Howard Hughes Medical Institute, Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA
* Author for correspondence (e-mail: gschupbach{at}molbio.princeton.edu)
Accepted 15 January 2004
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SUMMARY |
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Key words: Drosophila, Oogenesis, hnRNP, Gurken, mRNA localization, Nurse cell nuclear morphology, Hrp48
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Introduction |
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Squid (sqd), also known as Drosophila hrp40
(Matunis et al., 1992a;
Matunis et al., 1992b
;
Matunis et al., 1994
), encodes
an hnRNP that has been characterized mainly for its role in dorsoventral (DV)
axis formation during oogenesis where it plays an important role in
gurken (grk) mRNA localization and protein accumulation
(Kelley, 1993
;
Norvell et al., 1999
). All
Drosophila hnRNPs identified so far have structural similarities to
human hnRNP A/B proteins (Dreyfuss et al.,
1993
). Sqd contains the common hnRNP structural features
(Krecic and Swanson, 1999
;
Dreyfuss et al., 2002
): two
RNA-binding domains, auxiliary glycine-rich and M9-like domains, and existence
in multiple protein isoforms.
Grk is a TGF-like ligand that is produced in the oocyte and signals
to the EGFR (Epidermal Growth Factor Receptor) expressed in all follicle cells
that comprise the epithelium surrounding the germline cells. During
mid-oogenesis, establishment of the DV axis of the egg and embryo depends on
the precise spatial restriction of grk RNA and protein to the dorsal
anterior region of the oocyte to produce localized activation of EGFR
(Neuman-Silberberg and Schüpbach,
1993
; Neuman-Silberberg and
Schüpbach, 1994
). In sqd mutants, grk RNA
is mislocalized and translated around the entire anterior of the oocyte
producing ectopic EGFR activation and induction of excess dorsal cell fates,
which results in an expansion of dorsal appendage material around the anterior
circumference of the egg (Kelley,
1993
). The three Sqd isoforms (A, B and S) are generated by
alternative splicing and differ only in their extreme C-terminal regions. They
have different subcellular localization patterns and distinct roles in the
regulated nuclear export and localization of grk mRNA, as well as in
Grk protein accumulation (Norvell et al.,
1999
). A working model for the role of Sqd in the regulation of
Grk expression as proposed by Norvell et al.
(Norvell et al., 1999
)
suggests that SqdS associates with grk RNA in the oocyte nucleus to
facilitate regulated nuclear export and RNA localization within the cytoplasm,
where SqdA then associates with the grk transcript to facilitate
translational regulation. SqdB does not appear to play a role in DV
patterning.
To identify proteins that interact with Sqd to function in the regulation of Grk expression, we performed a yeast two-hybrid screen with Sqd and isolated the hnRNP, Hrb27C. We found that hrb27C mutants have DV defects as a result of mislocalized grk RNA. Thus, it appears that Hrb27C and Sqd function together to regulate Grk expression. We have also identified a physical interaction between Hrb27C and Ovarian tumor (Otu). Examination of otu mutants reveals that otu also plays a role in regulating grk mRNA localization. sqd, hrb27C and otu mutants also share a nurse cell chromosome organization defect, indicating that these proteins function together in other processes during oogenesis. mRNA biogenesis is a multi-step process, presumably involving many trans-acting factors. We report the identification of several proteins that interact with each other and regulate both grk RNA localization and nurse cell chromosome dynamics during oogenesis.
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Materials and methods |
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Generation of Sqd antibodies, immunoprecipitations and western analysis
The full-length SqdA-GST fusion described in Norvell et al.
(Norvell et al., 1999) was
purified and used for mouse monoclonal antibody production.
Immunoprecipitations were performed according to Van Buskirk et al.
(Van Buskirk et al., 2000
)
with the following modifications: a complete mini protease inhibitor cocktail
tablet (Roche) was used in lieu of other protease inhibitors in the lysis
buffer, 1U/µl RNAse inhibitor (Roche) was added to the lysis buffer,
lysates were not pre-cleared with pre-immune serum coated beads, and lysates
were rotated with the antibody coated beads for 60 minutes at 4°C. The
following antibodies were used: monoclonal anti-Sqd serum (8F3; 3:10
dilution), monoclonal anti-SpnF serum (10D8; 3:10 dilution) (U. Abdu,
unpublished), polyclonal anti-Otu (guinea pig against amino acids 1-338; 1:10
dilution) (Glenn and Searles,
2001
) or polyclonal anti-Odd skipped (1:10 dilution)
(Kosman et al., 1998
). For
RNAse treated samples, 1 µg/µl RNAse A was added to the lysis buffer
instead of RNAse inhibitor. NuPAGE Bis-Tris pre-cast gels (4-12%; Invitrogen)
were used and the samples were transferred to nitrocellulose (Amersham) using
the Xcell II blotting apparatus with standard protocol (Invitrogen), blocked
in TBST (Tris-buffered saline + 0.1% Tween-20) + 5% milk + 1% BSA, incubated
in anti-Hrp48 (Siebel et al.,
1994
) at 1:20,000 (or anti-Sqd at 1:100), washed and incubated in
HRP-conjugated anti-rabbit antibody (Vector) at 1:7500 or HRP-conjugated
anti-mouse antibody (Jackson ImmunoResearch) at 1:10,000. After washing, the
bands were visualized by the ECL-Plus chemiluminescent system (Amersham).
Germarial western analysis was performed according to Van Buskirk et al. (Van
Buskirk et al., 2002) with 20 germaria in 50 µl of loading buffer (5 M
Urea, 0.125 M Tris [pH 6.8], 4% SDS, 10% ß-mercaptoethanol, 20% glycerol
and 0.1% Bromophenol Blue). Samples were loaded on a 7% NuPAGE Tris-Acetate
pre-cast gel (Invitrogen), transferred, blocked, probed and detected as
described above using guinea pig anti-Otu (1:500 in TBST + 5% milk) or
monoclonal anti-Tubulin (1:250 in TBST + 5% milk; Sigma T-9026). HRP-anti
guinea pig or HRP anti-mouse (Jackson ImmunoResearch) was used at 1:2000 in
TBST.
Fly stocks
The following hrb27C alleles were generously provided by Don Rio
and the Bloomington Stock Center: hrb27C [10280, rF680, k16303,
k10413, 02647, k02814]. The following alleles are lethal P-element
insertions at various distances from the coding region:
hrb27C10280 (1.5 kb), hrb27CrF680 (1.8
kb), hrb27Ck16303 (2.2 kb),
hrb27Ck10413 (2.2 kb) and hrb27C02647
(3.3 kb) (Hammond et al.,
1997). There are no molecular data on
hrb27Ck02814; hrb27C377 is an
EMS allele produced by Mary Lilly. hrb27C FRT alleles
(FRT40A-377, -rF680, -02647, -10280,
-k02814) alleles were generously provided by Mary Lilly. The FLP-DFS
(yeast flipase recombination target-site specific recombinase-dominant female
sterile) system described by Chou and Perrimon
(Chou and Perrimon, 1992
;
Chou and Perrimon, 1996
) was
used to generate germline clones of hrb27C. Progeny from yw
hsFLP; ovoD FRT40A/CyO x FRT40A-Hrb27CX (x
one of the alleles listed above) were heat shocked at 37°C for 2
hours a day for 3 days during the second and third larval instar. The
sqd1 allele is a P-element insertion that specifically
disrupts germline expression during midoogenesis
(Kelley, 1993
;
Matunis et al., 1994
). The
isoform specific transgenes of sqd in a sqd1
mutant background were described in Norvell et al.
(Norvell et al., 1999
). The
otu alleles and Df(1)RA2/FM7, which uncovers otu,
were obtained from the Bloomington Stock Center. hfp9
(Van Buskirk and Schüpbach,
2002
) was the allele used for RT-PCR and western analysis. Otu104
flies that carry a transgene expressing only the 104 kDa isoform of Otu under
the control of the otu promoter
(Sass et al., 1995
) were
provided by Lillie Searles.
In situ hybridization
Ovaries were dissected in PBS and fixed for 20 minutes in 4%
paraformaldehyde in PBS with Heptane and DMSO. Subsequent steps were performed
as previously described (Tautz and
Pfeifle, 1989) using a grk RNA probe.
Immunohistochemistry
Ovaries were fixed for 20 minutes in 4% paraformaldehyde in PBS plus
Heptane. After several rinses in PBST (PBS + 0.3% Triton), ovaries were
blocked for 1 hour in 1% BSA + 1% Triton. After a 1 hour incubation in a 1:10
dilution of monoclonal anti-Grk sera (ID12)
(Queenan et al., 1999) in
PBST, the ovaries were washed in PBST overnight at 4°C. AlexaFluor
568-conjugated anti-mouse secondary (Molecular Probes) was used at 1:1000 in
PBST. During secondary incubation, DNA was stained with 1:10,000 Hoechst
(Molecular Probes). For DNA stain alone, ovaries were dissected and fixed as
described above, incubated in 1:10,000 Hoechst for 1 hour, washed and mounted.
Only stage 6 and older egg chambers were counted to assay the nurse cell
phenotype.
UV cross-linking analysis
Fresh ovarian lysate was prepared according to Norvell et al.
(Norvell et al., 1999) with
the addition of a complete mini protease inhibitor tablet (Roche) to the lysis
buffer. Radiolabeled RNA probes were made according to Norvell et al.
(Norvell et al., 1999
) using 1
µg of linearized (HindIII for grk and osk, XbaI
for nos) DNA template. The templates for transcription include the
158 bp HincII-HindIII fragment from grk genomic DNA
cloned into pBS, the 126 bp EcoRI-DraI fragment from
osk genomic DNA cloned into pBS, and the nos+6 construct
(Gavis et al., 1996
).
Unlabelled competitor RNAs were synthesized using Ampliscribe in vitro
transcription system (Epicenter) and the final product was purified by phenol
extraction and ethanol precipitation. For the binding reaction, lysate
equivalent to approximately three ovaries was incubated with 1.5 µl of
10x binding buffer [500 mM Tris-HCl (pH 8.3), 750 mM KCL, 30 mM
MgCl2], probe (5x105 to 1x106
cpm), and water to a final volume of 15 µl. The reactions were incubated
for 15 minutes on ice, crosslinked on ice at 999 mJ in a Stratalinker UV
crosslinker. The probe was digested for 15 minutes at 37°C with 7 units of
RNAse ONE (Promega) and 1 unit of RNAse H (Roche). The immunoprecipitations
were performed after RNAse digestion, by incubation of the entire reaction
with antibody-coated [anti-Hrp48 or anti-CycE (Santa Cruz#481)] protein A/G
beads for 1 hour at 4°C. For the competition experiments, 200-fold excess
competitor RNA was incubated with the binding reaction for 10 minutes on ice
prior to addition of the probe. The concentration of competitor was determined
by UV spectroscopy. After addition of loading buffer, the samples were boiled
for 5 minutes, resolved on a 10% Tris-HCl Ready Gel (BioRad) and visualized by
autoradiography.
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Results |
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As Sqd plays a role in both RNA localization and translation regulation of
grk RNA (Norvell et al.,
1999), we wanted to know if the same was true for Hrb27C. To
determine if the mislocalized RNA in hrb27C mutants is translated, we
analyzed Grk protein expression by whole-mount antibody staining. We did
observe egg chambers where Grk was mislocalized throughout the oocyte
cytoplasm and in a ring around the entire anterior of the oocyte
(Fig. 2I), indicating that at
least in some cases, mislocalized grk RNA is translated in the
absence of Hrb27C. In many egg chambers, Grk was diffuse throughout the oocyte
with an enrichment in the dorsal anterior region, but we also observed some
egg chambers where Grk protein was localized normally
(Fig. 2G,H). However, the
occasional observation of Grk protein on the ventral side of the egg chamber
clearly indicates a lack of translational repression in hrb27C
mutants. Presumably, the egg chambers in which Grk is mislocalized in a ring
would give rise to the completely dorsalized eggs laid by hrb27C
mutant females, as this is the cause of the dorsalized eggs laid by females
mutant for sqd.
To test whether Sqd and Hrb27C cooperate during oogenesis, we tested
genetic interactions between the two genes. For this experiment, we used the
few viable transheterozygous combinations of hrb27C alleles that
produce eggs. These mutant combinations produce mildly abnormal eggshell
phenotypes, which are less severe than those produced by germline clones. The
hypomorphic allele sqdk12 also shows mild phenotypes
(Kelley, 1993).
sqd1/sqdk12 transheterozygotes produce
a range of mutant eggshell phenotypes, but the majority of these eggs are wild
type. Mutations in hrb27C can enhance the eggshell phenotype of
sqd1/sqdk12; the eggs laid by females
doubly mutant for hrb27C and sqd are much more dorsalized,
18-37% of the eggs have a crown of dorsal appendage material
(Table 1). This non-additive
enhancement of the dorsalized phenotype is consistent with Sqd and Hrb27C
cooperating in the regulation of Grk expression.
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The ovarian tumor gene can rescue the nurse cell nuclear morphology of sqd mutants
Nurse cell chromosomes that fail to disperse are also observed in certain
alleles of ovarian tumor (otu)
(King et al., 1981;
King and Storto, 1988
;
Heino, 1989
;
Mal'ceva and Zhimulev, 1993
;
Heino, 1994
;
Mal'ceva et al., 1995
).
otu produces two protein isoforms, Otu98 and Otu104, by alternative
splicing of a 126 bp exon. Genetic and molecular analyses reveal distinct
requirements for each isoform during oogenesis
(Storto and King, 1988
;
Steinhauer and Kalfayan, 1992
;
Sass et al., 1995
;
Tirronen et al., 1995
). In
particular, a mutant that specifically disrupts the Otu104 product has
persistent polytene nurse cell chromosomes, suggesting that the 98 kDa Otu
isoform is not capable of mediating wild-type chromosome dispersion
(Steinhauer and Kalfayan,
1992
). This phenotype was also described for mutants in half
pint (hfp; pUf68 FlyBase). Hfp encodes a
polyU-binding factor and plays an important role in the alternative splicing
of otu. In hfp mutants, there is a dramatic decrease in the
levels of the Otu104-encoding transcript as seen by RT-PCR analysis of
germarial RNA, and Otu104 is not detectable on a western blot. The nurse cell
phenotype of hfp mutants can be rescued by a transgene that expresses
the 104 kDa Otu isoform under the control of the otu promoter
(Van Buskirk and Schüpbach,
2002
). To determine whether defects in Otu104 cause the polytene
phenotype of sqd mutants, we assayed the ability of the Otu104
transgene to rescue this defect in sqd mutants. In
sqd1 mutants, 98% of the egg chambers (stage 6 and older)
have nurse cells chromosomes that are not dispersed. When one copy of Otu104
is expressed in a sqd1 background, only 17% of the egg
chambers have persistent polytene nurse cell chromosomes, and expressing two
copies of Otu104 leaves only 8% of the egg chambers with polytene nurse cell
chromosomes (Fig. 4F).
As Otu104 can rescue the nurse cell phenotype of both hfp and sqd, we asked whether sqd affects the alternative splicing of otu, as is the case for hfp mutants. RT-PCR analysis of RNA isolated from sqd1 germaria reveals that otu is properly spliced in sqd mutants (Fig. 4G). To determine if Otu104 protein accumulates properly in sqd1 mutants, we performed a western blot on extracts prepared from germaria of wild-type and sqd1 mutant females. Western analysis reveals that although both Otu isoforms are present in sqd1; the level of the 104 kDa isoform appears reduced in sqd1 mutants compared with wild type (Fig. 4H). Though the decrease in the levels of Otu104 observed in sqd1 mutants is not as striking as that seen in hfp mutants, the decrease is consistent over many experiments. As a single copy of Otu104 can rescue the nurse cell phenotype of sqd mutants (Fig. 4F), and the polytene nurse cell phenotype is observed in females heterozygous for a deficiency that removes otu and even in females heterozygous for the Otu104-specific mutants, otu11 or otu13 (data not shown), it seems that the level of Otu104 is crucial for proper nurse cell chromosome dispersion.
Given that hrb27C mutants share the polytene nurse cell chromosome phenotype of sqd mutants, we assayed the ability of two copies of Otu104 to rescue the phenotype of a viable transheterozygous combination of hrb27C (hrb27C377/hrb27C02647). While the Otu104 rescue of the hrb27C phenotype was not as complete as that of sqd1 mutants, we consistently observed a qualitatively improved (less blob-like) nuclear morphology. The Otu104-partial rescue of the nurse cell dispersion defect of hrb27C suggests that both Sqd and Hrb27C may act with or through Otu104 to regulate chromosome dispersal.
Otu interacts with Hrb27C and affects DV patterning
As otu mutants share a similar nurse cell nuclear morphology
defect with sqd and hrb27C, we tested whether Otu physically
interacts with these proteins. Although we have not been able to detect a
physical interaction between Sqd and Otu, we do detect a physical interaction
between Otu and Hrb27C by co-immunoprecipitation
(Fig. 5A). This interaction is
RNA independent as RNAse treatment of the lysate prior to immunoprecipitation
does not disrupt it (data not shown). It is therefore possible that Otu could
be part of the same complex with Sqd and Hrb27C through its interaction with
Hrb27C. To evaluate a potential role for Otu in grk regulation, we
analyzed the eggshell phenotype of eggs laid by otu7 and
otu11 mutant females. Indeed, although only a few mature
eggs are produced, some of these eggs are clearly dorsalized with a crown of
dorsal appendage material (Fig.
5B). otu mutant eggs show a range of dorsalization
similar to the phenotypes of hrb27C mutants (see
Fig. 2). We also assessed
grk RNA localization in otu7 mutants by in situ
hybridization and found that grk RNA is mislocalized in 34%
(n=92) of the stage 9 egg chambers
(Fig. 5C). grk RNA is
also mislocalized in egg chambers from otu11 females.
Additional support for the role of Otu cooperating with Sqd in regulating Grk
expression derives from the fact that eggs laid by females expressing two to
four additional copies of Otu104 in a sqd mutant background have
extra appendage material, but not a complete crown (data not shown). These
results suggest that Otu could be in a complex with Hrb27C and Sqd that
regulates grk RNA localization and potentially translational
regulation.
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Discussion |
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Hrb27C and Sqd interact and regulate the localization of grk mRNA
We have identified a physical and genetic interaction between Hrb27C and
Sqd. The two proteins were previously biochemically purified as part of an
hnRNP complex from Drosophila cells
(Matunis et al., 1992a;
Matunis et al., 1992b
), but it
was not known if they interact directly nor had their RNA targets been
isolated. We originally identified the interaction in a two-hybrid screen and
we have confirmed it biochemically by co-immunoprecipitation. In vivo, this
interaction requires the presence of RNA; this result was unexpected because
the yeast two-hybrid constructs that originally revealed the interaction did
not contain the RRMs and presumably cannot bind RNA. The interaction may
require the full-length proteins to be in unique conformations that are
achieved only when they are bound to RNA. The truncated proteins used in the
yeast two-hybrid screen may be folded in such a manner that the
protein-protein interaction domains are exposed even in the absence of RNA
binding, making it possible for the interaction to occur in yeast.
We also observed a striking genetic interaction between Sqd and Hrb27C;
weak hrb27C mutants can strongly enhance the DV defects of weak
sqd mutants. This suggests that the physical interaction has in vivo
significance. The phenotypes of sqd and hrb27C place both
proteins in a pathway required for proper grk localization and
suggest a previously undetected role for Hrb27C. hrb27C mutants lay
variably dorsalized eggs, and in situ hybridization analysis reveals that
grk RNA is mislocalized. As the mislocalized RNA in hrb27C
mutants is translated, causing a dorsalized egg phenotype, it is likely that
Hrb27C also functions with Sqd in regulating Grk protein accumulation. A role
in the regulation of RNA localization and translation is novel for
Hrb27C/Hrp48; it has been previously described as functioning in the
inhibition of IVS3 splicing of P-element encoded transposase
(Siebel et al., 1994;
Hammond et al., 1997
). Hrb27C
has nuclear functions and has been observed in the nucleus and cytoplasm of
somatic and germline cells in embryos
(Siebel et al., 1995
). As Sqd
is localized to the oocyte nucleus
(Matunis et al., 1994
) where
it is thought to bind grk RNA
(Norvell et al., 1999
), we
favor a model where Hrb27C and Sqd bind grk RNA together and are
exported possibly as a complex into the cytoplasm.
Two unpublished studies have implicated Hrb27C in osk RNA
localization (J. Huynh, T. Munro, K. Litière-Smith and D. St Johnston,
personal communication) and translational regulation (T. Yano, S. Lopez de
Quinto, A. Shevenchenko, A. Shevenchenko, T. Matsui and A. Ephrussi, personal
communication). Translational repression of osk RNA until it is
properly localized is essential to prevent disruptions in the posterior
patterning of the embryo (Gavis and
Lehmann, 1992; Gavis and
Lehmann, 1994
; Kim-Ha et al.,
1995
; Webster et al.,
1997
). An analogous mechanism of co-regulation of mRNA
localization and translation appears to control Grk expression. Certain
parallels between osk and grk regulation are very striking.
Both RNAs are tightly localized and subject to complex translational
regulation; they also share certain factors that mediate this regulation. In
addition to Hrb27C, the translational repressor Bruno appears to be a part of
the regulatory complexes that are required for the proper expression of both
RNAs (Kim-Ha et al., 1995
;
Webster et al., 1997
;
Norvell et al., 1999
;
Filardo and Ephrussi, 2003
). It
will be interesting to determine if there are other shared partners that
function in the regulation of both RNAs, as well as identify the factors that
give each complex its localization specificity.
Otu plays a role in grk RNA localization and interacts with Hrb27C
Otu is also involved in the localization of grk mRNA and interacts
physically with Hrb27C. Though a definitive role of Otu in regulating Grk
expression has not been previously described, Van Buskirk et al. (Van Buskirk
et al., 2002) found that four copies of Otu104 are able to rescue the
grk RNA mislocalization defect of hfp mutants. Our analysis
of the hypomorphic alleles, otu7 and
otu11, reveals a requirement for Otu in localizing
grk RNA for proper DV patterning. Alternative splicing of the
otu transcript produces two protein isoforms: a 98 kDa isoform and a
104 kDa isoform that differ by the inclusion of a 126 bp alternatively spliced
exon (6a) in the 104 kDa isoform
(Steinhauer and Kalfayan,
1992). This alternatively spliced exon encodes a tudor domain, a
sequence element present in proteins with putative RNA-binding abilities
(Ponting, 1997
).
Interestingly, the otu11 allele, which contains a missense
mutation in exon 6a (Steinhauer and
Kalfayan, 1992
), shows the grk localization and
dorsalization defect, strongly suggesting that the tudor domain of Otu plays
an important function in grk localization. As otu mutants
also show defects in osk localization
(Tirronen et al., 1995
), it
appears that like several other factors, Otu is required for both grk
and osk localization. Additionally, Otu has been isolated from
cytoplasmic mRNP complexes (Glenn and
Searles, 2001
).
Hrb27C, Sqd and Otu function in nurse cell chromosome regulation
Surprisingly, we found an additional shared phenotype of hrb27C,
sqd and otu mutants. During early oogenesis, the endoreplicated
nurse cell chromosomes are polytene. As they begin to disperse, the
chromosomes are visible as distinct masses of blob-like chromatin that
completely disperse by stage 6 (King,
1970; Dej and Spradling,
1999
). The formation of polytene nurse cell chromosomes is due to
the endocycling that occurs during early oogenesis
(Dej and Spradling, 1999
) (for
a review, see Edgar and Orr-Weaver,
2001
). The mechanism of chromosome dispersal is hypothesized to
involve the degradation of securin by the anaphase-promoting complex/cyclosome
as a result of separase activity that cleaves cohesin
(Kashevsky et al., 2002
) (for
a review, see Nasmyth et al.,
2000
). The significance of chromosome dispersion is not clear, but
Dej and Spradling (Dej and Spradling,
1999
) suggest that it could facilitate rapid ribosome synthesis. A
defect in the dispersal of polytene chromosomes has been previously described
for otu mutants (King et al.,
1981
; King and Storto,
1988
), and we have found that both sqd and
hrb27C mutants also have nurse cell chromosomes that fail to
disperse. Weak double transheterozygous allele combinations of sqd
and hrb27C produce the persistent polytene nurse cell chromosome
phenotype that is not observed in either of the weak mutants alone. Thus,
genetic and physical interactions between these gene products suggest that
they function together in regulation of this process.
The failure to disperse nurse cell chromosomes has also been observed in
mutants of hfp where the nurse cell nuclear morphology defect is due
to improper splicing of Otu104 (Van
Buskirk and Schüpbach, 2002). As was shown for hfp,
expressing Otu104 in a sqd mutant background is able to rescue the
polytene nurse cell chromosome phenotype. Although Hfp affects otu
splicing, it is not clear how Sqd functions to mediate this phenotype, but
evidence supports a role in Otu104 protein accumulation. The transcript that
encodes Otu104 is clearly present in sqd mutants, as assayed by
RT-PCR, but the level of Otu104 protein is decreased. Our results suggest that
the level of Otu104 is crucial to maintaining proper nurse cell chromosome
morphology as the polytene phenotype of sqd mutants can be rescued by
expressing only one extra copy of Otu104 and the majority of egg chambers from
females heterozygous for otu13, otu11, or a
deficiency lacking otu, have nurse cell chromosomes that fail to
disperse.
The reduced level of Otu104 in sqd mutants raises the possibility
that Sqd could translationally regulate otu RNA and that the polytene
nurse cell phenotype may be a result of the decreased level of Otu104.
Alternatively, Sqd and Hrb27C could form a complex that recruits and
stabilizes Otu104 protein, and this complex in turn functions directly or
indirectly to regulate chromosome dispersion. An indirect role seems more
likely, given that Otu has never been observed in the nucleus
(Steinhauer and Kalfayan,
1992). As Otu104 can rescue the phenotype of sqd mutants
and we can co-precipitate Otu and Hrb27C, it seems plausible that Sqd and
Hrb27C interact with Otu in the nurse cell cytoplasm to affect an RNA target
that could then mediate chromosome dispersion.
Model for a RNP complex containing Sqd, Hrb27C, and Otu
Our genetic data support a model in which Sqd, Hrb27C, and Otu function
together in a complex that affects at least two processes during oogenesis: DV
patterning within the oocyte and mediation of nurse cell chromosome
dispersion. Although our biochemical data are consistent with this model, it
is also possible that Hrb27C and Sqd could form a complex that is distinct
from a complex containing Hrb27C and Otu. However, the in vivo genetic
interactions and mutant phenotypes reveal that all three proteins affect both
processes and lead us to favor a model in which all three proteins function in
one complex.
Our results allow us to expand upon the model proposed by Norvell et al.
(Norvell et al., 1999) for the
regulation of grk RNA expression
(Fig. 6). Sqd and Hrb27C
associate with grk RNA in the oocyte nucleus. Hrb27C and Sqd remain
associated with grk in the cytoplasm where Otu and possibly other
unidentified proteins associate with the complex as necessary to properly
localize, anchor and translationally regulate grk RNA. A likely
candidate to be recruited to the complex is Bruno, which interacts with Sqd
(Norvell et al., 1999
).
Although we do not know the RNA target of Sqd, Hrb27C and Otu in the nurse
cells, the proteins could form a complex composed of different accessory
factors to regulate localization and/or translation of RNAs encoding proteins
that affect chromosome morphology.
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ACKNOWLEDGMENTS |
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Carson, J. H., Cui, H., Krueger, W., Schlepchenko, B., Brumwell, C. and Barbarese, E. (2001). RNA trafficking in oligodendrocytes. Results Probl. Cell. Differ. 34, 69-81.[Medline]
Chou, T. B. and Perrimon, N. (1992). Use of a
yeast site-specific recombinase to produce female germline chimeras in
Drosophila. Genetics
131,643
-653.
Chou, T. B. and Perrimon, N. (1996). The
autosomal FLP-DFS technique for generating germline mosaics in Drosophila
melanogaster. Genetics
144,1673
-1679.
Dej, K. J. and Spradling, A. C. (1999). The
endocycle controls nurse cell polytene chromosome structure during Drosophila
oogenesis. Development
126,293
-303.
Dreyfuss, G., Matunis, M. J., Pinol-Roma, S. and Burd, C. G. (1993). hnRNP proteins and the biogenesis of mRNA. Annu. Rev. Biochem. 62,289 -321.[CrossRef][Medline]
Dreyfuss, G., Kim, V. N. and Kataoka, N. (2002). Messenger-RNA-binding proteins and the messages they carry. Nat. Rev. Mol. Cell. Biol. 3, 195-205.[CrossRef][Medline]
Edgar, B. A. and Orr-Weaver, T. L. (2001). Endoreplication cell cycles: more for less. Cell 105,297 -306.[Medline]
Filardo, P. and Ephrussi, A. (2003). Bruno regulates gurken during Drosophila oogenesis. Mech. Dev. 120,289 -297.[CrossRef][Medline]
Gavis, E. R., Curtis, D. and Lehmann, R. (1996). Identification of cis-acting sequences that control nanos RNA localization. Dev. Biol. 176, 36-50.[CrossRef][Medline]
Gavis, E. R. and Lehmann, R. (1992). Localization of nanos RNA controls embryonic polarity. Cell 71,301 -313.[Medline]
Gavis, E. R. and Lehmann, R. (1994). Translational regulation of nanos by RNA localization. Nature 369,315 -318.[CrossRef][Medline]
Glenn, L. E. and Searles, L. L. (2001). Distinct domains mediate the early and late functions of the Drosophila ovarian tumor proteins. Mech. Dev. 102,181 -191.[CrossRef][Medline]
Großhans, J., Schnorrer, F. and Nusslein-Volhard, C. (1999). Oligomerisation of Tube and Pelle leads to nuclear localisation of dorsal. Mech. Dev. 81,127 -138.[CrossRef][Medline]
Gunkel, N., Yano, T., Markussen, F. H., Olsen, L. C. and
Ephrussi, A. (1998). Localization-dependent translation
requires a functional interaction between the 5' and 3' ends of
oskar mRNA. Genes Dev.
12,1652
-1664.
Hammond, L. E., Rudner, D. Z., Kanaar, R. and Rio, D. C. (1997). Mutations in the hrp48 gene, which encodes a Drosophila heterogeneous nuclear ribonucleoprotein particle protein, cause lethality and developmental defects and affect P-element third-intron splicing in vivo. Mol. Cell. Biol. 17,7260 -7267.[Abstract]
Heino, T. I. (1989). Polytene chromosomes from ovarian pseudonurse cells of the Drosophila melanogaster otu mutant. I. Photographic map of chromosome 3. Chromosoma 97,363 -373.[Medline]
Heino, T. I. (1994). Polytene chromosomes from ovarian pseudonurse cells of the Drosophila melanogaster otu mutant. II. Photographic map of the X chromosome. Chromosoma 103, 4-15.[Medline]
Kashevsky, H., Wallace, J. A., Reed, B. H., Lai, C.,
Hayashi-Hagihara, A. and Orr-Weaver, T. L. (2002). The
anaphase promoting complex/cyclosome is required during development for
modified cell cycles. Proc. Natl. Acad. Sci. USA
99,11217
-11222.
Kelley, R. L. (1993). Initial organization of the Drosophila dorsoventral axis depends on an RNA-binding protein encoded by the squid gene. Genes Dev. 7, 948-960.[Abstract]
Kim-Ha, J., Kerr, K. and Macdonald, P. M. (1995). Translational regulation of oskar mRNA by bruno, an ovarian RNA-binding protein, is essential. Cell 81,403 -412.[Medline]
King, R. C. (1970). Ovarian Development in Drosophila melanogaster. New York: Academic Press.
King, R. C., Riley, S. F., Cassidy, J. D., White, P. E. and Paik, Y. K. (1981). Giant polytene chromosomes from the ovaries of a Drosophila mutant. Science 212,441 -443.[Medline]
King, R. C. and Storto, P. D. (1988). The role of the otu gene in Drosophila oogenesis. BioEssays 8, 18-24.[Medline]
Kosman, D., Small, S. and Reinitz, J. (1998). Rapid preparation of a panel of polyclonal antibodies to Drosophila segmentation proteins. Dev. Genes. Evol. 208,290 -294.[CrossRef][Medline]
Krecic, A. M. and Swanson, M. S. (1999). hnRNP complexes: composition, structure, and function. Curr. Opin. Cell Biol. 11,363 -371.[CrossRef][Medline]
Mal'ceva, N. I., Gyurkovics, H. and Zhimulev, I. F. (1995). General characteristics of the polytene chromosome from ovarian pseudonurse cells of the Drosophila melanogaster otu11 and fs(2)B mutants. Chromosome Res. 3, 191-200.[Medline]
Mal'ceva, N. I. and Zhimulev, I. F. (1993). Extent of polytene in the pericentric heterochromatin of polytene chromosomes of pseudonurse cells of otu (ovarian tumor) mutants of Drosophila melanogaster. Mol. Gen. Genet. 240,273 -276.[Medline]
Matunis, E. L., Matunis, M. J. and Dreyfuss, G. (1992a). Characterization of the major hnRNP proteins from Drosophila melanogaster. J. Cell Biol. 116,257 -269.[Abstract]
Matunis, M. J., Matunis, E. L. and Dreyfuss, G. (1992b). Isolation of hnRNP complexes from Drosophila melanogaster. J. Cell Biol. 116,245 -255.[Abstract]
Matunis, E. L., Kelley, R. and Dreyfuss, G. (1994). Essential role for a heterogeneous nuclear ribonucleoprotein (hnRNP) in oogenesis: hrp40 is absent from the germ line in the dorsoventral mutant squid. Proc. Natl. Acad. Sci. USA 91,2781 -2784.[Abstract]
Nasmyth, K., Peters, J. M. and Uhlmann, F.
(2000). Splitting the chromosome: cutting the ties that bind
sister chromatids. Science
288,1379
-1385.
Neuman-Silberberg, F. S. and Schupbach, T. (1993). The Drosophila dorsoventral patterning gene gurken produces a dorsally localized RNA and encodes a TGF alpha-like protein. Cell 75,165 -174.[Medline]
Neuman-Silberberg, F. S. and Schupbach, T.
(1994). Dorsoventral axis formation in Drosophila depends on the
correct dosage of the gene gurken. Development
120,2457
-2463.
Norvell, A., Kelley, R. L., Wehr, K. and Schupbach, T.
(1999). Specific isoforms of squid, a Drosophila hnRNP, perform
distinct roles in Gurken localization during oogenesis. Genes
Dev. 13,864
-876.
Ponting, C. P. (1997). Tudor domains in proteins that interact with RNA. Trends Biochem. Sci. 22, 51-52.[CrossRef][Medline]
Queenan, A. M., Barcelo, G., van Buskirk, C. and Schupbach, T. (1999). The transmembrane region of Gurken is not required for biological activity, but is necessary for transport to the oocyte membrane in Drosophila. Mech. Dev. 89, 35-42.[CrossRef][Medline]
Sass, G. L., Comer, A. R. and Searles, L. L. (1995). The ovarian tumor protein isoforms of Drosophila melanogaster exhibit differences in function, expression, and localization. Dev. Biol. 167,201 -212.[CrossRef][Medline]
Siebel, C. W., Admon, A. and Rio, D. C. (1995). Soma-specific expression and cloning of PSI, a negative regulator of P element pre-mRNA splicing. Genes Dev. 9, 269-283.[Abstract]
Siebel, C. W., Kanaar, R. and Rio, D. C. (1994). Regulation of tissue-specific P-element pre-mRNA splicing requires the RNA-binding protein PSI. Genes Dev. 8,1713 -1725.[Abstract]
Steinhauer, W. R. and Kalfayan, L. J. (1992). A specific ovarian tumor protein isoform is required for efficient differentiation of germ cells in Drosophila oogenesis. Genes Dev. 6,233 -243.[Abstract]
Storto, P. D. and King, R. C. (1988). Multiplicity of functions for the otu gene products during Drosophila oogenesis. Dev. Genet. 9, 91-120.[Medline]
Tautz, D. and Pfeifle, C. (1989). A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma 98, 81-85.[Medline]
Tirronen, M., Lahti, V. P., Heino, T. I. and Roos, C. (1995). Two otu transcripts are selectively localised in Drosophila oogenesis by a mechanism that requires a function of the otu protein. Mech. Dev. 52,65 -75.[CrossRef][Medline]
Van Buskirk, C., Hawkins, N. C. and Schupbach, T.
(2000). Encore is a member of a novel family of proteins and
affects multiple processes in Drosophila oogenesis.
Development 127,4753
-4762.
Van Buskirk, C. and Schupbach, T. (2002). Half pint regulates alternative splice site selection in Drosophila. Dev. Cell 2,343 -353.[Medline]
Webster, P. J., Liang, L., Berg, C. A., Lasko, P. and Macdonald,
P. M. (1997). Translational repressor bruno plays multiple
roles in development and is widely conserved. Genes
Dev. 11,2510
-2521.
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