SHOWCASE ON RESEARCH
Control of Eukaryotic Translation
1Molecular
2School
Thomas Preiss1,2
Genetics Program, Victor Chang Cardiac Research Institute, NSW 2010
of Biotechnology & Biomolecular Sciences and St Vincent's Clinical School,
University of New South Wales, NSW 2052
A common view holds that most control mechanisms
to regulate eukaryotic gene expression target the
primary step, namely transcription in the nucleus. In
contrast to this, it is becoming increasingly apparent
that controls acting on post-transcriptional steps of
mRNA metabolism, in particular at the level of
translation, are also of critical importance (Fig. 1).
Translation is carried out on the ribosomes and is
usually divided into three phases: (i) initiation, (ii)
elongation and (iii) termination. The initiation phase
represents all processes required for the assembly of a
ribosome at the start codon of the mRNA. The actual
polypeptide synthesis takes place during the elongation
phase. When ribosomes reach the stop codon this
signals termination − the dissociation of the completed
polypeptide and the ribosome from the mRNA. Why
control translation? The best answer to this question
probably is that it affords desirable complexity to gene
regulation. There are several features of translational
control that are particularly advantageous in certain
cellular situations. It is a fast response, which may
explain why it is commonly involved in cellular stress
responses. Translation can also be controlled locally in
areas distant from the nucleus, for instance to support
synaptic function in the nervous system. Furthermore, it
can operate in the absence of nuclear activity, a feature
of early development or the late stages of
erythropoiesis.
The Mechanism of Translation Initiation
Ribosomes cannot carry out their functions alone; they
depend on auxiliary factors that help them to engage the
mRNA template, to select the activated building blocks
for polypeptide synthesis and to mediate termination.
This is particularly true during translation initiation on
eukaryotic mRNAs (Fig. 2). This process depends on
the 5' m7G(5')ppp(5')N cap structure and the 3' poly(A)
tail of a typical mRNA and at least 12 eukaryotic
initiation factors (eIFs) (1, 2). Initiation begins with the
binding of several eIFs and other components to the
small (40S) ribosomal subunit. This complex is
recruited to the (capped) 5' end of the mRNA, then
'scans' the 5' untranslated region (UTR) of the mRNA
and recognises the start codon. Joining of a large (60S)
subunit completes the assembly of a complete (80S)
ribosome. The 40S subunit is primed for initiation
through binding of a ternary complex comprising eIF2,
Met-tRNAiMet and GTP. The mRNA is prepared by the
action of the eIF4 group of factors. eIF4E binds the cap
structure and eIF4A is an ATP-dependent RNA
helicase that is able, upon stimulation by eIF4B, to
unwind secondary structure in the cap-proximal
region of the mRNA (Fig. 2A). eIF4G is an adaptor
protein that interacts with eIF4E, eIF4A, and eIF3,
another 40S-bound factor. The poly(A) tail-binding
protein PABP also has a critical function at this stage: it
helps to recruit eIF4G and confers a pseudo-circular
conformation to the mRNA, the exact functional
significance of which still remains to be determined.
Scanning of the mRNA 5' UTR by the 40S subunit
requires contributions by several of the assembled eIFs
(Fig. 2B). Base-pairing between the start codon and
anticodon loop of the Met-tRNA i Met triggers GTP
hydrolysis by eIF2, dissociation of eIFs and 60S subunit
joining (Fig. 2C). A second GTP hydrolysis step by
eIF5B completes 80S ribosome assembly. An important
feature of eIF2 is that it requires eIF2B to exchange
bound GDP for GTP after each round of initiation.
Fig. 1. 'Strict Tempo' models
of eukaryotic gene regulation.
Contrary to an extreme view
(step diagram on the left), the
outcome of the gene
expression cascade does not
only depend on mechanisms
to control gene transcription.
Instead, virtually all aspects of
mRNA and protein
metabolism are subject to
controlling influences that can
affect the outcome of gene
expression in a quantitative
and qualitative manner (step
diagram on the right).
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SHOWCASE ON
RESEARCH
Control of Eukaryotic Translation
altering availability or function of eIFs, most
commonly eIF2 and eIF4E. mRNA-specific control
typically involves regulatory complexes that recognize
particular elements, usually in the 5' or 3' UTR of the
mRNA, and exclusively alter translation of the targeted
mRNAs. Regulatory elements found in the 5' UTR of
mRNAs include repressor protein binding sites,
inhibitory RNA structural features, upstream AUG
(uAUG) or upstream short open reading frames
(uORF) and internal ribosome entry sites (IRES).
Bound repressors and RNA structures serve as steric
'roadblocks' that hinder the normal progression of
initiation, while uAUGs and uORFs typically engage a
proportion of scanning ribosomes in non-productive
initiation events that lower translation of the main
ORF. IRES elements are complex RNA structures that
can recruit the translation initiation machinery directly
to internal positions on the mRNA, bypassing the need
for the cap structure at the 5' end of the mRNA. 3'
UTR elements often recruit regulatory complexes that
affect translation by forming a bridging interaction
with initiation intermediates at the 5' end.
Examples of translational control that illustrate aspects
of these generic descriptions are presented below.
Global Control
Fig. 2. The initiation phase of translation.
(A) An early step in initiation is the binding of the eIF4
factors to the cap structure followed by unwinding
of secondary structure in the mRNA 5' UTR. The
interaction of PABP with eIF4G aids this process
and leads to circularisation of the mRNA.
(B) The 40S ribosomal subunit, associated eIFs, MettRNAiMet, and GTP are recruited and this complex
moves along the mRNA in a 3' direction.
(C) Identification of the AUG start codon leads to the
release of eIFs, GTP hydrolysis and binding of the
60S ribosomal subunit. The simplified diagrams
only show a selection of participating eIFs.
Strategies for Translational Control
Initiation is usually the rate-limiting step of
translation and the most common target of regulatory
intervention. Translational control mechanisms may be
broadly divided into global and mRNA-specific types
of control (3). Global control affects the translation of
most cellular mRNAs and is usually achieved by
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A common means to achieve global control of
translation is through changes in the phosphorylation
state of eIFs or the regulators that act on them.
Mammalian cells contain several kinases that
phosphorylate the α subunit of eIF2, leading to a block
of the GDP/GTP exchange reaction and inhibition of
global translation. Each kinase is activated in response
to specific cellular stress conditions: PKR (protein
kinase activated by double-stranded RNA) is activated
during viral replication; GCN2 (general control nonderepressible 2) is stimulated by amino acid starvation;
PERK (PKR-like endoplasmic reticulum eIF2α kinase)
senses unfolded protein accumulation in the
endoplasmic reticulum; HRI (heme-regulated inhibitor)
reacts to heme depletion. Appropriate control of eIF2
and eIF2B is important for normal physiology and
mutations in the genes for PERK or eIF2B give rise to
serious human disease (4).
Extracellular cues such as insulin and growth factors
activate the PI3K/AKT/mTOR and Ras/MAPK
signaling pathways that also modulate translation (5,
6). Ras/MAPK signalling increases eIF4E
phosphorylation (and thus translation) through the
eIF4G-bound Mnk1/2 kinases. The mTOR pathway
leads to phosphorylation of the 4E-binding proteins, a
group of small regulatory proteins that mimic the part
of eIF4G that interacts with eIF4E (Fig. 3A).
Hypophosphorylated 4E-BPs bind to eIF4E and
competitively displace eIF4G, resulting in inhibition of
translation. Hyperphosphorylated 4E-BPs are released
from eIF4E, leading to activation of translation (7).
Translational control through these pathways is critical
for appropriate regulation of cell growth. Its
deregulation is involved in cancer biology (8) and
pathological cardiac hypertrophy (9, 10).
AUSTRALIAN BIOCHEMIST
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SHOWCASE ON
RESEARCH
Control of Eukaryotic Translation
Proteolytic cleavage of eIFs is also used to alter
cellular translation (11). During apoptosis, caspase-3
cleaves both mammalian isoforms of eIF4G at multiple
positions. Caspase-3 also cleaves the eIF4Ghomologous protein, death associated protein-5
(DAP5) near its C-terminus, which may activate its
function as a specialised initiation factor.
Although these global controls all affect general
translation, there is mounting evidence that they affect
the translation of specific mRNAs in a particular
manner. Such mRNAs may have structural features
and/or regulatory elements that make their
translation particularly sensitive or resistant to a
global change in cellular translation.
mRNA-Specific Control
The paradigm of mRNA-specific control in the
context of general eIF regulation is GCN4 mRNA,
which encodes a transcriptional activator of amino
acid biosynthesis genes in yeast (3, 12). The 5' region
of GCN4 mRNA contains four uORFs, which
collectively lead to low levels of Gcn4p synthesis
under normal conditions and, somewhat
paradoxically, increased levels during amino acid
starvation. This is explained by an elegant scenario: at
normal amino acid levels, ribosomes translate uORF 1,
which has properties that favour an unusual
resumption of scanning by ribosomal subunits. These
scanning complexes quickly rebind the ternary
complex comprising eIF2, Met-tRNAiMet, and GTP and
re-initiate translation at subsequent uORFs. uORFs 2-4
induce efficient dissociation of terminating ribosomes,
resulting in little initiation of translation at the main
GCN4 ORF. Amino acid starvation activates the kinase
Gcn2p, leading to eIF2α phosphorylation and
reduction of available ternary complexes. With regard
to GCN4 mRNA, this leads to longer 'recharging'
times of complexes scanning downstream of uORF 1.
This in turn lowers the rates of re-initiation at the
inhibitory uORFs 2-4 and increases initiation
frequency at the main ORF.
Several mRNA-specific translational regulators target
eIF4E function (7). For instance, during vertebrate
oocyte maturation, the cytoplasmic polyadenylation
control element-binding protein (CPEB) recruits the
eIF4E-binding protein Maskin to target mRNAs and
blocks their translation by establishing a repressive
interaction between the 3' UTR-bound repressor
complex and cap-bound eIF4E (Fig. 3B). CPEB-bound
dormant mRNAs also have short poly(A) tails. At the
appropriate time CPEB is phosphorylated, which
stimulates mRNA poly(A) tail elongation in the
cytoplasm. This enhances PABP-binding and
recruitment of eIF4G, leading to displacement of
Maskin from eIF4E and activation of translation. In
this way, CPEB and cytoplasmic polyadenylation are
central to an intricate network of translational
activation and repression of stored maternal mRNAs
in early development. Furthermore, there are
indications for a role of this control mechanism in
neuronal synaptic function.
Vol 36 No 3 December 2005
Fig. 3. Global and mRNA-specific mechanisms to
target eIF4E function.
(A) Hypophosphorylated 4E-BP binds to eIF4E and
competitively displaces eIF4G. mTOR
signalling leads to phosphorylation of 4E-BP
and release from eIF4E, allowing active
translation (symbolised by the magic wand).
(B) Masking and activation of maternal mRNAs.
CPEB binds the CPE (cytoplasmic
polyadenylation element) in the 3' UTR of
maternal mRNAs and forms a complex with
maskin, which in turn interacts with eIF4E.
This renders the mRNA translationally
inactive. Phosphorylation of CPEB leads to
recruitment of the cytoplasmic polyadenylation
machinery and elongation of the poly(A) tail
(magic wand). Binding between maskin and
eIF4E is reduced, clearing the way for
activation of translation (magic wand).
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SHOWCASE ON
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Control of Eukaryotic Translation
MicroRNAs (miRNAs) are an emerging class of
eukaryotic post-transcriptional regulators with roles in
a variety of cellular and developmental pathways (13,
14). Several hundred of these ~22 nucleotide RNAs are
known, they assemble into larger RNA-protein
complexes, and trigger either decay or inhibition of
translation of their mRNA targets. In plants, miRNAtarget interactions are often within the coding region
and are nearly perfectly complementary, which
triggers mRNA degradation. By contrast, animal
miRNA/target duplexes generally are interrupted by
gaps and mismatches and occur in the 3' UTR of
mRNAs, which leads instead to inhibition of
translation. This view stems from work on two
miRNAs − lin-4 and let-7, which were identified by
genetic studies as regulators of developmental timing
in Caenorhabditis elegans. Until recently almost nothing
was known about the mechanism by which miRNAs
regulate translation (15). New data indicates that they
target the function of eIF4E in initiation (16), ultimately
leading to a sequestration of silenced mRNAs into
cytoplasmic foci termed P-bodies (17).
Perspectives
Annu. Rev. Biochem. 68, 913-963
7. Richter, J.D., and Sonenberg, N. (2005) Nature 433,
477-480
8. Holland, E.C., Sonenberg, N., Pandolfi, P.P., and
Thomas, G. (2004) Oncogene 23, 3138-3144
9. Proud, C.G. (2004) Cardiovasc. Res. 63, 403-413
10. Hannan, R.D., Jenkins, A., Jenkins, A.K., and
Brandenburger, Y. (2003) Clin. Exp. Pharmacol.
Physiol. 30, 517-527
11. Holcik, M., and Sonenberg, N. (2005) Nat. Rev. Mol.
Cell Biol. 6, 318-327
12. Hinnebusch, A.G. (1997) J. Biol. Chem. 272, 2166121664
13. Bartel, D.P., and Chen, C.Z. (2004) Nat. Rev. Genet.
5, 396-400
14. Mattick, J.S. (2004) Nat. Rev. Genet. 5, 316-323
15. Pasquinelli, A.E., and Ruvkun, G. (2002) Annu. Rev.
Cell Dev. Biol. 18, 495-513
16. Humphreys, D.T., Westman, B.J., Martin, D.I.K., and
Preiss, T. (2005) Proc. Natl. Acad. Sci. USA in press
17. Liu, J., Valencia-Sanchez, M.A., Hannon, G.J., and
Parker, R. (2005) Nat. Cell. Biol. 7, 719-723
18. Beilharz, T.H., and Preiss, T. (2004) Brief Funct.
Genomic Proteomic 3, 103-111
Although much progress has been made in
understanding the mechanisms of global and mRNAspecific control of translation, there is still much work
to be done. We know quite well how translation of a
limited number of mRNAs is regulated and further
work on these examples will deepen our
understanding of the control mechanisms that operate
on them. Many new examples of regulated mRNA
translation will come to our attention as researchers
widen their horizons to include translational control as
an option for regulating the expression of their
favourite gene. Altered signalling to general translation
factors can have regulatory effects on specific mRNAs
that are difficult to predict. Here, a combination of
polyribosome purification and subsequent microarray
analyses has shown great promise in providing
information on changes in the translation state of the
cellular transcriptome in response to several such
triggers (18). In the future, such genome-wide
polysome profiling data will be integrated with data
from conventional transcriptome and proteome
profiling data to comprehensively describe changes in
gene expression.
References
1. Preiss, T., and Hentze, M W. (2003) Bioessays 25,
1201-1211
2. Sonenberg, N., and Dever, T.E. (2003) Curr. Opin.
Struct. Biol. 13, 56-63
3. Gebauer, F., and Hentze, M.W. (2004) Nat. Rev.
Mol. Cell Biol. 5, 827-835
4. Proud, C.G. (2005) Semin. Cell Dev. Biol. 16, 3-12
5. Gingras, A.C., Raught, B., and Sonenberg, N. (2004)
Curr. Top. Microbiol. Immunol. 279, 169-197
6. Gingras, A.C., Raught, B., and Sonenberg, N. (1999)
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