1 European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg,
Germany
2 Institut für Genetik, Heinrich-Heine-Universität Düsseldorf,
Universitätsstrasse 1, 40225 Düsseldorf, Germany
Author for correspondence (e-mail:
ephrussi{at}embl-heidelberg.de)
Accepted 4 December 2003
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SUMMARY |
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Key words: PKA-R1, oskar, Embryonic patterning, Drosophila, Oogenesis
![]() |
Introduction |
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At the posterior pole of the oocyte, Oskar plays a central role in abdomen
and germline formation. It directs the assembly of the pole plasm, which
contains factors responsible for abdomen formation, such as nanos,
and factors for the formation of the pole cells, the primordial germ cells
(Ephrussi et al., 1991;
Kim-Ha et al., 1991
). In
oskar loss-of-function mutants, no abdomen and germ cells are formed.
Conversely, mis-targeting of Oskar protein to the oocyte anterior
(Ephrussi and Lehmann, 1992
),
or overexpression of Oskar throughout the oocyte
(Smith et al., 1992
), results
in the formation of an ectopic abdomen and germ cells. These observations
reveal the importance of restricting oskar activity to the posterior
pole for correct anteroposterior patterning of the embryo.
Tight localization of Oskar protein is achieved in several steps (reviewed
by Riechmann and Ephrussi,
2001). oskar RNA is produced in the nurse cells and
transported in a translationally quiescent state into the oocyte, then
specifically to the oocyte posterior pole. There, localized oskar RNA
is translationally activated, the RNA and protein complex is anchored
(Vanzo and Ephrussi, 2002
),
and Oskar protein stabilized (Riechmann et
al., 2002
). It has been proposed that the polarized microtubule
network established by cell-cell communication between the oocyte and the
posterior follicle cells mediates transport of the RNA
(González-Reyes et al.,
1995
; Roth et al.,
1995
; Brendza et al.,
2000
). oskar RNA transport depends on cis-acting elements
in its 3'UTR (Kim-Ha et al.,
1993
), on the RNA-binding protein Staufen
(St Johnston et al., 1991
), on
components of the Exon-Junction Complex, such as Y14/Tsunagi
(Hachet and Ephrussi, 2001
;
Mohr et al., 2001
) and Mago
nashi (Micklem et al., 1997
;
Newmark et al., 1997
), and on
Barentsz, a protein enriched around the nurse cell nuclei and at the posterior
pole (van Eeden et al., 2001
).
Translational repression of the RNA during transport also requires cis-acting
elements in the oskar 3' UTR, and proteins such as Bruno and
HRP48 bind to these elements and are involved in this process
(Kim-Ha et al., 1995
;
Webster et al., 1997
) (T. Yano
and A.E., unpublished). Upon localization, translational repression of
oskar is alleviated. It has been shown that cis-acting elements in
the oskar 5'UTR (Gunkel et
al., 1998
) and Staufen protein
(Micklem et al., 2000
) are
involved in the activation of oskar translation at the posterior
pole. In addition, oskar RNA is subject to cytoplasmic
polyadenylation, which is mediated by ORB, the Drosophila CPEB
(cytoplasmic polyadenylation element binding protein) homolog, resulting in an
enhancement of oskar translation at the posterior pole
(Chang et al., 1999
;
Castagnetti and Ephrussi,
2003
).
In this report, we identify the type 1 regulatory subunit of
cAMP-dependent protein kinase (Pka-R1) as a key factor in the
restriction of Oskar protein to the posterior pole. Embryos derived from
Pka-R1 germline clones show a duplication of the posterior
structures, the so-called bicaudal phenotype. This phenotype is caused by the
premature and ectopic accumulation of Oskar protein in developing
Pka-R1 mutant oocytes, in which anteroposterior polarity and
oskar RNA localization occur normally. PKA-R1 is a component of the
PKA holoenzyme complex, a Serine/Threonine kinase that mediates various
developmental processes by controlling the activity of its target proteins.
PKA regulatory subunits control PKA activity by inactivating the catalytic
subunits in the holoenzyme complex and releasing them in response to the
second messenger cAMP (Taylor et al.,
1990). We show that PKA catalytic subunit activity is upregulated
in the Pka-R1 mutant, and that the maternal-effect patterning defect
is suppressed by reducing catalytic subunit gene dosage, indicating that
aberrant accumulation of Oskar results from misregulation of PKA activity. In
addition, we show that overexpression of PKA-R1 causes a moderate reduction in
PKA activity, resulting in a significant reduction in the amount of Oskar
protein produced at the posterior pole of the oocyte. These results reveal
that PKA mediates the spatial restriction of Oskar protein during oogenesis
and that precise regulation of PKA activity by PKA-R1 is crucial in this
process.
![]() |
Materials and methods |
---|
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---|
For the rescue of 18304 mutants by Pka-R1, an EST clone encoding
the RA isoform (LD43873) was subcloned into pCaTub67MatPolyA
(Micklem et al., 1997) (a gift
of D. Ferrandon), or into pUASp2
(Rørth, 1998
). The
rescue constructs were introduced into the germline of w1118 embryos,
according to standard procedures, and the ability of the transgenes to rescue
the 18304 maternal-effect bicaudal phenotype was tested by crossing them into
the 18304/Df(3L)ME107 and 18304/ E1 mutant backgrounds. In the case
of UASp2-RA, expression in the germline was under the control of the pCOGGAL4
driver (Rørth, 1998
).
For overexpression, expression of the RA isoform of UASp2-RA was achieved
using the actinGAL4 or nanosGAL4VP16 drivers
(Rørth, 1998
) in the
w1118 background. Fertility assays and cuticle preparation were
carried out according to Filardo and Ephrussi
(Filardo and Ephrussi,
2003
).
FRT screen
An X-linked P{w+mC=lacW} element was mobilized using
P{ry+t7.2=Delta2-3} and 1129 lethal P-insertion
chromosomes were isolated over a T(2;3)CyO DTS Ubx SuDCS compound
balancer (M. Erdélyi, A. Guichet, P. Závorszky and A.E.,
unpublished). Chromosomes bearing lethal mutations (n=928) were
outcrossed over Cyo or TM3 balancers, respectively, and kept
as stocks. The lethal chromosomes were recombined with chromosomes bearing two
FRT sites close to the centromere of each chromsome arm:
P{ry+t7.2=neoFRT}40,
P{w+mW.hs=FRT(whs)}G13 with FRT sites at 40A and
42B for the second chromsome; and P{FRT(whs)}2A,
P{neoFRT}82B with FRT sites at 79DF and 82B for the third chromosome
(Chou and Perrimon, 1996).
Recombinant chromosomes (n=665) were selected based upon resistance
to G418 and lethality. Germline clones were generated using X-linked
P{hsFLP}12 and autosomal P{ovoD1-18} insertions
with the appropriate FRT sites, as described previously
(Chou and Perrimon, 1996
).
RNA in situ hybridization and immunohistochemistry
Whole-mount antibody staining of ovaries and embryos was performed
according to Tomancak et al. (Tomancak et
al., 2000), and RNA in situ hybridization of ovaries was carried
out according to Filardo and Ephrussi
(Filardo and Ephrussi,
2003
).
Northern and western blotting
Preparation of ovarian extracts and western blot analysis were perfomed as
previously described (Markussen et al.,
1995). The following antibodies were used: rabbit anti-Oskar
(1:2000) (Vanzo and Ephrussi,
2002
); mouse monoclonal anti-
-Tubulin DM1A (Sigma, 1:2000);
and rabbit anti-human PKA-R1ß (Santa Cruz Biotech, 1:200). The
specificity of anti-human PKA-R1ß for the Drosophila homolog was
confirmed by its ability to recognize Drosophila RA and RB expressed
in bacteria. Extraction of ovarian RNAs and northern blot analysis were
carried out as previously described
(Castagnetti and Ephrussi,
2003
).
EMS mutagenesis
About 200 males of a w; P{FRT(whs)}2A ru h th st sr e
ca isogenized stock were fed with 1% sucrose containing 40 mM EMS (Sigma)
overnight. As the purpose was to isolate new alleles of an existing mutant, we
used high concentrations of EMS for the mutagenesis. Flies were allowed to
recover by feeding with fresh yeast paste overnight, and were crossed to
w; TM3/TM6b virgins. In the following generation, single w;
P{FRT(whs)}2A ru h th st sr e ca/TM3 or TM6b males
were crossed to 4 w; 18304/TM3 virgins. In a first screen,
2,500 lines were tested for semi-lethality in trans to 18304. Females
trans-heterozygous for these candidate lines and 18304 were then tested for
production of embryos with a bicaudal phenotype, and e Sb flies
(P{FRT(whs)}2A ru h th st sr e ca/TM3) were used for
establishing stocks.
Mapping and identification of the 18304 locus
The 18304 bicaudal phenotype was mapped between st (73A3) and
P{FRT(whs)}2A (79D-F) by meiotic recombination.
Deficiencies in the region were tested for complementation, and three
deficiencies in chromosomal region 77E-78A1, Df(3L)ri-XT106
Df(3L)ri-XT1, and Df(3L)ME107 failed to complement
the 18304 bicaudal phenotype. Although the distal breakpoint of
Df(3L)ri-XT106 was mapped to 77E2-8 and the proximal
breakpoint of Df(3L)ME107 was mapped to 77F3 by analysis of polytene
chromosomes (FlyBase), our tests revealed that they fail to complement the
lethality of each other (data not shown). As these two deficiencies failed to
complement 18304, we concluded that they have a small overlap in the region
77E2-77F3. To analyze the breakpoints of these deficiencies, first we screened
for RFLP markers specific to the 18304 chromosome in the region 77E8-78A1. To
identify such RFLPs, a series of genomic fragments in the region were
amplified by PCR [about 2 kb of genomic DNA was amplified approximately every
5 kb in the genomic region from DNA of wild-type and
18304/Df(3L)ri-XT1 (which removes the whole region under
analysis) adult flies]. The PCR products were digested with restriction
enzymes including SauIIIA1, RsaI and HaeIII, and
fragments showing a different digestion pattern in wild-type and 18304 mutant
DNA were identified. To precisely map the breakpoints of
Df(3L)ri-XT106 and Df(3L)ME107, genomic fragments
containing RFLPs characteristic of 18304 were amplified from
18304/Df(3L)ri-XT106 and 18304/Df(3L)ME107 adult
flies by PCR, and the products digested with restriction enzymes identifying
the polymorphisms. Absence of the genomic region from a deficiency was
revealed when only the pattern characteristic of 18304 was obtained, whereas
presence of the genomic region in a deficiency was revealed by a mixed pattern
of 18304 and wild-type fragments.
Kinase activity assay
Kinase assays were carried out essentially as described by Lane and
Kalderon (Lane and Kalderon,
1993). Protein concentration of extracts was determined using a
Bio-Rad protein assay kit (Bio-Rad).
![]() |
Results |
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|
|
To address which step of oskar regulation is affected in the
mutant, we first analyzed oskar RNA level and localization during
oogenesis. Northern blot analysis of ovarian poly(A)+ mRNA revealed
no detectable difference in the amount of oskar mRNA between wild
type and the mutant (Fig. 2I).
Examination of the spatial distribution of oskar RNA by in situ
hybridization showed that oskar RNA is localized correctly at the
posterior pole, and no appreciable difference between wild type and mutants
was observed (Fig. 2A,E). From
these results, we conclude that oskar RNA level and distribution are
not affected in 18304 mutant ovaries. Furthermore, the proper localization of
oskar RNA also indicates that the intrinsic anteroposterior polarity
of the egg chamber is correctly established in the mutants. Dorsoventral
polarity also appears normal in the mutant, as no defect in the structure of
the eggshells is apparent (Schüpbach,
1987) (data not shown).
|
18304 encodes Drosophila Pka-R1
We mapped the 18304 locus by meiotic recombination to an interval between
st (73A3) and P{FRT(whs)}2A (79D-F) on chromosome
3L. Three deficiencies in chromosomal region 77E-78A1,
Df(3L)ri-XT106, Df(3L)ri-XT1 and
Df(3L)ME107 are semi-lethal in trans to 18304, and escaper females
produce embryos that display a bicaudal phenotype. We mapped the breakpoint of
these deficiencies using RFLP markers specific to the 18304 chromosome (see
Materials and methods). A 25 kb candidate domain was determined by the
proximal breakpoint of Df(3L)ri-XT106 and the distal
breakpoint of Df(3L)ME107 (Fig.
3A). In parallel, to characterize the locus, we conducted an EMS
screen to isolate new alleles. One line, E1, was identified as an allele of
18304. The behavior of E1 in complementation tests was indistinguishable from
deficiencies uncovering the locus, indicating that E1 is a null allele (see
Table 1). E1 is lethal over the
3 deficiencies that fail to complement the 18304 maternal-effect phenotype. As
18304 is semi-lethal over these deficiencies, E1 appears to be a stronger
allele than 18304 with respect to lethality. About 10% of the eggs derived
from E1 germline clones develop into embryos showing patterning defects (head
deletion and bicaudal), and many fail to develop a cuticle, suggesting that El
has an effect on oogenesis in addition to its role in oskar
regulation. As the bicaudal phenotype was equally penetrant in embryos derived
from 18304 germline clones and in 18304 hemizygote embryos, 18304 appears to
be a strong, if not a null allele for this phenotype. Sequencing analysis of
18304 and E1 revealed point mutations in Pka-R1 gene. In both
alleles, the mutations are in residues highly conserved among different
organisms (Fig. 3D).
|
The Serine/Threonine kinase PKA is a key mediator of the second messenger
cAMP in signaling events required for various biological processes. The PKA
holoenzyme consists of a cAMP-binding regulatory subunit (R) and a catalytic
subunit (C). It is generally described as a hetero-tetrameric complex (R2-C2),
which consists of a dimer of two identical regulatory subunits with each
subunit bound to a monomeric catalytic subunit. Upon cAMP binding, the
regulatory subunits release the catalytic subunits, relieving the active
catalytic subunits from inhibition (Taylor
et al., 1990). In Drosophila, the DC0
(Pka-C1 - FlyBase) locus encodes a catalytic subunit that has the
highest homology to mammalian PKA, whereas two other genes, DC1 and
DC2 (Pka-C2 and Pka-C3, respectively - FlyBase),
might encode alternative catalytic subunits
(Lane and Kalderon, 1993
). Two
genes encode regulatory subunits (Pka-R1 and Pka-R2 -
FlyBase) (Kalderon and Rubin,
1988
; Park et al.,
2000
). Analyses of DC0 mutants have revealed that this
gene is the major source of PKA activity in Drosophila, and that PKA
is required at various stages for normal growth and development
(Lane and Kalderon, 1993
),
learning (Davis et al., 1995
)
and behavior (Majercak et al.,
1997
), and for the control of hh signaling (reviewed by
Kalderon, 1995
). In oogenesis,
it has been shown that the DC0 gene product controls polarization of
oocyte microtubules and the integrity of the actin cytoskeleton
(Lane and Kalderon, 1993
;
Lane and Kalderon, 1994
). A
null mutant of Pka-R2 has been isolated and characterized. This
mutant is viable and fertile, and although adults show defects in behavior,
and in formation and maturation of follicle cells in the ovary, no phenotype
regarding oskar regulation has been observed
(Park et al., 2000
). In the
case of the Pka-R1 locus, 2 mutant alleles, 7I5 and
11D4, have been identified. These alleles appear to be hypomorphic,
and are viable and fertile, with defects in olfactory learning
(Goodwin et al., 1997
). These
alleles showed no maternal-effect phenotype in trans to
Pka-R118304 or Pka-R1E1, or as
hemizygotes (data not shown).
To analyze the expression of PKA-R1 in the Drosophila ovary, we
performed western blotting using an antibody raised against human
PKA-R1ß. We detected a single band of 50 kD
(Fig. 3C), which is in
agreement with what has been proposed to correspond to RA
(Goodwin et al., 1997).
Consistent with this, bacterially expressed Drosophila RA migrates as
a 50 kD band on SDS gels, whereas RB and RD migrate as
40 kD bands (data
not shown). Thus it appears that, at the protein level, RA is the only
expressed PKA-R1 isoform in the ovary. These observations, and the results of
our genetic rescue of Pka-R1 using RA transgenes, suggest that RA is
the functional isoform with regard to the maternal-effect bicaudal phenotype.
Subcellular analysis of the distribution of PKA-R1 during oogenesis revealed
that PKA-R1 protein is present in the cytoplasm of nurse cells, of the oocyte
and of follicle cells, and that the protein accumulates at cell membranes
(Fig. 3E-G).
Upregulation of PKA activity in the Pka-R118304 mutant causes the maternal-effect bicaudal phenotype
To address the effect of Pka-R118304 on regulation of
PKA activity, we carried out kinase activity assays on protein extracts of
Pka-R118304 hemizygotes and wild-type flies.
Pka-R118304 mutant extracts show slightly elevated levels
(about 1.2-fold) of PKA activity in the absence of exogenous cAMP, indicating
an upregulation of PKA activity in the mutant. The mutant extracts show an
increase in PKA activity in response to an addition of exogenous cAMP, and
showed a 1.5- to 2-fold elevation in activity compared with wild-type extracts
(Fig. 4), revealing an excess
of total PKA catalytic subunit activity in the Pka-R118304
mutant.
|
|
|
![]() |
Discussion |
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Our analysis demonstrates a requirement for precise modulation of PKA activity in the Drosophila germline for correct spatial distribution of Oskar protein. We have shown that, in the Pka-R118304 mutant, where PKA activity is upregulated, Oskar protein is overexpressed and accumulates ectopically thoughout the oocyte, although oskar RNA localization and levels are normal. Dorsoventral patterning is correctly established. Anterior patterning also appears normal in the mutant, as revealed by the fact that Pka-R118304, oskar double mutants develop a normal head and thorax. In addition, we have shown that overexpression of the regulatory subunit causes a modest reduction in PKA activity, without affecting oskar RNA localization in the ovary. In such egg chambers, Oskar is underexpressed, suggesting that PKA activity is indeed required for Oskar expression. Taken together, our results suggest that, in addition to its role in the establishment of microtubule polarity and actin cytoskeleton integrity, PKA has a positive role in the regulation of Oskar protein expression at the posterior pole.
Regulation of PKA by PKA-R1 is required during Drosophila development
We observe that, in addition to defects in oogenesis, both of our
Pka-R1 alleles have reduced viability, indicating that the control of
PKA activity by PKA-R1 is required in several developmental processes. It has
been shown that Pka-R1 is expressed throughout the cell-body layer of
the central brain and optic lobes, and strongly accumulates in mushroom bodies
(Goodwin et al., 1997).
Analysis of hypomorphic alleles has revealed that Pka-R1 is involved
in olfactory learning (Goodwin et al.,
1997
) and courtship conditioning
(O'Dell et al., 1999
). The
novel Pka-R1 mutants we have described appear to be stronger alleles,
and their analysis should prove useful for investigating the role of
Pka-R1 in brain development and function. In addition, the role of
PKA in different process of development has been investigated by making use of
mutants in DC0, PKA-R2, and factors that modulate cAMP levels such as
dunce and rutabaga. For example, it has been demonstrated
that PKA antagonizes hh signaling by phosphorylating and inactivating
the downstream transcription factor Cubitus interruputus
(Wang et al., 1999
;
Price and Kalderon, 1999
). In
addition, PKA-mediated signaling was shown to be involved in learning and
behavior, and drug responses. Regulation of PKA by PKA-R1 is likely to be
crucial in these processes as well.
The Pka-R118304 mutant phenotype is caused by a defect in PKA repression
The increase in PKA activity in Pka-R118304 mutant
extracts and the suppression of both the semi-lethality and the
maternal-effect bicaudal phenotype by reduction of a functional copy of the
DC0 catalytic subunit reveals that the phenotype of
Pka-R118304 is due to its failure to repress PKA activity
in the mutant. Release of active PKA catalytic subunits from the inactive PKA
holoenzyme is controlled by cAMP levels. It is also known that free catalytic
subunits are more susceptible to proteolytic degradation than are catalytic
subunits in the holoenzyme complex (Park
et al., 2000). We observed an excess of PKA catalytic activity
both in the absence and the presence of exogenous cAMP in the mutant extract,
suggesting that upregulation of PKA catalytic subunit activity in
Pka-R118304 is due to a defect of mutant PKA-R1 in
inhibiting catalytic subunit activity. In addition, the mutant extract still
shows an increase in PKA activity in response to cAMP, which suggests the
existence of a holoenzyme complex in the mutant. This is likely to be the
case, as the point mutation in Pka-R118304 is in a
conserved arginine in the `inhibitory domain' that acts as a catalytic unit
pseudosubstrate (Gibson et al.,
1997
). However, it is also possible that an autoregulatory
feedback loop controlling expression or stability of catalytic subunits
contributes to the increase in total PKA activity.
A PKA-dependent mechanism regulates Oskar expression and anteroposterior patterning
The premature and ectopic accumulation of Oskar in
Pka-R118304 suggests a role for PKA-R1 in oskar
localization-dependent translation. An alternative explanation is that PKA-R1
is involved in the control of Oskar protein stability. In the case of C.
elegans, some germ plasm components are excluded from the somatic cells
by cullin-dependent degradation (DeRenzo et
al., 2003). A similar process might operate to restrict Oskar to
the posterior pole. This assumes the existence of a mechanism whereby
precociously and ectopically translated Oskar is degraded, and that this
process requires PKA-R1 and its inhibition of PKA activity. However, there is
no evidence to date of translation of oskar RNA prior to its
posterior localization (Markussen et al.,
1995
; Rongo et al.,
1995
; Gunkel et al.,
1998
), or of active degradation of mislocalized Oskar
(Ephrussi et al., 1991
;
Smith et al., 1992
;
Kim-Ha et al., 1995
;
Riechmann et al., 2002
).
We have previously shown that Oskar degradation is inhibited by
phosphorylation (Riechmann et al.,
2002). We also showed that both the protection and the degradation
machineries operate throughout the oocyte and not just at the posterior pole.
As nucleotide substitutions in the 3'UTR of oskar lead to its
ectopic translation and detectable accumulation
(Kim-Ha et al., 1995
), under
normal circumstances, oskar translational regulation seems fairly
tight, and is responsible for the specific accumulation of the protein at the
posterior. Therefore we speculate that the ectopic accumulation of Oskar in
PKA-R1 mutants reflects a role of PKA in activation of oskar
translation at the posterior pole, and that phosphorylation of translation
regulatory proteins by PKA might cause the release of oskar mRNA from
translational repression outside of the posterior domain.
Lane and Kalderon demonstrated that PKA is involved in the reception of the
signal from the posterior follicle cells for the establishment of oocyte
polarity at mid-oogenesis, specifically for the destabilization of the
posterior microtubule organizing center
(Lane and Kalderon, 1994). The
signal from the posterior follicle cells might activate PKA at the posterior
pole of the oocyte, and this local activation might in turn be responsible for
the localized activation of oskar expression. It has been shown that
the PKA holoenzyme can be targeted to specific subcellular domains by
association with A-kinase anchoring proteins (AKAPs)
(Huang et al., 1997
) (reviewed
by Colledge and Scott, 1999
), a
mechanism that has been proposed to regulate the spatial distribution of PKA
activity. It is tempting to speculate that, in wild-type egg chambers, PKA
holoenzyme complexes are targeted to specific AKAPs through PKA-R1, which
blocks phosphorylation of specific targets, thus preventing ectopic expression
of Oskar outside of the posterior domain. However, an alternative explanation
is possible, whereby it is not PKA, but rather a PKA target involved in
oskar activation that is asymmetrically distributed, with an
enrichment at the posterior pole - as is the case for oskar mRNA.
Uniformly moderate levels of PKA activity throughout the oocyte would result
in phosphorylation/activation of the target exclusively at the posterior pole;
over-activation of PKA throughout the oocyte would result in activation of the
target protein throughout the oocyte, which would be sufficient for ectopic
activation of Oskar expression outside of the posterior pole. To address these
possibilities, and to address directly the mechanism by which PKA controls the
spatial restriction of oskar, it will be important to visualize the
localization of the kinase activity, and to identify and determine the
subcellular localization of the targets of PKA in this process.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
![]() |
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