1129
Development 126, 1129-1138 (1999)
Printed in Great Britain © The Company of Biologists Limited 1999
DEV5231
Apontic binds the translational repressor Bruno and is implicated in
regulation of oskar mRNA translation
Yung S. Lie and Paul M. Macdonald*
Department of Biological Sciences, Stanford University, Stanford, CA 94305-5020, USA
*Author for correspondence (e-mail: [email protected])
Accepted 14 December 1998; published on WWW 15 February 1999
SUMMARY
The product of the oskar gene directs posterior patterning
in the Drosophila oocyte, where it must be deployed
specifically at the posterior pole. Proper expression relies
on the coordinated localization and translational control of
the oskar mRNA. Translational repression prior to
localization of the transcript is mediated, in part, by the
Bruno protein, which binds to discrete sites in the 3′
untranslated region of the oskar mRNA. To begin to
understand how Bruno acts in translational repression, we
performed a yeast two-hybrid screen to identify Brunointeracting proteins. One interactor, described here, is the
product of the apontic gene. Coimmunoprecipitation
INTRODUCTION
A wide variety of mechanisms serve to regulate gene expression.
Some of these act after transcription and control the stability,
distribution or translation of existing mRNAs. Posttranscriptional control is especially prominent during late
oogenesis and early embryogenesis, when transcriptional
activity is low or not detectable, and the vast majority of proteins
are synthesized from maternally contributed mRNAs. Control
mechanisms are often tailored for individual mRNAs, allowing
for specialized patterns of expression. This is particularly true
for mRNAs encoding proteins that direct important
developmental events and must appear only at the appropriate
times and, in some cases, only at certain positions in the egg or
embryo (reviewed by St Johnston, 1995; Curtis et al., 1995;
Macdonald and Smibert, 1996; Wickens et al., 1996).
Subcellular positioning is commonly achieved through a
combination of mRNA localization and translational regulation,
while the latter alone is usually sufficient for temporal control.
Both of these forms of post-transcriptional control are often
mediated by cis-acting regulatory elements in the 3′ untranslated
region (UTR) of the mRNA, and several of the factors that bind
such elements have been identified (Macdonald et al., 1995;
Kim-Ha et al., 1995; Smibert et al., 1996; Rivera-Pomar et al.,
1996; Chan and Struhl, 1997; Kelley et al., 1997; Bashaw and
Baker, 1997; Gebauer et al., 1998; Paillard et al., 1998; Hake
and Richter, 1994; Murata and Wharton, 1995; Zhang et al.,
1997; Deshler et al., 1997; Ross et al., 1997; Ostareck et al.,
experiments lend biochemical support to the idea that
Bruno and Apontic proteins physically interact in
Drosophila. Genetic experiments using mutants defective in
apontic and bruno reveal a functional interaction between
these genes. Given this interaction, Apontic is likely to act
together with Bruno in translational repression of oskar
mRNA. Interestingly, Apontic, like Bruno, is an RNAbinding protein and specifically binds certain regions of the
oskar mRNA 3′ untranslated region.
Key words: apontic, tracheae defective, bruno, arrest, oskar,
Translational regulation
1997). A central issue in understanding the biochemical
mechanisms responsible for such control events involves the
roles played by these RNA binding proteins. Of particular
interest is the question of how translation can be influenced by
the binding of a protein to a region of the mRNA, the 3′ UTR,
which is not traversed by ribosomes during protein synthesis.
Several mechanisms contribute to specialized forms of
translational regulation. The best understood, and probably the
simplest, involves the binding of a protein to regulatory
sequences in the 5′ UTR, as exemplified by the regulation of
ferritin mRNA translation by IRE-BP (reviewed in Klausner et
al., 1993). In this case translation is repressed because the bound
protein interferes with movement of the preinitiation complex
from the 5′ cap to the start codon. This type of mechanism does
not appear to require a specific interaction between the RNA
binding protein and other factors (Stripecke et al., 1994).
Another mechanism of translational regulation, which is
mediated through the 3′ UTR, involves changes in poly(A)-tail
length (Richter, 1996; Wickens et al., 1996). The logic
underlying this type of control is simple, although many of the
details are unknown. In brief, mRNAs with long or growing
poly(A) tails tend to be more efficiently translated than those
with short tails. Thus, enzymes that modify poly(A)-tail length
can control translation. 3′ UTR binding factors could either
possess such activities or recruit the appropriate enzymes to
specific mRNAs. It is less certain how the binding of a protein
to the 3′ UTR can affect translation in the absence of a change
in poly(A)-tail length, as has been observed for the lipoxygenase
1130 Y. S. Lie and P. M. Macdonald
mRNA in reticulocytes (Ostareck-Lederer et al., 1994; Ostareck
et al., 1997). However, it does seem likely that this type of
mechanism will involve interaction of the binding protein with
other protein factors, and so identification and characterization
of those factors should lead to mechanistic insights.
Many of the best characterized mRNAs under elaborate posttranscriptional control come from Drosophila, where the protein
products of maternal mRNAs dictate pattern along the
dorsoventral and anteroposterior body axes. One of these mRNAs
is oskar (osk), which encodes a spatial determinant required for
posterior patterning (Lehmann and Nüsslein-Volhard, 1986;
Ephrussi and Lehmann, 1992; Smith et al., 1992). The osk mRNA
is transcribed in the nurse cells of the ovary, rapidly transported
into the oocyte, and eventually localized to the posterior pole of
the oocyte (Kim-Ha et al., 1991; Ephrussi et al., 1991).
Translation of the osk mRNA is repressed during the early stages
of oogenesis and activated coincident with posterior localization
in the oocyte (Kim-Ha et al., 1995; Rongo et al., 1995; Markussen
et al., 1995). Translational repression requires cis-acting elements
within the osk mRNA 3′ UTR called BREs (Bruno response
elements), and the Bruno (Bru) protein, which specifically binds
these elements. Disruption of the Bru-osk mRNA interaction in
vivo allows translation prior to localization of the mRNA, and
Osk protein accumulates throughout the oocyte. As a
consequence, the entire oocyte and embryo acquire posterior
positional values, a lethal condition (Kim-Ha et al., 1995;
Webster et al., 1997). At present there is no evidence to suggest
that Bru-mediated translational repression involves changes in
polyadenylation. The poly(A) tail of the osk mRNA in ovaries is
short, and its length does not appear to vary with the translational
status of the mRNA (Webster et al., 1997). Thus the binding of
Bru protein to osk mRNA is likely to interfere with translation
by an as yet undefined mechanism. Here we describe the results
of a yeast two-hybrid screen to identify proteins that interact with
Bru and potentially contribute to translational regulation of osk
mRNA. One gene characterized in detail, apontic (apt), has
properties indicating that it encodes a protein that acts as a
corepressor of osk translation.
MATERIALS AND METHODS
Yeast two-hybrid interaction trap
We used the system of Gyuris et al. (1993). In this system proteinprotein interactions between bait (Bru fused to the LexA DNA binding
domain) and prey (unknown cDNA fused to a transcriptional activation
domain) constitute a protein assembly that binds to LexA operator
sequences and can function as a transcriptional activator. In the
appropriate yeast strain in which the leu2 gene has been placed under
control of LexA operator sequences, the bait-prey interaction allows
for growth on medium lacking leucine, thereby allowing selection of
positive prey cDNAs. A bait plasmid (pY3) was constructed that
contained the complete bru cDNA fused to the LexA DNA-binding
domain in the 2µ HIS3+ plasmid pEG202. Expression of protein from
this plasmid is under control of the ADH1 promoter. In preliminary
tests we found that this plasmid conferred substantial transcriptional
activation in the absence of any prey plasmid, making it difficult to use
this selection scheme to identify prey cDNAs encoding Bru-interacting
proteins. To address the problem of transcriptional activation by bait
alone, Brent and coworkers have constructed lacZ reporter plasmids
that can be used as an alternative method of detecting bait-prey
interactions. These plasmids contain the lacZ gene under the control
of variable numbers of LexA operator sequences, setting different
threshold levels for detection of transcriptional activation. We tested
several such reporter plasmids and found that in the yeast strain EGY48
(MATa,ura3, his3, trp1, LEU2::LexAop6-LEU2) containing plasmid
pRB1840 (a 2µ URA3+ plasmid that contains a single LexA operator
upstream of the lacZ gene), transcriptional activation conferred by the
Bru bait plasmid was below the level of detection by colorimetric plate
assay. The prey library was RFLY3, a gift from R. Brent. This library
was made from Drosophila melanogaster ovarian cDNAs in the 2µ
TRP1+ plasmid pJG4-5. Expression of the library cDNA fusion from
this vector is under control of the GAL1 promoter. The yeast strain
was transformed with pRB1840 and pY3 and then subsequently
transformed with RFLY3. 246,000 yeast transformants containing all
three plasmids were plated on media lacking histidine, tryptophan and
uracil, and containing 2% galactose and the substrate X-gal. Colonies
were visually screened for β-galactosidase activity. 89 blue colonies
were selected. In order to determine whether this activity was
dependent upon expression of the library cDNA, each colony was
subsequently retested for dependence of β-galactosidase activity on
galactose-containing medium. 56 colonies retained activity. All of
these were then tested for interaction specificity. Library plasmids were
individually transformed into several yeast strains, each containing a
different bait plasmid as well as the reporter plasmid pRB1840. True
interacting proteins were identified as those that activated lacZ
expression in the presence of the Bru bait plasmid and not in the
presence of Bcd (pRFHM-1) or Exu bait plasmids. 12 of the original
lacZ+ clones were specific in their interactions with Bru. DNA
sequencing indicated that these clones could be grouped into five
different classes. The apt class contained four clones with identical apt
cDNA sequence and variable numbers of EcoRI adaptors at their 5′
ends. These clones encode amino acids 429-490 of the Apt protein. All
were further tested for specificity of interaction with Bru using bait
plasmids expressing Tra, Tra-2, Rbp-1, Vasa or the N-terminal portion
of Vasa; activation was only observed with the Bru bait plasmid.
Recombinant Apt protein
The apt cDNA beginning at the internal SalI site was blunt-ended and
cloned into the blunt-ended BamHI site of the pET-3b vector (Novagen),
and this plasmid was transformed into the bacterial strain pLysS. The
resulting protein lacks the N-terminal 37 amino acids of Apt.
Expression was induced by the addition of 0.5 mM IPTG to log phase
cultures and subsequent growth for 2.5 hours at 37°C. Bacterial protein
was prepared by centrifugation of the culture at 4,000 rpm for 10
minutes at 4°C to pellet the cells. The pellet was resuspended in cold
TNE solution (50 mM Tris-Cl, pH 8.0, 250 mM NaCl, 2 mM EDTA)
and pelleted a second time by centrifugation. The pellet was frozen at
−80°C, then thawed in cold TNE with 2 mM β-mercaptoethanol, 1 mM
PMSF and 10 mM benzamidine. Lysozyme was added to about 0.2
mg/ml, and after a 10 minute incubation on ice Triton X-100 was added
to 1% and the mixture incubated for another 10 minutes on ice. The
lysate was then sonicated for 3×10-second pulses and this solution was
centrifuged through a 40% sucrose cushion (40% sucrose, 10 mM TrisCl, pH 8.0, 200 mM NaCl, 1 mM EDTA) for 30 minutes at 12,000 rpm
at 4°C. The resulting pellet was recovered and resolubilized in 8 M urea,
50 mM Tris-Cl, pH 8.0, 500 mM NaCl, 1 mM EDTA, 1 mM PMSF.
This solution was dialyzed overnight at 4°C into 50 mM Tris-Cl, pH
8.0, 500 mM NaCl, 1 mM PMSF and 10% glycerol. Soluble protein,
in which Apt was the major component, was recovered in the
supernatant after centrifugation for 15 minutes at 12,000 rpm at 4°C to
pellet insoluble proteins. This Apt protein was used both for UV crosslinking assays and for injection into rats for preparation of antiserum
(Josman Laboratories). The rat polyclonal serum specifically detects
Apt on western blots (data not shown) and was used for all
immunoprecipitations and whole-mount staining.
Immunoprecipitations
For immunoprecipitations, protein extracts were prepared by
homogenizing hand-dissected ovaries in extraction buffer (50 mM
Apontic binds translational repressor Bruno 1131
Hepes, pH 7.9, 150 mM NaCl, 1 mM EDTA and 0.5 mM PMSF) and
clearing the extract by centrifugation at 15,000 rpm for 12 minutes at
4°C. Glycerol was added to a final concentration of 20%. 25 µl extract
diluted with 75 µl of extraction buffer was preincubated with 5 µl serum
for 1 hour at 4°C. Samples were centrifuged 10 minutes at
15,000 rpm at 4°C to pellet aggregates. After transferal of
the supernatant to a fresh microfuge tube, 50 µl of a 1:1
slurry of equilibrated protein G-agarose beads
(Boehringer-Mannheim) to extraction buffer was added.
These samples were then rotated for 2 more hours at 4°C.
Beads were washed four times with cold 50 mM Tris-Cl,
pH 8.0, 250 mM NaCl and 1% Tween-20. 50 µl 2× protein
sample loading buffer was added to the washed beads;
samples were heated to 100°C for 5 minutes and
electrophoresed through 9% SDS-polyacrylamide gels.
Electrophoresed samples were transferred to nitrocellulose
(Idea Scientific), and subsequent analysis was by western
blotting. Proteins were detected by chemiluminescence
(Western Light, Tropix) using the primary antibody at a
1:2000 dilution for anti-BruA antiserum (Webster et al.,
1997) and the secondary goat anti-rabbit alkaline
phosphatase- conjugated antibody as per the
manufacturer’s protocol.
was added (along with competitor RNA for competition experiments).
Radiolabeled probes were made by in vitro transcription using different
regions of the osk 3′ UTR as templates (Kim-Ha et al., 1995). Other
RNA probes used in this assay were transcribed from the following
RNA and protein analysis
In situ hybridizations were performed as described by
Tautz and Pfeifle (1989) using RNA probes. Whole-mount
antibody stains were done with anti-Apt antibodies at a
1:100 dilution. Secondary antibodies for signal detection
were a goat anti-rat horseradish peroxidase conjugate or a
goat anti-rat Cy3 conjugate (Jackson ImmunoResearch
Laboratories). To visualize nuclei, fixed tissues were
stained for 5 minutes with 1 µg/ml DAPI and washed
thoroughly in 1× PBS, 0.1% Tween-20.
Fly stocks and manipulations
apt41 and apt167 both have missense mutations in the third
exon of the apt gene. The apt P element stock l(2)09049
contains a P element insertion just upstream of the first
intron of the apt gene (Gellon et al., 1997). tdfP∆3 and
tdfP∆4, which we refer to in the text as aptP∆3 and aptP∆4,
are both deletion mutants lacking 5′ regions of apt
(Eulenberg and Schuh, 1997). The aretQB72 allele was
used in all experiments described and contains a nonsense
mutation resulting in a stop codon in the protein at amino
acid 404 (Webster et al., 1997). w1118 flies were used for
immunohistochemistry, in situ hybridization and protein
extracts.
Germline clones were induced using the FLP-DFS
method (Chou and Perrimon, 1992). apt alleles were
recombined onto the P[FRT-G13, w+] chromosome. The
aptP∆3, P[FRT-G13, w+] recombinant fly stock was a gift
from R. Schuh. Female flies carrying a recombinant
chromosome were crossed to y, w, P[hs-Flp-1]; P[FRTG13, w+], P[ovoD1, w+] males. Second and third instar
larvae were heat-shocked at 37°C for 30 minutes, allowed
to recover at room temperature for 30 minutes, and then
heat-shocked a second time at 37°C for 30 minutes. Flies
were maintained at 25°C. Upon eclosion, virgin females
of the desired genotype were selected and placed in cages
with wild-type males to determine egg-laying ability.
Ovaries were hand-dissected, fixed and stained with
DAPI as described above.
UV cross-linking assay
0.5 µg bacterially expressed Apt protein was preincubated
with 10 µg yeast tRNA, and labeled probe (1×106 cpm)
Fig. 1. apt mRNA and protein expression patterns. Distribution of apt mRNA and
protein during oogenesis was detected by in situ hybridization and
immunohistochemistry in whole-mount ovaries (A-C). Early stage egg chambers
are toward the left, while later stage egg chambers are toward the right; each
individual egg chamber contains a cluster of nurse cells on the left and a single
oocyte on the right and is surrounded by a layer of somatic follicle cells. (A) In situ
hybridization. apt mRNA is detected at high levels in the follicle cells in the
germarium and decreases in abundance in these cells throughout the remainder of
oogenesis. apt mRNA accumulates in the germline cells and by stage 6 the
transcript becomes concentrated in the oocyte (shown as the darkly stained single
cell on the right of egg chambers). mRNA is also present in the nurse cells, which
are visualized as the cluster of cells with large polyploid nuclei towards the left of
each egg chamber. (B,C) Confocal images of whole-mount antibody stains,
focusing on the exterior (B) or the interior (C) of the ovariole. Apt protein can be
detected in both the germline and somatic cells of developing egg chambers. Apt is
expressed in the nuclei of all populations of follicle cells, including the border cells,
which are indicated by the open arrowhead. In the oocyte, protein is concentrated in
the nucleus (solid arrowhead) and is found at lower levels in the cytoplasm. Apt
protein is evenly distributed throughout both the cytoplasm and nuclei of the nurse
cells. (D,E) Double staining of a blastoderm-stage embryo prior to cellularization.
Nuclei are revealed by DAPI staining (E), and can be seen to line the periphery of
the embryo. Apt protein is detected by immunofluorescence and appears throughout
the embryo, in both the cytoplasm and the nuclei, but is not specifically
concentrated in the nuclei (D). By stage 5 of embryogenesis, Apt is localized to
nuclei and can no longer be detected in the cytoplasm (Eulenberg and Schuh,
1997).
1132 Y. S. Lie and P. M. Macdonald
templates: C78, the 5′-most 185 nucleotides (nt) of the nanos (nos) 3′
UTR; C84, the same 185 nt fragment with point mutations in the SREs
(smaug recognition elements); C88, multimerized 3× SRE (89 nt);
C90, multimerized point mutated 3× SRE (89 nt); pY17, the 3′ end of
vitelline membrane protein 32E (approx. 900 nt); pY84 (approx. 800
nt) and pY86 (approx. 800 nt), two uncharacterized cDNAs isolated in
the two hybrid interaction trap; and the pSP73 (Promega) polylinker
(105 nt). Reactions were incubated for 5 minutes at room temperature
and subsequently irradiated on ice with 105 erg/mm2 of UV light in a
Stratagene UV cross-linker. Following UV cross-linking, RNA was
digested with 30 µg RNase A for 15 minutes at room temperature.
Protein sample loading buffer was added, samples were heated to
100°C for 4 minutes, and proteins were resolved by SDS-PAGE on 9%
gels followed by autoradiography. Apt bound specifically to regions of
the osk 3′ UTR (see Fig. 5) and also bound all the other unrelated RNAs
tested (listed above) except for C88 and C90.
RESULTS
Bru-mediated repression of osk mRNA translation presumably
requires the interaction of Bru with other protein factors. To
identify candidate Bru binding partners, we performed a yeast
two-hybrid screen using the interaction trap system (Gyuris et
al., 1993). 12 Drosophila ovarian cDNAs passed initial tests
for evidence of a specific interaction of the encoded protein
with Bru and could be grouped into five classes (see Materials
and methods). For the class of cDNA (consisting of four nearly
identical clones) described in this report, we performed
additional tests in the two-hybrid system using a panel of other
proteins (see Materials and methods), which further
demonstrated specificity in the interaction of the cDNAencoded protein with Bru.
The cDNAs recovered from the two-hybrid screen were not
full length. Consequently, additional cDNAs were isolated
from an ovarian library and the longest one was sequenced.
This cDNA corresponds to a gene which was recently
identified independently in three different laboratories and has
been called apontic (apt) (Gellon et al., 1997; Su et al., 1999)
or tracheae defective (tdf) (Eulenberg and Schuh, 1997). We
refer to the gene and mutants as apt. Our cDNA (GenBank
AF027123) has minor differences relative to the other cDNA
sequences. These include several single nucleotide changes as
well as small insertions or deletions that do not change the
reading frame; all are likely to represent sequence
polymorphisms or errors introduced by reverse transcription.
The sequences also diverge in the 5′ region, resulting in an
additional six amino acids in the protein defined by our cDNA.
This divergence can be simply explained by use of an
alternative promoter and 5′ exon, an interpretation that is
consistent with the appearance in northern blot analysis of a
slightly smaller form of the mRNA during oogenesis and early
embryogenesis (data not shown; see also Fig. 4B of Gellon et
al., 1997).
Identification of apt in the two-hybrid screen provides
suggestive evidence that Bru and Apt proteins interact. In the
following sections we first describe the expression of apt in
ovaries, which shows that Bru and Apt are found in the same
subcellular compartments and thus may interact in vivo. We
then present biochemical and genetic evidence strongly
supporting the existence of a Bru-Apt interaction and
suggesting that Apt, like Bru, acts in regulation of osk mRNA
translation.
Fig. 2. Bru coimmunoprecipitates with Apt. Western blot probed
with anti-Bru antiserum. Lane 1, crude ovary extract (not
immunoprecipitated). Lane 2, ovary extract immunoprecipitated with
preimmune serum. Lane 3, ovary extract immunoprecipitated with
anti-Apt antiserum. The arrow on the right points to Bru, and the
positions of molecular mass markers in kDa are indicated on the left.
Bru is detected in the anti-Apt immunoprecipitate but not in the
preimmune serum immunoprecipitate. Coimmunoprecipitation was
not affected by pretreatment of the ovarian extract with RNase A
(data not shown). Thus Bru and Apt are unlikely to be tethered to one
another by virtue of binding to a common RNA, a possibility we
considered since both proteins have RNA binding activity (Kim-Ha
et al., 1995; this work). We confirmed that the anti-Apt antiserum
does not cross-react with Bru by conducting independent
immunoprecipitation experiments using in vitro translated Bru (data
not shown).
Patterns of apt expression are consistent with an
interaction with Bru
Previous reports on apt have focused primarily on its
expression in the embryo, and details of the ovarian expression
have not been described. We examined the mRNA expression
pattern of apt during oogenesis by in situ hybridization (Fig.
1A). Expression occurs in both the somatic follicle cells and
the germline nurse cells and oocyte. apt transcripts are detected
as early as stage 2A at low levels in the germarium and at
higher levels in the follicle cells. The amount of apt mRNA in
the soma decreases during the remainder of oogenesis, while
the level in the germline increases. apt mRNA becomes
concentrated in the oocyte and also accumulates in the nurse
cells at about stage 6. apt transcripts continue to be found in
both the oocyte and nurse cells throughout oogenesis.
To determine when and where Apt protein is expressed
during oogenesis, antisera directed against a recombinant Apt
protein were prepared and used for protein detection in wholemount ovaries by confocal microscopy (Fig. 1B,C). Apt protein
appears in both the germline and somatic cells of the ovary
throughout all stages of oogenesis. In the germline, Apt protein
is present in both cytoplasm and nuclei. Within the nurse cells
the protein is more concentrated in the cytoplasm, while in the
oocyte more protein is found in the nucleus. The protein,
however, is not localized to any subdomain within the
cytoplasm of either the nurse cells or the oocyte.
Although Apt protein is not strictly nuclear or cytoplasmic
in cells of the female germline, the protein is highly
concentrated in nuclei of the ovarian follicle cells (Fig. 1B,C)
and in post-cellularization-stage embryos (Eulenberg and
Schuh, 1997). The developmental differences in subcellular
location suggest that Apt may have functions, perhaps
different, in both nuclei and cytoplasm. Nuclear proteins
expressed from maternal mRNAs are sometimes present at
Apontic binds translational repressor Bruno 1133
high levels in the cytoplasm of early embryos. Examples
include the Bicoid, Caudal and Hunchback proteins, which
appear in both nuclei and cytoplasm shortly after egg laying.
As nuclear divisions progress and the density of nuclei
increases, nuclear localization of these proteins remains strong
while the fraction of protein in the cytoplasm diminishes
(Driever and Nüsslein-Volhard, 1988; Macdonald and Struhl,
1986; Mlodzik and Gehring, 1987; Tautz, 1988). Thus there
appears to be no early impediment to nuclear localization,
simply a paucity of nuclei. In contrast, the subcellular
distribution of Apt protein appears to be actively controlled in
early development. We monitored Apt protein in early
embryos, using DAPI staining of nuclei to define
developmental stages. Even after migration of nuclei to the
surface of the embryo, Apt protein remains evenly distributed
between nuclei and cytoplasm (Fig. 1D), unlike any of the
other examples described above. This unusual persistence of
Apt protein in the cytoplasm suggests the existence of a
mechanism to control its distribution, reinforcing the notion of
roles for Apt in both cytoplasm and nuclei.
Biochemical evidence for the Bru-Apt interaction
To support the idea that a physical interaction between Bru and
Apt occurs in Drosophila, we performed immunoprecipitation
experiments. Dissected ovaries were homogenized and
incubated with Protein G-agarose beads and either preimmune
serum or anti-Apt antiserum. Immunoprecipitated proteins
were then assayed by western blot analysis for the presence of
Bru protein (Fig. 2). Notably, Bru was coimmunoprecipitated
with anti-Apt antiserum but not with preimmune serum,
demonstrating that Bru and Apt interact in the ovarian extract.
Although we also performed the complementary
immunoprecipitations with anti-Bru antiserum, we were
unable to detect Apt protein in the Bru immunoprecipitates
(data not shown). It is possible that the epitope recognized by
the anti-Bru antibody is masked by the Bru-Apt interaction,
which would interfere with coimmunoprecipitation. In support
of this interpretation, we note that while much of Bru protein
is readily detectable by immunohistochemistry in whole mount
ovary preparations, the fraction of Bru protein colocalized with
osk mRNA at the posterior pole of the oocyte can be detected
only if the ovaries are pretreated with protease (Webster et al.,
1997). Thus, certain populations of Bru protein appear to have
epitopes not readily accessible to the anti-Bru antibodies.
apt and aret interact genetically
Physical interactions between proteins suggest but do not prove
that the proteins function together in vivo. We therefore looked
for a genetic interaction between the bru and apt genes, which
would argue that the physical interaction is important for
function. The arrest (aret) mutants are defective for Bru (i.e.
aret and bru are the same gene; Webster et al., 1997) and lead
to a developmental arrest early in oogenesis (Schüpbach and
Wieschaus, 1991; Castrillon et al., 1993; Webster et al., 1997).
For our analysis we used aretQB72, a strong allele that has an
internal stop codon (Webster et al., 1997). Mutants in apt are
zygotic lethal (Gellon et al., 1997; Eulenberg and Schuh,
1997), and some alleles also cause arrested oogenesis (below).
We used several different apt alleles for all analyses (see
Materials and methods), as the genetics of apt are complex and
different alleles have different effects (see below and
Discussion). To test for a genetic interaction between the aret
and apt mutants, we asked if reducing the dosage of both genes
would cause a phenotype. Females heterozygous for aretQB72,
heterozygous for any of the five apt alleles, or
transheterozygous for both aretQB72 and an apt allele, were
crossed to wild-type males, and the progeny embryos were then
examined for cuticular defects. In these crosses mothers
heterozygous for aretQB72 or for any allele of apt produce only
embryos with wild-type cuticles. In contrast, females
transheterozygous for aretQB72 and apt41, apt167, aptl(2)09049,
aptP∆3 or aptP∆4 produce a fraction of embryos with head
defects (Fig. 3B, Table 1).
Head defects can result from ectopic or excessive posterior
body patterning activity, as this activity interferes with
expression of the anterior body patterning morphogen, Bicoid
(Wharton and Struhl, 1991). Consequently, the observed head
defects could be explained if both Bru and Apt contribute to
repression of osk mRNA translation. Alternatively, the head
defects could result from a more direct effect on anterior
development, a possibility suggested by the genetic interaction
between apt and Dfd, a gene involved in head development,
and the fact that homozygous apt− embryos have head defects
Table 1. Dependence of the apt/aret maternal-effect
phenotype on nos gene dosage
Fig. 3. apt interacts genetically with aret. (A) Wild-type head
skeleton. (B) Reduced head skeleton of an embryo from an apt/aret
mother. The fraction of head-defective embryos produced by apt/aret
mothers is listed in Table 1. Note that the female was crossed to a
wild-type male, and so the embryo is not homozygous for either
mutant chromosome. Thus the phenotype cannot be attributed to a
lack of zygotic apt activity, a condition which causes head defects.
Maternal-effect phenotypes of this sort often result from
inappropriate expression of posterior patterning activities (Wharton
and Struhl, 1991). This is the case for four of the five apt alleles, as
these head defects were suppressed by additionally reducing the
dosage of nos (see Table 1).
Proportion of progeny
embryos with head defects (%)
apt allele
apt/aret;+/+
apt/aret;nosL7/+
41
167
l(2)09049
P∆3
P∆4
1.6
6.7
9.4
22.0
16.0
0
0
9.5
2.4
1.2
For each genotype, a minimum of 240 embryos were examined.
The above values represent the percentage of the total number of embryos
that were head-defective.
1134 Y. S. Lie and P. M. Macdonald
(Gellon et al., 1997). To distinguish between these possibilities
we determined the consequences of reducing nanos (nos) gene
dosage in mothers transheterozygous for aret and apt. nos
encodes a limiting component of the posterior patterning
activity (Lehmann and Nüsslein-Volhard, 1991; Wang and
Lehmann, 1991), and reduction of the nos gene dosage should
only affect anterior patterning defects arising from
misexpression of posterior patterning molecules. For
transheterozygotes of aretQB72 and four of the five apt alleles,
reducing nos dosage largely suppressed the head defects
phenotype (Table 1). Thus the head defects phenotype of these
transheterozygotes can be attributed, at least in large part, to
ectopic or excessive posterior body patterning activity, a
finding consistent with a role for apt in control of osk mRNA
translation. The fact that the phenotype of the
aptl(2)09049/aretQB72 transheterozygotes is not suppressed by
reducing nos dosage indicates that this allele affects apt
function differently than the other alleles (see Discussion).
Fig. 4. apt is required in the germline. Homozygous mutant
germlines were established by creating germline clones in females
heterozygous for apt mutations. Phenotypes were determined by
examining ovaries and, when possible, embryos. (A) A wild-type
ovariole stained with DAPI to reveal nuclei. For each of the
ovarioles, early stages are at the left. Morphologically similar ovaries
were found when the germline was homozygous for aptl(2)09049 or
apt167. In each case the females produced many eggs, and cuticles
from these were prepared for examination. Embryos from
homozygous aptl(2)09049 germlines were all wild type. However, a
small fraction (<1%) of embryos from homozygous apt167 germlines
had defective head skeletons (B). (C-E) Germline clone phenotype of
apt41 or aptP∆3. The ovaries shown here were DAPI-stained in order
to visualize nuclei. Development arrests at approximately stage 6,
after which the egg chambers degenerate (rightmost portions of
ovarioles). Some egg chambers have an aberrant number of nuclei;
examples with too many or too few nuclei are shown enlarged in D
and E, respectively.
apt ovarian phenotype
Earlier genetic analyses of apt have concentrated on the zygotic
phenotype (Gellon et al., 1997; Eulenberg and Schuh, 1997).
To define more completely the role of apt in the female
germline, we created females with apt− germline clones using
the FLP/DFS method (Chou and Perrimon, 1992). Ovaries
containing germline clones were dissected, stained with DAPI
to highlight nuclei, and examined for phenotype. Different apt
mutants display dramatically different ovarian phenotypes.
One allele, aptl(2)09049, is indistinguishable from wild type, as
females with aptl(2)09049 germline clones had phenotypically
wild-type ovaries (Fig. 4A) and laid eggs that developed into
fertile adults. Females with apt167 germline clones also had
phenotypically wild-type ovaries, but a small fraction of the
eggs laid developed into embryos with head defects (Fig. 4B).
In contrast, ovaries from females with apt41 or aptP∆3 germline
clones have phenotypes that are similar to one another and
severe: development is arrested in early oogenesis
(approximately stage 6), and the oocyte fails to differentiate
with all nuclei becoming polyploid. In addition, some of the
egg chambers have an abnormal number of nuclei (Fig. 4C-E).
This phenotype is highly unlikely to result from a background
mutation, as the apt41 and aptP∆3 alleles were induced with
different mutagens on different parental chromosomes (Gellon
et al., 1997; Eulenberg and Schuh, 1997). We conclude that apt
is necessary for oogenesis and that loss of apt activity leads to
a developmental arrest during oogenesis. Just as for aret
mutants, the arrest occurs too early to allow us to examine the
ovaries for defects in osk mRNA translation.
Apt binds specifically to regions of the osk mRNA 3′
UTR
The biochemical and genetic data presented here demonstrate
that Bru and Apt interact with one another and suggest that Apt
contributes to Bru-mediated translational repression of osk. To
begin to examine the mechanism of Apt function, we asked if
Apt, like Bru, can bind RNA. Recombinant Apt was expressed
in E. coli and tested for RNA binding activity in a UV crosslinking assay. In initial experiments we used substrate RNAs
corresponding to different parts of the osk mRNA 3′ UTR. Apt
binding to certain RNAs is easily detectable, while RNAs from
other parts of the 3′ UTR do not support binding (results
Apontic binds translational repressor Bruno 1135
summarized in Fig. 5A). We more rigorously confirmed the
apparent differences in binding affinities using competition
binding experiments. Binding of Apt to the osk AB region
RNA was tested in the presence of increasing amounts of
unlabeled competitor RNAs (Fig. 5B). Although the osk AB
RNA competes effectively, the other RNAs tested compete
only weakly. Thus Apt is an RNA binding protein, and it
displays substantial specificity in its binding activity.
Remarkably, the regions of the osk 3′ UTR bound by Apt, the
AB and C regions, are precisely those bound
by Bru. To determine if Bru and Apt have
the same RNA binding specificity, we tested
Apt binding to a series of RNAs used to map
the Bru binding sites, called BREs, within
the osk C region. Three of these RNAs retain
the BREs and are bound by Bru, while a
fourth RNA, C∆4, lacks the BREs and fails
to bind Bru (Kim-Ha et al., 1995). Apt binds
all four RNAs, including C∆4 (Fig. 5C),
indicating that Apt can bind to sites other
than BREs.
Subsequent binding experiments were
performed with a variety of other in vitro
transcribed RNAs. Apt binds detectably to
6 of 8 RNAs tested (data not shown; see
Materials and methods). We have been
unable to identify a sequence shared by all
of the bound RNAs. Thus, despite its
ability to efficiently discriminate between
different parts of the osk mRNA, Apt
appears to be relatively promiscuous in its
binding and may recognize many sites or
perhaps a structural feature common to
many RNAs.
repression of osk translation is Bicaudal C (Bic-C) (Saffman
et al., 1998). Bic-C mutants display a dominant maternaleffect phenotype in which osk translation initiates
prematurely and the embryos thus develop with defective
anterior patterning. Bic-C protein has RNA binding activity
but has not been shown to bind specifically to osk mRNA.
Another protein suggested to act in repression is p50, which
was identified by virtue of its binding to osk mRNA, but for
which genetic confirmation of such a role has not been
DISCUSSION
Proper control of osk mRNA translation is
essential and requires strict coordination
with localization of the transcript to
the posterior pole of the oocyte. Not
surprisingly, translational regulation of osk
mRNA appears to be complex, and several
factors involved in repression and
activation have already been identified. The
evidence for a direct role in osk translation
is strongest for Bru, a protein that binds
specifically to regulatory sequences in the
osk mRNA 3′ UTR (Kim-Ha et al., 1995;
Webster et al., 1997). Bru is also expected
to bind and regulate additional mRNAs, as
mutants lacking functional Bru protein
display defects in both oogenesis and
spermatogenesis
(Schüpbach
and
Wieschaus, 1991; Castrillon et al., 1993)
that cannot be attributed solely to problems
in osk mRNA metabolism. Consistent with
this idea, Bru has been shown to bind at
least one other mRNA, gurken (Kim-Ha et
al., 1995). A second protein that acts in
Fig. 5. Apt binds to specific regions within the osk mRNA 3′ UTR. (A) Diagram of the
osk mRNA 3′ UTR. The AB and C regions of the osk 3′ UTR contain multiple copies of
BREs and bind Bru. The RNA between the AB and C regions, corresponding to the
DraI-XbaI restriction fragment, lacks BREs and does not bind Bru. Also shown are
deletion mutant RNAs of the C region, constructed previously to define the Bru binding
sites (Kim-Ha et al., 1995). Each transcript was tested for binding to Apt, with the results
summarized at right and documented in (B) and (C). (B) Competition binding
experiments using the osk AB region RNA as a probe. Lane 1 shows the binding of Apt
in the absence of competitor RNA. Three different unlabeled competitor RNAs were
added at 30-, 100-, 300- or 600-fold excess (lanes 2-5, 6-9, 10-13). Competitor AB is the
osk AB region, which competes very efficiently. Competitor DraI-XbaI is the RNA
corresponding to the DraI-XbaI fragment separating the osk AB and osk C regions; this
competes much less efficiently than the osk AB RNA. Competitor bcd is a portion of the
bicoid mRNA localization signal and shows the lowest affinity for Apt, competing with
the lowest efficiency. Positions of molecular mass (kDa) markers are indicated to the left
of the panel. (C) Binding experiments using deletion mutants of the osk C region RNA.
None of the mutants eliminates Apt binding, although some variation in binding
efficiency is apparent. The position of Apt is marked by the arrow on the right. The
binding assays shown in B and C are from the same experiment and were exposed by
autoradiography for equal lengths of time.
1136 Y. S. Lie and P. M. Macdonald
obtained (Gunkel et al., 1998). We now add apt to this roster
of proteins and genes implicated in negative regulation of
translation.
Apt protein in cytoplasm and nucleus
It has been suggested that Apt functions as a transcription
factor during embryogenesis, perhaps acting as a cofactor for
certain Hox genes (Gellon et al., 1997; Eulenberg and Schuh,
1997). Two types of evidence have been presented to support
this conclusion. First, the Apt protein is highly concentrated in
nuclei during most of embryogenesis, which strongly
implicates a nuclear function. Second, the predicted structure
of the Apt protein includes domains similar to those found in
certain transcription factors (Eulenberg and Schuh, 1997). One
is a short region enriched in glutamine residues, which may
serve as a transcriptional activation domain. This by itself does
not strongly support a role as a transcription factor, as similar
glutamine-rich regions are found in a wide variety of
Drosophila proteins, some of which are not involved in
transcriptional regulation. The other domain is a potential bZIP
motif. One part of this motif, the leucine zipper, is clearly
present in Apt and may imply that the protein homodimerizes
or forms a heterodimer with another protein in vivo. The
second part of the bZIP motif, a flanking basic region, appears
in an unusual form: certain amino acids known to be involved
in DNA binding are present, but these are positioned much
closer to the leucine zipper than in any other characterized
bZIP domain. In addition, there are few basic amino acids
(Eulenberg and Schuh, 1997). Consequently, Apt is either a
rather unusual example of a bZIP transcription factor, or it may
be a related protein whose function in the nucleus is less
certain.
Apt is not always nuclear. Apt protein is persistently retained
in the cytoplasm of early stage embryos even after other
maternally provided proteins have shifted to the nuclei. This
evidence for programmed control of the subcellular location of
Apt suggests a requirement for Apt in the cytoplasm of early
embryos. Although this type of control could serve to prevent
Apt from functioning in the nucleus at this stage of
development, this seems unlikely, as Apt is not excluded from
the nuclei but simply is not concentrated there. In the nurse
cells of the ovary Apt protein is partitioned primarily to the
cytoplasm. This phenomenon – cytoplasmic distribution in
nurse cells of a protein that is nuclear in most other tissues –
is not unusual. Other examples include hnRNP40 (Squid)
(Matunis et al., 1994) and Sex lethal (Bopp et al., 1993).
Furthermore, a number of other nucleic acid binding proteins,
including TfIIIA and the Y box proteins, have distinct
functions in the cytoplasm and the nucleus (reviewed by
Ladomery, 1997).
One possibility for how the subcellular distribution of Apt
may be controlled is suggested by differences in apt mRNAs.
The use of alternate 5′ exons leads to variation at the amino
terminus of the protein. Exon choice appears to vary during
development, with one form of the mRNA found primarily
among maternal transcripts while other forms are ubiquitous
or most prevalent among zygotic transcripts. This pattern
correlates well with the changing distribution of Apt protein:
cytoplasmic Apt protein is synthesized largely or entirely from
maternal mRNAs, while nuclear protein is synthesized from
both maternal and zygotic mRNAs. Thus one form of the
protein could be targeted to the nucleus and the other form to
the cytoplasm.
Role of apt in osk mRNA translation
Evidence implicating apt in the control of osk translation is
indirect. Biochemical experiments indicate that Bru and Apt
proteins interact physically but provide no insight into the
significance of the association. The genetic evidence – head
defects among progeny of mothers transheterozygous for apt
and aret mutations – reveals a functionally significant
interaction between the apt and aret genes but does not specify
the exact nature of the interaction. Nevertheless, given the
established role for Bru in repression of osk mRNA translation,
one likely explanation is that Bru and Apt both act in this
process. Consequently, reducing the amount or activity of both
Bru and Apt proteins could lead to a modest derepression of
osk translation. This interpretation is supported by the
sensitivity of the phenotype to reduction of nos gene dosage.
Curiously, for one of the apt mutants, the P element insertion
allele aptl(2)09049, the genetic interaction with aret is not
suppressed by reduction of nos gene dosage. The same allele
has no ovarian phenotype when tested by germline clonal
analysis. We know of no simple explanation for the behavior
of this allele, although it seems possible that insertion of the P
element affects only one form of the apt transcripts, perhaps
leaving the ovarian-enriched transcript intact. Notably,
aptl(2)09049 is lethal when homozygous or in combination with
other apt alleles, but is viable in trans to a deficiency that
removes the apt gene (Gellon et al., 1997; W. McGinnis,
personal communication; data not shown), further indicating
that it is an unusual allele.
Although the genetic interaction of aret and apt supports a
role for apt in repression of osk mRNA translation, this
function may not be essential. One of the apt mutants that
shows a nos-sensitive interaction with aret, apt167, has only a
modest phenotype in germline clonal analysis: a small fraction
of the embryos from the homozygous mutant germlines display
head defects. While this phenotype is consistent with a partial
relaxation of the controls on osk activity, it is inconsistent with
a complete derepression of osk mRNA translation. (Note that
although homozygous apt− embryos have defects in head
development, the embryos obtained from the homozygous
mutant germlines were fertilized by wild-type males and are
thus heterozygous for apt167.) Could apt167 be a weak allele?
This seems somewhat unlikely (but not impossible) as it
displays a stronger genetic interaction with aret than does
apt41, which has a strong arrested oogenesis phenotype in
homozygous mutant germlines. Another possibility is that apt
performs a redundant or partially redundant role in repression
of osk mRNA translation. An appealing feature of this model
is that a candidate exists for a protein with overlapping
function. Gunkel et al. (1998) recently described a protein, p50,
that also binds to the regions of the osk mRNA bound by Apt;
Apt and p50 could have similar roles in regulation of osk
expression. The gene encoding p50 has not been identified, so
genetic tests of this model are not yet possible.
Our demonstration that Apt is an RNA binding protein is
somewhat unexpected, as none of the well-characterized RNA
binding motifs (Burd and Dreyfuss, 1994) appear in the
predicted protein sequence. The ability of Apt to discriminate
in its binding to certain regions of the osk mRNA 3′ UTR is
Apontic binds translational repressor Bruno 1137
striking, but its significance is uncertain, especially given the
binding of Apt to a wide variety of other RNAs. In further
characterization of apt function, it will be of interest to
determine whether Apt RNA binding activity is important for
proper regulation of osk mRNA translation, or if the interaction
of Apt and Bru proteins is sufficient. Notably, Apt protein does
not colocalize with Bru and osk mRNA to the posterior pole
of the oocyte, raising the possibility that displacement of Apt
from Bru may allow translational activation.
We thank R. Brent and R. Finley for yeast strains, two-hybrid
libraries and plasmids, and helpful advice; V. Heinrichs and A.
Nakamura for bait plasmids; A. Harris, L. Luo, W. McGinnis, R.
Schuh, T. Schüpbach and the Berkeley Drosophila Genome Project
for fly stocks; W. McGinnis for discussions and sharing unpublished
information; B. Holley for assistance with confocal imaging; D.
Guarnieri and T. Lee for discussions regarding generation of germline
clones; C. Smibert for UV cross-linking probes; and P. Lasko for
BruA antibodies. In addition, we thank E. Arn, A. Harris, B. Holley,
R. Mancebo, C. Smibert and P. Webster for discussions and criticism
of the manuscript. This work was supported by NIH grant GM54409
to P. M. M.
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Genomic sequence of the region containing apt (GenBank
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contains 5′ sequences provided by an exon located
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