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Contrasting mechanisms of regulating translation
of specific Drosophila germline mRNAs at the
level of 5 -cap structure binding
P. Lasko*1 , P. Cho†, F. Poulin†2 and N. Sonenberg†
*Department of Biology, McGill University, 1205 Avenue Docteur Penfield, Montréal, QC, Canada H3A 1B1, and †Department of Biochemistry and
McGill Cancer Centre, 3655 Promenade Sir William Osler, Montréal, QC, Canada H3G 1Y6
Abstract
Translational control is a key genetic regulatory mechanism underlying the initial establishment of the major
spatial axes of the Drosophila embryo. Many translational control mechanisms target eIF4E (eukaryotic
initiation factor 4E), an initiation factor that recognizes the 5 -cap structure of the mRNA. Cap recognition
by eIF4E, in complex with eIF4G, is essential for recruitment of the mRNA to the small ribosomal subunit.
One established mechanism for repressing translation involves eIF4E-binding proteins, which competitively
inhibit the eIF4E–eIF4G interaction. Our group has uncovered a novel mechanism for repression in which an
eIF4E cognate protein called d4EHP, which cannot bind eIF4G, binds to the 5 -cap structure of cad mRNA
thus rendering it translationally inactive. These two related, but distinct, mechanisms are discussed and
contrasted in this review.
Translational control is a key mechanism underlying the
initial establishment of the major spatial axes of the Drosophila embryo. Proteins encoded by several mRNAs, including oskar (osk), nanos (nos) and caudal (cad), are essential
for anterior–posterior patterning. In order to carry out their
functions in development, Osk, Nos and Cad proteins must
be restricted in space to the future posterior of the embryo. To
achieve asymmetric distribution of the proteins, translation of
all these mRNAs is repressed in regions of the embryo from
where the relevant protein needs to be excluded. Mechanisms
also exist to localize and enrich the concentrations of osk and
nos mRNAs at the posterior pole, where the proteins they
encode are required. Unlike osk and nos, cad mRNA is uniformly distributed. Translational repression of osk, nos and
cad mRNAs occurs at the mRNA 5 -cap structure recognition step, but two distinct mechanisms that target different
components of the cap-binding complex are involved. For
osk and nos, regulatory proteins that bind eIF4E (eukaryotic
initiation factor 4E) are recruited to the mRNA. These
proteins competitively inhibit the interaction between eIF4E
and eIF4G, which is an essential step of cap-dependent
translation initiation. In contrast, our group has uncovered a
different mechanism that regulates cad mRNA, whereby an
eIF4E-related cap-binding protein is recruited to the 5 -cap
structure [1]. This protein, called 4EHP (eIF4E-homologous
protein), cannot bind eIF4G and therefore acts in a dominantKey words: Bicoid (Bcd), 5 -cap structure, eukaryotic initiation factor 4E-homologous protein
(4EHP), Drosophila, eukaryotic initiation factor 4E (eIF4E), translation.
Abbreviations used: Bcd, Bicoid; BBR, Bcd-binding region; Bru, Bruno; cad, caudal; eIF4E,
eukaryotic initiation factor 4E; 4EHP, eIF4E-homologous protein; nos, nanos; osk, oskar; Smg,
Smaug; SRE, Smg-response element; UTR, untranslated region.
1
To whom correspondence should be addressed (email [email protected]).
2
Present address: Department of Integrative Biology, University of California, Berkeley, CA
94720-3140, U.S.A.
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negative manner by associating with the cap and blocking its
association with the cap-binding complex. These two related
but distinct mechanisms of translational repression will be
discussed in detail and contrasted below.
Translational repression of osk through
recruitment of the eIF4E-binding
protein Cup
osk mRNA is translationally repressed outside of the pole
plasm, and this repression is alleviated for the small proportion [2] of osk RNA that is localized. The interaction
of Bruno (Bru) and Apontic (Apt) proteins with specific
sequences [BREs (Bru-response elements)] in the osk 3 -UTR
(3 -untranslated region) contributes to translational repression of unlocalized osk mRNA ([3–5], reviewed in [6]).
Bru-mediated translational repression of osk involves its association with Cup, an eIF4E-binding protein [7,8]. As such,
Cup blocks the recruitment of eIF4G to eIF4F, the complex
that binds the 7-methylguanosine cap present at the 5 -end of
most eukaryotic mRNAs and is required for their translation.
Thus an association between osk and Cup through Bru
renders osk translationally inactive.
In the posterior pole, repression of osk translation is
alleviated and translation is activated; these may be distinct
processes [9]. Several molecules have been implicated in
these processes and several models have been proposed.
Staufen is required for osk localization [10,11] and for its
translational activation [12] through an unknown mechanism.
Perhaps more relevant to alleviating Cup-mediated translational repression is the activity of Orb, the Drosophila
homologue of CPEB (cytoplasmic polyadenylation elementbinding protein). Orb and the 3 -UTR of osk RNA interact,
Stem Cells and Development
and in orb mutants osk translation is reduced, suggesting
that cytoplasmic polyadenylation might underlie activation
of osk translation [13]. Although experiments in extracts that
support Bru-mediated regulation indicate that changes in
poly(A) tail length are not involved in translational control of
osk [5], a more recent report indicates that Orb and a poly(A)
tail of at least 150 residues are necessary for Osk accumulation
in vivo [14].
nos translation is also repressed by Cup
Like osk, translation of nos is regulated through the interaction of proteins with cis-acting elements present in its 3 UTR. Also similarly to osk, unlocalized nos, which makes up
over 90% of the total [2] is translationally repressed, and
this repression is alleviated in the pole plasm. In early
embryos, nos is translationally repressed outside the pole
plasm through an interaction between Smaug (Smg) protein
and two specific sequences in the 3 -UTR called SREs (Smgresponse elements) [15–17]. Recent evidence indicates that
Smg, like Bru, functions by interacting with the eIF4E-binding protein Cup [18]. Thus Smg specifically recruits nos
mRNA into a translationally inactive state, blocking translation by interacting with eIF4E and sequestering it away
from the cap structure at the 5 -end of the mRNA.
In addition to its translational repressor activity, which
requires eIF4E binding, Smg has an important function in the
degradation of unlocalized maternally derived mRNAs in
the early embryo [19]. This phenomenon has been studied by
examining Hsp83 mRNA, which is concentrated to the pole
plasm by a mechanism that involves its stabilization at the
posterior and its degradation elsewhere. In smg mutant
embryos, Hsp83 mRNA is not degraded outside the pole
plasm. The function of Smg in this process is to recruit the
CCR4–POP2–NOT deadenylase complex (where CCR4
stands for carbon catabolite repressor 4) to Hsp83 mRNA.
Interestingly, Smg has no effect on Hsp83 (heat-shock protein 83) translation, nor are Cup or the SRE involved in Smgmediated effects on RNA stability.
cad translation is repressed by a novel
d4EHP-dependent mechanism, not by
sequestering eIF4E
Our work has identified a novel mode of mRNA-specific
translational inhibition, which does not involve competition
between eIF4G and an eIF4E-binding protein such as Cup,
for eIF4E. These two modes of translational inhibition are
compared graphically in Figure 1. The competition in this
case is for binding to the 5 -cap structure itself. It has
long been established that a posterior-to-anterior gradient
of Cad protein is established in early embryogenesis from
uniformly distributed maternal cad mRNA, and this gradient
is essential for posterior patterning [20]. Establishment of
this Cad gradient requires Bicoid (Bcd), which mediates capdependent translational repression of cad mRNA dependent
on the BBR (Bcd-binding region), an element in its 3 -UTR
Figure 1 Two contrasting mechanisms of cap-dependent
translational regulation of specific mRNAs
(A) d4EHP-mediated repression of cad translation. The RNA-binding
protein Bcd is recruited to a regulatory element (BBD) in the cad 3 -UTR.
Bcd binds d4EHP (4EHP), which in turn binds to the cap structure at the
5 -end of cad mRNA. Since d4EHP cannot bind eIF4G, the cap-binding
complex composed of eIF4G (4G), eIF4E (4E) and eIF4A (4A) is not
recruited to cad mRNA and translation initiation is therefore blocked.
(B) eIF4E-binding protein-mediated repression of nos translation. The
RNA-binding protein Smg is recruited to a regulatory element (SRE)
in the nos 3 -UTR. Smg recruits the eIF4E-binding protein Cup, which
competes with eIF4G for binding to eIF4E. The Cup–eIF4E interaction
prevents assembly of the cap-binding complex at the 5 -end of nos
mRNA.
[21–24]. Thus as for eIF4E-binding protein-mediated translational repression, specificity for a particular mRNA is
conferred by an RNA-binding protein (in this case, Bcd) that
interacts with an element located within its 3 -UTR.
An eIF4E-related protein called human 4EHP was described previously [25]. While 4EHP has 5 -cap structurebinding activity, it does not interact with eIF4G, suggesting
that it could function as a negative regulator of translation
[25,26] as it cannot function in ribosome recruitment.
The Drosophila genome encodes an orthologue of 4EHP
(d4EHP), which we showed has biochemical activities similar
to human 4EHP. To understand the function of 4EHP in
development, a hypomorphic allele was created by imprecise
excision of a nearby P-element. This produced a small deletion which removed part of the d4EHP gene but which
still allowed low-level expression from a cryptic AUG codon
located within the first intron. Homozygotes for this d4EHP
allele are viable, but approximately half of the embryos
produced by homozygous females fail to hatch into larvae
and exhibit patterning defects reminiscent of the progeny of
bcd females. This suggested that d4EHP has a role in Bcdmediated developmental processes.
We observed no effect of the d4EHP mutation on the
transcriptional activation function of Bcd, as measured by
monitoring zygotic hb expression. However, we observed a
clear defect in translational regulation of cad mRNA, in that
Cad protein, which in wild-type embryos is only detectable in
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Biochemical Society Transactions (2005) Volume 33, part 6
the posterior half of the embryo, was apparent throughout the
entire length of many embryos produced by d4EHP mutant
females.
By immunoprecipitation from embryonic extracts and
from cultured mammalian cells transfected with constructs
expressing epitope-tagged proteins, a physical interaction
between Bcd and d4EHP was demonstrated. Conversely,
Bcd and deIF4E did not show detectable interaction in these
assays. The co-transfection assay was used to map residues
in d4EHP required to bind the 5 -cap structure and to bind
Bcd. Mutant forms of d4EHP that were abrogated either
for Bcd binding or for cap binding could not confer translational regulation of cad in embryos produced by d4EHP
mutant mothers, whereas wild-type d4EHP expressed from a
transgene in the same way fully rescued the Cad gradient and
embryonic viability. Thus d4EHP must interact with both
the 5 -cap structure and Bcd to carry out its developmental
function. In a similar manner, individual residues in Bcd were
identified that are essential for d4EHP binding. While Bcd
contains a sequence motif related to the consensus eIF4Ebinding domain [27], we determined that a critical residue
for d4EHP binding (Tyr66 ) is distinct from this domain and
a tyrosine residue that is invariant and essential in eIF4Ebinding domains (Tyr68 ) is dispensable for d4EHP binding.
Furthermore, mutant forms of Bcd that cannot interact with
d4EHP are unable to confer translational repression of cad
mRNA in vivo, while wild-type or mutant forms of Bcd that
do not affect d4EHP binding can rescue this phenotype of
embryos produced by bcd females.
In a final experiment to demonstrate that the d4EHP–Bcd
interaction is required for the BBR-mediated inhibition of
cad translation, capped reporter mRNAs containing BBR
sequences in their 3 -UTRs were used as templates for in vitro
translation reactions. These assays were performed with
mouse Krebs-2 cell-free translation extracts, because their
activity is highly cap-dependent [28] and they do not express
endogenous Bcd. We found that Bcd or d4EHP has no
significant effect on translation of a BBR-containing mRNA
when added to these extracts individually, but when added
together they repress translational activity by approx. 60%,
depending on a BBR in the sense strand.
Inhibition of cad mRNA translation by the d4EHP–Bcd
complex demonstrates for the first time the involvement of a
cellular cap-binding protein other than eIF4E in cap-dependent translational control. Furthermore, it provides a new
molecular mechanism governing the formation of morphogenetic gradients in the early Drosophila embryo. 4EHPmediated translational repression is potentially more potent
than that mediated by eIF4E-binding proteins such as Cup,
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which have to compete with eIF4G for binding to eIF4E.
This is because the eIF4E–eIF4G interaction is very stable
[29].
In summary, we have uncovered a novel mechanism for
achieving spatially regulated expression of a protein, which
in turn is essential for embryonic patterning and development. We expect that future research will uncover numerous additional examples of 4EHP-mediated translational
repression, in Drosophila and in other systems.
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Received 17 June 2005