorylation of 4EBP1
results in increased affinity for eIF4E and reduces eIF4F complex
availability. Depletion of 4E-BP1 by RNAi has shown that this is
the primary mechanism for the shutdown in translation following
ionizing radiation in nontransformed cells (Braunstein et al.,
2009). Signaling to mTOR, and thus 4EBP1, upon DNA damage
appears to occur through a p53-dependent pathway requiring
transcriptional activation of Sestrin1 and Sestrin2, which in turn
activate AMP-responsive protein kinase (AMPK), which then
activates the repressor of mTOR TSC2 (Budanov and Karin,
2008). Recently, an additional pathway has been indentified in
which ATM directly activates LKB1/AMPK leading to a decrease
in mTOR activity through TSC2, independently of p53 (Alexander
et al., 2010).
However, mTOR inhibition and 4EBP1 dephosphorylation do
not take place following all types of DNA damage. In fact,
mTOR activity is increased upon exposure to UVB (Brenneisen
et al., 2000). UV exposure causes DNA damage by creating
cyclopyrimidine dimers and 6-4 photoproducts which require
excision and repair by the nucleotide excision repair (NER)
pathway (Shuck et al., 2008). Under these conditions, global
translation is inhibited not through 4EBP1, but eIF2 a phosphorylation at serine 51, thus reducing the level of ternary complex
and resulting in an overall decrease in translation initiation
(Deng et al., 2002; Powley et al., 2009). This phosphorylation of
eIF2 a is the critical mechanism for inhibiting protein synthesis
in response to UV as cells carrying a nonphosphorylatable
version of eIF2 a (Ser51 to Ala51) or lacking an upstream kinase,
GCN2, fail to shut down translation (Deng et al., 2002). A recent
study has shown that the DNA damage response kinase,

DNA-PKcs, is the critical link between the DNA damage

machinery, GCN2, and the translational apparatus following
UV-induced DNA damage, since inhibition or loss of this kinase
results in a failure to inhibit translation (Powley et al., 2009).
Whereas both eIF4F complex disruption and low ternary
complex concentration lead to the global translational repression following DNA damage, certain mRNAs contain elements,
normally within the 50 UTR or 30 UTR, which allow selective and
specific regulation of these mRNAs. For example, mRNAs that
contain IRESs that are used following DNA damage include
those that encode p53, the antiapoptotic proteins BCL2 and
XIAP (which block the release of cytochrome c from the mitochondria and directly inhibit caspase function, respectively),
and BAG-1 (Dobbyn et al., 2008). Thus, following etoposide
treatment the IRESs found in the 50 UTRs of p53 and BCL2 allow
translation of these mRNAs when cap-dependent translation is
inhibited (Sherrill et al., 2004; Yang et al., 2006). The IRES found
in the 50 UTR of p53 interacts with the ITAF polypyrimidine tract
binding protein (PTB), a nuclear protein that translocates to the
cytoplasm in response to etoposide treatment, thus activating
the p53 IRES element. Depletion of PTB by RNAi reduces both
the expression levels of p53 and IRES activity following etoposide treatment (Grover et al., 2008). Similarly, the IRES in the
50 UTR of XIAP mRNA allows the synthesis of XIAP upon exposure to ionizing radiation. Interestingly, the negative regulator
of p53, MDM2, appears to act as an ITAF for the XIAP IRES,
directly interacting with the IRES element in the cytoplasm
following its translocation from the nucleus (Gu et al., 2009).
Serine hydroxymethyltransferase 1 (SHMT1) is the ratelimiting enzyme in the thymidylate biosynthesis pathway
required during cell division and DNA repair. The 50 UTR of
SHMT1 possesses an IRES that allows synthesis of SHMT1
following UVC treatment; however, regions in the 30 UTR are
also required for full activity following UVC exposure (Fox
et al., 2009; Fox and Stover, 2009). Two ITAFs, hnRNPH2 and
CUGBP1, have been shown to be particularly important in the
translation of the SHMT1 mRNA following UVC exposure.
Following treatment with UVC, the levels of hnRNPH2 increase
while CUGBP1 redistributes from the nucleus to the cytoplasm.
These proteins interact with the 50 UTR and 30 UTR of the SHMT1
mRNA, respectively, resulting in circularisation of the mRNA and
an activation of translation (Fox et al., 2009; Fox and Stover,
2009). An emerging common theme is a shift in the subcellular
localization of proteins that function as ITAFs in response to
different types of DNA damage, thereby allowing selective translation of those IRES-containing mRNAs that they regulate
(Dobbyn et al., 2008).
A genome-wide screen of mRNAs that are selectively translated following exposure to UVB identified those that encode
proteins with essential roles in the DNA damage response
(Powley et al., 2009). These include vital components of the
NER pathway, XPA, ERCC5, ERCC1, OGG1, and DDB1. Examination of these mRNAs found a high proportion that contain
at least uORF in their 50 UTR. The ability of the 50 UTRs of these
DNA repair enzymes to maintain translation in response to
UVB was dependent upon the presence of uORFs (Powley
et al., 2009). Although, as discussed above, it is known that
uORF-containing mRNAs, e.g., ATF4, are activated under
Molecular Cell 40, October 22, 2010 2010 Elsevier Inc. 233

Molecular Cell

Review
conditions of eIF2a phosphorylation (Vattem and Wek, 2004), it is
not solely this mechanism that is used following UVB exposure
to permit translation of selected mRNAs. Thus translation of
the uORF-containing DNA repair enzymes was not activated
following salubrinal treatment, which leads to increased phosphorylation of eIF2a, demonstrating that specific activation of
translation following UVB requires other factors than eIF2a phosphorylation alone (Powley et al., 2009).
Hypoxia
With the notable exceptions of the anaerobic Loricifera, all metazoans require a plentiful supply of oxygen to all their tissues
(Danovaro et al., 2010), and mechanisms have evolved to protect
cells from reductions in oxygen concentration (Majmundar et al.,
2010). Hypoxia causes a decrease in global translation rates to
50% of those seen under normal conditions (Connolly et al.,
2006; Thomas and Johannes, 2007). During hypoxia, translational regulation is more extensive than transcriptional control
(Koritzinsky et al., 2005), with different mechanisms required
for reprogramming translation in acute versus prolonged hypoxia
(Koritzinsky et al., 2007). Phosphorylation of eIF2 a is a rapid
consequence of hypoxic stress, reducing the availability of
competent initiation complexes. EIF2 a phosphorylation in
hypoxia is effected by the endoplasmic reticulum-based kinase
PERK, which itself is activated by hyperphosphorylation,
possibly in response to the increase in mitochondrial ROS
present in hypoxic cells (Liu et al., 2008). Also in common with
other stress responses, it is becoming apparent that the translation of a number of stress response mRNAs is increased despite
eIF2 a phosphorylation and consequent ternary complex
shortage. The presence of uORFs in, for example, ATF4, is
responsible for this increase in expression under stress conditions, and it is becoming clear that a similar mechanism is likely
to be more widely utilized by stress response genes (Vattem and
Wek, 2004).
mTOR is inhibited under more sustained hypoxic conditions,
leading to the dephosphorylation and activation of the eIF4E
binding proteins, and, via inhibition of S6 kinase, a decrease in
eIF4B and eIF4A activity, with the consequent inhibition of
cap-dependent translation (Connolly et al., 2006). EIF4E activity
is further reduced during prolonged hypoxia by its relocalization
by 4E transporter protein (Koritzinsky et al., 2006). The efficiency
of translation elongation is also reduced in the absence of mTOR
activity, since both elongation factor eEF2 and ribosomal protein
S6K are activated by mTOR. During hypoxia, mTOR is thought
to be inhibited via the tuberous sclerosis complex 1 and 2
(TSC1/2) in response to a shortage in energy production, and
also by REDD1, which is a transcriptional target of HIF1 (Wouters
and Koritzinsky, 2008). The inhibition of mTOR signaling during
hypoxia reduces the translation of terminal oligopyrimidine
tract (TOP)-containing mRNAs (which encode ribosomal proteins and factors that control protein biosynthesis) and reduces
translation.
Perhaps the most important protein regulators of the hypoxic
response are the hypoxia-inducible factors (HIFs) (Keith and
Simon, 2007). HIFs are transcription factors that directly regulate
the expression of over 70 targets in response to a reduction in
oxygen concentrations to coordinate a stress response involving
changes in the expression of hundreds of proteins. HIFs act as
234 Molecular Cell 40, October 22, 2010 2010 Elsevier Inc.

a heterodimer of an a and a b subunit. Although the b subunit

is constitutively expressed, the levels of three closely related
HIF-1 a subunits are sensitive to oxygen levels, allowing a rapid
and diverse response to reduced oxygen concentrations. Under
normal conditions, the HIF-1 a subunits are rapidly degraded
due to the hydroxylation of two proline residues by prolyl hydroxylases 13 (PHD13), which leads to binding of the VHL protein
and consequent ubiquitinylation (Lang et al., 2002; Mazumdar
et al., 2009). However, during hypoxia, in addition to increased
protein stability, translation of HIF-1 a is also increased, indicating that the HIF-1 a mRNA can escape the global translational
shutdown that occurs in response to reduced oxygen concentrations. It has been proposed that the HIF-1 a mRNA contains an
IRES which allows continued expression in a cellular environment in which cap-dependent translation is inhibited (Lang
et al., 2002). However, the presence of cryptic promoter activity
in the HIF-1 a 50 UTR has cast a certain amount of doubt on this
hypothesis (Bert et al., 2006; Young et al., 2008). The observation
that the ITAF, PTB, can specifically interact with the HIF-1
a 50 UTR, and increase HIF-1 a expression under hypoxic conditions, suggests a cap-independent maintenance of HIF-1
a protein expression (Schepens et al., 2005). Furthermore,
a number of other ITAFs including HuR, nucleolin, and hnRNPA2
also interact with HIF-1 a mRNA to increase translation (Masuda
et al., 2009).
Vascular endothelial growth factor (VEGF) promotes the development of increased vasculature and is therefore another important protein in the coordination of a defense against hypoxia
(Mac Gabhann and Popel, 2008). In addition to being a transcriptional target of HIF-1, VEGF also contains two IRESs that enable
continued expression in translationally repressed hypoxic cells
(Bornes et al., 2004; Bornes et al., 2007). The two IRESs give
rise to VEGF proteins which differ at their N termini: IRES A
allows translation from the canonical AUG start codon, whereas
IRES B initiates protein synthesis at an upstream site, leading to
a longer VEGF isoform. Other proteins known to be important in
responding to hypoxia, including FGF2 and PDGF, also contain
IRESs, suggesting that a switch from cap-dependent to capindependent translation may be important for cell survival under
hypoxic conditions (Le and Maizel, 1997; Sella et al., 1999).
The interior of a tumor is likely to be severely hypoxic, and
cancerous cells which have mutations in hypoxic response
genes to enable increased survival, and promotion of vasculature, will have a significant selective advantage, allowing an
increased ability to proliferate and metastasize. For these
reasons, proteins such as HIF1 a and VEGF have become attractive targets in the development of anticancer drugs (Kenneth and
Rocha, 2008).
Summary
Although the shutdown of protein synthesis that is brought about
by different cell stresses is, in general, mediated either by the
modulation of recruitment of the 48S complex to the mRNA
and/or by reducing the amount of ternary complex that is available, there are many different mechanisms that are used by individual mRNAs to override the repression of protein synthesis. It is
now fundamental to our understanding of posttranscriptional
control of gene expression to define the precise mechanisms

Molecular Cell

Review
by which subsets of mRNAs are collectively regulated under
a variety of conditions. This information will be, in the longer
term, important for developing new targets in diseases that
range from cancers to diabetes.

Cherkasova, V.A., and Hinnebusch, A.G. (2003). Translational control by TOR

and TAP42 through dephosphorylation of eIF2 alpha kinase GCN2. Genes
Dev. 17, 859872.

ACKNOWLEDGMENTS

Clemens, M.J., Bushell, M., Jeffrey, I.W., Pain, V.M., and Morley, S.J. (2000).
Translation initiation factor modifications and the regulation of protein
synthesis in apoptotic cells. Cell Death Differ. 7, 603615.

Thanks to Drs. M. MacFarlane and I. Cannell for critically reading the manuscript. A.E.W. is a BBSRC Professorial Fellow, and M.B. is a MRC Senior
Fellow.
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