1 Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle,
WA 98109, USA
2 Howard Hughes Medical Institute, Seattle, WA 98109, USA
3 Department of Zoology, University of Washington, Seattle, WA 98195, USA
* Author for correspondence (e-mail: jpriess{at}fhcrc.org)
Accepted 19 July 2004
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SUMMARY |
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Key words: ATX-2, GLD-1, MEX-3, PAB-1, Germline, Translational regulation
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Introduction |
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The yeast ataxin 2-related protein, Pbp1p, is a non-essential protein. By
contrast, the C. elegans and D. melanogaster orthologs have
been shown to be essential proteins. Loss or overexpression of
Drosophila Datx2 leads to diverse defects such as female sterility,
tissue degeneration and lethality. These abnormalities have been proposed to
result from a defect in regulation of the actin cytoskeleton
(Satterfield et al., 2002).
Depletion of C. elegans ATX-2 was reported to cause embryonic
lethality; however, the basis for this lethality has not been analyzed
(Kiehl et al., 2000
).
We show that ATX-2 functions in the postembryonic development of the C.
elegans germline. The germline contains numerous mRNAs whose expression
is temporally and/or spatially regulated
(Wickens et al., 2000). For
example, temporal control of TRA-2 protein translation is essential for the
production of both sperm and oocytes: C. elegans is a
self-fertilizing hermaphrodite that produces sperm briefly during the last
larval (L4) stage of development, and then switches to produce only oocytes
during adulthood. TRA-2 protein promotes female development, and repression of
tra-2 mRNA translation is required to allow spermatogenesis during
the L4 stage (Goodwin et al.,
1993
). Another example of regulated translation in the germline is
the spatial control of expression of the yolk receptor RME-2. Germ cells in
the hermaphrodite gonad undergo sequential developmental programs as they move
proximally toward the uterus (Schedl,
1997
). At the distalmost end of the gonad, germ cells proliferate
and remain undifferentiated. As germ cells move proximally, they enter
meiosis; cells in the medial part of the gonad are in the pachytene stage of
meiosis. In the proximal gonad, germ cells undergo terminal differentiation as
either sperm or oocytes (Fig.
1A). While rme-2 mRNA is broadly expressed in both the
medial and proximal regions of the adult gonad, RME-2 protein is expressed
only in the proximal part of the gonad, where germ cells undergo oogenesis
(Fig. 5A)
(Grant and Hirsh, 1999
;
Lee and Schedl, 2001
). A key
regulator of mRNA translation in the gonad is the GLD-1 protein
(Francis et al., 1995a
). GLD-1
contains a KH-domain and is a member of the STAR (signal transduction and
activation) family of RNA-binding proteins
(Jones and Schedl, 1995
;
Vernet and Artzt, 1997
). GLD-1
represses translation of several mRNAs, including tra-2 and
rme-2 (Jan et al.,
1999
; Lee and Schedl,
2001
). GLD-1 has been shown to bind directly to at least some of
its mRNA targets; however, the precise mechanism of GLD-1-mediated repression
remains unknown.
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Materials and methods |
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LGI: dpy-5(e61), fer-1(hc13)
(Ward and Miwa, 1978),
gld-1(q485) and gld-1(q361)
(Francis et al., 1995a
),
gld-2(q497) (Kadyk and Kimble,
1998
), mex-3(or20)
(Draper et al., 1996
),
unc-13(e51)
LGII: tra-2(q122gf) (Schedl
and Kimble, 1988)
LGIII: glp-1(oz112gf) (Berry et
al., 1997), unc-32(e189)
LGIV: fem-1(hc17) (Nelson et
al., 1978), fem-3(q20gf)
(Barton et al., 1987
),
him-8(e1489), rme-2(b1008) (Grant
and Hirsh, 1999
)
LGV: fog-2(q71) (Schedl and
Kimble, 1988).
Unless noted, mutations are referenced by Hodgkin
(Hodgkin, 1997). Transgenic
strains were lag-2::GFP JK2003(qEx233) (kindly provided by
J. Kimble), vit-2::GFP DH1033
(Grant and Hirsh, 1999
) and
atx-2::3xmyc; this construct (atx-2 genomic fragment with a
triple-myc sequence inserted at the 3' end, plus 1 kb of the 5'
UTR and 0.7 kb of the 3' UTR) was integrated into the genome by
microparticle bombardment as described by Praitis et al.
(Praitis et al., 2001
).
atx-2 RNAi experiments
To assure effective depletion of ATX-2, animals were subjected to
atx-2(RNAi) from the fourth larval stage onwards. As adults, these
initially produced some viable progeny that, with continued exposure to
atx-2 dsRNA, grew into sterile atx-2(RNAi) adults. We
obtained comparable results by injecting, soaking or feeding dsRNA; the
feeding technique (Timmons and Fire,
1998) was used for the experiments here. atx-2 RNAi was
not efficient at 20°C; unless described otherwise, all RNAi experiments
were performed at 25°C (24.8-25.2°C). Temperature-sensitive (ts)
fem-1(hc17) and fem-3(q20gf) mutants were exposed to
atx-2 RNAi as L4 animals at 20 or 15°C, respectively, and the L1
progeny were transferred to 25°C.
gld-1(q485)
Unc progeny from gld-1(q485)unc-13(e51) heterozygotes [strain
BS3156] were analyzed.
gld-1(q361)
One-third of the non-Dpy, non-Unc progeny of
gld-1(q361)/dpy-5(e61)unc-13(e51) animals [strain BS87] subjected to
atx-2(RNAi) had well proliferated germlines that contained very few
or no sperm. We presume these were gld-1(q361);atx-2(RNAi)
animals.
gld-2(q497)
One-third of the non-Dpy, non-Unc progeny of
gld-2(q497)/dpy-5(e61)unc-13(e51) animals subjected to
atx-2(RNAi) lacked pachytene nuclei in their germlines. We presume
these were gld-2(q497);atx-2(RNAi) animals.
Gain-of-function (gf) glp-1(oz112) mutants
Unc progeny from glp-1(oz112gf)unc-32(e189) heterozygotes [strain
BS860] were analyzed.
tra-2(q122gf)
Feminized tra-2(q122gf) hermaphrodites were mated with
tra-2(q122gf) males on atx-2 dsRNA feeding plates, and the
hermaphrodite progeny were analyzed for the presence of sperm/oocytes. In
control experiments, sperm-defective fer-1(hc13) ts hermaphrodites
grown at restrictive temperature were mated with N2 males on
atx-2(RNAi) plates, and the hermaphrodite progeny were examined for
sperm/oocytes.
Antibodies, immunolocalization and in situ hybridization
Anti-ATX-2 mouse monoconal antibodies mAbP1G12 and mAbP6D2 were generated
following published procedures (Wayner et
al., 1989) using His-tagged N-terminal half of ATX-2. Other
antibodies/antisera used in this study were 1CB4 mAb
(Okamoto and Thomson, 1985
),
anti-ph-H3 (Crittenden et al.,
2002
; Hendzel et al.,
1997
), anti-HIM-3 (Zetka et
al., 1999
), anti-GLP-1
(Crittenden et al., 1994
),
anti-RME-2"INT" (Grant and
Hirsh, 1999
), anti-MEX-3
(Draper et al., 1996
),
anti-MEX-5 (Schubert et al.,
2000
) and anti-GLD-1 (Jones et
al., 1996
). Worms were prepared for immunolocalization according
to Lin et al. (Lin et al.,
1998
). In situ hybridization was performed essentially as
described by Lee and Schedl (Lee and
Schedl, 2001
). The fragment of rme-2 cDNA used to create
an antisense probe corresponds to amino acids 10-482
(Grant and Hirsh, 1999
).
Immunoprecipitation and identification of ATX-2 and PAB-1 by mass-spectrometry
One-step IP was performed as described by Ciosk et al.
(Ciosk et al., 1998). Following
in-gel digestion (Shevchenko et al.,
1996
), samples were desalted, dried, resuspended in 5 µl of
0.1% TFA and analyzed by LC MS/MS with a ThermoFinnigan LCQ mass spectrometer
(Gatlin et al., 1998
). Data
were collected in the data-dependent mode in which a MS scan was followed by
MS/MS scans of the three most abundant ions from the preceding MS scan. The
MS/MS data were searched against the NCBI non-redundant and the C.
elegans protein databases. The matches were scored by SEQUEST and
identifications were considered valid if the identified protein contained at
least two peptides with Xcorr scores above 2.0 and did not appear in a control
sample.
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Results |
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ATX-2 is a cytoplasmic protein that forms a complex with poly(A)-binding protein
Previous studies have shown that human ataxin 2 is a cytoplasmic protein
that may be enriched in the Golgi apparatus
(Huynh et al., 2003), and that
Datx2, the Drosophila ortholog of ataxin 2, may function in actin
filament formation (Satterfield et al.,
2002
). To see if ATX-2 is required for actin cytoskeleton
organization in C. elegans, we stained atx-2(RNAi) gonads
for microfilaments but did not detect obvious abnormalities (data not shown).
To determine the localization pattern of ATX-2, we generated mouse monoclonal
antibodies against ATX-2. On a western blot, anti-ATX-2 Abs produced a
prominent signal (Fig. 1F, left
arrow). Extracts from transgenic worms with a Myc-tagged allele of
atx-2 showed an additional, slightly larger band, as expected
(Fig. 1F, right arrow). The
ATX-2 signal was greatly reduced in comparable extracts from worms subjected
to atx-2 RNAi (Fig.
1G). Thus, the antibodies specifically recognize ATX-2 in worm
extracts. In whole-mount preparations of wild-type hermaphrodites and males,
the anti-ATX-2 Abs stained throughout the cytoplasm in many, if not all, cell
types, with slightly stronger staining in the gonad
(Fig. 1H; data not shown). This
staining is specific to ATX-2, as the gonads of atx-2(RNAi) worms did
not stain (Fig. 1I), nor did
somatic cells, with the exception of neurons; several studies by others have
shown that neurons in C. elegans are particularly refractive to RNAi.
We conclude that ATX-2 is a widely expressed protein enriched in the cytoplasm
but not in the Golgi apparatus or in filamentous structures.
To identify proteins that might interact with ATX-2 in vivo, we
immunopurified ATX-2 from extracts of adult worms. Silver-stained ATX-2
immunoprecipitates (IPs) consistently showed three major bands
(Fig. 1J, arrows).
Microsequencing showed that the two closely spaced top bands were ATX-2. The
third, lower band was identified as PAB-1, one of the two C. elegans
cytoplasmic poly(A)-binding proteins
(Mangus et al., 2003). In
control experiments, we found that both ATX-2 and PAB-1 were absent from an IP
prepared with an unrelated antibody (Fig.
1J, lane 3), and that atx-2(RNAi) caused a significant
reduction in the amount of purified PAB-1 and of the lower ATX-2 band
(Fig. 1J, lane 5); the lower
ATX-2 band appears to be specific to the gonad (R.C., M.D.P. and J.P.,
unpublished). We conclude that ATX-2 forms a complex with PAB-1, suggesting a
possible role for ATX-2 in PAB-1-mediated processes.
To address the function of PAB-1, we subjected animals to pab-1 RNAi. Depletion of PAB-1 did not prevent development of most somatic tissues, except that the vulva protruded abnormally in adults (Fig. 1K, arrowhead). By contrast, development of the germline was highly abnormal; adult gonads were very small and contained few, undifferentiated germ cells (Fig. 1K). Thus, PAB-1 appears to be required primarily for development of the gonad; depletion of both PAB-1 and PAB-2, a second C. elegans cytoplasmic poly(A)-binding protein, caused additional defects in somatic development (data not shown). The observation that the phenotype of PAB-1-depleted animals (or both PAB-1 and PAB-2-depleted animals) is much more severe than the phenotype of ATX-2-depleted animals suggests that ATX-2 does not have an essential role in all activities mediated by poly(A)-binding proteins.
The small size of atx-2(RNAi) gonads is due to reduced expression of GLP-1/Notch in the germline
To determine the cause of the small size of atx-2(RNAi) gonads, we
first counted the number of mitotic germ cells in the distal region of the
gonad using an antibody that recognizes a mitosis-specific form of histone H3
(Hendzel et al., 1997;
Crittenden et al., 2002
). We
found that the number of mitotic figures was approximately half that of the
wild-type number (Fig. 2A).
This finding suggests that atx-2(RNAi) gonads are small because
mitotic proliferation in the germline is reduced. To determine whether ATX-2
is required for normal proliferation irrespective of the sexual identity of
the germline, we depleted ATX-2 from hermaphrodites with either masculinized
or feminized germlines, and from wild-type males. In all these cases,
proliferation was reduced (Fig.
2A), suggesting that reduced proliferation and masculinization of
the germline are independent consequences of depleting ATX-2.
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Masculinization of the atx-2(RNAi) germline is caused by continuous repression of TRA-2 translation
Wild-type gonads depleted of ATX-2 are masculinized and produce only sperm,
while fem-1 mutant gonads depleted of ATX-2 are feminized and produce
only oocytes. This suggests that ATX-2 is not essential for spermatogenesis or
oogenesis per se, but rather for the switch from spermatogenesis to oogenesis.
FEM-3 and TRA-2 are key regulators of this switch and promote spermatogenesis
and oogenesis, respectively. The protein levels of FEM-3 and TRA-2 in the
germline are under tight translational control
(Goodwin and Ellis, 2002).
During the last larval stage (L4), translation of tra-2 mRNA is
repressed (TRA-2 Off); this allows FEM-3 protein to promote spermatogenesis so
the L4 germline has a `male' identity. In adults, the germline switches into a
`female' mode; this switch requires repression of fem-3 mRNA and
translation of TRA-2 protein (TRA-2 On). Thus, the masculinization of
ATX-2-depleted germlines could result from the failure to repress FEM-3, or
from the failure to express TRA-2. Alternatively, the masculinization of
ATX-2-depleted germlines could result from expression of FEM-3 and TRA-2
simultaneously, as masculinizing activity of FEM-3 is dominant over feminizing
activity of TRA-2 (Barton et al.,
1987
; Schedl and Kimble,
1988
).
Translational repression of tra-2 mRNA and fem-3 mRNA is
mediated by distinct proteins. Genetic and biochemical data suggest that GLD-1
participates in translational repression of tra-2 mRNA by binding two
sequences in the 3'UTR called TGEs
(Goodwin et al., 1993;
Jan et al., 1999
). To see if
masculinization of atx-2(RNAi) germline could be caused by a failure
in translating TRA-2, we used tra-2(q122), a weak gain-of-function
mutation with a deletion of one of the two TGE sequences
(Goodwin et al., 1993
;
Schedl and Kimble, 1988
). If
tra-2 mRNA was present but translationally repressed in
atx-2(RNAi) animals, the tra-2(q122gf) mutation should allow
TRA-2 protein expression, thus promoting oogenesis at the expense of
spermatogenesis. Indeed, we found that the gonads of
tra-2(q122gf);atx-2(RNAi) animals contained oocyte-like germ cells
(compare Fig. 3B,D), and did
not contain sperm (Fig. 3A,C). Because the germline is fully feminized upon forced translation of TRA-2 in
tra-2(q122gf);atx-2(RNAi) gonads, FEM-3 is likely to be repressed in
these gonads. This observation suggests that the masculinization of
ATX-2-depleted germlines represents a failure in TRA-2 translation.
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The GLD-1-mediated pathway of entry into pachytene is compromised in atx-2(RNAi) gonads
In addition to roles in the regulation of glp-1 mRNA and
tra-2 mRNA, GLD-1 has been shown to be involved in the entry of germ
cells into pachytene, although the mRNA target(s) for this process have not
been identified. Genetic studies have shown that the requirement for GLD-1 in
pachytene entry is redundant with GLD-2, a poly(A)-polymerase
(Kadyk and Kimble, 1998). In
wild-type gonads, the presence of pachytene nuclei can be determined by the
distinctive thread-like appearance of DAPI-stained chromosomes
(Francis et al., 1995a
).
Although gld-1 or gld-2 single mutants contain pachytene
nuclei (Francis et al.,
1995a
), no pachytene nuclei are present in gld-1;gld-2
double mutants (Kadyk and Kimble,
1998
).
To test if GLD-1 was able to promote pachytene in gld-2 mutants
depleted of ATX-2, we asked if gld-2;atx-2(RNAi) germlines contained
pachytene nuclei by DAPI. We also examined expression of HIM-3, a core
component of meiotic chromosomes (Fig.
4A) (Zetka et al.,
1999). In control experiments, we found that pachytene nuclei were
present in the gonads of atx-2(RNAi), gld-2/+;atx-2(RNAi),
gld-1;atx-2(RNAi) and gld-2 adults
(Fig. 4B-D; data not shown)
(Hansen et al., 2004
). By
contrast, we found that atx-2;gld-2 gonads did not contain any
pachytene nuclei. Germ cell nuclei in atx-2;gld-2 gonads did not
resemble pachytene nuclei when stained by DAPI
(Fig. 4E), and contained only
low levels of HIM-3 that did not associate with chromosomes
(Fig. 4F). This result is
consistent with the hypothesis that the function(s) of GLD-1 in promoting
pachytene entry is compromised in the absence of ATX-2.
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The second abnormality in fem-1:atx-2(RNAi) animals was the
presence of RME-2 in the distalmost gonad
(Fig. 5B). This distalmost
RME-2 also was present in masculinized atx-2(RNAi) gonads
(Fig. 5D) and male
atx-2(RNAi) gonads (data not shown). Because rme-2 mRNA has
not been reported to be present in the distalmost region of wild-type
hermaphrodite or male gonads, we addressed this issue using in situ
hybridization. We often detected a very low level of staining with probes for
rme-2 mRNA in the distalmost regions of wild-type hermaphrodite
gonads, but never in the distalmost regions of control rme-2(RNAi)
gonads (data not shown). Because the gonad is a syncytium, we do not know
whether rme-2 mRNA is transcribed by the distalmost germ nuclei, or
originates from more proximal nuclei. GLD-1 is not thought to repress RME-2 in
the distalmost hermaphrodite gonad or in the male gonad. GLD-1-depleted
hermaphrodites do not express RME-2 in the distalmost gonad
(Lee and Schedl, 2001), and
RME-2 is not expressed in GLD-1-depleted males (data not shown), even though
low levels of rme-2 mRNA are present in the male germline (T. Schedl,
personal communication). These observations suggest that the distalmost germ
cells, irrespective of the sexual identity of the germline, have a
GLD-1-independent mechanism to prevent expression of RME-2, and that this
mechanism requires ATX-2.
Regulation of RME-2 expression in the distalmost part of the gonad
A possible candidate for a role in the distalmost repression of RME-2 is
the KH-domain protein MEX-3. MEX-3 has been shown to repress translation of
pal-1 mRNA in the proximal germline and in the embryo
(Draper et al., 1996; Hunter et
al., 1996). Interestingly, MEX-3 also is present at low levels in the
distalmost part of the germline where its function has not been investigated
(Fig. 5E) (B. Draper and J.P.,
unpublished). We examined RME-2 expression in mex-3 mutants and in
animals depleted of MEX-3 by RNAi. In both hermaphrodites and males, we found
that RME-2 was expressed inappropriately in the distalmost region of the gonad
(Fig. 5F) (data not shown). We
then examined rme-2 mRNA expression by in situ hybridization, and
could detect no differences in the levels of distalmost rme-2 mRNA
between mex-3 hermaphrodites and wild-type hermaphrodites
(Fig. 5G). Thus, MEX-3 appears
to have a role in regulating rme-2 mRNA translation, rather than
transcription, in the distalmost gonad. Because the distalmost gonad consists
of mitotic germ cells, we would predict that glp-1(gf) gonads that
consist entirely of mitotic cells (Berry et
al., 1997
) should have widespread MEX-3 expression and lack RME-2
expression. As expected, we found that glp-1(gf) gonads expressed
MEX-3 but not RME-2 (data not shown). Removing MEX-3 from these gonads should
allow expression of RME-2; indeed, we found that RME-2 was widely expressed in
the gonads of glp-1(gf);mex-3(RNAi) animals
(Fig. 5H,I).
Because MEX-3 is expressed in the distalmost region of the wild-type gonad,
where GLD-1 is scarce, but not in more proximal regions that contain high
levels of GLD-1, we considered the possibility that GLD-1 might regulate MEX-3
expression, or that MEX-3 might regulate GLD-1 expression. We found that
gld-1 animals expressed MEX-3 throughout the gonad
(Fig. 6B). By contrast, GLD-1
expression appeared normal in mex-3 mutant gonads (data not shown).
These results suggest that GLD-1 activity in the medial, pachytene region of a
wild-type gonad prevents expression of MEX-3, thus limiting MEX-3 to the
distalmost region of the gonad (Fig.
6E). Although we do not know if GLD-1 directly binds to and
represses translation of mex-3 mRNA, we found several possible
GLD-1-binding sites in the 3'UTR of mex-3
(Ryder et al., 2004) (data not
shown).
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Discussion |
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Some of the atx-2(RNAi) phenotypes suggest a simple loss of translational regulation. First, MEX-3 is expressed throughout glp-1(gf)atx-2(RNAi) gonads, but is unable to repress the expression of RME-2. Second, expression of GLD-1 and RME-2 in the pachytene germ cells of ATX-2-depleted feminized gonads [fem-1;atx-2(RNAi) animals] suggests a loss of GLD-1-dependent repression. Third, the absence of pachytene entry in gld-2;atx-2(RNAi) double mutants is consistent with a loss of GLD-1-mediated repression.
Other phenotypes of atx-2(RNAi) animals are more complex, and
suggest an alteration in, rather than loss of, GLD-1-mediated repression.
Although reduced expression of GLP-1 in atx-2(RNAi) gonads may be
explained by the abnormally high levels of distalmost GLD-1, GLP-1 expression
is also reduced in feminized fem-1;atx-2(RNAi) animals that contain
only low levels of GLD-1 in the distalmost gonad. The repression of
tra-2 mRNA also appears abnormal in atx-2(RNAi) gonads;
tra-2 mRNA is `locked' in the repressed state. Our genetic
experiments suggest that repression of tra-2 mRNA in
atx-2(RNAi) gonads occurs through a mechanism that requires TGE
elements in the 3'UTR of tra-2 mRNA, as in wild-type
development, and that at least some proteins that contribute to normal
TGE-mediated repression appear to be functioning in atx-2(RNAi)
gonads. First, removal of the GLD-1-binding partner FOG-2 promotes oogenesis
in atx-2(RNAi) gonads, as it does in otherwise wild-type gonads.
Second, a semi-dominant mutant form of GLD-1 encoded by the
gld-1(q361) allele that has weak feminizing effect on the germline
(Francis et al., 1995a)
markedly reduced spermatogenesis in atx-2(RNAi) germlines (R.C.,
M.D.P. and J.P., unpublished). GLD-1 has been shown to be part of a larger
complex called DRF that associates with the TGE elements
(Goodwin et al., 1993
;
Jan et al., 1999
). Because
GLD-1 produced from the gld-1(q361) allele is unable to bind TGEs
(Jan et al., 1999
), the
reduction of spermatogenesis in gld-1(q361);atx-2(RNAi) germlines
could reflect the formation of DRF with decreased ability to bind
tra-2; this would lead to expression of TRA-2 and a decrease in
spermatogenesis. One interpretation of the above results is that an aberrant
DRF complex assembles on the tra-2 3'UTR in the absence of
ATX-2, locking tra-2 mRNA in the repressed state. Alternatively, DRF
might be normal, but some feature of tra-2 mRNA (or its associated
proteins) could be altered by the absence of ATX-2, resulting in an abnormal
response to repressor binding.
Translational regulation in the stem-cell compartment of the gonad
The distalmost gonad can be considered a stem cell niche, where germ nuclei
actively divide but do not commit to either meiosis or differentiation. We
have shown that MEX-3 is required to prevent these mitotic germ cells from
expressing RME-2. The observation that rme-2 mRNA levels in the
distal gonad appear identical in wild-type gonads and mex-3 mutant
gonads suggests that MEX-3 does not regulate transcription of the
rme-2 gene. Instead, we favor the hypothesis that MEX-3 directly or
indirectly regulates rme-2 mRNA translation, analogous to its role in
regulating translation of pal-1 mRNA in the embryo. By preventing the
expression of proteins such as RME-2, that are characteristic of
differentiating oocytes, MEX-3 may contribute to the totipotency of the distal
germ cells.
Does human ataxin 2 function in translational regulation?
The ataxin 2-binding protein A2BP1 is an ortholog of an RNA-binding protein
from C. elegans called SPN-4. Intriguingly, SPN-4 has been recently
shown to associate with, and promote translation of, glp-1 mRNA in
the early embryo (Ogura et al.,
2003). Thus, both ATX-2 and SPN-4 appear to function in
translational regulation, suggesting that the human counterparts may also have
roles in translation. Moreover, SPN-4, ATX-2 and GLD-1 activities merge on the
same glp-1 mRNA to regulate translation. Although we do not know if
glp-1 translation in the embryo requires ATX-2, those observations
suggest that translation of some mRNAs might be regulated by a conserved set
of proteins: GLD-1/SPN-4/ATX-2 in worms, and a GLD-1 ortholog/A2BP1/ataxin 2
in mammals. The mouse orthologs of GLD-1, called QKI-5, -6, and QKI-7, are
different splice variants encoded by the quaking (qk) gene.
QKI proteins are known for their function in stability and export of mRNAs
encoding proteins, such as myelin basic protein (MBP), that are required for
myelination in the central and peripheral nervous systems
(Vernet and Artzt, 1997
;
Larocque et al., 2002
).
Interestingly, QKI-6 was shown in vivo to functionally substitute for GLD-1 in
translational repression of tra-2 mRNA
(Saccomanno et al., 1999
).
This result led to the hypothesis that QKI proteins might function as
repressors of MBP translation (Larocque et
al., 2002
). If true, it would be interesting to test if
translational regulation of MBP required ataxin 2 and A2BP1 proteins.
Ataxin-2-related proteins may constitute a family of PABP-binding proteins
Our studies provide the first in vivo evidence that an ataxin 2-related
protein forms a complex with a poly(A)-binding protein (PABP). Because
two-hybrid analysis showed that an ataxin-2-related protein from lower
eukaryotes (Pbp1p) interacts with PABP, the association with poly(A)-binding
proteins may be a conserved feature of ataxin-2 family members. PABPs
influence many aspects of mRNA metabolism such as mRNA translation, transport
and stability (Mangus et al.,
2003). We showed that depletion of PAB-1, the binding partner of
ATX-2, had much more severe consequences for germline development than had
depletion of ATX-2, suggesting that ATX-2 does not play an obligate role in
all PABP activities, and may instead have specialized functions.
A common role of PABPs is to promote polyadenylation by stabilizing the
poly(A) tail. The length of the poly(A) tail plays an important role in
regulating translation of many mRNAs
(Richter, 2000).
Interestingly, the presence of a poly(A) tail is essential for TGE-mediated
translational repression in Xenopus
(Thompson et al., 2000
).
However, there is no evidence that the length of the rme-2 or
tra-2 mRNA poly(A) tail plays a role in GLD-1-mediated repression
(Lee and Schedl, 2001
). In
addition to binding the poly(A) tail, PABPs throughout the animal kingdom have
been shown to bind eIF4G, a component of the 5' cap-binding translation
initiation factor eIF4F that also contains eIF4A and eIF4E
(Gingras et al., 1999
;
Sachs, 2000
). This binding is
thought to result in bridging the 5' end of mRNA with its 3'
poly(A) end. Such a `closed loop' conformation is thought to facilitate
translation. In mammals, a protein related to eIF4G called Paip1 mimics eIF4G
binding to both eIF4A and PABP, and enhances translation
(Craig et al., 1998
;
Roy et al., 2002
). Another,
unrelated PABP-binding protein called Paip2 competes with Paip1 for the
binding to PABP, and is able to prevent PABP association with the poly(A) tail
(Khaleghpour et al., 2001a
;
Khaleghpour et al., 2001b
).
Thus, Paip1 and Paip2 have set precedence for proteins that are not part of
the core translational machinery yet influence translation through their
association with PABP. Because ATX-2 interacts with PAB-1 and affects
translational regulation, an interesting possibility is that ATX-2 might alter
the ability of PABP to interact with components of the core translational
machinery. This could influence the architecture of the mRNA, for example the
conformation of mRNA or composition of its associated translational factors,
which might result in altered response to the UTR-binding repressors such as
GLD-1 or MEX-3.
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ACKNOWLEDGMENTS |
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