Cup Blocks the Precocious Activation of the Orb
Li Chin Wong*¤, Paul Schedl
Department of Molecular Biology, Princeton University, Princeton, New Jersey, United States of America
Translational regulation of localized mRNAs is essential for patterning and axes determination in many organisms. In the
Drosophila ovary, the germline-specific Orb protein mediates the translational activation of a variety of mRNAs localized in
the oocyte. One of the Orb target mRNAs is orb itself, and this autoregulatory activity ensures that Orb proteins specifically
accumulate in the developing oocyte. Orb is an RNA-binding protein and is a member of the cytoplasmic polyadenylation
element binding (CPEB) protein family. We report here that Cup forms a complex in vivo with Orb. We also show that cup
negatively regulates orb and is required to block the precocious activation of the orb positive autoregulatory loop. In cup
mutant ovaries, high levels of Orb accumulate in the nurse cells, leading to what appears to be a failure in oocyte
specification as a number of oocyte markers inappropriately accumulate in nurse cells. In addition, while orb mRNA is
mislocalized and destabilized, a longer poly(A) tail is maintained than in wild type ovaries. Analysis of Orb phosphoisoforms
reveals that loss of cup leads to the accumulation of hyperphosphorylated Orb, suggesting that an important function of
cup in orb-dependent mRNA localization pathways is to impede Orb activation.
Citation: Wong LC, Schedl P (2011) Cup Blocks the Precocious Activation of the Orb Autoregulatory Loop. PLoS ONE 6(12): e28261. doi:10.1371/
Editor: Christos Samakovlis, Stockholm University, Sweden
Received August 30, 2011; Accepted November 4, 2011; Published December 2, 2011
Copyright: ? 2011 Wong, Schedl. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was initially supported by grant number GM56937 from National Institutes of Health until it was terminated in 2009. Subsequent support
came from National Institutes of Health grant number GM043432. The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
¤ Current address: Smilow Neuroscience Program, New York University School of Medicine, New York, New York, United States of America
In eukaryotic cells, cytoplasmic polyadenylation is used to
activate the on-site translation of localized mRNAs. Polyadenyl-
ation is thought to depend upon two key elements in the 39 UTR
of the mRNA. The first is the AAUAAA motif, which is bound by
the cleavage and polyadenylation specificity factor (CPSF) while
the second is the U-rich cytoplasmic polyadenylation element
(CPE) which is bound by the cytoplasmic polyadenylation element
binding protein (CPEB) , . The evolutionarily conserved
CPEBs are RNA-recognition motif (RRM)-type RNA-binding
proteins and they have been found in species ranging from
nematodes to humans. One of the founding members of the CPEB
family is the Drosophila germline-specific protein Orb . Orb
plays a critical role in the development of the female germline and
is required for mRNA localization and translational regulation
throughout much of oogenesis , .
Phenotypic analysis of strong orb alleles indicates that the
formation of the 16-cell cyst, the specification of the oocyte and
the proper expression of the TGF-a signaling molecule Gurken
(Grk) at the posterior of pre-vitellogenic egg chambers requires
orb activity –. In the hypomorphic allele orbmel, these early
steps in oogenesis appear normal; however, the specification of
both the anterior-posterior (AP) and dorsal ventral (DV) polarity
axes in vitellogenic egg chambers is disrupted. Orb protein is
thought to function in AP axis specification by binding to oskar
(osk) mRNA after it is localized to the posterior pole of the oocyte
and activating its translation by a mechanism involving
polyadenylation , , . In the DV polarity pathway, Orb
is required for the localized translation of grk mRNA at the
dorsal-anterior corner of the oocyte. In orbmelovaries, grk mRNA
is mislocalized and little or no Grk protein is produced. Like
other mutations that disrupt grk signaling, orbmeleggs have
ventralized chorions that either have fused or lack dorsal
respiratory appendages , .
Another Orb regulatory target is its own mRNA. Orb is
required to localize orb mRNAs to sites in the oocyte cortex and
to promote their on-site translation. As is the case for osk mRNA,
it is thought to act by binding to target sequences in the orb 39
UTR andactivating polyadenylation
autoregulatory activity ensures that high levels of Orb specifically
accumulate in the oocyte, which is the compartment that
requires orb activity. Since orb mRNA is synthesized in the nurse
cells, and must be transported through the nurse cells into the
oocyte and then localized within the oocyte to the cortex, there
must be mechanisms in place that prevent the precocious
activation of the orb positive autoregulatory loop. Previous
studies have shown that the Drosophila Fragile X mental
retardation protein dFMR1 downregulates orb mRNA transla-
tion in nurse cells ; however, the effects of dfmr1 mutations
on Orb protein expression, and on oogenesis in general, are
relatively modest and it seems likely that other factors may play
more central roles in blocking the premature activation of the orb
positive autoregulatory loop.
One candidate for a gene that prevents the precocious
activation of the orb positive autoregulatory loop is fs(2)cup (cup).
PLoS ONE | www.plosone.org1December 2011 | Volume 6 | Issue 12 | e28261
cup was discovered in a screen for female sterile mutations .
Strong loss-of-function Class I alleles arrest oogenesis prior to the
onset of vitellogenesis and they accumulate many small, round and
abnormal-looking egg chambers . Moderate Class II alleles
progress farther; the egg chambers appear to take up yolk and
have a more elongated shape. Oogenesis in Class III alleles is
relatively normal up until stage 9–10 when the oocyte stops
growing and this gives rise to cup-like chorions. The Cup protein
has been shown to function as a translational repressor of several
mRNAs including one of the known orb targets, osk , .
While the mechanism of repression is not fully understood, Cup
has been shown to interact directly with three other translation
factors, the cap-binding initiation factor eIF4e, and the RRM-type
RNA-binding proteins Bruno and Smaug –.
These interactions involve different domains of the Cup
protein. For example, Cup-eIF4E interactions are mediated by
a canonical and a non-canonical eIF4E binding motif in the Cup
N-terminus, while the C-terminal end of the Cup protein
mediates interactions with Bruno –. Though Cup has
no known RNA binding activity, protein-protein interactions with
Bruno (or Smaug) would function to recruit Cup to mRNAs like
osk and orb that contain sequence motifs recognized by the Bruno
protein. Cup is thought to inhibit the translation of these mRNAs
by binding to eIF4E and sequestering it from interacting with
eIF4G –. This prevents the assembly of the eIF4F
initiation complex (consisting of eIF4A, eIF4E and eIF4G) and
the loading of the 40S ribosomal subunit at the 59 end of the
mRNA –. Consistent with this model, the translation of
osk mRNA is prematurely upregulated in several cup hypomorphic
mutant combinations and Osk protein can be detected in stage 6–
7 egg chambers , . Moreover, in older mutant chambers,
translation appears to be activated at the anterior of the oocyte
insteadof the posterior. Further
premature activation of osk mRNA translation is also observed
in a cup mutant, cupD212, which lacks the canonical high affinity
eIF4E binding motif . Interestingly, however, though the
interaction of the mutant CupD212protein with eIF4E is expected
to be compromised, it is only a very weak Class III allele. This
observation suggests that the regulatory activities of cup during
oogenesis are likely to include other functions besides sequester-
ing the eIF4E translation factor.
In this paper, we present evidence that one of the other
functions of the cup gene is to prevent the premature activation of
the orb positive autoregulatory loop. In wild type ovaries, the orb
autoregulatory loop is activated in the oocyte ensuring that high
levels of Orb protein accumulate in the compartment where its
activity is required. In contrast, in cup mutants the autoregulatory
appears to be precociously activated and high levels of Orb
protein accumulate in the nurse cells. Our data suggest that Cup
employs at least two different though likely overlapping
mechanisms to prevent the premature activation of the orb
autoregulatory loop. The first is to limit the poly(A) tail length of
orb mRNAs. In cup mutants orb mRNAs have longer poly(A) tails
than in wild type. The second is to limit the accumulation of
hyperphosphorylated (activated) Orb protein isoforms. In wild
type ovaries, there are two Orb isoform populations, hypo- and
hyperphosphorylated, that differ in the extent of phosphorylation
and in their activity . Most of the Orb protein in wild type
ovaries is hypophosphorylated. In contrast, in cup ovaries there is
a shift in the isoform distribution and most the Orb protein is
hyperphosphorylated. In addition to these effects on orb
expression and post-translational modifications, we find that
cup is required for the proper localization and stability of orb
Cup associates with Orb in vivo
To identify components of the machinery that regulates Orb
activity or localization, we searched for proteins that associate with
Orb in vivo. For this purpose, we tested candidate proteins that
were detected in a previous mass spectrometry analysis on Orb-
immunoprecipitated wild type ovary extracts . To enrich for
proteins that are associated with Orb because they are in the same
protein complexes rather than being linked together via an RNA
bridge, we immunoprecipitated in the presence of RNase A. The
proteins recovered from both the Orb and Dorsal immunopre-
cipitates were then analyzed by MudPIT, a mass spectrometry
technology used for identifying proteins in complex mixtures (;
see also ). Altogether, ,170 proteins were detected in Orb but
not Dorsal immunoprecipitates. Proteins that were only found in
Orb immunoprecipitates include over 30 ribosomal proteins,
PABP, the Drosophila Gld2-homolog Wisp, five predicted RNA
helicases, multiple RNA binding proteins, components of the
siRNA machinery and proteins involved in decapping and RNA
turnover like Me31B, Trailer Hitch, Not4, Enhancer of decapping
and Bicaudal-C. There were also several proteins (e.g., Encore,
Didum, Ovarian tumor and Oskar) implicated in mRNA
localization in Drosophila ovaries. We also found Cup and one of
its known partners, the initiation factor eIF4E .
To confirm the physical association between Cup and Orb, we
immunoprecipitated ovarian extracts with either Cup or Orb
antibodies and then analyzed the immunoprecipitates by Western
blotting. Orb, but not HA antibody is able to pull-down Cup
protein (Fig. 1A). The total amount of lysate, which was used in
the pull-down, and then loaded in the IP lane, was approximately
ten-fold greater than the lysate which was loaded in the extract
lane. Thus, only about 10% of the Cup protein is pulled down by
Orb antibody. While IP efficiency could account for this
difference, another factor that is likely to be important is that
Orb protein is largely restricted to the oocyte. By contrast, Keyes
and Spradling  have shown that while Cup is somewhat
enriched in the oocyte especially in early stages of oogenesis, there
are nevertheless substantial amounts of Cup protein in nurse cells.
In the converse experiment, we found that Cup antibody co-
immunoprecipitates Orb protein (Fig. 1B). This association also
does not depend upon an RNA bridge as RNase A treatment does
not disrupt the Cup-Orb association. In fact, the amount of Orb
pulled down in Cup immunoprecipitates seems to be greater when
RNAse is present during the immunoprecipitation, compared to
when RNAse is not present. These findings may indicate that the
epitopes recognized by the Cup antibodies are occluded in native
Orb-Cup complexes that contain mRNAs (and in some cases
polyribosomes; ). When these complexes are treated with
RNase, they may rearrange or lose factors that prevent
immunoprecipitation of Orb with Cup antibodies.
cup negatively regulates orb
To determine if the physical association between orb and cup is
functionally important, we tested for genetic interactions. orb is
weakly haploinsufficient for its activity in the establishment of DV
polarity in the developing egg chamber, and 5–10% of the eggs
laid by females heterozygous for the null allele orb343have
ventralized chorions due to a defect in the production of the Grk
ligand. It is possible to exacerbate this haploinsufficiency by
introducing a dominant negative transgene HD19G. HD19G
expresses an hybrid mRNA that contains b-galactosidase protein
coding sequence fused to the 39 UTR of orb mRNA . The orb
39 UTR in the chimeric mRNA competes with the 39 UTR of the
Cup Negatively Regulates Function of Orb
PLoS ONE | www.plosone.org2December 2011 | Volume 6 | Issue 12 | e28261
ovaries were equilibrated back to PBSTT by incubation in
decreasing concentrations of hybridization buffer in PBSTT. The
ovaries were blocked in 1% BSA/PBSTT for 30 minutes then
incubated in 1:5000 a-DIG for 90 minutes at room temperature,
then rinsed in PBSTT and washed in alkaline wash. The ovaries
were developed in 2% NBT/BCIP in alkaline wash.
Quantitative real-time PCR
Approximately ten pairs of ovaries were hand-dissected for each
experiment. Previtellogenic stages were separated from the post-
vitellogenic stages; only the previtellogenic stages were used in
these experiments and the post-vitellogenic stages were discarded.
Total RNA was isolated using Trizol and reverse-transcribed.
Quantitative real-time PCR was performed using primers specific
for orb and osk. To normalize the amount of mRNA in the ovaries,
actin specific primers were used as a control and as a proxy of
relative total amounts of mRNA in the wild type and cup ovaries.
The relative total amount of mRNA in the cup ovaries were
calculated as a ratio of the actin mRNA in cup mutants to the
amount of actin mRNA in the wild type ovaries. The relative
amount of orb and osk mRNA in the cup ovaries is calculated as a
ratio of amounts of orb and osk mRNA to the amounts of orb and
osk mRNA in the wild type ovaries. To compare the relative
amounts of orb and osk mRNA in the mutants relative to the wild
type, the ratio of the relative amounts of orb and osk mRNA in cup
mutants to the relative total amount of actin mRNA is calculated.
nation. Green: Encore. Red: Orb. WT: In wild type ovaries,
Encore and Orb are concentrated in the presumptive oocyte in
newly formed 16 cell cysts in the germarium. During early pre-
vitellogenic stages the localization pattern is further refined so that
only the oocyte has high levels of these two proteins. In the
germarium of cup8ovaries, Encore and Orb are distributed in most
of the cells in the 16-cell cyst (see arrow). The two proteins are also
not properly concentrated into the presumptive oocyte in many
older mutant egg chambers. Instead, several cells have high levels
of Encore and Orb (see arrowheads).
cup mutants have defects in oocyte determi-
tails of orb mRNA from wild type and cup1355(as indicated) were
analyzed using the anchored poly(A)-tail assay . Anchor
primers for reverse transcription and amplification from the
poly(A) tail were as described in , while the nested orb specific
primers (F2 and F4) were derived from the orb 39 UTR. The
amplification products were analyzed on an agarose gel and
visualized with ethidium bromide.
Poly(A) tails are elongated in cup1355. Poly(A)
the ventralization phenotype of HD19Gorb343. Females
that were trans-heterozygous for HD19Gorb343and five different
alleles of cup mutants were generated by crossing HD19G orb343/
TM3Ser females (n=10 in each cross) with cup/CyO males.
Independent crosses were set up and scored at 18uC and 25uC.
The number of embryos scored at each temperature is shown in
the table. While approximately 20–30% of embryos laid by
HD19G orb343/+ females were ventralized, suppression of this
phenotype was seen in trans-heterozygotes of all five alleles.
Approximately 2.5–7.3% of embryos laid by cup mutant
transheterozygotes were ventralized at 18uC. Suppression was
weaker but still very obvious and statistically significant at 25uC,
where 8.6–20.4% of embryos were ventralized.
cup negatively regulates orb and suppresses
We thank Trudi Schu ¨pbach, Craig Smibert and Allan Spradling for
reagents. Immunofluorescence imaging was performed in the Confocal and
Electron Microscopy Core Facility, with technical assistance from Joe
Goodhouse. Quantitative real-time PCR was performed at the SynSeq
Core Facility. We greatly appreciate helpful comments from the
Conceived and designed the experiments: LCW PS. Performed the
experiments: LCW PS. Analyzed the data: LCW PS. Wrote the paper:
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