Department of Biology, Unit of Zoology, University of Fribourg, 1700 Fribourg, Switzerland
Author for correspondence (e-mail:
Alessandro.Puoti{at}unifr.ch)
Accepted 2 March 2004
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SUMMARY |
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Key words: Cyclophilin, mog-6, fem-3, gld-3, mep-1, Germline development
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Introduction |
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Here we report that mog-6 (cyp-4 WormBase) codes
for a divergent cyclophilin widely expressed in somatic and germline nuclei.
All cyclophilins harbor a conserved central binding domain (CBD) necessary for
Cyclosporin A (CsA) binding and protein folding
(Fischer et al., 1989;
Handschumacher et al., 1984
).
We found that MOG-6 binds to MEP-1, and provide evidence that the CBD in MOG-6
is neither necessary for MEP-1 binding nor for its role in germline sex
determination.
Besides the sperm/oocyte decision, germ cells in the hermaphrodite also undergo a decision between mitotic proliferation and meiosis. We show that in the absence of gld-3, loss of mog-6 causes germ cells to proliferate mitotically instead of differentiating into sperm, indicating that mog-6 functions in the decision between mitosis and meiosis.
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Materials and methods |
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Northern blotting and RT-PCR
PolyA-enriched RNA and total RNA were isolated from synchronized animals as
described (Puoti and Kimble,
1999). Total RNA was treated by DNase for 30 minutes at 37°C
(Stratagene). For northern analyses, polyA-enriched RNA or total RNA was
resolved on a denaturing gel, blotted and probed with the entire corresponding
cDNA, unless stated otherwise. For act-1, a 250 nt gene-specific
fragment was used. CeIF that is expressed at constant levels during
development was used as a loading control
(Roussell and Bennett, 1992
).
The gld-3 probe corresponded to the 5' end of the cDNA (nt
1-766).
For RT-PCR experiments, single-stranded cDNA was synthesized at 37°C using 1.5 µg of total RNA, 100 ng of random primers and 400 units Superscript II reverse transcriptase (Gibco BRL). 1/20 of each sample was used for PCR. For positive controls, 14 ng of genomic DNA and 44 ng of oligo(dT)-primed single-stranded cDNA from wild-type worms were used (Fig. 5A, lanes 1, 2, 7, 8, 13, 14, 19 and 20). Primer sequences are available upon request.
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Protein-protein interaction assays
For yeast two-hybrid interaction assays, the coding sequences for the MOG-6
and derivatives thereof were introduced in frame with the LexA DNA binding
domain in vector pBTM116 (Bartel and
Fields, 1995). The coding sequences of mep-1 derivatives
were cloned into pACTII in frame with the Gal4 activation domain
(Bai and Elledge, 1997
). All
constructs were fully sequenced. Two-hybrid assays were performed in strain
L40 (ura-) using 1 mg/ml of X-Gal
(Bai and Elledge, 1997
).
Typically, filters were incubated for 2 hours at 30°C. Interactions
reported on Fig. 4A were
confirmed in the opposite configuration (i.e. with MOG-6 as a Gal4 ACT
fusion), except for MEP-1(SH) and MEP-1(FL), because LEXA::MEP-1(SH) caused
autoactivation of the reporter transgene, and LexA::MEP-1(FL) was not
expressed in yeast (data not shown). The presence of fusion proteins was
verified by western blotting.
|
RNA interference
mog-6/mIn1[dpy-10(e128)mIs14 GFP] hermaphrodites were grown at
25°C on HT115 bacteria producing both sense and antisense gld-3
RNA strands (nt 327 to 766) (Timmons and
Fire, 1998). DAPI-stained adults were scored for germline
defects.
Nucleotide sequence accession number
The mog-6 cDNA sequence data have been submitted to the
DDBJ/EMBL/GenBank databases under accession number AF421146.
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Results |
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pAP6 was predicted to contain one single open reading frame (ORF) of 1575
nucleotides, 1239 of which were deleted in the mog-6(q465) allele.
The deletion started 600 nucleotides downstream of the initiator codon and
ended 61 nucleotides downstream of the stop codon
(Fig. 1E). Along with the
rescue analysis, the presence of this deletion strongly indicated that pAP6
corresponded to mog-6. A mog-6 cDNA containing a coding
region of 1575 nucleotides and UTRs of 1 and 50 nucleotides on the 5'
and 3' end, respectively, was isolated. Although the cDNA was missing
two nucleotides (A and T) in the 5' UTR, as well as the spliced leader,
it nevertheless contained the entire ORF for the following reasons: (1) a
splice acceptor site was located five nucleotides upstream of the translation
initiation codon in the mog-6 gene; (2) the initiation codon was
within a very good consensus sequence for translational start in C.
elegans; and (3) the first methionine of MOG-6 and the following amino
acids were conserved in closely related homologs
(Page and Winter, 1998).
mog-6 codes for a protein that belongs to the family of
cyclophilins (Fig. 2A).
Cyclophilins are small proteins that bind Cyclosporin A (CsA)
(Handschumacher et al., 1984)
and catalyze protein folding (Lang et al.,
1987
). Cyclophilins are characterized by a conserved CBD that is
required for both CsA-binding and protein-folding activities. In
mog-6(q465), the CBD and the C-terminus were deleted
(Fig. 1E). To date, 17
cyclophilins have been identified in C. elegans
(Ma et al., 2002
;
Page et al., 1996
;
Zorio and Blumenthal, 1999
).
MOG-6 is a divergent cyclophilin in that it contains N-terminal and C-terminal
extensions that are unique to MOG-6. Furthermore, MOG-6 contains only 12 out
of 15 conserved residues that bind CsA (shown in bold in
Fig. 2A)
(Page et al., 1996
). Thus,
MOG-6 differs from the prevailing cyclophilins such as human CypA or C.
elegans CYP-3 or CYP-7. MOG-6 has previously been described in the
context of a wide characterization of C. elegans cyclophilins
(Page et al., 1996
). In this
study, MOG-6 was named CYP-4 and had moderate protein-folding activity.
hCyP-60, the human ortholog of MOG-6 is 46% identical and was isolated as a
protein that binds to the proteinase inhibitor peptide eglin c
(Wang et al., 1996
). In
addition to the N- and C-terminal extensions, hCyP-60, MOG-6 and their close
nematode orthologs share most residues of the CBD
(Page and Winter, 1998
) (boxed
in Fig. 2A). Furthermore, no
closer homologs of hCyP-60 were found in the worm genome, indicating that
MOG-6 is its C. elegans ortholog. Using anti-MOG-6 polyclonal
antibodies, we detected a 64 kD protein in both wild-type and masculinized
fem-3(gf) worms. The 64 kD protein most likely corresponded to native
MOG-6, since it was of the expected size and was absent in mog-6
mutants (Fig. 2B). The level of
MOG-6 was reduced in the fem-3(gf) mutant, possibly because MOG-6 was
also expressed in oocytes and embryos, which are absent in masculinized
animals (see below).
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To follow the distribution of the MOG-6 protein in somatic tissues, we fused mog-6 to the green fluorescent protein (gfp) gene (Fig. 3B). The mog-6::gfp reporter gene was expressed in many somatic cell nuclei, including those of the intestine, the pharynx and the uterus (Fig. 3B). A nuclear expression of MOG-6 was expected, because MOG-6 contains a nuclear localization signal (Fig. 2A).
Transgenes that are expressed as multiple copies are silenced in the C.
elegans germline (Seydoux and Schedl,
2001). Therefore in the germline, absence of MOG-6::GFP cannot be
taken as indicative of mog-6 expression. Moreover, northern analysis
and the Mog-6 mutant phenotype strongly indicated that MOG-6 was present in
the germline. We therefore followed MOG-6 expression in the germline using
anti-MOG-6 antibodies. Immunolocalization showed that MOG-6 was expressed in
many nuclei of the germline and the soma
(Fig. 3C,F). The germline of
the C. elegans hermaphrodite is a syncytium made of two U-shaped arms
(one arm is shown in dark gray in Fig.
3E). Mitotic divisions occur in the distal portion of the
germline, while the proximal part contains mature gametes. We observed that in
L4 larvae MOG-6 was ubiquitously expressed in the mitotic and meiotic regions
but was absent in the proximal zone (Fig.
3C). Spermatozoa are formed from spermatocytes that divide twice
to become haploid spermatids. Nuclei start to condense in secondary
spermatocytes and are fully condensed in mature sperm
(L'Hernaut, 1997
). Co-staining
with DAPI showed that MOG-6 was not expressed in primary and secondary
spermatocytes, spermatids and mature sperm
(Fig. 3C,D,E), but was present
throughout the whole germline of a feminized adult
(Fig. 3H,I). fem-3 is
required for spermatogenesis and is therefore expected to be expressed in
sperm precursors. As a consequence, the distribution of MOG-6 correlates with
its proposed role in fem-3 repression. Consistent with the expression
of MOG-6::GFP, we also found MOG-6 in intestinal nuclei
(Fig. 3C, white arrows) and in
somatic and germline nuclei of larvae at different developmental stages
(Fig. 3G). Staining with
anti-MOG-6 antibodies was absent in a mog-6(q465) mutant (not
shown).
MOG-6 interacts with MEP-1
Previous studies have identified MEP-1 as a nuclear zinc finger protein
that physically interacts with MOG-1, MOG-4 and MOG-5
(Belfiore et al., 2002). The
nuclear coexpression of MOG-6 and MEP-1
(Fig. 3F) prompted us to check
whether MOG-6 was able to physically interact with MEP-1. For this, we fused
MOG-6 to the DNA-binding domain of LexA and tested its ability to interact
with a Gal4(AD)MEP-1 fusion protein in the yeast two-hybrid system
(Fields and Song, 1989
). We
found that MOG-6 interacted with full-length MEP-1 (MEP-1(FL)) and MEP-1(SH)
(Fig. 4A, top rows). MEP-1(SH)
lacks a large portion of its N-terminus but still retains the seven zinc
fingers (Belfiore et al., 2002
)
(Fig. 4C, row 2). We found that
MOG-6 did not bind to any of the other MOG proteins, nor to FBF, NOS-3 or
GLD-3 (Fig. 4A, bottom rows).
An independent yeast two-hybrid screen with MOG-6 as a bait confirmed the
importance of the interaction between MOG-6 and MEP-1, since more than 70% of
the cDNAs recovered were mep-1 clones (data not shown). Yeast
two-hybrid interactions were confirmed in a GST pulldown assay. MOG-6 was
retained on full-length or short MEP-1, but neither on GST alone nor on FBF
fused to GST (Fig. 4B). To
exclude the possibility that an RNA derived from the in-vitro translation
system could bridge MEP-1 and MOG-6 through unspecific protein-RNA
interactions, we treated the in-vitro synthesized MOG-6 protein with RNAse
before incubation with GST-MEP-1. In all cases, the RNAse did not reduce the
amount of radiolabeled protein that was retained (data not shown).
MEP-1 interacts with MOG-6 through two distinct domains
We used MEP-1 deletions to identify the domains through which MEP-1
interacts with MOG-6. MEP-1 is composed of an N-terminal extension and two
sets of three and four zinc fingers, respectively, that surround a
glutamine-rich domain (Q-domain) (Fig.
4C, row 1). The short MEP-1 isoform (row 2) was sufficient for
MOG-6 binding but a deletion of the last 35 residues abolished this
interaction (Fig. 4C, row 4).
The same deletion had no effect on the interaction between MOG-6 and MEP-1(FL)
(row 3), suggesting that MOG-6 and MEP-1(FL) bound through two independent
domains, one located at the C-terminus and the other at the N-terminus. This
idea was supported, in that N-terminal portions of MEP-1 did bind to MOG-6
(rows 9 to 12). However, MOG-6 binding was suppressed if fewer than 233
residues of the MEP-1 N-terminus were retained (233 residues in row 12; 155 in
row 13). The shortest portion of the MEP-1 N-terminus that was sufficient for
MOG-6 binding consisted of 111 amino acids (row 17). N- or C-terminal
deletions of this fragment abolished the interaction with MOG-6 (rows 15 and
16).
To study the MOG-6 binding site in MEP-1(SH), we tested the effect of C-terminal deletions. MEP-1(SH) interacted with MOG-6 (row 2) but did not if either the last 35 residues of its C-terminus (row 4) or the Q-domain and zinc fingers I to III were deleted (row 6). To ask whether the seven zinc fingers of MEP-1(SH) were all necessary for the interaction with MOG-6, we deleted the first cluster of zinc fingers of MEP-1(SH) (row 20). The corresponding protein bound to MOG-6, provided that the C-terminus was intact (compare rows 20 and 22). Deletion of the Q-domain and the first cluster of zinc fingers abolished MOG-6 binding (row 21). However, deletion of the first set of zinc fingers did not interfere with MOG-6 binding, as long as the Q-domain was intact (rows 18-20). In summary, we found that MOG-6 and MEP-1 interacted through two distinct domains in MEP-1, one that was located within residues 122 to 233 of MEP-1 (row 17, red box) and one that included the Q-domain, the second cluster of zinc fingers and the C-terminus (row 20, red box).
The CBD in MOG-6 is dispensable for sex determination and MEP-1 binding
Cyclophilins bind CsA and catalyze protein folding through the CBD
(Kallen et al., 1991;
Spitzfaden et al., 1992
). We
wondered whether the CBD was necessary for MOG-6 function in worms. We
therefore attempted to rescue mog-6 with mutated forms of the
mog-6 gene. We tested three mutant MOG-6 proteins in which residues
crucial for CsA binding and protein folding were changed to alanine.
Substitution of R323, F328 and M329 did not
affect mog-6 rescue (Fig.
4D, row 2). Similarly, L390, H394 and
I325 were not essential for MOG-6 function (rows 3 and 4).
Importantly, a MOG-6 protein with a deleted CBD (AA275-434) still rescued
mog-6 (row 5), while the CBD alone did not (row 6). Furthermore, if
taken separately, the C-terminal (AA435-523) and N-terminal domains of MOG-6
(AA1-274) did not rescue mog-6 on their own (rows 7 to 9). Our
findings suggest that both the N- and C-termini of MOG-6, but not the CBD, are
necessary and sufficient for MOG-6 function in vivo. The question that now
arises is how does MOG-6 act on fem-3 to regulate the sperm/oocyte
switch? Since the CBD was dispensable for MOG-6 function, we propose that
MOG-6 is unlikely to bind CsA or catalyze protein folding to achieve the
sperm/oocyte switch. However, we have shown that MOG-6 bound to MEP-1 and,
therefore, one possibility was that MEP-1 binding could be essential for MOG-6
function. We therefore tested different MOG-6 derivatives for MEP-1 binding.
We found that the CBD was not required for MEP-1 binding, since a MOG-6
protein that was missing the CBD still bound efficiently to MEP-1 (row 5,
right), whereas the CBD alone did not (row 6). If taken separately, the N- and
C-termini of MOG-6 did not bind MEP-1 efficiently (rows 8 and 9). In summary,
we found that both N- and C-terminal extensions of MOG-6 were necessary for
MEP-1 binding and MOG-6 function.
No abnormal general splicing was detected in mog-6 mutants
The findings that some cyclophilins are involved in pre-mRNA splicing
(Bourquin et al., 1997;
Horowitz et al., 1997
;
Teigelkamp et al., 1998
) and
that MOG-1, -4 and -5 are orthologs of well-characterized splicing factors
(Puoti and Kimble, 1999
;
Puoti and Kimble, 2000
) led us
to consider the possibility that mog-6 might control fem-3
via regulated splicing. To this end, we analyzed several transcripts including
fem-3, fbf, nos-3 and gld-3. We analyzed each transcript by
RT-PCR using primers that span the entire coding region. For fem-3, a
2.15 kb product was expected from unspliced RNA
(Fig. 5A, lane 1). A 1.17 kb
product that corresponded to fully spliced fem-3 was obtained with
poly(A)-enriched RNA from wild-type animals
(Fig. 5A, lane 2), as well as
from both wild-type (lane 6) and mog-6 mutants (lane 4). The 1.17 kb
product was absent if no reverse transcriptase was used, indicating that it
corresponded to reverse-transcribed RNA (lanes 3 and 5). For fbf-2,
the expected sizes of the unspliced and fully spliced RNAs were 2.1 and 1.83
kb, respectively (Fig. 5A,
lanes 7 and 8). A 1.83 kb product was obtained from either mog-6 or
wild-type worms (lanes 10 and 12), indicating that fbf-2 was
correctly spliced in mog-6 animals. A 2.59 kb product was obtained
for the fully spliced nos-3 mRNA, while the genomic fragment that
contained four introns yielded a 3.07 kb PCR product. Again, no splicing
defects were detected in nos-3 (lanes 16 and 18). Since fem-3,
fbf-2 and nos-3 are maternal mRNAs
(Ahringer et al., 1992
;
Zhang et al., 1997
;
Kraemer et al., 1999
), one
possibility was that the RT-PCR products could have been generated from
maternal RNAs that were provided by the heterozygous mother. To rule out this
possibility, we analyzed the ceh-13 mRNA, which is zygotically
expressed in early embryos and which is not contributed maternally
(Wittmann et al., 1997
).
Splicing of ceh-13 was normal in mog-6 mutants, since only
one 0.66 kb product that corresponded to fully spliced ceh-13 mRNA
was detected (Fig. 5, lanes 20
to 24). Splicing was also verified by northern blotting. The RNAs examined
included mog-6, fbf, nos-3, ges-1 and actin-1. ges-1 is
expressed in the soma from the 200-cell stage onward
(Aamodt et al., 1991
). The
mog-6 mRNA was absent in the mog-6 mutant, and no transcript
containing the corresponding deletion was detected, indicating that it is
degraded (Fig. 5B, top).
Germline-specific transcripts fbf and nos-3 were of the same
sizes in mog-6 and wild-type animals, indicating that they were
completely spliced (Fig. 5B).
Since fbf and nos-3 are abundant in embryos and oocytes, we
expected a reduced signal in mog-6 extracts
(Kraemer et al., 1999
;
Zhang et al., 1997
).
ges-1 and actin mRNAs also migrated to the expected size for fully
spliced messengers (Fig. 5B,
bottom). gld-3 produces at least two major transcripts
(Eckmann et al., 2002
). Even
if less abundant, probably because of the absence of embryos and oocytes, the
large and small gld-3 transcripts were detected in mog-6
extracts and were of the expected sizes
(Fig. 5C). In summary, among
the transcripts analyzed, we did not detect abnormal products, indicating that
general splicing occurred normally in mog-6 animals.
mog-6 is not required for the correct localization of other fem-3 regulators
FBF, GLD-3 and NOS-3 have been shown to function as cytoplasmic
trans-acting regulators of the fem-3 mRNA
(Eckmann et al., 2002;
Zhang et al., 1997
;
Kraemer et al., 1999
). One
possibility is that MOG-6 could repress fem-3 by regulating the
expression of such trans-acting factors. We therefore verified if
GLD-3, FBF and NOS-3 were correctly expressed in mog-6 animals. In
wild-type animals, GLD-3 is expressed in the germline cytoplasm throughout the
mitotic, transition and pachytene zones, in oocytes and primary spermatocytes
(Eckmann et al., 2002
). We
stained masculinized fem-3(gf) germlines and found that GLD-3 was
expressed throughout the mitotic and meiotic regions, but not in secondary
spermatocytes (Fig. 6A,B). A
similar expression pattern was observed in masculinized mog-6
animals, with the one difference that the transition zone and pachytene region
were larger than in fem-3(gf) animals
(Fig. 6C,D), possibly because
the latter produced more sperm that filled the distal germline. FBF was
present throughout the germline cytoplasm and was enriched in the mitotic
region in hermaphrodites and males
(Crittenden et al., 2002
;
Zhang et al., 1997
)
(Fig. 6E). We found that FBF
expression was essentially similar in masculinized mog-6 mutants and
wild-type hermaphrodites, as it was detected in the mitotic and transition
zones and decreased in germ cells that differentiate into oocytes or sperm
(Fig. 6E,G). Like in
masculinized mog-6 hermaphrodites, FBF was not detected in the
spermatogenic region in wild-type males, although it was present in the
mitotic and meiotic regions (not shown). The localization of NOS-3 was similar
to that of FBF, and we did not observe differences of NOS-3 expression in
wild-type versus mog-6 hermaphrodites (data not shown).
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Role of mog-6 in the mitosis versus meiosis decision
Earlier studies have shown that mog-1(0) mutants produce fewer
germ cells than normal (Puoti and Kimble,
1999). Similarly, mog-6 animals also made reduced amounts
of germ cells (1511±117, n=22 worms analyzed) compared with
wild-type hermaphrodites that contain 2400 germ cells
(Kimble and White, 1981
). Even
more significantly, fog-1;mog-6 animals produced fewer oocytes
(2.5±0.7, n=45 gonadal arms analyzed) than fog-1
mutants (15.5±2.8, n=21), indicating that mog-6 is
required for robust germline proliferation.
In addition to their roles in germline sex determination, nos, fbf
and gld-3 are also required for germline survival
(Subramaniam and Seydoux,
1999; Crittenden et al.,
2002
; Eckmann et al.,
2002
; Kraemer et al.,
1999
). We examined mog-6 gld-3 double mutants to ask
whether mog-6 acted upstream of gld-3 in germline sex
determination, and whether both genes interacted for germline proliferation.
We found that mog-6/mIn1;gld-3(RNAi) heterozygotes made no sperm and
accumulated oocytes (Fig. 7A).
mog-6 animals made only sperm and had smaller germlines than
fem-3 gain-of-function mutants
(Fig. 7B) (Barton et al., 1987
;
Graham et al., 1993
).
Surprisingly, germlines of mog-6(q465) gld-3(RNAi) animals were
neither masculinized nor feminized, but tumorous (Tum), as they were filled
with mitotically dividing germ cells (Fig.
7C-E; Table 1).
Germline tumors were never observed in mog-6 single mutants (0%,
n>1000). A very low incidence of Tum phenotypes was observed in
gld-3(q730) or gld-3(RNAi) animals: 1% Tum (n=163)
and 4% Tum (n=933), respectively. Germline tumors were also found in
other mog mutants that were subjected to gld-3(RNAi)
(Table 1). A completely
penetrant Tum phenotype was observed in a genetic mog-6(q465)
gld-3(q730) double mutant (Table
1). We asked whether the Tum phenotype was due to a masculinized
germline, or rather to a defective mog gene. To this end, we analyzed
the effect of gld-3(RNAi) in a masculinized fem-3(gf)
germline, and found a majority of maculinized germlines, some partial
feminization, but no significant amounts of Tum germlines
(Table 1, last row). This
finding suggests that gld-3 and mog are synthetically
required for meiosis.
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Discussion |
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MOG-6 is an unusual cyclophilin that binds to MEP-1
We found that MOG-6 interacted with MEP-1, which also binds to three other
MOG proteins (Belfiore et al.,
2002). Surprisingly, the MEP-1-MOG-6 interaction required both the
N- and C-terminal domains of MOG-6, but not the conserved CBD. Because the CBD
was not necessary for MOG-6 in the sperm/oocyte switch as well, we propose
that rather than the conserved CBD, the N- and C-terminal extensions of MOG-6
are essential for MOG-6 function and that this function might require the
interaction between MOG-6 and MEP-1. Furthermore, MOG-6 has a reduced
protein-folding activity (Page et al.,
1996
). We therefore propose that MOG-6 might not function as a
normal cyclophilin in the sperm/oocyte switch.
What is the molecular role of MOG-6 in fem-3 regulation? At
present, we cannot answer this question in detail, but we nevertheless provide
important insights. In this study, we show that MOG-6 is expressed in the
nucleus of many somatic and germline cells, except in sperm and sperm
precursors. The same expression pattern has been observed for MEP-1
(Belfiore et al., 2002), thus
giving the opportunity for MEP-1 and MOG-6 to bind. An earlier study showed
that in somatic tissues, mog-6 was able to repress a transgene
carrying the fem-3 3'UTR
(Gallegos et al., 1998
). The
somatic expression of MOG-6 now explains the action of MOG-6 on the
transgene.
Role of mog-6 in germline proliferation
Maternal mog-6 is needed for embryogenesis
(Graham et al., 1993), thus
suggesting a more general role of MOG-6 than solely germline sex
determination. Like gld-3, mog-6 is required for robust germline
proliferation (Eckmann et al.,
2002
). However, gld-3 mog-6 double mutants fail to
produce sperm and develop tumorous germlines. The same phenotype was observed
whenever the gld-3 mutation was combined with mog-1, -4 or
-5, suggesting that gld-3 and mog may function
synthetically in the decision between mitosis and meiosis. At present, it is
not clear whether mitotic proliferation occurs as a result of defective
meiotic progression (Francis et al.,
1995
; Subramaniam and Seydoux,
2003
), or as a result of a defective meiotic entry. Our data is
consistent with the recent finding that both nos-3 gld-2 and
nos-3 gld-3 double mutants also develop germline tumors
(Hansen et al., 2004
). The
molecular role of the RNA-binding protein GLD-3 is not clear, but one
possibility is that GLD-3 might bind to GLD-2 to control poly(A) tail
extension of targets RNAs such as gld-1
(Hansen et al., 2004
;
Wang et al., 2002
). The
implication of MOG in the mitosis/meiosis decision is not clear either. The
MOG proteins could function synthetically with GLD-3 to control the expression
of the gld-1 RNA, but many other possibilities remain. Previous
studies have shown that fbf, nos, gld-2 and gld-3 function
not only in germline sex determination but also in the decision between
meiosis and mitosis. We show now that at least mog-1, -4, -5 and
-6 also play a dual role in germline fates.
Possible molecular roles of MOG-6
Many molecular functions have been attributed to cyclophilins, including
RNA processing (Horowitz et al.,
1997; Teigelkamp et al.,
1998
). We checked whether mog-6 was required for the
splicing of several mRNAs but did not detect unprocessed RNAs. A similar
result was obtained in mog-1 null mutants
(Puoti and Kimble, 1999
). If
general pre-mRNA splicing was normal in mog-6 mutants, what might be
the role of mog-6 in fem-3 regulation? One possibility is
that MOG-6 might indeed be required for splicing, but that its function would
be redundant with that of other proteins, perhaps another cyclophilin or MOG
protein. Alternatively, mog-6 might be necessary for splicing
selected RNAs that are different from those tested. Many other possibilities
remain. For instance, MOG-6 could be required for the localization of other
factors such as FBF, NOS-3 and GLD-3 that in turn act on fem-3 in the
cytoplasm. However, we did not observe an altered expression pattern of FBF
and GLD-3 in mog-6 mutants. Another possibility is that MOG-6 is
required for the biological activity of FBF, NOS-3 and GLD-3 rather than for
their expression.
How does the MOG-6 protein control the sperm/oocyte switch? Its maternal
requirement for embryonic development
(Graham et al., 1993), its
ubiquitous expression and its roles in both germline sex determination and
proliferation indicate that fem-3 is unlikely to be the unique target
of mog-6. The identification of other proteins that genetically or
physically interact with MOG-6, as well as the discovery of additional targets
of mog-6, are likely to bring more insights.
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ACKNOWLEDGMENTS |
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Footnotes |
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