Download The role of IRES trans-acting factors in regulating translation initiation

Post-Transcriptional Control: mRNA Translation, Localization and Turnover
The role of IRES trans-acting factors in regulating
translation initiation
Helen A. King*†, Laura C. Cobbold† and Anne E. Willis†1
*School of Pharmacy, Centre for Biomolecular Science, University Park, University of Nottingham, Nottingham NG7 2RD, U.K., and †MRC Toxicology Unit,
Hodgkin Building, Lancaster road, University of Leicester, Leicester LE1 9HN, U.K.
Abstract
The majority of mRNAs in eukaryotic cells are translated via a method that is dependent upon the recognition
of, and binding to, the methylguanosine cap at the 5’ end of the mRNA, by a set of protein factors termed
eIFs (eukaryotic initiation factors). However, many of the eIFs involved in this process are modified and
become less active under a number of pathophysiological stress conditions, including amino acid starvation,
heat shock, hypoxia and apoptosis. During these conditions, the continued synthesis of proteins essential
to recovery from stress or maintenance of a cellular programme is mediated via an alternative form of
translation initiation termed IRES (internal ribosome entry site)-mediated translation. This relies on the
mRNA containing a complex cis-acting structural element in its 5’-UTR (untranslated region) that is able
to recruit the ribosome independently of the cap, and is often dependent upon additional factors termed
ITAFs (IRES trans-acting factors). A limited number of ITAFs have been identified to date, particularly for
cellular IRESs, and it is not yet fully understood how they exert their control and which cellular pathways
are involved in their regulation.
Cap-dependent translation initiation
Translation of mRNA into protein involves three stages:
initiation, elongation and termination. The initiation step
is tightly regulated to allow the cell to respond efficiently
to a given stimulus and is the rate-limiting step. Capdependent initiation relies upon recognition of the m7 GpppN
(7-methylguanosine) cap at the 5 end of the mRNA by a
complex of canonical initiation factors (Figure 1). eIF (eukaryotic initiation factor) 4F, which comprises eIF4A (a DEADbox RNA helicase), eIF4E (the cap-binding protein), and
eIF4G (a multi-domain scaffold protein), recognizes
and binds the 5 -cap. This is then able to recruit the 40S
ribosomal subunit along with ternary complex (GTP-bound
eIF2 and charged methionine initiator-tRNA) as part of
the 43S initiation complex, via interactions between eIF3
(a large multi-subunit protein) and eIF4G (Figure 1). Once
assembled, the complex is referred to as the 48S initiation
complex: this scans along the 5 -UTR (untranslated region) of
the mRNA with the helicase eIF4A assisting in resolution
of any complex and potentially inhibitory secondary
structure. Scanning continues until an AUG in optimal
‘Kozak’ consensus context is encountered [1]. Release of the
initiation factors is triggered by eIF5-mediated hydrolysis of
the GTP bound to eIF2. This disassembly of the complex
Key words: eukaryotic initiation factor (eIF), internal ribosome entry site (IRES), IRES trans-acting
factor (ITAF), translation initiation, untranslated region (UTR).
Abbreviations used: Apaf-1, apoptotic peptidase-activating factor 1; Bag-1, Bcl-2-associated
athanogene-1; BiP, immunoglobulin heavy-chain-binding protein; eIF, eukaryotic initiation factor;
4E-BP, eIF4E-binding protein; EMCV, encephalomyocarditis virus; IRES, internal ribosome entry
site/segment; ITAF, IRES trans-acting factor; PABP, poly(A)-binding protein; PTB, polypyrimidinetract-binding protein; Unr, upstream of N-Ras; UTR, untranslated region.
1
To whom correspondence should be addressed (email [email protected]).
Biochem. Soc. Trans. (2010) 38, 1581–1586; doi:10.1042/BST0381581
allows the 60S ribosomal subunit to join to the 40S and form
the complete 80S ribosome. The ribosome then enters the elongation phase of translation. (For detailed reviews of the
mechanism of cap-dependent translation initiation, see [2–4].)
Several features in the 5 -UTR of an mRNA are inhibitory
to the progress of the scanning ribosome, including uAUGs
(upstream AUGs), a long length of UTR and a high degree of
secondary structure. However, a number of cap-dependent
mechanisms exist to overcome the effects of these inhibitory
elements, including ribosomal shunting, leaky scanning and
termination-reinitiation [5,6].
There are a number of situations in which cap-dependent
initiation is compromised, due either to cleavage of one or
more of the canonical initiation factors including eIF4G,
eIF4b and eIF3 [7], or a change in the phosphorylation state
of the factors and their binding partners.
During poliovirus infection, viral protease 2A is able to
cleave eIF4G at Arg486 -Gly487 , which separates the eIF4Ebinding site on eIF4G from the eIF4A- and eIF3-binding
sites. This abolishes the eIF4E-binding function of eIF4G
and, as a consequence, inhibits cap-dependent translation
[8]. Interestingly, a similar situation occurs during apoptosis,
where eIF4G is cleaved by effector caspases into three
fragments termed C-, M- and N-FAG (C-terminal, middle
and N-terminal fragment respectively), which separates
the PABP [poly(A)-binding protein] binding site from the
eIF4A-, eIF3- and eIF4E-binding sites [9].
Cap-dependent translation can also be inhibited
by phosphorylation of eIF2 on the α subunit, or
hypophosphorylation of 4E-BP (eIF4E-binding protein).
Phosphorylation of eIF2 occurs during many stress
conditions, including amino acid starvation and hypoxia, as
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Figure 1 Cap-dependent translation initiation complex
Figure 2 Methods of cap-dependent translation inhibition
The 5 -cap is recognized by eIF4E as part of the eIF4F complex, which
in turn recruits the 43S initiation complex including the 40S ribosomal
(a) Hypophosphorylation of 4E-BP due to serum starvation or picornavirus infection allows it to compete for binding to eIF4E with eIF4G,
subunit to form the 48S initiation complex at the cap (see the text
for details). The mRNA is circularized via interactions between eIF4G
at the 5 -end and PABP at the 3 -end. The 48S complex scans through
causing eIF4F levels to become limiting. When hyperphosphorylated,
for example during growth conditions, 4E–BP is no longer able to bind
eIF4E. (b) Phosphorylation of the α subunit of eIF2 by kinases such as
the 5 -UTR until an AUG in optimal context is encountered, where the
complex stalls, the initiation factors dissociate, and the 60S ribosomal
subunit joins to form the translation elongation competent 80S ribosome.
PKR (double-stranded-RNA-dependent protein kinase) cause it to bind
with stronger affinity to its guanine-nucleotide-exchange factor eIF2B,
resulting in levels of ternary complex becoming limiting.
Meti , initiator methionine.
well as during stages of the standard cell cycle, such as mitosis.
Phosphorylation of eIF2α at Ser51 increases its affinity for its
guanine-nucleotide-exchange factor eIF2B, so that it remains
associated with eIF2B and is no longer recycled into a new
ternary complex. In this way, the amount of ternary complex
becomes limiting for cap-dependent initiation (Figure 2b)
[5]. Hypophosphorylation of 4E-BP occurs during heat
shock, serum starvation and picornavirus infection. When
hyperphosphorylated, 4E-BP cannot bind eIF4E. However,
when hypophosphorylated, 4E-BP competes for binding
to eIF4E with eIF4G, and consequently sequesters eIF4E
away from the eIF4F complex. This results in levels of eIF4F
becoming limiting and cap-dependent translation being
inhibited (Figure 2a) [5].
IRES (internal ribosome entry
site)-mediated translation
The first IRESs were originally identified in members of
the picornaviridae [10,11]. This virus family have positivesense RNA genomes which are uncapped, yet are translated
efficiently in eukaryotic cells, therefore it was hypothesized
that they may be translated via a cap-independent method.
Subsequently it was shown that the 5 -UTRs of EMCV
(encephalomyocarditis virus) and poliovirus are able to drive
translation initiation by recruiting the ribosomal subunit
directly, independent of active eIF4F [10,11] (Figure 3a).
Eukaryotic IRESs were later identified, the first in the
mRNA encoding BiP (immunoglobulin heavy-chain-binding
protein) [12]. It was observed that translation of BiP is
maintained during poliovirus infection, when several of
the canonical initiation factors are cleaved as discussed
above. Bicistronic IRES vectors were used to demonstrate a
functional IRES in the BiP 5 -UTR, where expression of the
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Authors Journal compilation first cistron is under 5 -cap-dependent translational control,
and the second cistron is under IRES-dependent control
[12]. The bicistronic vector assay has received some criticism,
due to the possibility of apparent IRES-mediated expression
of the second cistron arising from aberrant splicing, the
presence of a cryptic promoter or ribosomal read-through
from the first cistron [13]. However, with the correct control
experiments in place, these possibilities can be reliably ruled
out, as demonstrated in [14].
Since these initial discoveries, the list of mRNAs known to
contain IRES elements, and hence have the ability to utilize
cap-independent translation, has been growing steadily, and
in silico analyses estimate that up to 10 % of cellular mRNAs
may contain an IRES element [15]. Importantly, the protein
products of IRES-containing mRNAs tend to be involved
in control of cell growth or cell death [16]. The presence of
an IRES element in such mRNAs allows their translation to
be either maintained or up-regulated under conditions where
cap-dependent translation is inhibited. Furthermore, they are
known to require a combination of both specific canonical
initiation factors and auxiliary trans-acting factors for their
function.
ITAFs (IRES trans-acting factors)
The activity of a given IRES can vary greatly between
different cell lines, due to variation in the availability of
ITAFs. For example, it was found that translation mediated
by the IRES of both poliovirus and rhinovirus is weak in the
Post-Transcriptional Control: mRNA Translation, Localization and Turnover
Figure 3 IRES-mediated translation initiation
(a) An IRES is a complex structural element within the 5 -UTR that is able to recruit the 43S initiation complex in a
cap-independent manner. Specific proteins termed ITAFs are required for this recruitment. (b) Bag-1 mRNA contains
an IRES which has been shown to require both PCBP1 and PTB for function. Initially, the protein PCBP1 binds to a
stem–loop in the IRES, which modifies the loop such that two PTB proteins can bind, which in turn allows recruitment
of the 40S ribosomal subunit. In this example, the ITAFs are behaving as RNA chaperones. Adapted from [32] with
c American Society for Microbiology, Molecular and Cellular Biology, vol. 24, 2004, pp. 5595–5605,
permission. Copyright doi:10.1128/MCB.24.12.5595-5605.2004.
rabbit reticulocyte lysate system; however, their activity is
restored by addition of a ribosome salt wash from HeLa cells
[17]. This cell-type variability is also true for cellular IRES:
the activity of the c-myc IRES was tested in a wide range
of cell lines, and was found to be 20-fold more active in HeLa
cells than in MCF7 cells, owing to the differing availability
of ITAFs between these two cell lines [18].
The mechanism of action of ITAFs is not fully understood,
but it is thought that they act either as RNA chaperones,
changing or stabilizing the secondary structure of the IRES
to allow further proteins or the 40S ribosomal subunit to bind
(Figure 3b), or as adaptor proteins, acting as anchors to which
other proteins or the 40S ribosomal subunit could bind [16].
Both cellular and viral IRESs require the action of ITAFs,
and indeed many compete for the same proteins. The
ITAF requirements of numerous IRESs have been examined
(Table 1, and for a more comprehensive list, see [19]). The
ITAF that has been most extensively studied is the multi-
functional RNA-binding protein PTB (polypyrimidinetract-binding protein). In addition to a role in translation,
this protein is also involved in mRNA splicing, stability
and localization within the cell [20]. The data suggest that
the majority of cellular IRESs require PTB for function,
and PTB was shown to be particularly important for the
control of IRESs which are active during apoptosis [21,22].
The regulation that an ITAF confers upon an IRES may be
either a positive or a negative one. For example, whereas PTB
regulates both p27Kip1 and BiP IRES, its binding enhances
p27Kip1 IRES activity, but down-regulates BiP IRES activity
[23–25].
Interestingly, the mRNAs that encode ITAFs have also
been shown to contain IRESs. For example, Unr (upstream of
N-Ras), an ITAF known to positively regulate several IRESs,
is encoded by an mRNA that itself contains an IRES which
is negatively regulated by Unr protein in an auto-regulatory
negative-feedback loop, as well as by PTB [26–28].
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Table 1 Examples of known trans-acting factors, and the IRESs
which they regulate
The list of known IRESs and the ITAFs which regulate them has
been growing since IRESs were first identified. ITAFs for both viral
and cellular IRESs are listed. There are many other ITAFs and IRESs
which have not been included owing to space restrictions. For a
more comprehensive list of IRESs, see http://www.iresite.org/ [19].
FMDV, foot-and-mouth disease virus; HAV, hepatitis A virus; HCV,
hepatitis C virus; hnRNP, heterogeneous nuclear ribonucleoprotein;
HRV, human rhinovirus; IGF-1R, insulin-like growth factor 1 receptor;
PCBP, poly(rC)-binding protein; PDGF2, platelet-derived growth factor 2;
PV1, poliovirus 1; TMEV, Theiler’s murine encephalomyelitis virus; XIAP,
Table 1 (Continued)
ITAF/trans-acting factor
IRES
Reference
HIV1
[56]
BiP
EMCV
[57]
[58]
HCV
XIAP
Coxsackievirus B3
[59]
[54]
[51]
HCV
[60]
X-linked inhibitor of apoptosis; YB1, Y-box-binding protein 1.
ITAF/trans-acting factor
IRES
Reference
PTB (hnRNPI)
EMCV
FMDV
TMEV
[36]
[37]
[38]
PV1
HAV
BiP
[39]
[40]
[24]
Apaf-1
Bag-1
[41]
[41]
IGF-1R
p27kip1
Unr
[42]
[23]
[28]
Mnt
Myb
p53
[22]
[22]
[43]
Cytochrome P450 (CYP1b1)
SetD7
Cyclin T1
[21]
[21]
[21]
Cat-1
TMEV
Apaf-1
[44]
[45]
[46]
Unr
HRV
Apaf-1
Unr
[47]
[46]
[26]
ITAF45
hnRNPA1
FMDV
Cyclin D1
[38]
[48]
hnRNPE2 (PCBP2)
c-myc
PV1
Coxsackievirus B3
[49]
[50]
[51]
c-myc
HRV
PV1
[49]
[52]
[50]
c-myc
PDGF2/c-sis
XIAP
[49]
[53]
[54]
c-myc
Cat-1
c-myc
[49]
[44]
[34]
GRSF
YB1
c-myc
c-myc
c-myc
[34]
[34]
[34]
La
PV1
[55]
nPTB (neural PTB)
hnRNPE1 (PCBP1)
hnRNPC1/C2
hnRNPL
PSF
p54nrb
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Authors Journal compilation Significant advances have been made in identifying how
ITAFs regulate viral IRESs. For example, it is known that
PTB regulates the EMCV IRES by stabilizing its threedimensional structure, thus acting as an RNA chaperone [29].
Moreover, research in the viral IRES field has been aided
by structural similarities between the IRESs of viruses that
harbour different primary sequences. One such example is
the presence of a large stem–loop structure at the 3 -end of
the 5 -UTR in both the HCV (hepatitis C virus) and CSFV
(classical swine fever virus) IRESs, which acts as the ribosome
landing site in both cases [30,31].
For the cellular IRESs identified and studied to date, there
has been no structural or sequence parallels observed, making
it challenging to extrapolate findings from one IRES to the
next [31]. However, a few interactions between cellular IRESs
and their cognate ITAFs have been defined. In particular, the
Apaf-1 (apoptotic peptidase-activating factor 1) and Bag-1
(Bcl-2-associated athanogene-1) IRESs have been extensively
studied in this regard, and the data suggest that ITAFs
remodel the structures of these two IRESs so that they
attain the correct conformation for interaction with the 40S
ribosomal subunit [27,32]. Thus the Bag-1 IRES requires
the ITAF PCBP1 [poly(rC)-binding protein 1] (as an RNA
chaperone) to bind to domain II and open up an adjacent
structure in domain III, and then PTB (again as an RNA
chaperone) to bind and expose the ribosome landing site
[32] (Figure 3b). Likewise, the Apaf-1 IRES requires Unr
to bind first and open up the secondary structure, which then
allows two nPTB (neural PTB) molecules to bind, which
in turn exposes the ribosome landing site [27]. Similarly,
the secondary structure of the XIAP (X-linked inhibitor of
apoptosis) IRES has been predicted and the IRES is known
to require the ITAFs PTB, La and hnRNP (heterogeneous
nuclear ribonucleoprotein) C1/C2 for function [33].
Finally, it has been shown that mutations in cellular IRESs
can affect their interaction with ITAFs and alter their activity.
For example, in multiple myeloma-derived cell lines and
in patients with this disease, the c-myc IRES was found
to contain a single C>T base substitution. This mutation
results in an increase in IRES-mediated translation of c-myc
and hence a large increase in the amount of c-myc protein.
Thus dysregulated cell proliferation was shown to be due
to an increased affinity of two ITAFs, PTB and YB1 (Y-boxbinding protein 1), for the mutated IRES [34,35]. By reducing
Post-Transcriptional Control: mRNA Translation, Localization and Turnover
the levels of these ITAFs, it was shown that it was possible
to reduce the expression of c-myc and cell proliferation
[34,35]. Several other ITAFs required by c-myc have also
been identified and are shown in Table 1.
Although work has been carried out on individual IRESs,
there has as yet been no large-scale screen of IRESs and their
trans-acting factor requirements.
Overview and perspectives
Cap-dependent translation initiation is the main mechanism
by which translation-competent ribosomes are recruited
to cellular mRNAs. However, this mechanism is not able to
function under certain normal cellular conditions, such
as during mitosis, as well as during physiological stress
conditions, owing to a number of control mechanisms which
modify the canonical initiation factors, thereby reducing their
availability and/or activity. During these times, the cell must
still be able to produce protein in order to overcome the
period of stress or cell cycle and either emerge intact or
undergo programmed cell death. Many proteins that are
essential to these outcomes contain an IRES in the 5 -UTR of
their respective mRNAs.
IRESs have different requirements for both the canonical
initiation factors and auxiliary factors termed ITAFs. The
levels of these ITAFs vary between cell lines, explaining
the cell-line-dependence of IRESs in terms of activity. Finetuning of ITAF levels is likely to allow the cell exquisite
control over the activity of different IRESs.
Work is being undertaken to identify and classify ITAFs
in order to allow control over IRES-mediated protein
expression. In particular, in our laboratory, we are interested
in the IRESs that are active during apoptosis, a cellular
process subverted in many diseases. Control over IRESmediated expression would potentially allow us to dictate cell
fate, which has far-reaching implications for many diseases
including cancers and neurological diseases.
Funding
A.E.W. holds a Professorial Fellowship from the Biotechnology and
Biological Sciences Research Council. H.A.K. and L.C.C. are funded
by a grant from the Biotechnology and Biological Sciences Research
Council.
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Received 7 June 2010
doi:10.1042/BST0381581