Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA
Author for correspondence (e-mail:
hunter{at}mcb.harvard.edu)
Accepted 30 March 2004
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SUMMARY |
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Key words: GLD-1, PAL-1, Translational control, C. elegans, STAR/Maxi-KH Domain, Post-initiation repression
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Introduction |
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Studies on translational control mechanisms have repeatedly converged on
regulatory elements in the 5' and 3' untranslated regions (UTRs).
Through both genetic and biochemical studies, numerous examples of UTR
regulatory elements and their associated trans-acting factors have been
discovered (reviewed by Wickens et al.,
1996). One of the best-understood examples of 5'
UTR-mediated repression is iron response element (IRE)-mediated repression of
ferritin synthesis. Under low cellular iron levels, the iron regulatory
protein binds the IRE in the ferritin mRNA 5' UTR and is thought to
sterically inhibit recruitment of the small ribosomal subunit
(Gray and Hentze, 1994
;
Muckenthaler et al., 1998
).
3' UTR-mediated control is more prevalent than 5' UTR-mediated
control in developmentally regulated genes
(Wickens et al., 1996
). The
key role of the 3' UTR is not surprising, given that the 3' UTR is
less evolutionarily constrained than both the coding sequence and the 5'
UTR; the coding sequence must code for a functional polypeptide, while the
5' UTR must have a secondary structure that is amenable to ribosome
scanning. Recent data suggest factors bound to 3' UTRs may control
translation in part, by regulating the interaction of eIF4E, the 5' cap
binding protein, with eIF4G, the adaptor protein that binds eIF4E and recruits
the small ribosomal subunit via its interaction with eIF3. For example, in
Xenopus, many maternal mRNAs are regulated by a 3' UTR element
called the CPE (cytoplasmic polyadenylation element) and the CPE binding
protein, CPEB (Mendez and Richter,
2001
). A ternary complex of CPEB, Maskin and eIF4E is hypothesized
to circularize mRNAs and repress translation initiation by masking the eIF4G
binding site on eIF4E (Stebbins-Boaz et
al., 1999
). Similarly, in Drosophila, Bicoid is
hypothesized to simultaneously bind eIF4E and the caudal 3'
UTR, thereby preventing recruitment of eIF4G to caudal mRNA
(Niessing et al., 2002
).
Patterning of the C. elegans embryo relies upon the asymmetric
distribution of maternal regulatory proteins among early blastomeres.
Translational control is implicated in asymmetric expression of maternal
PAL-1, the Caudal homolog. Like Caudal, PAL-1 is expressed in the posterior of
the embryo where it functions in posterior patterning
(Hunter and Kenyon, 1996;
Edgar et al., 2001
). PAL-1
protein is first detected in posterior blastomeres beginning at the four-cell
stage, even though pal-1 mRNA is present in all cells of the early
embryo (Hunter and Kenyon,
1996
). Translational control of PAL-1 expression is suggested by
the ability of the pal-1 3' UTR to restrict the expression of a
lacZ reporter RNA to posterior blastomeres; lacZ RNA
injected into hermaphrodite gonads is expressed in all blastomeres, whereas
lacZ::pal-1 3' UTR RNA is expressed primarily in posterior
blastomeres (Hunter and Kenyon,
1996
).
Control of maternal PAL-1 expression must begin in the germline to ensure
that embryos do not inherit PAL-1 protein. The gonad consists of two U-shaped
arms in which presumptive oocyte nuclei progressively mature while moving from
the distal to the proximal region of each arm
(Fig. 1A). In the distal
region, syncytial nuclei progress from mitosis to meiotic pachytene, while in
the proximal region, nuclei complete meiosis I and become cellularized as they
mature into oocytes. pal-1 mRNA is present throughout the germline
(Nematode Expression Pattern DataBase (NEXTDB),
http://nematode.lab.nig.ac.jp),
yet PAL-1 protein is observed only transiently; low levels are observed in
immature oocytes found in the bend of the gonad arm
(Fig. 1A) (D.M. and C.P.H.,
unpublished). mex-3 is hypothesized to repress pal-1
translation in mature oocytes and early embryos. PAL-1 is present at high
levels in oocytes and all cells of early embryos that lack maternal
mex-3, and expression of lacZ::pal-1 3' UTR RNA is not
restricted to posterior blastomeres in mex-3 mutant embryos
(Hunter and Kenyon, 1996).
Interestingly, mex-3 encodes a putative RNA binding protein,
containing two KH (K homology) domains, that is expressed cytoplasmically in a
pattern complementary to that of PAL-1
(Draper et al., 1996
). Hence,
MEX-3 may directly mediate the translational repression of pal-1 in
oocytes and early embryos.
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Materials and methods |
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Reporter RNA experiments
All pal-1 3' UTR sequence was cloned into pJK370 which bears
an NLS::lacZ coding sequence and a poly(A) tract of 30 residues.
Residue 1 in Fig. 2 corresponds
to the third residue following the pal-1 stop codon. Capped RNAs were
synthesized essentially as described previously
(Evans et al., 1994) and
poly(A)+ purified using Oligotex kits (Qiagen) according to manufacturer's
instructions, with the exception that RNA was not denatured before incubation
with Oligotex resin. RNA concentration was determined spectrophotometrically,
and RNA integrity was verified by electrophoresis. Two or more preparations of
RNA were injected for each construct. RNAs (50 nM) were injected into the
distal gonad arm, and worms were stained with X-gal
(Fire et al., 1990
) after a
6-hour recovery at 25°C. In situ hybridizations were performed 75-90
minutes following injections using a lacZ antisense probe as
described previously (Seydoux and Fire,
1995
). Strong signal in the in situ hybridization experiments was
defined as being visible through a Nomarski filter at 100x.
|
Gradients
Sucrose gradient buffer contained 50 mM Tris-HCl (pH 8.5), 25 mM KCl, 10 mM
MgCl2, and 1% protease inhibitor cocktail (Sigma, P8215) (EDTA was
added to 0.1 M to some gradients). Linear gradients were made by overlaying
5.5 mls 0% sucrose gradient buffer on 5.5 mls 56% sucrose gradient buffer in a
14x89 mm tube. Tubes were first placed horizontally for 2 hours at room
temperature, and then placed vertically for 30-45 minutes at 4°C (the
linearity of the gradients made by this method was confirmed by measuring the
refractive index of multiple fractions). 0.5 ml was removed from the gradient
tops before sample loading. Polysome extracts were prepared by grinding frozen
worms to a fine powder using a mortar and pestle on dry ice. The resulting
worm powder was added to 3-4 volumes of ice-cold buffer consisting of 0.2 M
Tris-HCl (pH 8.5), 50 mM KCl, 25 mM MgCl2, 1 mM DTT, 2 mM EGTA, 0.2
mg/ml heparin, 500 U/ml RNasin, 2% PTE, and 1% protease inhibitor cocktail
(EDTA was added to 0.1 M in some samples). Samples were centrifuged for 10
minutes at 16,000 g and supernatant was layered on a sucrose
gradient. Gradients were centrifuged at 4°C for 1.5 hours at 40,000 rpm in
a SW-41 rotor. 0.2 or 0.5 ml fractions were collected manually from the top of
the gradients and RNA was extracted using Trizol (Gibco). Absorbance (260 nm)
measurements were made on an aliquot of each purified RNA fraction. Pooled
fractions were analyzed for pal-1 or mex-3 mRNA using
Ambion's RPAIII kit. Metrizamide gradient analysis was performed as described
by Olsen and Ambros (Olsen and Ambros,
1999). Ribosomal fractions were identified by the index of
refraction as described by Olsen and Ambros
(Olsen and Ambros, 1999
). Only
the top 90% of the gradient was analyzed because of the high metrizamide
viscosity at the bottom. Fractions analyzed by western blot were concentrated
by TCA precipitation.
Other methods
Antibody staining was performed as described in Huang et al.
(Huang et al., 2002). The
biotin pull-down assays were performed essentially as described by Lee and
Schedl (Lee and Schedl, 2001
),
except that 4 picomoles of each RNA were used and worms were fragmented using
a mortar and pestle as described above.
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Results |
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Several lines of evidence suggest that the absence of distal germline expression observed following injection of lacZ::3' UTR RNA is due to translational repression, rather than RNA degradation or transport into the proximal germline. First, similarly strong ß-galactosidase signal was detected in oocytes and embryos following injection of lacZ::3' UTR RNA and lacZ RNA, suggesting that the lacZ::3' UTR RNA was not subject to significant degradation. Second, ß-galactosidase was detected in oocytes as late as 8 hours post-injection of lacZ::3' UTR RNA (signal in oocytes is first detected 70 minutes post-injection). Since germline nuclei are continuously passing from the distal to the proximal gonad arm, this result suggests that the RNA translated in the proximal germline at the end of the 8-hour period was present in the distal germline throughout much of the incubation period. Finally, lacZ and lacZ::3' UTR RNA were detected in the distal germline by in situ hybridization in a comparable percentage of gonad arms (90%, n=31 and 79%, n=63, respectively) (Fig. 1B,C, insets).
It is possible that the detected lacZ::3' UTR RNA was
degraded and not competent for translation, therefore we determined the
efficiency with which unstable RNA can be detected. Since uncapped RNAs are
translated inefficiently and degraded rapidly in multiple systems (reviewed by
Mitchell and Tollervy, 2000),
we injected uncapped lacZ RNA and found that it was not expressed in
the germline (n=16) and was inefficiently detected by in situ
hybridization. Only 33% (n=27) of injected gonad arms exhibited
signal, and of these, only one (4% of total) exhibited strong signal. This is
in contrast to the high percentage of gonad arms that exhibited a strong in
situ signal following injection of lacZ or lacZ::3'
UTR RNA (89% and 62%, respectively). These data indicate that degraded RNA
yields a minimal in situ signal, and suggest that the lacZ::3'
UTR RNA is intact, but translationally repressed.
Multiple pal-1 3' UTR elements inhibit germline expression
To identify regions of the pal-1 3' UTR responsible for this
translational repression activity, we performed deletion analysis of the
lacZ::3' UTR RNA. For ease of discussion, the first, middle,
and last thirds of the 3' UTR have been designated A, B and C,
respectively. These thirds average 175 nucleotides in length and they overlap
by approximately 18 nucleotides (Fig.
1E). Region C has minimal germline repression activity, as full
germline repression is maintained when region C is deleted (construct AB,
Fig. 1E), and lacZ RNA
bearing region C alone is expressed in the distal germline of 92%
(n=53) of injected gonad arms.
In contrast, both region A and region B are necessary for complete distal germline repression. lacZ::3' UTR RNA is never expressed in the distal germline, whereas lacZ::3' UTR RNAs lacking region A or B (constructs BC and AC) are expressed in the distal germline in 31% and 60% of gonad arms, respectively. Importantly, like the lacZ::3' UTR RNA, these two constructs (and all other deletion constructs) were expressed in the proximal germline, thus showing that functional reporter RNAs were injected. The germline repression activities of region A and B are further supported by the observation that constructs that contain either region A or B in isolation were sufficient to repress distal germline expression of lacZ reporter RNA in approximately two-thirds of gonad arms.
A minimal germline repression element (GRE) represses translation
Deletion of region B from the lacZ::3' UTR construct
impaired repression more significantly than deletion of region A, therefore we
further mapped repression activity within region B. We found that the second
half of B (B2) lacks repression activity, while the first half (B1) robustly
inhibits germline expression; lacZ::B1 RNA was expressed in the
distal germline in only 2% (n=63) of gonad arms
(Fig. 1D,E). Hence, B1, a
107-nucleotide element (Fig.
1F), is both necessary and sufficient for robust germline
repression and hereafter B1 will be referred to as the germline repression
element, or GRE. Importantly, lacZ RNA bearing two copies of the GRE
is efficiently detected in the distal germline by in situ hybridization,
suggesting that the GRE represses translation, as opposed to regulating RNA
stability or localization (Fig.
1D, inset).
GLD-1 mediates the translational repression activity of the GRE
To understand how the GRE promotes translational repression, we sought to
identify trans-acting factors that mediate its activity. A clear candidate is
GLD-1, a cytoplasmic RNA binding protein (STAR/Maxi-KH domain protein) that
represses the expression of at least four genes
(Jones and Schedl, 1995;
Jones et al., 1996
;
Jan et al., 1999
;
Lee and Schedl, 2001
;
Xu et al., 2001
;
Marin and Evans, 2003
). GLD-1
is expressed in the distal, but not the proximal germline, and thus is
expressed at the right time and place to be a pal-1 repressor
(Jones et al., 1996
). To
assess the genetic interaction between GLD-1 and the GRE, we constructed a
transgenic line in which expression of a GFP::histone H2B fusion protein is
driven by a germline promoter (pie-1) and under the regulation of two
tandem copies of the GRE (GH::2X GRE). The expression pattern of this reporter
recapitulated that of the lacZ::GRE reporter, as nuclear GFP was
observed throughout the proximal germline, but not the distal germline
(Fig. 2B). Importantly, a
similar transgene that differs only by the presence of the pie-1
3' UTR instead of the pal-1 GREs is expressed in nuclei
throughout both the distal and proximal germline
(Fig. 2A), indicating that the
lack of distal germline expression of the GFP::2X GRE reporter is due to the
repression activity of the GRE. In addition, upon long exposures (several
seconds) of dissected GH::2X GRE distal gonad arms using a CCD camera, we
noted weak cytoplasmic GFP expression (Fig.
2D), which is discussed below.
Following bacterially mediated RNA interference (RNAi) of gld-1 in
GH::2X GRE worms, GFP was detected in virtually all nuclei of the distal
germline (Fig. 2C), indicating
that GLD-1 acts through the GRE to inhibit distal germline expression. Because
gld-1 null adults have a proximal germline tumor which is formed by
nuclei that exit meiotic pachytene and proliferate mitotically
(Francis et al., 1995a), we
considered the possibility that the ectopic GH::2X GRE expression in the
distal germline was a secondary consequence of the aberrant proximal germline
development. However, we eliminated this explanation because we observed
ectopic reporter expression at the extreme distal ends of the adult gonad,
where germline development appears normal, and because we observed ectopic
expression in gld-1 L4 larvae, which do not yet have a tumorous
germline (Francis et al.,
1995a
) (data not shown). To determine whether gld-1
controls the stability or the translation of GH::2X GRE RNA, we quantified the
amount of GH::2X GRE in wild type and gld-1 RNAi worms. Duplicate
RNase protection assays indicated that the amount of reporter RNA and
endogenous RNA was not significantly changed following gld-1 RNAi,
suggesting that GLD-1 represses translation as opposed to destabilizing the
RNA (Fig. 2E).
GLD-1 also represses the expression of endogenous PAL-1 in the distal
germline, as ectopic PAL-1 immunofluorescence was detected in the distal
germline of adult gld-1 null mutants (q485) and RNAi worms
(Fig. 3B). This ectopic PAL-1
signal was restricted to small clusters of nuclei instead of being distributed
throughout the distal germline, like the ectopic GFP expression of GH::2X GRE
reporter following gld-1 RNAi. We hypothesized that derepression of
endogenous pal-1 translation was incomplete in gld-1 mutants
because of additional repression elements, and asked whether mex-3,
which functions to repress PAL-1 expression in the proximal germline, is
ectopically active in the distal germline of gld-1 mutants. Indeed,
we observed ectopic MEX-3 throughout the distal germline of gld-1
null mutants (q485) and RNAi worms (n=59), and we observed a
clear increase in ectopic PAL-1 in the distal germline of mex-3(RNAi);
gld-1(RNAi) worms (Fig.
3C,E). In 100% of gld-1 (q485) and RNAi worms, between
0-50 distal germline nuclei express PAL-1 (n=63), whereas in 71% of
gld-1(RNAi); mex-3(RNAi) worms, between 50-200+
distal germline nuclei express PAL-1 (n=41). Hence, in wild-type
worms, gld-1 inhibits both PAL-1 and MEX-3 expression in the distal
germline, while MEX-3 inhibits PAL-1 expression in the proximal germline.
Interestingly, a leaky switch from GLD-1 to MEX-3-mediated repression may
account for the transient PAL-1 expression in the bend of the gonad arm
(Fig. 1A), which is precisely
where GLD-1 levels decline and MEX-3 levels rise
(Draper et al., 1996;
Jones et al., 1996
).
|
|
For these experiments, we used worm samples that contained abundant
germline pal-1 mRNA, but no PAL-1 protein hermaphrodites of
the last larval stage (L4), which synthesize maternal mRNAs but do not yet
produce oocytes, and young adult hermaphrodites, which have just completed the
L4 molt and do not yet produce oocytes. Germline enrichment of pal-1
mRNA in L4s and adults is supported by RNase protection experiments indicating
that very little pal-1 mRNA is present in glp-4 (bn2ts) L4s
and adults, which have a greatly reduced germline
(Beanan and Strome, 1992)
(Fig. 5A). Duplicate
comparisons of wild-type and glp-4 worms indicate that approximately
75% of pal-1 mRNA from wild-type L4s and 95% of pal-1 mRNA
from adults is associated with the germline.
|
Similar results were obtained from L4 larval extracts fractionated on a
metrizamide gradient (Fig. 5C).
pal-1 mRNA was found in the same fraction as monosomes and polysomes
of all sizes (fraction four), whose position is inferred both from the total
RNA peak (ribosomal RNAs) and from the index of refraction of C.
elegans ribosomes under these conditions
(Olsen and Ambros, 1999). In
addition, to determine whether GLD-1 may mediate post-initiation repression,
we assayed metrizamide gradient fractions by immunoblot and found that GLD-1
protein from adults was enriched in the ribosome containing fraction
(Fig. 5D).
gld-1 as a general repressor in the distal gonad arm
Because gld-1 inhibits the distal germline expression of the
pal-1 regulator MEX-3, we asked whether gld-1 also inhibits
the distal germline expression of proteins that repress PAL-1 expression in
the early embryo: SPN-4, MEX-5 and MEX-6
(Huang et al., 2002). SPN-4 is
an RRM protein (Gomes et al.,
2001
), while MEX-5 and MEX-6 are 70% identical CCCH zinc finger
proteins with overlapping functions
(Schubert et al., 2000
).
Because the individual functions of MEX-5 and MEX-6 have not been defined with
respect to PAL-1 regulation, we refer to these proteins collectively as
MEX-5/6. SPN-4 and MEX-5/6 are expressed in the proximal, but not distal,
gonad arms of wild-type adults (N. N. Huang and C.P.H., unpublished)
(Schubert et al., 2000
)
(Fig. 6A,C). In gld-1
null mutants (q485), we observed that 80% (n=25) and 100%
(n=28) of gonad arms exhibited ectopic SPN-4 and MEX5/6 expression,
respectively (Fig. 6B,D).
Hence, gld-1 directly or indirectly represses the distal germline
expression of all three proteins known to repress PAL-1 expression at later
developmental stages.
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Discussion |
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The STAR/Maxi-KH domain protein GLD-1 mediates the translational repression activity of the GRE. GLD-1 is specifically precipitated from worm extracts by GRE RNA, and GLD-1 represses the distal germline expression of both PAL-1 and a GH::2X GRE reporter, without destabilizing the respective mRNAs. GLD-1 could be the only regulator of the GRE, since the GH::2X GRE reporter is ectopically expressed in all distal germline nuclei following gld-1 RNAi, and removal of mex-3 activity in addition to gld-1 activity does not noticeably increase the level of ectopic expression (data not shown). In contrast, repression of full-length pal-1 mRNA may require regulators in addition to GLD-1, as ectopic PAL-1 is detected only in a subset of distal germline nuclei in gld-1 mutants. Moreover, even following reduction of ectopic mex-3 activity through gld-1; mex-3 double RNAi, PAL-1 is still not detected in all nuclei. This apparently incomplete derepression of PAL-1 expression could be due to the failure to eliminate all GLD-1 and MEX-3 protein via double RNAi, or due to the up-regulation of another PAL-1 repressor in the germline of gld-1 (RNAi) worms. Indeed, we found that SPN-4 and MEX-5/6, which repress PAL-1 expression in embryos, are ectopically expressed in the distal germline following gld-1 RNAi, and they may contribute to PAL-1 repression. Alternatively, there may be additional protein(s) that normally contributes to PAL-1 repression in the distal gonad arm.
GLD-1 is homologous to a sub-family of KH domain proteins known as the GSG
or STAR domain family (Jones and Schedl,
1995), whose members include the evolutionarily conserved Quaking
protein, mammalian Sam68 and SF1 and Drosophila How
(Vernet and Artzt, 1997
). The
200 amino acid STAR domain consists of an enlarged KH RNA-binding domain
(maxi-KH domain) flanked by conserved residues on both sides
(Vernet and Artzt, 1997
).
While the functions of these family members are not well understood, they have
been implicated in various aspects of RNA metabolism, including mRNA splicing,
nuclear export and translation (Arning et
al., 1996
; Larocque et al.,
2002
; Saccomanno et al.,
1999
). Other than GLD-1, only one family member, the mouse Quaking
I isoform 6, has thus far been implicated as a translational regulator, and
this is based on its ability to repress tra-2 expression when
expressed in C. elegans
(Saccomanno et al., 1999
).
Specificity of GLD-1 regulation
GLD-1 was previously shown to repress the expression of tra-2, rme-2,
mes-3 and glp-1 mRNAs (Jan
et al., 1999; Lee and Schedl,
2001
; Xu et al.,
2001
; Marin and Evans,
2003
). GLD-1 can directly bind the 3' UTRs of all these
mRNAs, yet minimal binding sites with translational repression activity in
vivo have been described only for tra-2 and glp-1
(Goodwin et al., 1993
;
Marin and Evans, 2003
). The
GRE is the third element that mediates gld-1 repression in vivo, yet
it does not share obvious primary or secondary structures with the previously
identified elements, and a comparison of all known and putative binding sites
reveals only limited homology between two targets, raising the question of how
GLD-1 regulates many disparate mRNAs. GLD-1 may specifically bind mRNAs in
vivo in cooperation with co-factor(s). For example, the novel F-box-containing
protein, FOG-2, is implicated as a co-factor specifically in the regulation of
tra-2 translation. FOG-2 forms a ternary complex with GLD-1 and the
tra-2 3' UTR (Clifford et
al., 2000
). fog-2 is not required for pal-1
repression, as no ectopic PAL-1 was observed in the germline of fog-2
mutants (data not shown). Alternatively, GLD-1 may be guided to its targets by
small non-coding RNAs or microRNAs (miRNAs). The C. elegans miRNA
lin-4 is hypothesized to repress lin-14 and lin-28
expression after translation initiation by binding imperfect complementary
elements in 3' UTRs of these mRNAs
(Olsen and Ambros, 1999
;
Seggerson et al., 2002
). In
addition, the Drosophila and mouse homologs of the Fragile X
translational repressor associate with the miRNA mir2b and the
non-coding RNA BC1, respectively
(Caudy et al., 2002
;
Ishizuka et al., 2002
;
Zalfa et al., 2003
). In
support of the hypothesis that miRNAs may participate in GLD-1-mediated
repression, we have observed ectopic PAL-1 expression in the distal germline
of worms with impaired miRNA processing (data not shown).
Post-initiation translational repression
There are four explanations for our observation that translationally
repressed pal-1 mRNA co-fractionates with ribosomes: (1)
pal-1 mRNA could be a component of large heterogeneous
ribonucleoprotein particles (mRNPs) that have the density of ribosomes as well
as the sedimentation coefficients of polysomes of various sizes; (2)
pal-1 mRNA could be in a mRNP associated with ribosomes actively
translating other messages; (3) PAL-1 protein could be degraded
co-translationally in a manner dependent on the pal-1 3' UTR
but independent of PAL-1 protein sequence (since lacZ and
gfp reporters are accurately regulated); or (4) pal-1 mRNA
may be associated with ribosomes that have slowed or stalled while translating
it. The post-initiation repression mechanism is the simplest explanation, and
it is the only mechanism that also explains the cytoplasmic GFP signal we
observed in the distal germline of animals expressing the GH::2X GRE transgene
(Fig. 2D). Reversing the order
of the two protein coding regions in this transgene never resulted in GFP
signal in the distal germline, but these constructs were poorly expressed in
the proximal germline (data not shown). Like pal-1 mRNA, GLD-1 also
co-fractionates with ribosomes, and our working hypothesis is that GLD-1 is a
component of a post-initiation repression complex in the distal germline. This
derives from the fact that GLD-1 directly or indirectly represses the distal
germline expression of at least eight proteins (four described in this paper)
and that for at least two of these, PAL-1 and MEX-3, post-initiation
repression is implicated.
Although polysome analysis has been performed on one other GLD-1 target,
tra-2 (Goodwin et al.,
1993), it is unclear as to whether GLD-1 represses TRA-2
expression before or after translation initiation. For example, the polysome
analysis was performed on adults, yet GLD-1 represses tra-2
translation in larvae (Jan et al.,
1999
). Also, the interpretation of the data is hindered by the
fact that tra-2 activity is regulated both in the soma and germline
of males and hermaphrodites (reviewed by
Kuwabara and Perry, 2001
). All
these populations of mRNA were analyzed together in the polysome analysis, but
GLD-1 is required only for the repression of tra-2 mRNA in the
hermaphrodite germline (Francis et al.,
1995a
; Francis et al.,
1995b
; Jan et al.,
1999
). The trans-acting factor(s) required for repression in the
male soma and germline are unknown and the mechanism of repression may be
different.
There is a small but growing list of mRNAs subject to post-initiation
repression, yet GLD-1 is the one of the first trans-acting proteins to be
implicated in this process. For example, the mRNAs encoding C.
elegans LIN-14 and LIN-28 co-fractionate with polysomes when the proteins
are not detectable, but no trans-acting proteins involved in repression have
been identified (Olsen and Ambros,
1999; Seggerson et al.,
2002
). Similarly, roughly half of Drososphila nanos mRNA
is found associated with polysomes in embryos with no detectable protein, and
although the RNA-binding protein Smaug can repress Nanos expression, it is not
known whether it represses the polysomal or sub-polysomal population of
nanos mRNA (Simbert et al.,
1996
; Dahanukar et al.,
1999
; Clark et al.,
2000
). Despite the growing number of examples, the mechanisms of
post-initiation repression remain unknown. The identification of GLD-1 as a
putative regulator of post-initiation repression may provide an inroad for the
molecular dissection of this process.
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ACKNOWLEDGMENTS |
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Footnotes |
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