1 Genome Biology Laboratory, Center for Genetic Resource Information, National
Institute of Genetics, Mishima 411-8540, Japan
2 Department of Physiology, Tokyo Women's Medical University School of Medicine,
Tokyo 162-8666, Japan
* Present address: Department of Pharmacology, Yokohama City University School
of Medicine, Yokohama 236-0004, Japan
Author for correspondence (e-mail:
ykohara{at}lab.nig.ac.jp)
Accepted 11 March 2003
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SUMMARY |
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Key words: Caenorhabditis elegans, Maternal mRNA, Translational control, RNA-binding protein, 3' UTR
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INTRODUCTION |
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The embryogenesis of C. elegans is highly stereotyped
(Sulston et al., 1983), and
many maternal genes play important roles in the process
(Kemphues and Strome, 1997
;
Schnabel and Priess, 1997
). A
fertilized egg, P0, produces a large anterior blastomere, AB, and a
small posterior blastomere, P1. par genes determine the
anterior-posterior polarity of the one-cell stage embryos. The AB blastomere
produces the ABa and ABp blastomeres, and the P1 blastomere
produces the EMS and P2 blastomeres. The early ABa and ABp
blastomeres are identical and exchangeable
(Priess and Thomson, 1987
);
however, in the late four-cell stage, the ABp has a different cell fate
determination from that of ABa. For this determination, a Notch-like receptor,
GLP-1, and a Delta-like ligand, APX-1, are required
(Mello et al., 1994
).
GLP-1 translation is regulated temporally and spatially. In spite of the
presence of abundant levels of the maternal glp-1 mRNA in oocytes and
all blastomeres up to the eight-cell stage of C. elegans embryos,
GLP-1 is first detected in the anterior AB blastomere
(Evans et al., 1994). At the
four-cell stage, GLP-1 is detected only in the anterior blastomeres ABa and
ABp, and not in the posterior EMS and P2. A study using a reporter
RNA revealed that the 3' UTR of glp-1 mRNA (369 bases) is
important for the appropriate temporal and spatial translation of GLP-1
(Evans et al., 1994
): a
125-base temporal control region (TCR) located at the 3' end of the
3' UTR is required for the suppression of glp-1 translation in
oocytes and one-cell stage embryos. A 66-base spatial control region (SCR)
located in the middle of the UTR is required for the suppression of
glp-1 translation in the posterior blastomeres. Moreover, the
glp-1 SCR is highly conserved in related species
(Rudel and Kimble, 2001
).
Taken together, these findings suggest that unknown regulators bind the TCR or
SCR to control glp-1 mRNA translation.
APX-1 also seems to be translationally regulated. The apx-1 mRNA
is provided maternally and is present in every early blastomere. APX-1 is
first detectable in the posterior P1 blastomere at the two-cell
stage (Mickey et al., 1996).
At the four-cell stage, APX-1 is expressed in the EMS and P2
blastomeres, mainly at the membrane of P2, but never in the
anterior blastomeres, ABa and ABp. The molecular mechanism that underlies the
regulation of apx-1 mRNA translation is completely unknown.
A maternal gene product, POS-1, is involved in the translational regulation
of APX-1, because APX-1 is not detected in pos-1 embryos in spite of
the abundant apx-1 mRNA (Tabara
et al., 1999). Mutations in the pos-1 gene result in
maternal-effect embryonic lethality with abnormal cell-fate determination and
cell divisions. pos-1 embryos have little pharyngeal tissue, no
intestine, no germ cells and extra hypodermis. In addition, the P2,
P3, and P4 germline blastomeres have abnormally short
cell-cycle periods with little asymmetry. The POS-1 protein has two TIS11-type
CCCH zinc motifs and is observed in the cytoplasm of the posterior
blastomeres, including the germline blastomeres, as a temporary component of
the P granules (Tabara et al.,
1999
). However, the molecular function of POS-1 is largely
unknown.
In this paper, we report that POS-1 binds the 3' UTR of glp-1 mRNA and negatively regulates the translation of the maternal glp-1 mRNA in the posterior blastomeres. In addition, a newly identified POS-1-interacting protein, SPN-4, which has an RNP-type RNA-binding domain, also binds the 3' UTR of glp-1 mRNA, and is required for the translation of glp-1 mRNA in the anterior blastomeres. We propose that POS-1 cooperates with SPN-4 to control the maternal glp-1 mRNA translation in early embryos.
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MATERIALS AND METHODS |
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Tri-hybrid analysis
A tri-hybrid analysis was performed essentially according to Putz et al.
(Putz et al., 2000). pRevRX
was used for the expression of the hybrid RNA. For the expression of the GAL4
activation domain (AD) fusion proteins, pACT2 (for AD-POS-1), pGADT7 [for
AD-POS-1(C147Y)] and pACT (for AD-SPN-4) were used. POS-1(C147Y) has a
mutation at codon 147 C (TGC) to Y (TAC) in the
pos-1 ORF. This mutation was originally found in
pos-1(ne51), and disrupts the second zinc finger (CCCH to YCCH)
(Tabara et al., 1999
). The
mutation did not affect protein-protein interactions between POS-1 and POS-1
or SPN-4 in our two-hybrid analysis. Yeast strain CG-1945 was co-transformed
with hybrid RNA and AD fusion protein expression plasmids. Hybrid proteins
were expressed as GAL4 activation-domain fusion proteins. RevM10 was fused to
the GAL4 DNA-binding domain as a fusion protein to trap the hybrid RNA. If the
hybrid protein bound the hybrid RNA, GAL4-mediated transcription activation of
the HIS3 reporter gene occurred, which was detected by the extent of the
growth of the transformants on a selection medium containing appropriate
concentrations of 3-amino 1,2,4-triazole (3-AT). To analyze their
interactions, transformants were plated on a non-selection medium (SD-LW) and
a selection medium (SD-LWH+3-AT) lacking histidine and including
3-aminotriazole (3-AT; 50 mM for POS-1, 10 mM for SPN-4) to select for a
higher level of HIS3 activation.
Identification of POS-1-interacting proteins
Approximately 1,000,000 clones of a two-hybrid cDNA library that was kindly
provided by R. Barstead were screened. Y190 was used as the host strain. For
the expression of the full-length POS-1 bait, pAS2-1 (Clontech) was used.
Library screening was performed as described by the manufacturer (Clontech
Catalog Number K1604-1). The open reading frames of the pip genes
were determined by sequencing the positive cDNA clones from the two-hybrid
screen. The pip-1 ORF is identical to the predicted gene ZC404.8
(The C. elegans Sequencing
Consortium, 1998). The results of RT-PCR indicated the presence of
an SL1 splice leader sequence in the transcripts. The cDNA sequence of
spn-4 has been deposited in the DDBJ under the Accession Number
AB052819.
In vitro binding
Each PCR-amplified cDNA fragment was subcloned into the pGEX-KG, pGEX-5X-2
or pMAL-c2 vector. Fusion proteins were induced in E. coli with 1 mM
IPTG at 37°C for 3 hours. GST fusion proteins were bound with Glutathione
Sepharose 4B beads (Pharmacia Biotech) in cold 25 mM Tris HCl (pH 7.5), 150 mM
NaCl, 5 mM DTT, 1 mM MgCl2, 0.2% NP-40
(Simske et al., 1996). The
beads were washed with the same cold buffer, and mixed with extracts of the
E. coli that produced the MBP fusion proteins. The beads were washed
several times with the same cold buffer. Each sample was fractionated by 8%
SDS-PAGE and analyzed by western blotting using ECL (Pharmacia Biotech). We
used an anti-MBP antiserum (New England Biolabs) for the primary antibody, and
HRP-conjugated anti-rabbit IgG (Pharmacia Biotech) for the secondary
antibody.
RNA-mediated interference
EST clones yk458c6 (GenBank Accession Number, C48581 for spn-4),
yk61h1 (GenBank Accession Number, AB006208 for pos-1) and yk320h8
(GenBank Accession Number, C63438 for glp-1) were used as templates
for double-stranded RNA synthesis. Using the PCR-amplified insert, both RNA
strands were simultaneously synthesized with T3 and T7 RNA polymerase
(Promega), and the RNA mixture was heat-denatured and annealed to form
double-stranded RNA. Microinjection of the RNA was performed as described
(Mello et al., 1991;
Fire et al., 1998
) at a
concentration of 3
4 mg/ml in TE.
Antibodies and immunostaining
To raise anti-SPN-4 antibodies, the region corresponding to the C-terminal
111 amino acids of SPN-4 was amplified from the cDNA clone and subcloned into
a His tag vector, pET15b. The fusion protein produced in E. coli was
purified with Ni-NTA Agarose (Qiagen). Two rabbits were immunized with the
fusion protein. Essentially the same staining pattern was observed with the
two sera.
The anti-GLP-1 antibody was a gift from J. Kimble. The anti-POS-1 antibody
was from our stock (Tabara et al.,
1999). mabK76 (Strome and
Wood, 1982
) was from DSHB (Developmental Studies Hybridoma Bank,
University of Iowa, USA).
For all the antibodies, fixation and staining were performed essentially as
described by Zwaal et al. (Zwaal et al.,
1996), using Cy3- or Cy5-conjugated secondary antibodies
(Pharmacia Biotech). Specimens were observed on a confocal laser-scanning
microscope (Zeiss, LSM510).
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RESULTS |
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POS-1 specifically interacts with the spatial control region (SCR) in
the glp-1 mRNA 3' UTR
The effect of the pos-1 mutation on GLP-1 expression led us to
examine the interaction between POS-1 and the glp-1 mRNA 3'
UTR. For this purpose, we used a yeast tri-hybrid system
(Putz et al., 2000), which is
a modification of the two-hybrid system. In this system, a target RNA is
expressed as a hybrid RNA with the RRE (Rev responsive element). A target
protein is fused to the activation domain of GAL4, and the DNA-binding domain
is fused with HIV-1 RevM10, which binds RRE. If the target protein binds the
target RNA, transcriptional activation occurs and is detected by the same
procedure as in the two-hybrid system.
For the target RNA, we used a 294 bp region from the 3' UTR
(Fig. 2A). This region includes
the SCR and part of the TCR. The very 3'-end region has several motifs
for the processing of mRNA including polyA addition. These motifs could cause
disturbance in the assay through some complex formation; therefore, we removed
the very 3'-part of TCR. The tri-hybrid analysis showed that POS-1
specifically interacted with the 294-base RNA region, as shown in rows 1 and 5
in the `WT, S column' of Fig.
2C. To identify the interacting region within the 294-base RNA
region, we subdivided the 294-base region into three parts, 5' to
3': the 138-base proximal region (non-SCR and non-TCR region), the
66-base SCR and the 78-base subregion of the TCR
(Fig. 2A). As shown in the
`POS-1 WT' column of Fig. 2C,
we found that POS-1 preferentially interacted with the 66-base SCR. We found
that POS-1 also interacted with the 78-base RNA region within the TCR, but
much more weakly than with the SCR. The SCR is essential for the suppression
of glp-1 translation in the posterior blastomeres
(Evans et al., 1994); thus, the
physical interaction between POS-1 and SCR is consistent with the idea that
POS-1 negatively regulates its translation.
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A POS-1 interacting protein, PIP-1, is a RNP-type RNA-binding protein
and identical with SPN-4
To analyze the POS-1 function in more detail, we identified
POS-1-interacting proteins from a C. elegans yeast two-hybrid cDNA
library using the full-length POS-1 as bait. As positive cDNA clones, we
identified POS-1, MEX-3 (Draper et al.,
1996), F38H4.9, which encodes a catalytic subunit of protein
phosphatase 2A (PP2A) and ZC404.8, which encodes an RNP-type RNA-binding
protein. We confirmed that these proteins bound directly to POS-1, using an in
vitro GST pull-down assay (Fig.
3A). The fact that POS-1 was recognized in our two-hybrid screen
suggested that POS-1 forms a homodimer or a homomultimer. MEX-3 is a KH-type
RNA-binding protein, and is required for the control of maternal
pal-1 mRNA translation (Draper et
al., 1996
; Hunter and Kenyon,
1996
); therefore, POS-1 may also be required for the control of
the pal-1 mRNA. We found that the catalytic subunit of PP2A, F38H4.9,
corresponds to the let-92 gene, and that let-92 is required
for embryonic cell divisions rather than for translational regulation (K.-i.O.
and Y. K., unpublished). Here, we focused on a novel protein, ZC404.8, and
named it PIP-1, for POS-1 interacting protein. This gene was independently
identified and named spn-4 by Gomes et al.
(Gomes et al., 2001
) and Huang
et al. (Huang et al., 2002
).
Therefore, we call it spn-4 in this paper. Diagrams of the protein
and gene are shown in Fig.
3B,C.
|
We examined the expression of GLP-1 in spn-4 early embryos. The
result was the inverse of the expression in pos-1 mutants. There was
no GLP-1 expression in any of the blastomeres in the spn-4 mutant
(Fig. 1E) and
spn-4(RNAi) (data not shown). The GLP-1 expression in the distal
region of the adult gonads (Crittenden et
al., 1994) was not affected in the mutants (data not shown). These
results strongly suggest that SPN-4 positively regulates the translation of
maternal glp-1 mRNA in the anterior half of early embryos.
To analyze the relationship between pos-1 and spn-4, we tested the expression of GLP-1 in double mutant embryos for these genes. The pattern of GLP-1 expression in the double mutants was essentially the same as in pos-1 embryos (Fig. 1F). The same results were obtained with the double RNAi embryos (data not shown). These results indicate that pos-1 is genetically epistatic to spn-4 with respect to GLP-1 expression.
SPN-4 specifically interacts with a subregion of the TCR in the glp-1
mRNA 3' UTR
We analyzed whether there was an interaction between SPN-4 and the
glp-1 mRNA 3' UTR using the yeast tri-hybrid system. We found
that SPN-4 specifically interacted with the same 294-base RNA within the
glp-1 3'UTR as did POS-1 (rows 1 and 5, column `SPN-4, S' in
Fig. 2C). We found that SPN-4
interacted with the 78-base RNA region within the TCR but not with the 66-base
SCR, as shown in the `SPN-4' column of Fig.
2C. These results suggest that SPN-4 directly binds the 78-base
region within the 125-base TCR, and activates the translation. The 125-base
TCR was originally identified as a temporal control region for translational
repression; however, deletion of the TCR disrupts the spatial regulation of
translation as well as the temporal regulation
(Evans et al., 1994).
Therefore, we think that the 125-base TCR contains both a temporal inhibition
region and a spatial activation region (probably within the 78-base
region).
Localization of mRNA and protein of spn-4
In situ hybridization analysis revealed that spn-4 mRNA was
abundant in early embryos (Fig.
4). The mRNA was present at the same level in all blastomeres up
to the 4-cell stage (Fig.
4A-C). Afterwards, it persists in the P blastomere and its sister,
and then just the germ lineage (Fig.
4D-H). The mRNA was also present in the adult gonads (data not
shown).
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DISCUSSION |
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Negative regulation by POS-1 through its zinc fingers
We showed that GLP-1 was ectopically translated in the posterior
blastomeres of pos-1 embryos, although its temporal regulation did
not seem to be affected. Using the yeast tri-hybrid system, we also found that
POS-1 specifically interacted with the glp-1 mRNA SCR, and that a
missense mutation in the second CCCH zinc finger of POS-1 (2nd CCCH
to YCCH) disrupted this interaction. These results indicate that
POS-1 directly binds the glp-1 SCR and suppresses the translation of
maternal glp-1 mRNA in the posterior blastomeres.
Our preliminary studies using the tri-hybrid system showed that POS-1
interacted with various maternal mRNAs and that a mutant version of POS-1 (2nd
CCCH to YCCH) disrupted this interaction (N.K. and Y.K.,
unpublished). Thus, the zinc fingers of POS-1 are likely to play an important
role in the translational regulation of other mRNAs, probably by binding to
their 3' UTRs. The importance of zinc fingers in translational
regulation has been suggested in a study of another protein. Tenenhaus et al.
(Tenenhaus et al., 2001)
reported that a maternal gene product, PIE-1, which has a similar zinc-finger
structure to POS-1 is required for the normal expression of NOS-2 from
maternally provided mRNA. In this case as well, the second CCCH finger of
PIE-1 was important for the normal expression of NOS-2.
Positive regulation by the POS-1-interacting protein, SPN-4
We showed that a POS-1-interacting protein, SPN-4, has an RNP-type
RNA-binding motif. Proteins containing the RNP-type RNA-binding motif are
widely present in organisms from bacteria to animals, and bind RNA with a wide
range of affinities and specificities (Burd
and Dreyfuss, 1994). A protein bearing an RNP-type motif that is
highly homologous to SPN-4 is C. elegans FOX-1 (54.5%/77 amino acids)
(Hodgkin et al., 1994
). FOX-1
is a nuclear protein that post-transcriptionally reduces the level of XOL-1,
which acts as a sex determination switch
(Nicoll et al., 1997
). FOX-1
binds poly(A), poly(G) and poly(U) RNA in vitro
(Skipper et al., 1999
). By
analogy, SPN-4 probably binds directly to RNA through its RNP motif.
Analysis of the deletion mutant, tm291, showed that the spn-4 mutation caused strict maternal-effect embryonic lethality, indicating that spn-4 is essential only for embryogenesis. In contrast to pos-1 mutants, GLP-1 was never expressed in the spn-4 mutant. The mutant embryos had almost no pharynx and about half had no intestinal valve cells. GLP-1 is required for the differentiation of both the ABa and ABp blastomeres. Half of the pharynx is derived from ABa, and the intestinal valve cells are derived from ABp; therefore, these phenotypes are consistent with the lack of GLP-1 expression in these embryos. We showed that SPN-4 preferentially interacted with the 78-base RNA region within the 125-base TCR of the glp-1 mRNA 3' UTR using the yeast tri-hybrid system. These results suggest that SPN-4 positively regulates the translation of the maternal glp-1 mRNA in the anterior blastomeres by directly binding to the 78-base RNA region.
The 125-base TCR was originally identified as a temporal control region for
translational repression (Evans et al.,
1994); however, the deletion of the TCR results in the temporal
misexpression of the reporter in oocytes and all cells of early embryos
(Evans et al., 1994
). Thus, the
125-base TCR could contain a spatial control region as well as the temporal
control region. We think that the 78-base region contains a spatial control
region for the expression in the anterior blastomeres that is regulated by
binding with SPN-4. We also showed that POS-1 interacts with the TCR, although
much more weakly than with the SCR. POS-1 may compete with SPN-4 for the same
binding region, or the 78-base region may have another site for POS-1
binding.
A model for the translational regulation of maternal glp-1 mRNA by
POS-1 and SPN-4
The expression patterns of GLP-1 in spn-4;pos-1 double mutant
embryos were essentially the same as in pos-1 embryos, indicating
that pos-1 is genetically epistatic to spn-4 with respect to
GLP-1 expression. An attractive explanation of the epistasis results would be
that SPN-4 participates in restricting POS-1 to the posterior, however, this
is not the case because POS-1 was distributed apparently normally in
spn-4 mutant embryos (and vice versa). Based on these experimental
observations, we made a working model for the translational regulation of the
maternal glp-1 mRNA by POS-1 and SPN-4, as shown in
Fig. 7.
|
In a pos-1 mutant, the suppression of translation in the posterior embryo is released, leading to the ectopic translation of glp-1 mRNA in the posterior embryo. In a spn-4 mutant, the activation in the anterior end does not occur and low levels of POS-1 that may be present in the anterior cells can inhibit glp-1 mRNA translation; thus, there is no translation of glp-1 mRNA in the anterior embryo. In a pos-1; spn-4 double mutant, neither suppression nor activation occurs, and the maternal glp-1 mRNA is translated in an unregulated fashion, resulting in its expression in all blastomeres.
GLP-1 is not translated in oocytes or one-cell embryos, which have abundant SPN-4 but no POS-1. During this period, some factor(s) are expected to bind to the TCR to repress the translation. Thus, the above model explains the translational regulation after the release of the temporal control.
If this model is correct, the ectopic expression of POS-1 in anterior cells
should suppress GLP-1 translation. Schubert et al.
(Schubert et al., 2000)
reported that GLP-1 was not detected in mex-5;mex-6 embryos at the
four-cell stage. In this mutant, POS-1 is mislocalized and present in all
blastomeres. This finding supports our model, because ectopic POS-1 in the
anterior blastomeres would be expected to suppress translation. However,
Crittenden et al. (Crittenden et al.,
1997
) reported that in par-1 embryos, GLP-1 was detected
in all blastomeres, in a manner similar to pos-1 embryos. We examined
POS-1 expression in par-1(RNAi) embryos, and found that POS-1 was
abundant in all blastomeres (data not shown). This does not appear to be
consistent with our model, however, as the par-1 mutation disrupts
the anteroposterior polarity at the very beginning of embryogenesis, it might
well affect the transition from temporal control to spatial control, leading
to some unregulated status. Alternatively, if a post-translational
modification, for example, phosphorylation, is necessary for POS-1 to be
active, the par-1 mutation might affect this process.
How does POS-1 suppress the translation of glp-1 mRNA in posterior
blastomeres? It is generally thought that the elongation of the poly A tail of
mRNA leads to its active translation
(Seydoux, 1996;
Richter, 1999
;
Wickens et al., 2000
).
Interestingly, we have detected poly A tails of two different sizes in
glp-1 mRNAs after the two-cell stage, and found that the longer poly
A tail is present only in the anterior cell AB where glp-1 is
translated, but not in the posterior P1, where it is not (S. Onami
and Y.K., unpublished). POS-1 may shorten the poly A tail of the
glp-1 mRNA in the posterior blastomeres. There are interesting
parallels between POS-1 and mammalian TTP. Like POS-1, TTP has two CCCH-type
zinc fingers. TTP can bind to an AU-rich element (ARE) in the 3' UTR of
the TNFa mRNA to promote deadenylation and destabilization of the mRNA
(Lai et al., 1999
). The TTP
zinc CCCH finger is important for this binding. As to SPN-4, although there is
no link between SPN-4 and poly A length regulation, an attractive hypothesis
is that the poly A tail length of the maternal glp-1 mRNA is
determined by the balance between POS-1 and SPN-4 activity.
Translation of other maternal mRNAs are also regulated by POS-1 and
SPN-4
In the case of apx-1, the pos-1 mutation causes the
disappearance of its expression; thus, POS-1 regulates it positively
(Tabara et al., 1999). This is
the inverse of the glp-1 case. Interestingly, our preliminary
experiments showed that SPN-4 negatively regulated the temporal and spatial
translation of maternal apx-1 mRNA (K.-i.O. and Y.K., unpublished).
This is also the inverse of the glp-1 case. Recently, Gomes et al.
(Gomes et al., 2001
) reported
that SPN-4 negatively regulates the translation of maternal skn-1
mRNA in the anterior blastomeres. Huang et al.
(Huang et al., 2002
) have
reported that SPN-4 negatively regulates the translation of maternal
pal-1 mRNA in anterior blastomeres. In these cases, SPN-4 negatively
controls the translation of the mRNAs. Our preliminary studies using the yeast
tri-hybrid analysis showed that SPN-4 specifically interacts with the 3'
UTRs of apx-1, skn-1 and pal-1 mRNA (N.K. and Y.K.,
unpublished); therefore, we think that SPN-4 directly controls the translation
of these maternal mRNAs. In the SCR and TCR of glp-1 mRNA, two sets
of NRE (Nanos Response Element)- like motifs
(Murata and Wharton, 1995
) are
observed, and in the TCR, a TGE (TRA-2 and GLI Element)-like motif
(Jan et al., 1997
) is
observed. Interestingly, the TGE-like motif is present in the 3' UTRs of
the SPN-4 regulating genes, pal-1 and skn-1, as well as
apx-1. The NRE-like motif is found in apx-1 and
pal-1 3' UTRs, but not in skn-1. Work is in progress
to examine whether these motifs are functionally important for the
translational control. POS-1 and SPN-4 may change their function depending on
the target mRNAs, and in combination with other factors.
POS-1 and SPN-4 have other functions in regulating embryogenesis
The phenotypes of the pos-1 and spn-4 mutations are
rather pleiotropic, suggesting that the roles of POS-1 and SPN-4 are not
restricted to regulating the translation of the maternal glp-1
mRNA.
pos-1 embryos have little pharyngeal tissue, lack an intestine and
germ cells, and the P2, P3, and P4
blastomeres have abnormally short cell-cycle periods, with little asymmetry
(Tabara et al., 1999). Except
for the anterior pharynx, which is derived from the anterior ABa blastomere,
these affected tissues and cells are all derived from the posterior
blastomeres P2 or EMS (Sulston
et al., 1983
), which express GLP-1 ectopically in the
pos-1 mutants. However, the ectopic expression of GLP-1 probably does
not cause the phenotype, because its ligand, APX-1, is not expressed in
pos-1 embryos. Indeed, pos-1(RNAi);glp-1(RNAi) double RNAi
embryos still lack intestine (data not shown).
On the other hand, we and Gomes et al.
(Gomes et al., 2001) observed
that the terminal spn-4 embryos have almost no pharynx, extra germ
cells and (for half of the embryos) no intestine. Except for the anterior
pharynx, these affected tissues and cells are derived from the posterior
blastomeres P2 or EMS. Although GLP-1 is not detectable in
spn-4 mutants, its absence is unlikely to be responsible for these
effects, because GLP-1 is not required for the differentiation of the
posterior blastomeres (Priess et al.,
1987
). These results suggest that POS-1 and SPN-4 may cooperate to
regulate the translation of other maternal mRNAs that are required for the
differentiation of the posterior blastomeres. This regulation might occur at
the P granules, where both POS-1 and SPN-4 are present.
SPN-4 but not POS-1 was present in oocytes. Many maternal mRNAs accumulate
in oocytes, and the translation of some of them is temporally and spatially
regulated (Kemphues and Strome,
1997; Schnabel and Priess,
1997
). spn-4 is also required for normal cytokinesis and
spindle orientation in early embryos
(Gomes et al., 2001
) (this
study). Thus, SPN-4 could regulate the translation of various maternal mRNAs
in oocytes and very early embryos independent of POS-1, either by itself or in
association with another factor. We also show that SPN-4 is a temporal
component of the P granules. P granules are important for germline development
(Kawasaki et al., 1998
;
Amiri et al., 2001
); therefore,
the extra germ cells observed in spn-4 embryos may result from the
loss of the SPN-4 function in the P granules. For example, SPN-4 may repress
the activity of a determinant for germ cells in the P granules.
POS-1 and SPN-4 seem to have many functions and many targets. More factors are expected to work in translational regulation. These factors should constitute a network; therefore, the identification and characterization of other regulators and target mRNAs is needed to understand the nature of the translational control of maternal mRNA.
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ACKNOWLEDGMENTS |
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