EMBO
reports
© 2001 European Molecular Biology Organization EMBO reports vol. 2 | no. 10 | pp 899–904 | 2001 899
RNA and sex determination in Caenorhabditis
elegans
Post-transcriptional regulation of the sex-determining tra-2 and fem-3 mRNAs in
the Caenorhabditis elegans hermaphrodite
Alessandro Puoti+, Paolo Pugnale, Marco Belfiore, Anne-Catherine Schläppi & Zarifja Saudan
Department of Biology, University of Fribourg, Rue du Musée 10, 1700 Fribourg, Switzerland
Received April 10, 2001; revised August 9, 2001; accepted August 21, 2001
The Caenorhabditis elegans hermaphrodite sequentially
produces sperm and oocytes from a single pool of precursors.
Therefore, the hermaphrodite’s germ line is the site of two
major cell fate decisions: a germ cell precursor first undergoes
a mitosis/meiosis decision and then a sperm/oocyte decision.
While the mitosis/meiosis decision is governed by Notch/GLP-1
signalling, the sperm/oocyte decision relies on post-transcriptional
regulation of two key mRNAs, tra-2 and fem-3. This review
focuses on factors that are required for the silencing of these
mRNAs, which results in the sequential production of sperm
and oocytes. Most factors that regulate the expression of tra-2
and fem-3 are homologous to proteins involved in RNA
regulation in yeast, mammals or Drosophila, suggesting that at
least some of the molecular mechanisms regulating the two
worm mRNAs have been conserved throughout evolution.
Introduction
During the last two decades, numerous examples have shown
that post-transcriptional regulation of gene expression is important
in many organisms, particularly during embryogenesis and
larval development (Wickens et al., 2000). Post-transcriptional
regulation can occur at any step between transcription and
translation. Thus it can affect RNA processing, RNA transport,
stability or translation initiation. Post-transcriptional control not
only allows a quicker response to the environment compared
with de novo transcription, but also permits regulated gene
expression in the absence of transcription (Mathews et al.,
2000). This may be particularly important during early embryogenesis,
when transcription is often repressed. Furthermore,
regulation at the RNA level allows precise control of protein
dosage, as is the case for mammalian tumour necrosis factor-α
and Caenorhabditis elegans xol-1, which are regulated both
transcriptionally and post-transcriptionally (Han et al., 1991;
Nicoll et al., 1997).
Post-transcriptional regulation has been studied in many
organisms, including C. elegans, in which several genes that are
post-transcriptionally regulated have been identified. These
genes code for the heterochronic RNAs lin-14, lin-28 and lin-41
(Lee et al., 1993; Moss et al., 1997; Slack et al., 2000), the xol-1
RNA that is responsible for primary sex determination (Nicoll et
al., 1997; Skipper et al., 1999), the maternal RNAs glp-1 and
apx-1 that are required for embryonic polarity (Evans et al.,
1994; Mickey et al., 1996) and the germline sex determination
fem-3 and tra-2 mRNAs (feminization; sexual transformer; Ahringer
and Kimble, 1991; Goodwin et al., 1993). This review focuses on
insights recently gained from studies on germline cell fate decisions
mediated by the regulation of the tra-2 and fem-3 RNAs whose
products play distinct roles in specifying gametes as sperm or
oocytes. In both cases, regulation is achieved via elements that are
present in the 3′ untranslated regions (UTRs) of the RNAs.
Caenorhabditis elegans exists in two sexual forms, males and
hermaphrodites. Hermaphrodites develop through four larval
stages before reaching adulthood. They produce their first germ
cells during the fourth larval stage (L4), and these differentiate
into sperm. Approximately 160 sperm are made in each of the
two gonadal arms at this time. After the L4-to-adult molt,
spermatogenesis is turned off and all of the gametes that form
subsequently develop into oocytes (for a review see Schedl,
1997). The sequential production of two fundamentally different
germ cells requires post-transcriptional repression of two
+Corresponding author. Tel: +41 26 300 8904; Fax: +41 26 300 9741; E-mail: Alessandro.Puoti@unifr.ch
900 EMBO reports vol. 2 | no. 10 | 2001
A. Puoti et al.
review
mRNAs, tra-2 and fem-3; the former must be repressed for
spermatogenesis, and the latter for oogenesis (Figure 1). While
TRA-2 is a large transmembrane protein that has some similarity
to the Patched receptor (Kuwabara and Kimble, 1995), no functional
motifs have been found in the FEM-3 protein. One model
proposes that FEM-3 is a cytoplasmic protein that binds to the
intracellular portion of membrane-bound TRA-2 (Mehra et al.,
1999) and that the release of FEM-3 from TRA-2 in response to
the extracellular ligand HER-1 results in male development
(Kuwabara and Kimble, 1992). In this model, the sperm/oocyte
switch depends on the physical interactions between the TRA-2
and FEM-3 proteins and thus requires a finely tuned balance
between TRA-2 and FEM-3. Regulation at the RNA level might
be critical to achieving the proper stoichiometry.
Repression of tra-2 for the
onset of spermatogenesis
Genetic studies have shown that tra-2 promotes female development
in that tra-2 loss-of-function (lf ) mutations transform XX
animals into non-mating pseudomales that do not produce
oocytes (Hodgkin and Brenner, 1977). Insight into tra-2 regulation
came from analysis of tra-2 gain-of-function (gf ) mutants
that do not produce sperm during L4, but instead produce only
oocytes (Doniach, 1986; Figure 1). tra-2 expression is regulated
by two repeated elements in the 3′UTR that were identified
through dominant tra-2 (gf) mutations. These elements, the TGEs
(for tra-2 and Gli Elements, formerly named DREs), consist of
two repeats of 28 nucleotides that are separated by a fournucleotide
spacer and that are disrupted by small deletions in
tra-2 (gf ) alleles (Goodwin et al., 1993). Single copies of TGEs
are also found in the 3′UTR of other mRNAs such as the tra-2
mRNA from Caenorhabditis briggsae and the human oncogene
GLI, suggesting that regulation through the TGE is conserved in
other species (Jan et al., 1997).
Factors that bind directly to the TGEs or that negatively regulate
tra-2 have been sought and have provided insights into the
mechanisms that control tra-2 expression. RNA gel shift experiments
performed with crude worm extracts revealed a binding
activity—the direct repeat factor (DRF)—which specifically
Fig. 1. Development of the germ line in wild-type and mutant C. elegans hermaphrodites. The hermaphroditic gonad consists of two arms that extend during
development and are connected through the vulva (top). The L3 worm is outlined with its anterior to the left. The right gonadal arm is boxed and shown to scale in
L4 larvae and adults. Undifferentiated germ cell nuclei are shown as black dots, while mature sperm and oocytes are shown in blue and pink, respectively.
Development of a wild-type hermaphrodite follows the thick arrows. Repression is shown by an inverted T. Suppression of repression is shown by a crossed
inverted T. During the L3 stage, no mature gametes are present and the gonad arms start to reflex (top). At L4, wild-type hermaphrodites produce sperm (right)
while tra-2 (gf) mutants make oocytes (left). The onset of spermatogenesis is dependent on repression of tra-2 through its 3′UTR. Wild-type adults contain both
sperm and oocytes because spermatogenesis switches to oogenesis after the L4-to-adult molt (bottom, centre). This switch requires repression of the fem-3 mRNA
through its 3′UTR. While tra-2 (gf) mutants continue oogenesis, leading to a feminized germ line (Fog, bottom, left), fem-3 (gf) mutants do not switch to oogenesis
and accumulate excess sperm (Mog, bottom, right).
EMBO reports vol. 2 | no. 10 | 2001 901
RNA and sex determination in C. elegans
review
recognizes one TGE in the tra-2 3′UTR (Goodwin et al., 1993).
DRF is a good candidate for a post-transcriptional repressor of
tra-2, and its components are being characterized. These
potentially include the GLD-1 (germ line development) protein,
which was identified in a yeast three-hybrid screen as a specific
binding factor for the TGEs (Jan et al., 1999). In addition, the
F-box-containing protein FOG-2 (feminization of the germ line)
was found to interact directly with GLD-1 and has been
proposed to act as a bridge to assemble a tra-2 mRNA–multiprotein
complex, which could repress tra-2 translation (Clifford
et al., 2000). GLD-1, a cytoplasmic protein that belongs to the
STAR (signal transduction and activation of RNA) family has a
single KH RNA-binding motif that is required for inhibition of
tra-2 translation through the TGEs (Figure 2A; Jan et al., 1999).
Consistent with their role in regulating tra-2 activity, GLD-1 and
FOG-2 are both required for spermatogenesis and are expressed
in the hermaphrodite germ line (Jones and Schedl, 1995; Clifford
et al., 2000).
In another approach, a genetic screen for loss-of-function
alleles that mimic a Tra-2 (gf ) phenotype identified the laf-1
gene (lethal and Fog). Like gld-1, laf-1 is required for tra-2
repression and works through the tra-2 3′UTR. laf-1 is needed for
spermatogenesis and for the repression, in vivo and in vitro, of
transgenes bearing TGEs in their 3′UTRs (Goodwin et al., 1997).
In addition, double mutant analyses indicate that laf-1 acts
upstream of tra-2, as would be predicted for a regulator of tra-2.
The molecular identity of laf-1 is not known yet, and hence
direct binding of LAF-1 to the tra-2 3′UTR has not been tested.
Future experiments will determine whether or not LAF-1 is
another component of DRF.
Although polyribosome analysis has shown that tra-2 is regulated
at the level of translation, and that repression via its 3′UTR
acts through the TGE by shortening of the tra-2 mRNA poly(A)
tail (Thompson et al., 2000), the precise action of DRF on tra-2
remains to be elucidated.
In addition to regulation at the RNA level, the activity of TRA-2
might also be controlled post-translationally. In fact, tra-3 has
been shown to encode a calpain-like protease that cleaves TRA-2
and generates a peptide with a feminizing activity that could
bind and inactivate FEM-3 (Barnes and Hodgkin, 1996;
Goodwin et al., 1997; Sokol and Kuwabara, 2000).
Repression of fem-3 for the
sperm/oocyte switch
fem-3 is necessary for spermatogenesis in both the male and the
hermaphrodite; fem-3(lf) mutants never produce sperm (Kimble
et al., 1984). Conversely, fem-3(gf) mutants do not switch to
oogenesis and thus accumulate excess sperm (Barton et al.,
1987). These findings indicate that fem-3 inhibition is required
for the sperm/oocyte switch.
The first evidence that fem-3 might be regulated at the RNA
level came from fem-3(gf) mutants that were obtained as extragenic
suppressors of fem-1(lf) and fem-2(lf) (Barton et al., 1987).
The molecular lesions in all 19 fem-3(gf) alleles are point mutations
or small deletions in the 3′UTR, and disrupt a five-nucleotide
element named the PME (point mutation element; Ahringer
and Kimble, 1991). Thus, the PME may act as a cis-acting
repressor of fem-3. In addition to its localization in the 3′UTR,
three additional lines of evidence argue that the PME is responsible
for the post-transcriptional rather than transcriptional regulation
of fem-3. First, the steady-state levels of fem-3 mRNA are
similar in wild-type animals and fem-3(gf) mutants. Secondly, a
fem-3 RNA-binding activity can be titrated by expressing a
heterologous RNA that contains the wild-type fem-3 3′UTR,
resulting in masculinized germ lines in hermaphrodites. Such
masculinization does not occur if a gain-of-function mutated
fem-3 3′UTR is used, indicating that the wild-type fem-3 3′UTR
is specifically bound by one or several factors that are required
for the sperm/oocyte switch (Ahringer and Kimble, 1991).
Thirdly, the somatic expression of a reporter construct bearing a
heat shock promotor, the lacZ gene and the 3′UTR of fem-3 is
normally repressed in wild-type worms, but it is de-repressed if
a mutation disrupts the PME in the transgene (Gallegos et al.,
Fig. 2. Trans-acting regulators of tra-2 and fem-3. (A) tra-2 regulation. The
GLD-1 protein binds specifically to the TGEs in the 3′UTR of the tra-2
mRNA. FOG-2 interacts with RNA bound to GLD-1. Although a single GLD-1
protein is shown here, the TGEs could be bound by several molecules of GLD-1.
LAF-1 is an additional factor that regulates tra-2 either by binding directly to
tra-2 or by acting on GLD-1 or another putative factor (X). Some or all of the
trans-acting factors that regulate tra-2 might form a protein complex named
DRF. (B) fem-3 regulation. The model shows FBF and its interacting partner
NOS-3 forming a cytoplasmic complex on the fem-3 mRNA. FBF specifically
recognizes the PME through its eight Puf repeats, while NOS-3 binds RNAs
non-specifically. Although the function of NOS-3 is redundant with those of
NOS-1 and NOS-2, the latter proteins have not been included in this complex
since they do not bind to FBF. On the other hand, at least three nuclear MOG
proteins might act on fem-3 either directly or indirectly, via the FBF/NOS-3
complex and/or additional factors (Y). This action may involve RNA
processing and RNA binding.
902 EMBO reports vol. 2 | no. 10 | 2001
A. Puoti et al.
review
1998). In contrast to the distribution of tra-2, that of fem-3
mRNA in polyribosomes has not been studied so far, and as a
consequence, it remains to be shown whether fem-3 is actually
regulated at the level of RNA processing, nuclear export, stabilization
or translation. Furthermore, good antibodies against TRA-2
and FEM-3 have not been obtained so far. Thus, it has not been
shown that the protein levels of TRA-2 and FEM-3 actually
change during gametogenesis.
Factors that act in trans to fem-3 via its 3′UTR and that are
required for the regulation of the sperm/oocyte switch have been
sought via both genetic and biochemical means. The rationale
behind the genetic screens that were undertaken was that, if
such trans-acting factors are required for fem-3 repression, a
mutation leading to the loss of the repressor should generate a
phenotype that is similar to Fem-3(gf ). Genetic screens identified
six mog genes (masculinization of the germ line) that act
upstream of fem-3 and are required for the sperm/oocyte switch
in hermaphrodites (Graham and Kimble, 1993; Graham et al.,
1993). Additional evidence for mog function in fem-3 regulation
came from the observation that protein expression from the
lacZ::fem-3 3′UTR transgene was higher in each of the six mog
mutants than in wild-type worms, indicating that the mog genes
are required for fem-3 repression through its 3′UTR in vivo
(Gallegos et al., 1998). Of the six mog genes, mog-1, mog-4 and
mog-5 were cloned and found to encode nuclear proteins of a
particular DEAH-box RNA helicase subfamily. These proteins
are the C. elegans homologues of yeast splicing factors Prp16p,
Prp2p and Prp22p, respectively, suggesting that fem-3 regulation
could involve pre-mRNA splicing as well (Puoti and Kimble,
1999, 2000).
In addition to the genetic approach, yeast two- and threehybrid
screens were used to identify trans-acting factors that
either bind to known fem-3 regulators via protein–protein interactions
or that bind directly to the fem-3 3′UTR, respectively. To
identify factors that bind specifically to the fem-3 3′UTR, Zhang
et al. (1997) used a yeast three-hybrid screen for RNA-binding
proteins. Specifically, two 37-nucleotide tandem repeats
containing the PME were used as RNA bait. This screen yielded
two distinct proteins, FBF-1 and FBF-2 (fem-3 binding factor),
which share 91% amino acid identity and that are probably
redundant. RNA interference directed against both fbf-1 and
fbf-2 led to masculinized hermaphrodites that accumulate
excess sperm and do not switch to oogenesis, suggesting that fbf
is required for the switch from spermatogenesis to oogenesis
(Zhang et al., 1997).
In another approach, protein–protein interaction screens using
the yeast two-hybrid system identified additional trans-acting
factors. The fem-3-binding protein FBF-1 was used as a bait for
interacting proteins (Kraemer et al., 1999). The C. elegans
protein NOS-3 (Nanos) was identified as a binding partner for
both FBF-1 and FBF-2 in the yeast two-hybrid system and in
vitro. NOS-3 binds directly to FBF proteins but has an additional
general RNA-binding activity that is not required for its binding to
FBF. NOS-3 contains two unusual zinc fingers in its C-terminus
and has at least two orthologues in C. elegans, NOS-1 and NOS-2,
which are required for germ-line viability (Subramaniam and
Seydoux, 1999). Similarly to FBF, the three nos genes are likely
to have redundant functions since sterility or occasional excess
sperm was only observed in nos double or triple mutants
(Kraemer et al., 1999; Subramaniam and Seydoux, 1999).
FBF-like and NOS-like repressors
FBF is a member of a large protein family that is characterized by
the presence of eight Puf (Pumilio and FBF) repeats of ∼37 amino
acids and its Drosophila counterpart is the Pumilio (Pum) protein
(Zhang et al., 1997). While FBF represses fem-3 expression by
acting on the fem-3 3′UTR, Pum is necessary to repress the translation
of the hunchback (hb) mRNA through binding to the NREs
(Nanos response elements), which are located in the hb 3′UTR
(Wharton and Struhl, 1991). Repression of hb involves deadenylation
of the mRNA and requires the formation of a protein
complex that includes at least Pum, Nanos (Nos) and the NREs
(Wharton and Struhl, 1991; Wreden et al., 1997). In Drosophila,
the interaction between Nos and Pum is dependent on the presence
of the hb mRNA (Sonoda and Wharton, 1999). Additional
components such as the Drosophila ‘Brain Tumor’ (Brat) protein,
which forms a quaternary complex by associating with hb RNAbound
Pum and Nos (Sonoda and Wharton, 2001), might be
required for the repression of hb mRNA. Intriguingly, one Brat
homologue in C. elegans is LIN-41, which is required for the
switch from L4 to adult and which post-transcriptionally regulates
the heterochronic mRNA lin-29 (Slack et al., 2000).
Drosophila nos encodes a general RNA-binding protein with
two unusual CCHC zinc fingers that are conserved in the C.
elegans NOS-1, NOS-2 and NOS-3 proteins. Unlike Drosophila
Nos and Pum, C. elegans FBF and NOS-3 associate even in the
absence of an RNA molecule. Moreover, no interaction has so
far been reported between LIN-41 and either FBF or NOS-3.
However, the sequence similarities between NOS-3 and
Drosophila Nos and their cooperation with FBF and Pum,
respectively, suggest that similar molecular mechanisms govern
fem-3 and hb repression.
Drosophila Nos was first discovered to be essential, together
with Pum, for posterior patterning of the Drosophila embryo
(Lehmann and Nusslein-Volhard, 1991), while FBF and NOS-3
were identified as regulators of fem-3 and hence implicated
in the sperm/oocyte switch in C. elegans hermaphrodites.
However, recent studies have shown that Pum is also required
for germ cell development and migration in the embryo and for
maintenance of the germline stem cells in the adult ovary
(Forbes and Lehmann, 1998; Asaoka-Taguchi et al., 1999). Similarly,
the C. elegans nos and fbf genes might also have functions
beyond those in the sperm/oocyte switch since nos-1 to -3, and
some C. elegans pum homologues including fbf, appear to be
required for the proliferation and survival of germ cells (Kraemer
et al., 1999; Subramaniam and Seydoux, 1999).
Concerning cis-acting elements, Tadauchi et al. (2001) have
shown that Mpt5p, a Puf family member in Saccharomyces cerevisiae
also regulates mRNA through 3′UTR elements, in this
case, that of the HO mating type switch gene. Like Pum, yeast
Mpt5p negatively regulates HO by destabilizing the mRNA,
perhaps by promoting its deadenylation (Tadauchi et al., 2001).
Interestingly, sequence conservation is also found in the 3′UTRs
of fem-3, hb and HO mRNAs. In fact a four-nucleotide sequence
(UUGU) is found twice in each NRE, once in the HO 3′UTR and
once in the fem-3 3′UTR, where it almost overlaps with the
PME (ucUUGU; Zamore et al., 1997; Tadauchi et al., 2001).
Together with the similarities found between Puf family
members and their interaction with Nos-like proteins, the
identification of a partially conserved cis-acting element
EMBO reports vol. 2 | no. 10 | 2001 903
RNA and sex determination in C. elegans
review
suggests that Puf/Nos-dependent RNA regulation may represent
an ancient mechanism for post-transcriptional regulation of gene
expression.
Future perspectives
The cis-acting regulatory elements in fem-3 and tra-2 were
discovered 10 years ago. At present, several factors that are
required for the regulation of tra-2 and fem-3 are known, and
their initial characterization indicates that the regulation of the
two mRNAs in C. elegans involves trans-acting factors and cisacting
elements that may have similar or related functions in
other species. Although a molecular function has been proposed
for most of the trans-acting regulators that have been identified,
the molecular mechanisms through which they regulate tra-2
or fem-3 are not well understood. Searches for additional
components, the establishment of in vitro systems, and comparisons
with other model systems such as Drosophila and yeast are
likely to bring the answers.
Acknowledgements
We thank Betsy Goodwin and Adrian Streit for comments on
the manuscript. Thanks to H. Gachoud for preparing the
figures. A.P. is supported by grants 3130-054989.98 and
3100-055384.98 from the Swiss National Science Foundation.
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DOI: 10.1093/embo-reports/kve209
Front from left: Paolo Pugnale, Marco Belfiore,
Zarifja Saudan & Anne-Catherine Schläppi.
Back in the centre: Alessandro Puoti