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First published online 16 April 2008
doi: 10.1242/dev.013656
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Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur 208016, India.
* Author for correspondence (e-mail: subbu{at}iitk.ac.in)
Accepted 18 March 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Caenorhabditis elegans, Translational control, RNA-binding protein, 3'UTR, Germ cells, Nanos
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Dean et al.,
2002). Similarly, the translational control of maternal mRNAs such
as glp-1, apx-1 and pal-1 are essential for fate
specification of certain blastomeres of Caenorhabditis elegans embryo
(Evans and Hunter, 2005).
Genetic studies in flies point to the functioning of cascades of
translational control during embryogenesis
(Kuersten and Goodwin, 2003).
For example, development of posterior structures depends on the restriction of
hunchback translation to the anterior by Nanos
(Sonoda and Wharton, 1999).
Nanos translation, in turn, is restricted to the posterior by the action of
Smaug and Oskar (Dahanukar et al.,
1999; Smibert et al.,
1999). Once again, translational control restricts Oskar to the
posterior (Gunkel et al.,
1998). However, barring a few examples of translational cascades
characterized in Drosophila, their role during embryogenesis still
remains largely unexplored. Protein factors involved in the translation of
several maternal mRNAs are not known. Similarly, the target mRNAs for many
maternal RNA-binding proteins have yet to be identified. Identification of
these will be essential to obtain a complete picture of translational control
in development.
The development of primordial germ cells (PGCs) in C. elegans is a
good example of an embryonic process involving complex translational control.
In this organism, the maternal components required for germ line development
are sequestered to a single cell at the first embryonic division itself
(Seydoux and Strome, 1999).
However, the formation of PGCs is postponed to a later stage. This is because,
as shown in Fig. 1, the
posterior lineage, which preserves germline-specific maternal components,
gives rise to various somatic lineages during the first four divisions.
Therefore, the maternal mRNAs essential for the activation of germ
cell-specific developmental programs must remain translationally quiescent
through various developmental events from oocyte until the formation of the
germline founder cell P4, which is born at the 28-cell stage.
Although the CCCH-type zinc finger protein PIE-1 has been shown to be
essential for RNA maintenance in germline blastomeres
(Tenenhaus et al., 2001), it
is not clear how the translational quiescence is maintained.
The maternal mRNA encoded by nos-2, a C. elegans member
of the nanos family of germ cell regulators, is currently the only
known mRNA whose translation is specifically activated in P4
(Subramaniam and Seydoux,
1999; D'Agostino et al.,
2006). Earlier results have shown that the translation of
nos-2 is repressed from oocytes until 28-cell embryo, and that this
repression requires the functions of three distinct 3'UTR elements. It
has also been shown that the CCCH-finger protein, POS-1, is essential for the
activation of translation in P4
(D'Agostino et al., 2006).
Here, we report the identification of four additional maternal RNA-binding
proteins, namely OMA-1, OMA-2, MEX-3 and SPN-4, which suppress nos-2
translation in successive stages: OMA-1 and OMA-2 (suppress in oocytes), MEX-3
(in early embryo) and SPN-4 (in germline blastomeres). We find that these
proteins suppress translation by directly binding to nos-2
3'UTR. Furthermore, our results presented here suggest that POS-1
activates nos-2 translation in P4 by competing out SPN-4
for binding to nos-2 3'UTR. Thus, temporal changes in the
concentration of these maternal RNA-binding proteins appear to mediate the
PGC-specific activation of nos-2 translation.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), except that
all transgenic lines were kept at 25°C to avoid silencing of transgene
expression in the germline (Strome et al.,
2001). Transgenes were introduced into unc-119(-) strain
by biolistic bombardment as described
(Praitis et al., 2001), with
the following modifications: 1 µm tungsten particles were used as the micro
carrier with 1500-psi rupture discs. Mutant versions of the transgene were
created by PCR and inserted into the plasmid, pKS111His
5, which
contains the GFP:H2B:nos-2 3'UTR
(D'Agostino et al., 2006). The
following strains were used: EU769, spn-4(or25) unc-42(e270) V/nT1[let-?(m435)] (IV;V); JJ1014, mex-3(zu155) dpy-5(e61)/hT1 I; pos-1(zu148) unc-42(e270)/hT1 V; JJ462, +/nT1 IV; pos-1(zu148) unc-42(e270)/nT1 V.
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). Other target open reading frames (ORFs) were PCR amplified,
inserted into the RNAi feeding vector, L4440 and introduced into E.
coli HT115. These E. coli clones were used for inducing RNAi by
the feeding method (Timmons et al.,
2001) in transgenic worms carrying pKS111His5.
Protein expression and purification
Full-length ORFs of mex-3 was PCR-amplified and inserted at the
BamHI site of pMAL-c4E, which expresses the inserted ORF as a fusion
protein with the maltose-binding protein (MBP) (New England Biolabs). The ORF
of spn-4 was cloned between EcoRI and XhoI sites,
and the ORFs of oma-1 and oma-2 between EcoRI and
NotI sites of pGEX-4T1 vector. The pos-1 ORF was inserted
between EcoRI and BamHI sites of pGEX-2T. The pGEX vectors
express the cloned ORF as GST fusion protein (GE Lifesciences). Cloning
techniques, including PCR, were carried out following standard protocols
(Sambrook et al., 1989).
The transformants were grown in LB medium at 37°C until 0.5 OD at 600 nm before induction with 0.05 mM IPTG for 2 hours at 16°C. Cells were collected by centrifugation and lysed in lysis buffer [20 mM HEPES (pH 7.4), 0.5 M NaCl, 5 mM DTT, 0.02% Tween 20, 0.1 mM PMSF] by incubation on ice with 0.5 mg/ml of lysozyme, followed by 3 rounds of freeze-thaw cycles. The lysates were treated with 20 µg/ml of DNase I and cleared by centrifugation. Fusion proteins were purified from clear supernatants by affinity chromatography using either amylose resin (MBP:MEX-3) or glutathione-agarose (GST fusions) following manufacturers' protocols (MBP, New England Biolabs; GST, GE Lifesciences). Purified proteins were concentrated by ultrafiltration, added with glycerol to a final concentration of 50% and stored at -20°C.
Electrophoretic mobility shift assay
Radiolabeled RNA fragments used for EMSA were prepared by in vitro
transcription of DNA template using T7 RNA polymerase (Fermentas) with
-32P CTP (specific activity: 3000 Ci/mmole) following
standard protocols (Sambrook et al.,
1989). Full-length transcripts were purified from urea gel and
quantitated using a liquid scintillation counter. Template DNAs were generated
by PCR amplification using appropriate primers from pKS111His5. The T7
promoter sequence was incorporated to DNA templates through the forward PCR
primer. Required mutations were also introduced through PCR primers. A 360 bp
cDNA fragment encoding the splicing factor (GenBank accession # AW828516) of
Meloidogyne incognita, a parasitic nematode, was used as template for
generating the non-specific unlabeled RNA. This RNA is not GC rich and, using
the M fold RNA folding program (Zuker,
2003), we found that it does not form long stretches of stable
double-stranded structures (data not shown). Unlabeled RNA was prepared in the
same manner as above except that the -32P CTP was replaced
with CTP.
Binding reactions were carried out by incubating the appropriate RNA and protein in RNA-binding buffer [5 mM HEPES (pH 7.5), 25 mM KCl, 2 mM MgCl2, 1 mM EDTA, 2 mM DTT, 3.5% glycerol, 0.25 mg/ml yeast tRNA] at room temperature (RT) for 20 minutes. RNA was denatured by first incubating at 75°C for 10 minutes and then at 37°C for a further 10 minutes, before adding to the binding reactions. All lanes contained identical amounts of RNA and protein, except where indicated. For competitions, protein was incubated simultaneously with radiolabelled RNA and indicated amounts of unlabeled RNA. The reaction mixtures were eletrophoresed at +4°C at 200 V on a 16 x 20 cm non-denaturing polyacrylamide gel in TBE buffer. The concentration of acrylamide-bisacrylamide mix in these gels was 3.5% in the case of MEX-3, 6% in the case of OMA-1 and OMA-2 fusion proteins, and 7.5% in the case of POS-1 and SPN-4 fusion proteins. Duration of electrophoresis varied, depending on the size of the RNA, from 4 to 20 hours. Following electrophoresis, the gel was dried and exposed to phosphor imager screen and imaged using a phosphor imager (Personal Molecular Imager FX, BioRad). Intensity of radioactive bands were quantitated using the Quantity One software (BioRad).
Pull down assay
This assay, similar to the affinity purification of fusion proteins
described above, depends on the affinity of GST and MBP for their
corresponding ligands, glutathione and amylose, respectively. For binding
experiments with POS-1, glutathione-agarose beads were first washed three
times in distilled water, then five times in RNA-binding buffer (RBB). Washed
beads were incubated with GST::POS-1 at +4°C for 20 minutes with gentle
agitation. Protein-bound beads were incubated with RNA in RBB for 20 minutes
at room temperature. After the incubation period, the beads were collected by
brief centrifugation and washed five times with RBB. The GST::POS-1 protein
was eluted from beads with 20 mM glutathione and the bound RNA was separated
by phenol:chloroform extraction. The RNA was then precipitated and separated
on a 6% acrylamide gel containing 8 M urea. The gel was dried and exposed to
phosphor imager screen as described earlier. Binding experiments with MEX-3
were performed in a similar manner, except that amylose resin and maltose were
used as the solid matrix and eluant, respectively.
Immunofluorescence
Embryos permeabilized by the freeze-crack method and fixed in formaldehyde
were immunostained as described
(Subramaniam and Seydoux,
1999). The following primary antibodies were used: anti-POS-1
(Tabara et al., 1999) and
anti-SPN-4. Anti-SPN-4 antibodies were obtained by affinity purification of
polyclonal antiserum of rabbits immunized with GST:SPN-4. Immunofluorescence,
as well as the GFP fluorescence from embryos was imaged using a fluorescence
microscope (Zeiss Axioskop) and CCD camera (Axiocam HRm). Immunofluorescence
signal intensities were quantitated by measuring pixel density of deconvoluted
z-stack images using Axiovision software.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). This
screen identified four genes, namely oma-1, oma-2, mex-3 and
spn-4, the downregulation of which by RNAi resulted in misexpression
of GFP:H2B (Fig. 2A,B). In the
non-RNAi control embryos, GFP:H2B was not detected in any of the blastomeres
until the 28-cell stage. Similar to endogenous NOS-2 expression, GFP:H2B first
appeared at the 28-cell stage in the germline founder cell P4. By
contrast, in the case of oma-1(RNAi) oma-2(RNAi) `double mutants',
significantly higher levels of GFP:H2B were first detected in oocytes. As
OMA-1 and OMA-2 are essential for oocyte maturation, their absence leads to
oocyte arrest (Detwiler et al.,
2001). To test whether the increased GFP:H2B expression was merely
a result of accumulation of GFP in the arrested oocytes, we introduced the
GFP:H2B:nos-2 3'UTR transgene into tra-2(q122) worms,
another mutant in which the unmated hermaphrodites accumulate oocytes
(Barton et al., 1987), and
examined the expression of GFP:H2B in their oocytes. As shown in
Fig. 2A, these oocytes did not
show any increase in the level of GFP:H2B over wild-type control. By contrast,
removal of OMA-1 and OMA-2 proteins in these worms by RNAi led to a dramatic
increase in the level of GFP:H2B in their oocytes. Expression of GFP:H2B in
oma-1(RNAi) and oma-2(RNAi) `single mutants' were almost at
the level of non-RNAi control oocytes, which is probably a result of
functional redundancy between these two nearly identical genes
(Fig. 2A). We conclude OMA-1
and OMA-2 function redundantly to suppress nos-2 translation in the
oocyte.
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;
Gomes et al., 2001). From
these results, we conclude mex-3 and spn-4 are essential for
the suppression of nos-2 translation in the early embryo and probably
not essential in oocyte.
POS-1 de-represses, rather than activates, nos-2 translation
Earlier we had shown that the CCCH-type zinc-finger protein POS-1 is
required for the activation of nos-2 translation in the primordial
germ cells (PGCs) (D'Agostino et al.,
2006). The POS-1 protein could function either by activating
translation or by relieving the translational repression by repressors such as
MEX-3 and SPN-4. To distinguish between these two possibilities, we determined
the epistatic relationship among these genes. If POS-1 were to be required for
the activation of nos-2 translation, then the ectopic GFP:H2B
expression observed in embryos lacking MEX-3 or SPN-4 should be dependent on
POS-1. However, if it functions as a derepressor, then GFP:H2B expression in
mex-3(RNAi) or spn-4(RNAi) embryos would not be dependent on
POS-1. To ensure complete absence of POS-1 protein, we used the null allele,
pos-1(zu148) (Tabara et al.,
1999), rather than pos-1(RNAi), in these epistasis
analyses. The pattern of GFP:H2B observed in mex-3(RNAi) pos-1(zu148)
embryos was similar to that of mex-3(RNAi) and that in
spn-4(RNAi) pos-1(zu148) embryos was similar to that of
spn-4(RNAi) (Fig. 2C),
indicating that the POS-1 protein is not required for nos-2
translation in the absence of MEX-3 or SPN-4. We conclude that POS-1
derepresses, rather than activates, nos-2 translation.
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). As
shown in Fig. 3, all five
proteins retarded the electrophoretic mobility of nos-2 RNA. In case
of MEX-3, SPN-4, OMA-1 and OMA-2, incubation with 50-fold molar excess of
unlabeled nos-2 3'UTR RNA completely abolished the shift of the
radiolabeled RNA, whereas a similar molar excess of non-specific unlabeled RNA
did not affect this mobility shift (Fig.
3A). In the case of POS-1, although the non-specific RNA did
reduce the radioactive signal corresponding to mobility shift, its effect was
far less than the unlabeled nos-2 3'UTR RNA
(Fig. 3B). To validate these
results further, we performed an alternative RNA-binding assay for POS-1 and
MEX-3. This assay depends on the affinity of the fusion tags GST and MBP for
their corresponding ligands covalently linked to a solid matrix (see Material
and methods for details). Even in this assay, 50-fold molar excess of
unlabeled nos-2 3'UTR RNA significantly reduced the amount of
radiolabeled RNA bound to POS-1 or MEX-3. By contrast, a similar molar excess
of a non-specific unlabeled RNA did not alter binding of the radiolabeled RNA
(Fig. 3C). We used PUF-8,
another RNA-binding protein, as a negative control; this protein did not bind
nos-2 3'UTR RNA in either of the in vitro binding assays (data
not shown). Based on the above results, we conclude that the five proteins
identified in our RNAi screen can specifically and directly bind to the
nos-2 3'UTR in vitro. As OMA-1 and OMA-2 share a high degree of
sequence homology and their electrophoretic mobility shift patterns were
identical, we tested only OMA-2 in the subsequent EMSAs. We introduced a series of deletions in the 200 bp minimal nos-2 3'UTR and tested them in EMSA to identify the specific sequences that are responsible for the interaction. Deletion of any part of the minimal UTR abolished or significantly reduced the binding of MEX-3, SPN-4 and POS-1 (data not shown), indicating that the entire sequence of the minimal UTR may be essential for efficient interaction of these three proteins. Alternatively, it is also possible that the distance between different sequence elements within the UTR, rather than the whole of the minimal UTR sequence, is crucial for proper interaction. To address this, we substituted 30 bp stretches with a non-specific sequence [(TG)15] of the same length and tested them in EMSA (Fig. 4). Of the five substitutions tested, SubB and SubC significantly reduced the mobility shift by MEX-3 and SPN-4 proteins (Fig. 4B,C), suggesting that the wild-type sequences of SubB and SubC are crucial for the binding of these two proteins. These results were further confirmed by the pull-down assay described above. In this assay, unlabeled SubC substitution did not compete with labelled wild-type RNA as efficiently as the unlabeled wild-type RNA for binding to MEX-3. Consistently, the binding of radiolabeled SubC substitution was poorer when compared with the wild type (Fig. 4F). Surprisingly, none of the substitutions had an appreciable effect on POS-1 binding (data not shown). By contrast, OMA-2 bound the region defined by SubD and SubE substitutions as efficiently as the 200 bp 3'UTR (Fig. 4D). Consistent with this, in the substitution analysis, only SubE significantly reduced OMA-2 binding (Fig. 4E). The binding of SubE substitution was significantly weaker in the pull-down assay as well (Fig. 4F). These results indicate that the SubE region is sufficient for OMA-2 interaction with nos-2 3'UTR.
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)]. To test whether these two repeats are essential for the
RNA-protein interactions, we replaced these repeats with (TG)4 and
tested in EMSA. Mutations in only one of either direct repeats reduced the
mobility shift by MEX-3, which was further weakened when both repeats were
simultaneously mutated (Fig.
5B). These results indicate that the two direct repeats are
essential for the binding of MEX-3. In the case of SPN-4, although the double
mutant and the DR1 mutant significantly reduced the shift, DR2 mutations did
not affect the mobility shift, indicating a crucial role for DR1 in SPN-4
binding (Fig. 5B). Together
these results suggest that both MEX-3 and SPN-4 may bind the same region of
nos-2 3'UTR.
Binding to nos-2 3'UTR is essential for the translational suppression by MEX-3, SPN-4 and OMA-2
If the direct repeats DR1 and DR2 are required for the interactions with
MEX-3 and SPN-4 proteins, and if these proteins controlled nos-2
translation by direct interaction with nos-2 3'UTR, then DR1
and DR2 mutations should have the same effect on nos-2 translation as
that of the removal of these proteins. To test this, we prepared
GFP:H2B:nos-2 3'UTR transgene constructs with the same DR1 and
DR2 mutations used in the EMSA experiments and generated transgenic lines
expressing the mutant constructs. Mutations in either one of the repeats led
to weak GFP:H2B expression in all cells and stronger expression in a few cells
at the posterior of the embryo - a pattern identical to the
spn-4(RNAi) embryos. Although DR2 mutations did not affect the in
vitro binding of SPN-4 to nos-2 3'UTR, these results indicate
that both the direct repeats are probably crucial for the interaction in vivo,
where potential competitors are probably present (see below). By contrast,
GFP:H2B expression was uniformly stronger in all cells of the embryo when both
direct repeats were simultaneously mutated - a pattern strikingly similar to
the removal of MEX-3 (Fig. 5C).
This observation is remarkably consistent with the EMSA results described in
the previous section, in which the double mutant RNA showed considerably
weaker interaction with MEX-3 than did either of the single mutants. In
summary, the removal of MEX-3 and SPN-4 or mutations in the RNA sequence that
is essential for their binding both have very similar effects on
nos-2 translation. From these results, we conclude MEX-3 and SPN-4
suppress the translation of nos-2 mRNA by directly binding to
nos-2 3'UTR.
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),
which is contradictory to the effect of removal of OMA-1 and OMA-2 by RNAi. As
the SubE region contains a potential polyadenylation signal and the cleavage
site, we reasoned that mutations that affect this entire region would probably
interfere with the core translational machinery, leading to complete absence
of translation. To reveal potential subdomains within SubE that might be
crucial for OMA-2 interaction more specifically, we generated RNA probes
containing smaller substitutions (10-bp) of SubE and tested them in EMSA with
GST:OMA-2. As shown in Fig. 6B,
SubE-27 more severely reduced the mobility shift than the other
substitutions. Based on this, we generated a GFP:H2B:nos-2
3'UTR transgene construct carrying SubE-27 substitution and
introduced into worms. Quite remarkably, these transgenic worms strongly
expressed GFP:H2B in oocytes that showed striking similarity to the expression
pattern in oma-1(RNAi) oma-2(RNAi)
(Fig. 6C). These results
indicate that OMA-1 and OMA-2 suppress nos-2 translation in oocytes
by directly binding to the SubE region of nos-2 3'UTR.
POS-1 competes with SPN-4 for binding to nos-2 3'UTR
Two lines of evidence suggest a potential competition between SPN-4 and
POS-1 for binding to nos-2 3'UTR. First, RNAi epistasis
described earlier indicates that POS-1 acts to relieve the translational
repression by SPN-4. Second, both these proteins are present in the germline
blastomeres until P4 is born
(Tabara et al., 1999;
Ogura et al., 2003).
Therefore, we decided to test this potential competition more directly. For
this, we added both proteins simultaneously to the binding reactions and
observed the changes in electrophoretic mobility shift patterns when the
relative concentrations of these two proteins were varied. As shown in
Fig. 7A, an increase in the
POS-1 to SPN-4 ratio decreased the intensity of the band corresponding to the
RNA-SPN-4 complex with a concomitant increase in the intensity of RNA-POS-1
complex. If binding of one protein was independent of the other, then a `super
shift' resulting from the simultaneous binding of both proteins should have
been observed. By contrast, we observed partitioning of RNA between the two
proteins in a concentration-dependent manner, indicating that POS-1 and SPN-4
may indeed compete with each other for binding nos-2 3'UTR.
Next, we quantified the fluorescence intensities of P3 and
P4 cells in embryos immunostained with antibodies against POS-1 and
SPN-4, and calculated the POS-1 to SPN-4 ratio in these cells. Significantly,
this ratio in P4 was about ninefold higher than P3
(Fig. 7B,C). Taken together,
these results suggest that the higher POS-1 to SPN-4 ratio in P4
enables POS-1 to overcome the nos-2 translation repression by
SPN-4.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), acts
as a derepressor, rather than as an activator, of nos-2 translation.
Results of our in vitro binding studies show that POS-1 and SPN-4 compete with
each other for binding to the same region of nos-2 3'UTR.
Significantly, the POS-1: SPN-4 ratio increases about ninefold in
P4 over its mother P3. Based on these results, we
propose that the translational status of nos-2 in the embryonic
germline depends on the relative concentrations of POS-1 and SPN-4. According
to our model, the POS-1: SPN-4 ratio is below the threshold required for
translational activation in earlier stages, which subsequently increases above
this threshold in P4, resulting in the activation nos-2
translation.
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). Our
results show that the nos-2 mRNA is one of their direct targets.
First, simultaneous removal of OMA-1 and OMA-2 leads to nos-2
translation in oocytes. Second, both proteins interact in vitro with a short
region of nos-2 3'UTR in a sequence-specific manner. Finally, a
mutation in this region (27) that severely reduces OMA-2 binding
activates translation in vivo. In addition to these two proteins, at least one
other protein must be involved in translation repression in oocytes, for
mutations in a stem-loop at the 5' region of nos-2 3'UTR
abolishes this repression (D'Agostino et
al., 2006) and neither of these proteins interacts with the stem
loop. Surprisingly, MEX-3 and SPN-4, two other RNA-binding proteins that
interact with nos-2 3'UTR and suppress translation in the
embryo (see below), are unable to suppress nos-2 translation in the
oocyte, although both are present in oocytes
(Draper et al., 1996;
Ogura et al., 2003).
Translational repression in the early embryo
Absence of GFP:H2B in the somatic blastomeres of transgenic embryos
indicates that the translational repression mechanisms must operate in these
cells until the 16-cell stage, by which nos-2 mRNA is degraded in
these cells. At least four proteins seem to be involved in this repression:
depletion of the two nearly identical CCCH-finger proteins MEX-5 and MEX-6
(Schubert et al., 2000;
D'Agostino et al., 2006), the
KH-domain protein MEX-3 or the RRM protein SPN-4 results in the activation of
translation in all cells of the early embryo. A couple of observations
indicate that the role of MEX-5 and MEX-6 on nos-2 translation is
probably mediated via their effects on the expression of POS-1 and MEX-3: (1)
embryos lacking MEX-5 and MEX-6 express POS-1 ectopically in the anterior
cells (Schubert et al., 2000)
and this ectopic POS-1 is essential for the misexpression of GFP:H2B observed
in these embryos (Schubert et al.,
2000; D'Agostino et al.,
2006); and (2) the level of MEX-3 is significantly lower in
embryos lacking MEX-6 (Huang et al.,
2002). While MEX-3 directly interacts with nos-2
3'UTR, it is not clear whether MEX-5 or MEX-6 bind nos-2
3'UTR. We have earlier reported that the nos-2 mRNA degradation
in the somatic cells is delayed in mex-5(RNAi) mex-6(RNAi) embryos
(D'Agostino et al., 2006). A
similar delay has also been observed in mex-3(RNAi) embryos (M.R. and
K.S., unpublished). By contrast, MEX-3 and SPN-4 appear to play a more direct
role in the control of nos-2 translation. These proteins physically
interact with nos-2 3'UTR in vitro, and mutations in the
3'UTR that disrupt this interaction show strikingly similar effects on
translation in vivo as the RNAi depletion of these proteins, indicating that
they probably interact with the 3'UTR in vivo and that this interaction
is essential for the translation control of nos-2 mRNA.
At the two-cell stage, both MEX-3 and SPN-4 appear to be essential to
suppress nos-2 translation, for absence of either one leads to
activation of GFP:H2B expression. Both these proteins are present in both
cells of the two-cell embryo and they have been shown earlier to interact
physically (Huang et al.,
2002). Therefore, it is possible they act together to suppress
nos-2 translation at the two-cell stage. However, at later stages,
they appear to function independently. For example, at the four-cell stage,
although MEX-3 is primarily present only in the anterior two cells
(Draper et al., 1996), SPN-4 is
restricted to the posterior two cells
(Ogura et al., 2003). This
spatial restriction of MEX-3 appears to restrict its translational repressor
activity to the anterior cells, at least in the case of pal-1 mRNA
(Huang et al., 2002). In a
similar fashion, MEX-3 may repress nos-2 mRNA only in the anterior
cells. However, as the accumulation of SPN-4 on P granules of
mex-3(RNAi) embryos is significantly reduced (S.J. and K.S.,
unpublished), we think MEX-3 may also have an indirect role on the repression
of nos-2 translation in the posterior cells. This might explain the
expression of GFP:H2B in all cells of mex-3(RNAi) embryos. Consistent
with its spatial distribution pattern, SPN-4 appears to repress nos-2
translation primarily only in the posterior cells. In spn-4(RNAi)
embryos, the levels of GFP:H2B was significantly higher in the posterior cells
when compared with the anterior cells. Low levels of GFP:H2B seen in the
anterior cells probably results from perdurance of the protein produced at the
two-cell stage of these embryos. Thus, these proteins appear to act together
at the two-cell stage and independently at later stages to repress
nos-2 translation.
Activation of nos-2 translation in the germline founder cell
Activation of nos-2 translation in the germline founder cell
P4 depends on the presence of POS-1: although nos-2 mRNA
is present in pos-1(RNAi) embryos until the birth of PGCs, NOS-2
protein is not detected at any stage during embryogenesis
(D'Agostino et al., 2006).
However, POS-1 is not required for nos-2 translation in the absence
of the repressors MEX-3 and SPN-4. Similarly, premature activation of
translation caused by a 3'UTR mutation does not require POS-1
(D'Agostino et al., 2006).
These observations clearly indicate that POS-1 functions as a derepressor,
rather than as an activator, of nos-2 translation. Surprisingly, even
though POS-1 protein is continuously present in the P lineage starting from
the two-cell stage (Tabara et al.,
1999), it does not activate nos-2 translation until the
28-cell stage. One possible explanation for this is that POS-1 requires an
unknown P4-specific factor for its derepressor activity.
Alternatively, the ratio of POS-1 concentration to that of a repressor such as
SPN-4 may determine the translational status and this ratio in P4
probably tilts in favour of derepression. Our results support the second
model: (1) in vitro, both SPN-4 and POS-1 bind to nos-2 3'UTR,
and POS-1 competes with SPN-4 for binding to nos-2 3'UTR in a
concentration-dependent manner; and (2) quantitation of immunofluorescence
signals indicate that POS-1: SPN-4 ratio increases in the P lineage. We
propose that the POS-1: SPN-4 ratio increases in P4 above the
threshold required for the activation of nos-2 translation. Genetic
mutants or other means that alter this ratio will be essential to validate
this model.
|
;
Huang et al., 2002);
glp-1, which is negatively regulated by POS-1 and positively
regulated by SPN-4 (Ogura et al.,
2003); skn-1, which is negatively regulated by SPN-4
(Gomes et al., 2001); and
apx-1, which is positively regulated by POS-1
(Tabara et al., 1999). In
addition, MEX-3 suppresses the translation of rme-2 in the germline
stem cells of adult gonad (Ciosk et al.,
2004). Of these, the translational regulation by direct binding of
the 3'UTR has been demonstrated only in the case of glp-1 mRNA.
SPN-4 and POS-1 proteins bind at different sites within glp-1
3'UTR and mediate opposite effects on translation. Although SPN-4 binds
the temporal control region (TCR) and promotes translation, POS-1 binds the
spatial control region (SCR) and suppresses translation. Asymmetric
distribution of the two proteins in the two-cell embryo - SPN-4 is present in
both cells, but POS-1 is restricted to the posterior cell - ensures
restriction of glp-1 translation to the anterior
(Ogura et al., 2003).
Comparison of the translation control of glp-1 and nos-2
mRNAs reveal striking diversity in the translation regulation mediated by
these two proteins. Although the relative concentration of SPN-4 and POS-1 is
crucial for the translation of both these mRNAs, the final outcome is
opposite: a higher POS-1: SPN-4 ratio suppresses glp-1
(Ogura et al., 2003), but
activates nos-2. It is not clear at the moment how they promote
translation of one mRNA while inhibiting the translation of another. Some
clues emerge from the comparison of the 3'UTR sequences of
glp-1 and nos-2. There are some important differences
between these two 3'UTRs. First, both SPN-4 and POS-1 bind distinct and
relatively short regions of the glp-1 3'UTR. By contrast, they
require the entire 200 bp of nos-2 3'UTR for maximal binding.
Second, the two 8 bp direct repeats, which are crucial for SPN-4 binding of
nos-2 3'UTR, are not present in the SPN-4 binding element (TCR)
of glp-1 3'UTR. Finally, 3'UTRs of the two mRNAs do not
share any significant similarity at the sequence or secondary structure level.
Based on these observations, we propose the final outcome of translation
regulation depends on the type of 3'UTR sequence these proteins bind.
Binding of one specific 3'UTR sequence could lead to association with an
additional protein factor that might positively influence the translation
machinery, while the binding of a different RNA sequence could lead to
association with a different protein factor that might negatively influence
the translational machinery. Identification of protein partners of SPN-4 and
POS-1, and additional target mRNAs with which these two proteins directly
interact, will be helpful to test this hypothesis.
Significantly, MEX-3, SPN-4 and POS-1 have been shown to interact among
them (Huang et al., 2002;
Ogura et al., 2003). In
addition, these three proteins and the nos-2 mRNA associate with P
granules (Draper et al., 1996;
Subramaniam and Seydoux, 1999;
Tabara et al., 1999;
Ogura et al., 2003).
Presently, it is not clear whether these interactions play any role on the
translation control of nos-2 or any other mRNA. Experiments focused
on determining the importance of these interactions will be an interesting
challenge and will help us understand the mechanism(s) by which these proteins
differently influence the translation of different target mRNAs. Such an
understanding will help us explain the role of P granule-like structures
present in the germ cells of many organisms.
Translation regulation of nanos gene family members
Members of the nanos gene family are the evolutionarily conserved
regulators of germ cell development
(Kobayashi et al., 1996;
Forbes and Lehmann, 1998;
Subramaniam and Seydoux, 1999;
Koprunner et al., 2001;
Tsuda et al., 2003). In
addition to their function, even the basic aspects of the regulation of their
expression have been conserved: (1) the Drosophila, C. elegans and
zebrafish members are controlled at the translation level by mechanisms that
require 3'UTR; (2) in both Drosophila and C. elegans,
translation repression in oocytes and embryos are mediated by two distinct
regions of the 3'UTR (Forrest et
al., 2004; D'Agostino et al.,
2006). However, there is at least one major difference between the
translation regulation of Drosophila nanos and C. elegans
nos-2. The protein factors that control these two mRNAs do not share
either sequence or functional (other than the regulation of nanos)
similarity. The worm proteins OMA-1 and OMA-2, which bind nos-2
3'UTR and suppress translation in oocytes, are CCCH-type zinc-finger
proteins and are essential for oocyte maturation
(Detwiler et al., 2001). By
contrast, the fly protein Glorund, which binds nanos 3'UTR and
suppress translation in oocytes, is a hnRNP family protein and does not appear
to be essential for oocyte maturation
(Kalifa et al., 2006).
Similarly, the fly protein Smaug, which represses nanos translation
in embryos, is essential for nuclear divisions
(Dahanukar et al., 1999). By
contrast, MEX-3 and SPN-4, which repress the worm nos-2 in the
embryo, do not resemble Smaug at the sequence level and are not involved in
cell division. Consistently, the cis-elements of the two 3'UTRs also do
not share sequence similarity. These differences possibly reflect the
fundamental difference in the process of embryogenesis in these two species.
The fly zygote undergoes a series of nuclear divisions and forms a
multinucleate syncytium. During the ensuing cellularization, the first cells
to form are the PGCs, known in the fly as pole cells. By contrast, the worm
zygote does not form a syncytium. Instead, it undergoes an asymmetric cell
division generating a larger anterior cell called AB and a smaller posterior
cell called P1. Although P1 inherits the maternally
synthesized germ cell components, unlike the fly pole cells, P1 is
not a PGC. As mentioned earlier, the P lineage produces one somatic daughter
at each of first four rounds of cell division before becoming committed to PGC
fate (Fig. 1). Therefore, the
developmental contexts in which PGCs arise in these two species are different.
Consequently, the RNA-binding proteins available at these different contexts
for the translation control of nanos mRNA may not be similar. In
addition, at least some of the mechanistic details may also have diverged. For
example, although Smaug mediates translation repression by blocking
translation initiation (Nelson et al.,
2004), it also promotes mRNA degradation by recruiting
deadenylation complex (Semotok et al.,
2005). Whereas such a mechanism may operate in the somatic
blastomeres of worm embryo, an additional mechanism that does not involve RNA
degradation is essential in the P lineage to suppress translation, as
nos-2 mRNA is preserved in this lineage until the birth of PGCs.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/10/1803/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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