Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA
* Author for correspondence (e-mail: lgavis{at}molbio.princeton.edu)
Accepted 21 September 2004
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SUMMARY |
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Key words: nanos, Translational control, Translational regulation, Translational repressor, Drosophila, Oogenesis, Embryogenesis, Maternal mRNA
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Introduction |
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Synthesis of Nos at the posterior of the embryo requires localization of
maternal nos mRNA to the posteriorly localized germ plasm
(Gavis and Lehmann, 1992;
Wang et al., 1994
). When
localization of nos RNA is abolished by mutations in genes necessary
for formation of the germ plasm, such as oskar (osk) and
vasa (vas), nos translation is repressed and the
resulting embryos lack abdominal segments
(Gavis and Lehmann, 1994
).
Posterior localization of nos is inefficient, however, as the vast
majority of nos RNA fails to become localized and is distributed
throughout the embryo (Bergsten and Gavis,
1999
). Translational repression of this unlocalized pool of
nos mRNA is thus essential to restrict production of Nos protein to
the posterior.
A major theme in post-transcriptional regulation of developmentally
relevant mRNAs is its reliance on cis-acting regulatory elements located
within 3' untranslated regions (3'UTRs)
(Kuersten and Goodwin, 2003).
The mechanisms by which many of these elements function are ill defined,
however. Both posterior localization and translational repression of
nos RNA require cis-acting sequences in the nos 3'UTR
(Gavis and Lehmann, 1994
).
Translational repression of unlocalized nos is mediated by a 90
nucleotide translational control element (TCE), the function of which requires
formation of two stem-loops (II and III)
(Dahanukar and Wharton, 1996
;
Gavis et al., 1996
;
Smibert et al., 1996
;
Crucs et al., 2000
). Stem-loop
II contains a binding site for the Smaug (Smg) protein that has been
designated as the Smaug Recognition Element (SRE)
(Smibert et al., 1996
;
Crucs et al., 2000
). Mutation
of the SRE disrupts TCE function and loss of Smg results in ectopic
nos activity, indicating that Smg is a repressor of nos
translation (Dahanukar and Wharton,
1996
; Smibert et al.,
1996
; Dahanukar et al.,
1999
). Although stem-loop III is also required for TCE function,
existing evidence suggests that it acts independently of Smg. First, mutations
that disrupt base pairing in stem-loop III disrupt TCE-mediated translational
repression without affecting Smg binding. Second, the retention of TCE
function when stem-loops II and III are separated by a large spacer suggests
that the two regions of the TCE are recognized independently
(Crucs et al., 2000
). It is not
known, however, whether the two stem-loops act coordinately or make distinct
contributions to TCE function.
As a maternal RNA, nos is synthesized by the ovarian nurse cells,
and then enters the oocyte where it becomes localized to the posterior late in
oogenesis (Wang et al., 1994;
Forrest and Gavis, 2003
). Many
maternal mRNAs required for early embryonic development are maintained in a
deadenylated and translationally silent state during oogenesis. Translation of
these mRNAs is activated after fertilization by cytoplasmic polyadenylation
(Wickens et al., 2000
;
Mendez and Richter, 2001
). By
contrast, nos does not undergo a fertilization-dependent change in
polyA tail length (Sallés et al.,
1994
), suggesting that activation of nos translation may
not be temporally regulated. Although nos is translated in the nurse
cells (Wang et al., 1994
), the
issue of whether nos mRNA, either localized or unlocalized, is
translated in the oocyte remains unresolved. As Smg accumulates only after
fertilization (Dahanukar et al.,
1999
; Smibert et al.,
1999
), translational repression rather than activation of
nos may be temporally controlled.
We have now investigated regulation of nos RNA during oogenesis using a GFP-Nos fusion protein to monitor Nos translation. We find that translation of nos RNA becomes repressed at late stages of oogenesis but is activated selectively at the oocyte posterior upon localization of nos to the germ plasm. Neither Smg nor the SRE in TCE stem-loop II are required for repression of unlocalized nos RNA in the oocyte. By contrast, this repression specifically requires TCE stem-loop III. These results demonstrate that the spatial control of nos translation essential for anteroposterior patterning is initiated during oogenesis and requires a distinct ovarian repressor. Furthermore, they decipher the structural complexity of the TCE by showing that the two stem-loops correspond to temporally separable regulatory functions. Finally, we provide evidence that protein degradation contributes to spatial restriction of Nos protein during oogenesis.
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Materials and methods |
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Construction of transgenes and transgenic lines
Pnosgfp-nos and Pnosgfp-nos-tub3'UTR
The plasmid pBS-PnosGFP was created by joining a genomic
fragment containing the nos promoter and complete 5'UTR, with a
PCR engineered NcoI site at the position of the nos ATG, to
the NcoI-NotI fragment of pEGFP-N1 (Clontech), containing
the EGFP coding region, in pBS-SK (Stratagene). For fusion of
nos-coding sequences to EGFP, a genomic fragment containing the
nos coding region, 3'UTR, and 3' flanking DNA, was
modified by PCR to create a SmaI site in place of the ATG codon. The
nos fragment was joined at this SmaI site to an end-filled
BsrGI site overlapping the final EGFP codon, connecting the final
EGFP codon to the second Nos codon with the insertion of a glycine codon in
between. For Pnosgfp-nos-tub3'UTR, the
nos 3'UTR was substituted by the -tubulin
3'UTR as previously described (Gavis
and Lehmann, 1994
). Both transgenes were inserted into the CaSpeR4
P element vector (Thummel and Pirrotta,
1992
).
Transgenes were introduced into y w67c23 embryos by P
element-mediated germline transformation
(Spradling, 1986) and multiple
independent transgenic lines were isolated. A single copy of the
gfp-nos transgene was tested for complementation of the
nosBNX2.1 mutation. To generate 6 x
gfp-nos, two independent second chromosome gfp-nos
insertions were recombined. Flies homozygous for the both the recombinant
chromosome and an X chromosome gfp-nos insertion contain six copies
of the gfp-nos transgene.
The nos-tub3'UTR
(Gavis and Lehmann, 1994),
nos-tub:TCE (Gavis et al.,
1996
; Crucs et al.,
2000
), nos-tub:TCEIIA, nos-tub:TCEIIIA,
nos-tub:TCEIIIGC/GC, and nos-tub:TCEIIIA/U^C72
(Crucs et al., 2000
) transgenes
and transgenic lines have been previously described. The
nos-tub:TCE[SRE] transgene is identical to
nos-tub:TCE, except for the mutation of two nucleotides required for
Smg binding (SRE)
(Smibert et al., 1996
).
Direct GFP imaging and immunofluorescence
All images were captured with a Zeiss LSM 510 confocal microscope. To
analyze GFP-Nos distribution during oogenesis, ovaries from well-fed females
carrying either two (2x) or six (6x)copies of the gfp-nos
transgene were dissected in Schneider's insect culture medium (GIBCO-BRL).
Ovaries were quickly rinsed once in PBS, fixed for 15 minutes in 4%
paraformaldehyde/PBS, rinsed five times for 5 minutes in PBST (PBS/0.1%
Tween-20), and then incubated in the dark for 30 minutes in 1:250
Rhodamine-Phalloidin:PBST (Molecular Probes). Stained ovaries were washed
twice for 5 minutes in PBST, three times for 5 minutes in PBS, and mounted in
PBS under slight pressure using a #1.5 square glass coverslip (Corning).
To visualize Vas and GFP-Nos simultaneously, ovaries from 6
x gfp-nos females were dissected as above, fixed for 15 minutes
in 4% EM grade formaldehyde (Polysciences), rinsed in PBS/0.3% Triton X-100,
and incubated for 3 hours in PBT (PBS/0.3% Triton X-100/1% BSA) with 4% v/v
normal goat serum (NGS). The ovaries were then immunostained with 1:10,000
rabbit -Vas antibody (gift of P. Lasko) in PBT/4% NGS overnight at
4°C, washed for 2 hours with several changes of PBT/4% NGS, and incubated
for 2 hours with 1:500 Alexa-Fluor 568 goat
-rabbit antibody (Molecular
Probes) in PBT/4% NGS. The secondary antibody was preabsorbed overnight
against 0- to 2-hour-old embryos prior to use. Stained ovaries were washed for
several hours with PBS/0.3% Triton X-100, followed by PBS. To label DNA,
Hoescht dye (5 µg/ml final concentration) was added during the final PBS
washes.
Northern blot analysis
Ovaries were dissected from well fed females in PBS, washed once with PBS,
frozen in liquid N2, and stored at 80°C. Extraction of
RNA from frozen ovaries and northern blotting were performed according to
Bergsten and Gavis (Bergsten and Gavis,
1999). The blot was probed simultaneously with
32P-labeled probes for nos and rp49 RNAs as
previously described (Bergsten and Gavis,
1999
). Labeled bands were quantitated by phosphorimaging.
Immunoblot analysis
For analysis of HA-Nos levels during oogenesis, ovaries were dissected from
well-fed females in Schneider's medium and both stage 10 and stage 14 egg
chambers were carefully separated. Both total ovary and isolated egg chambers
were rinsed once in Schneider's medium, once in PBS, then frozen in liquid
N2 with minimal residual PBS. Embryos (0-2 hours) were
dechorionated and washed thoroughly before freezing in liquid N2.
Thawed tissue was homogenized in SDS lysis buffer containing 5 M urea
(Gavis et al., 1996), boiled
for 5 minutes, spun for 5 minutes in a microfuge, and the supernatants
resolved on a 10% SDS-PAGE gel. Proteins were transferred to PVDF membrane
(Millipore) and immunoblotting was carried out in 10 mM Tris-HCl pH 7.5/150 mM
NaCl/2% nonfat dry milk. Final antibody concentrations were 1:1000 rat
-HA (Roche), 1:20,000 mouse
-Snf (gift of P. Schedl), 1:2000
HRP-goat
-rat (Jackson Immunologicals) and 1:5000 HRP-sheep
-mouse (Amersham). Protein was visualized by chemiluminescence
(Roche).
In vitro translation assay
Luciferase plasmids
Luciferase reporter plasmids were constructed in a derivative of
pSP64poly(A) (Promega) that encodes a 25 nucleotide poly(A) sequence followed
by a unique NsiI site (kindly provided by D. Chagnovich and R.
Lehmann). Each reporter contains the entire nos 5'UTR fused to
the coding region of the firefly luciferase gene (Promega) and one of the
following 3'UTRs: -tubulin
(Gavis and Lehmann, 1994
),
three tandem copies of a wild-type nos TCE (Bergsten et al., 1999) or
three tandem copies of a mutant nos TCE (TCE:SRE)
(Smibert et al., 1996
);
TCEIIA, or TCEIIIA (Crucs et al.,
2000
).
In vitro transcription
Templates for in vitro transcription were prepared by digestion of
luciferase reporter plasmids with NsiI, followed by treatment with T4
DNA polymerase. Capped transcripts were generated with the mMessage mMachine
kit (Ambion). Unincorporated nucleotides and excess cap analog were removed by
a G-50 spin column (Pharmacia), and the RNA was purified by phenol extraction
and ethanol precipitation.
Translationally active embryo extract
Embryonic extracts were prepared as described previously
(Clark et al., 2000). Briefly,
fresh 0- to 2-hour-old embryos were homogenized on ice in 1 volume of Buffer A
(10 mM HEPES pH 7.5/5 mM DTT/0.5 mM PMSF). The extract was cleared by
microfuge centrifugation for 5 minutes at 4°C and supplemented with 1/9
volume of Buffer B (100 mM HEPES pH 7.5/1 M potassium acetate/10 mM magnesium
acetate/50 mM DTT). After centrifugation for 10 minutes at 4°C, 0.8 U
RNasin (Promega) and 200 µg creatine phosphokinase (Sigma) were added to
the supernatant, which was then frozen in aliquots in liquid nitrogen and
stored at 80°C.
In vitro translation reaction
Reactions (20 µl) contained 10 µl of extract, 0.1 nM luciferase
reporter RNA, 15 mM creatine phosphate (Sigma) and 4 µl of 5 x
translation buffer (125 mM HEPES pH 7.5/7.5 mM magnesium acetate/1 mM
spermidine/12.5 mM DTT/125 µM amino acids/6 mM ATP/1.5 mM GTP). Reactions
were incubated for 45 minutes at 28°C. Translation reactions in rabbit
reticulocyte extract (Promega) were carried out for 45 minutes using the
manufacturer's protocol. Enzymatic assays for luciferase were performed using
a substrate mix recommended by Promega. For each RNA, luciferase activity
produced by translation in Drosophila embryonic extract was
normalized to the value obtained after translation in reticulocyte extract, to
control for differences in quantity or quality of the RNAs.
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Results |
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Nos protein accumulates at the oocyte posterior upon localization of nos RNA
Synthesis of Nos protein at the posterior of the embryo requires
association of nos RNA with the posteriorly localized germ plasm.
However, localization of nos to the germ plasm is accomplished
earlier, during stages 11-13 of oogenesis, after nos is transferred
or `dumped' into the oocyte by the actin-dependent contraction of the nurse
cells (Forrest and Gavis,
2003). To determine whether translation of nos RNA is
initiated during oogenesis upon localization to the posterior, or only after
fertilization, we examined the distribution of GFP-Nos in late stage egg
chambers (Fig. 3). During stage
11, GFP-Nos synthesized in the nurse cells is dumped along with nos
RNA into the enlarging oocyte (Fig.
3A,D) and by stage 12, this protein is distributed uniformly
throughout the oocyte (Fig.
3B,E). During stage 13 (Fig.
3C,F), the level of GFP-Nos in the bulk ooplasm decreases (compare
Fig. 3C with 3B). As the oocyte
volume changes little between stages 12 and 13, this decrease is probably due
to protein degradation. This conclusion is indeed borne out by immunoblot
analysis (see below).
|
|
To determine whether translation of unlocalized nos RNA in late oocytes is repressed by the nos TCE, we compared the amount of Nos protein in oocytes from females carrying either the nos-tub3'UTR or nos-tub:TCE transgene (Fig. 1B). These transgenes, which differ only in the presence of the nos TCE within their 3'UTRs, produce similar levels of unlocalized RNA encoding functional, hemagglutinin epitope-tagged Nos protein (HA-Nos; Fig. 5A). Immunoblot analysis revealed accumulation of HA-Nos protein in stage 14 oocytes from nos-tub3'UTR females (Fig. 5B), consistent with analysis of gfp-nos-tub3'UTR oocytes (Fig. 4C). By contrast, little or no HA-Nos protein is detected in stage 14 oocytes from nos-tub:TCE females (Fig. 5B). nos-tub:TCE RNA is translated at earlier stages, however, as HA-Nos protein is present in total ovarian extract and stage 10 oocytes (Fig. 5B,C). Together, these results demonstrate that the nos TCE mediates translational repression of unlocalized nos RNA in late stage oocytes.
|
TCE stem-loops act differentially during oogenesis and embryogenesis
Although TCE-mediated repression initiates during oogenesis, Smg, the only
known TCE-binding factor and repressor of nos translation, is not
present in the ovary (Dahanukar et al.,
1999; Smibert et al.,
1999
). We have previously shown that mutations in TCE stem-loop
III disrupt translational repression of unlocalized nos RNA without
affecting the ability of Smg to bind to the SRE in stem-loop II
(Crucs et al., 2000
). As
repression during oogenesis must be mediated by a factor other than Smg, TCE
stem-loop III is a potential target for this factor. Alternatively, an ovarian
repressor may also recognize the SRE or may interact with a different motif in
stem-loop II.
Previous analyses of sequence and structural requirements for TCE function
in vivo examined the effect of TCE mutations on nos regulation using
phenotypic assays, in which defects in the anteroposterior pattern of the
larval cuticle provide a measure of nos activity
(Dahanukar and Wharton, 1996;
Gavis et al., 1996
;
Smibert et al., 1996
).
Consequently, these studies could not distinguish TCE-mediated repression
occurring during oogenesis from repression during embryogenesis. To determine
how the TCE mediates repression during oogenesis, we assayed the effects of
TCE mutations directly on Nos protein levels both in late oocytes and in
embryos, using nos-tub:TCE transgenes bearing mutant TCEs
(Fig. 1C). For all transgenic
lines used, comparable RNA expression levels were confirmed by northern
blotting (Fig. 5A)
(Crucs et al., 2000
).
Two mutations that alter stem-loop II and binding of Smg protein, TCEIIA
and SRE (Smibert et al.,
1996; Crucs et al.,
2000
), have no effect on TCE-mediated repression during oogenesis,
as the mutant TCEs still prevent HA-Nos protein accumulation in late oocytes
(Fig. 5B). Similarly, HA-Nos
protein cannot be detected in stage 14 oocytes from nos-tub:TCE
ovaries mutant for smg (Fig.
5D). By contrast, nos-tub:TCEIIA and
nos-tub:TCE[SRE] embryos show a dramatic increase
in the amount of HA-Nos over nos-tub:TCE embryos
(Fig. 5E) and a similar
increase occurs in nos-tub:TCE embryos mutant for smg (data
not shown). Thus, both stem-loop II and smg function are limited to
embryogenesis, consistent with the restricted expression of Smg protein.
Furthermore, the loss of anterior structures observed in larval cuticle
preparations of nos-tub:TCEIIA and
nos-tub:TCE[SRE] embryos
(Crucs et al., 2000
) (data not
shown) must result from excess Nos produced during embryogenesis.
Strikingly, mutation of stem-loop III (TCEIIIA) results in production of
HA-Nos in late oocytes (Fig.
5B), indicating that translation of nos-tub:TCEIIIA RNA
is not repressed. Phenotypic analysis showed that mutations that retain
base-pairing within TCE stem-loop III but alter the sequence of the distal
region of the stem (TCEIIIA/U^C72, and TCEIIIGC/GC, see
Fig. 1 legend) also compromise
TCE function, indicating that both the sequence and structure of stem-loop III
contribute to its activity (Crucs et al.,
2000). Indeed, these mutations disrupt repression of unlocalized
nos RNA during oogenesis, as HA-Nos is detected on immunoblots of
transgenic stage 14 oocytes (see Fig. S2 in the supplementary material). Thus,
TCE stem-loop III acts in a sequence- and structure-dependent manner to
repress translation during oogenesis. The complete lack of anterior structures
observed in cuticle preparations of nos-tub:TCEIIIA embryos
(Crucs et al., 2000
) indicates
that repression by stem-loop III during oogenesis is crucial for embryonic
development.
TCE stem-loop III mediates smg-independent repression during oogenesis
Although HA-Nos protein is also present in nos-tub:TCEIIIA embryos
(Fig. 5E), this protein may
derive solely from unregulated translation of nos-tub:TCEIIIA RNA
during oogenesis. To determine whether stem-loop III is required for
repression during embryogenesis as well as oogenesis, we took advantage of an
in vitro translation assay based on a preblastoderm embryo extract that
recapitulates TCE-mediated repression
(Clark et al., 2000). Capped
and polyadenylated luciferase reporter RNAs bearing either the control
tub 3'UTR, or three tandem copies of a wild-type or mutant TCE,
were used to program the embryonic extract and translation was monitored using
a luciferase activity assay.
As expected, we found that the SRE and stem-loop II are essential for TCE-mediated repression in vitro, as mutation of these sequences yielded luciferase levels comparable to that obtained with the tub 3'UTR (Table 1). By contrast, mutation of stem-loop III had little effect in this assay, indicating that the presence of HA-Nos in nos-tub:TCEIIIA embryos is due largely to perdurance of protein synthesized during oogenesis. Although the slight but significant decrease in repression observed for the TCEIIIA mutant suggests that stem-loop III plays a minor role during embryogenesis, this stem-loop acts primarily to promote Smg-independent translational repression of nos in late oocytes.
|
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Discussion |
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Achieving the restricted Nos protein distribution in the embryo requires
that translational activity of nos in the oocyte be spatially
regulated. However, the only known repressor of nos translation, Smg,
is absent from the ovary (Dahanukar et al.,
1999; Smibert et al.,
1999
). We have resolved this dilemma by showing that a distinct,
Smg-independent mechanism mediates translational repression of unlocalized
nos mRNA in late oocytes. Failure to repress nos in late
oocytes, as exemplified by the behavior of the nos-tub:TCEIIIA
transgene, results in unrestricted production of Nos protein that perdures to
embryogenesis. The resulting embryos die, lacking anterior structures
(Crucs et al., 2000
). Thus, by
showing that the program for spatially restricted synthesis of Nos operates
during oogenesis, our results reveal how temporal demands are reconciled with
spatial constraints on nos translation needed for embryonic
patterning.
Temporal modulation of nos repression through differential recognition of TCE stem-loops
The elucidation of temporally distinct functions of the two TCE stem-loops
explains the enigmatic structural complexity of this regulatory element. We
have previously shown that both stem-loops retain function, despite their
separation by a 52-nucleotide spacer, suggesting that they operate
independently (Crucs et al.,
2000). Indeed, phylogenetic analysis of the nos
3'UTR reveals that TCE stem-loops II and III are not juxtaposed in all
Drosophilid species (R.A.J. and E.R.G., unpublished), indicating that
the distance between stem II and III is not under tight evolutionary
constraint.
After fertilization, Smg binds to TCE stem-loop II to mediate repression in
the preblastoderm embryo (Smibert et al.,
1996; Dahanukar et al.,
1999
; Crucs et al.,
2000
). We do not know if the ovarian repressor remains bound to
stem-loop III in the embryo, but its function is superceded by Smg. A minor
requirement for stem-loop III in the embryo suggested by our in vitro
translation experiments may reflect the need to maintain the ovarian
repression mechanism until Smg reaches sufficient levels in the embryo.
Accordingly, the requirement for stem-loop III would decrease over time after
fertilization. A more significant contribution by stem-loop III might have
been missed, however, if the stem-loop III-dependent repressor is unstable in
the embryonic extract.
The smg mutant phenotype indicates that nos is not the
only target of Smg in the embryo (Dahanukar
et al., 1999). Although the ovarian repressor has not yet been
identified, we have recently isolated a candidate ovarian stem-loop III
binding factor (Y. Kalifa, T. Huang and E.R.G., unpublished) that appears to
regulate multiple maternal mRNAs. Thus, it seems likely that nos has
evolved to co-opt existing stage-specific regulatory proteins for its
advantage. We have previously shown that the nos TCE can repress
translation in subsets of cells in both the central and peripheral nervous
systems (Clark et al.,
2002
; Ye et al.,
2004
). Although the repressors are not known in these cases
either, it is possible that the ability to interact with yet additional
proteins will underlie the multifunctionality of the TCE.
Other RNAs may use a similar strategy of recognition by stage-specific
factors to maintain translational regulation across developmental transitions.
In the Drosophila oocyte, translational repression of unlocalized
osk mRNA occurs through the interaction of Bruno (Bru) with specific
sequence motifs in the osk 3'UTR
(Kim-Ha et al., 1995). As Bru
is not present in the embryo (Webster et
al., 1997
; Lie and Macdonald,
1999
), where the majority of osk mRNA remains unlocalized
(Bergsten and Gavis, 1999
), an
embryonic repressor may be required to maintain the repression initiated by
Bru. Intriguingly, the existence of binding sites for multiple, distinct
microRNAs within individual 3'UTRs
(Lewis et al., 2003
;
Stark et al., 2003
) suggests a
similar paradigm for controlling translation through multiple developmental
stages or in different tissues.
Multiple modes of regulation operate during oogenesis for spatial restriction of Nos
The translational quiescence of unlocalized nos in late oocytes
contrasts sharply with its translational activity in the nurse cells.
Deposition of both actively translated nos mRNA and the previously
synthesized Nos protein into the oocyte during nurse cell dumping presents a
challenge for restricting Nos to the posterior of the oocyte. Although we
cannot determine whether nos is repressed in oocytes prior to stage
10, our results indicate that the majority of nos RNA, which enters
the oocyte during dumping, must switch from a translationally active state in
the nurse cells to an inactive state in the oocyte. This switch could be
mediated by interaction of nos with an ovarian repressor restricted
to the oocyte. Alternatively, a repressor may bind to nos RNA in the
nurse cells, but become activated during or after passage into the oocyte.
We have previously shown that translationally repressed nos RNA is
associated with polysomes, indicating that repression is imposed at a late
step in the translation cycle (Clark et
al., 2000). However, recent evidence that Smg interacts with Cup
to prevent recruitment of eIF-4G by eIF-4E suggests that translation is
blocked at the initiation step (Nelson et
al., 2004
). The identification of a Smg-independent mechanism for
translational repression during oogenesis may explain these divergent results.
Indeed, a post-initiation mechanism may be ideally suited to rapidly repress
polysomal nos RNA entering the oocyte from the nurse cells.
In addition to translationally active nos RNA, substantial amounts of Nos protein enter the oocyte during nurse cell dumping. Perdurance of this protein to embryogenesis would probably disrupt anterior development. We find, however, that Nos protein entering the oocyte from stage 10 nurse cells is cleared from the oocyte by stage 13. Nos protein made in the nurse cells may therefore be specifically targeted for degradation. Alternatively, Nos might have a short half-life regardless of its site of synthesis. Despite considerable effort, we have not detected ubquitinated forms of Nos protein, although the transient nature of ubquitinated intermediates may preclude detection. Similarly, we have not detected a genetic interaction between mutations in numerous components of the ubiquitin degradation pathway and nos transgenes. Thus, how Nos is degraded remains an unanswered question. Regardless of mechanism, however, continuous translation of wild-type nos RNA at the posterior pole or of unlocalized nos-tub3'UTR RNA throughout the oocyte would result in accumulation of Nos protein. Thus post-translational control of Nos protein stability as well as post-transcriptional regulation of nos RNA contribute to the correct spatial distribution of Nos in the early embryo.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/131/23/5849/DC1
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