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First published online December 12, 2006
doi: 10.1242/10.1242/dev.02687
1 Department of Developmental Genetics, Deutsches Krebsforschungszentrum, Im
Neuenheimer Feld 280, D-69120 Heidelberg, Germany.
2 Laboratoire de Biologie Moléculaire de la Drosophile,
Département de Biologie Moléculaire, Institut Pasteur, Paris
F-75015, France.
3 Developmental Biology Unit, European Molecular Biology Laboratory, Heidelberg
D-69117, Germany.
* Author for correspondence (e-mail: j.anne{at}dkfz.de)
Accepted 10 October 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Drosophila oogenesis, Protein arginine methyltransferase, Pole plasm, Nuage, capsuléen
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). All
germ cells throughout the animal kingdom contain in their cytoplasm a distinct
cloud-like structure termed the nuage
(Eddy, 1975).
One system with a high potential for understanding the assembly and role of
the nuage is Drosophila oogenesis. The Drosophila egg
chamber consists of a germ line cyst generated from a single cystoblast by
four successive mitotic divisions and surrounded by a monolayer of somatic
follicle cells. Due to incomplete cytokinesis, the germ cells remain connected
to each other through specialized cytoplasmic bridges. The oocyte derives from
one of the germ cells, while the remaining 15 cells differentiate into nurse
cells (Spradling, 1993)
(Fig. 2A). The nuage is
concentrated in the perinuclear cytoplasm of nurse cells and can be associated
with nuclear pores. It appears as a dense fibrous organelle unbound by
membrane and often associated with mitochondrial clusters
(Mahowald, 1971). Moreover,
the nuage interfaces with sponge bodies, which are abundant RNA-rich particles
present in the cytoplasm of nurse cells and, to a lesser degree, in the oocyte
(Wilsch-Bräuninger et al.,
1997).
The majority of the components identified in the nuage are also present in
the pole plasm of the oocyte, and more particularly in the polar granules. The
pole plasm constitutes the determinant that is both necessary and sufficient
to induce germ cell formation during early embryogenesis
(Illmensee and Mahowald,
1974). The first step in pole plasm formation is the transport of
oskar (osk) transcripts synthesized in nurse cell nuclei to
the posterior pole of stage 8 oocytes
(Ephrussi et al., 1991;
Kim-Ha et al., 1991). At this
location Osk is synthesized and serves as an anchor to initiate polar granules
assembly (Ephrussi and Lehmann,
1992; Smith et al.,
1992; Snee and Macdonald,
2004). In addition to Osk the polar granule components include
Vasa (Vas) (Hay et al., 1988;
Lasko and Ashburner, 1990),
which interacts directly with Osk
(Breitwieser et al., 1996),
Tudor (Tud) (Bardsley et al.,
1993) and a number of transiently localized factors that are
mainly necessary for osk mRNA transport and translation. During late
oogenesis and early embryogenesis, the polar granules are maintained at the
posterior pole. At the time of blastoderm formation, they are sequestered in
the pole cells, the primordial germ cells of the fly, in which they coalesce
into a smaller number of large particles
(Mahowald, 1968), ultimately
disappear and are replaced by the nuage
(Mahowald, 1971). This
structure appears to evolve from components of the pole plasm and persists
only in established germ cells. The only identified nuage-specific component
absent from the polar granules is Maelstrom (Mael) which shuttles between the
nucleus and the cytoplasm (Findley et al.,
2003).
Among these proteins, Vas plays a cardinal role in the formation of the
nuage (Findley et al., 2003).
In vas ovaries the nurse cells are devoid of nuage at the
ultrastructural level (Liang et al.,
1994). The function of Tud in the nuage remains unknown but Tud
may play a role in the assembly or modification of specific RNP complexes, as
indicated by its requirement for the transfer of mitochondrial ribosomal RNAs
from the mitochondria to the polar granules
(Amikura et al., 2001). The
presence of shared components reinforces the view that the nuage and the polar
granules are closely related structures, in which components, such as Vas and
Aubergine (Aub), may dissociate from the nuage to reassemble into the polar
granules (Snee and Macdonald,
2004).
The mechanisms by which nuage components become assembled at the perinuclear region of the nurse cells remains, however, to be identified. Here we report that the catalytic activity of the Capsuléen (Csul) protein-arginine methyltransferase is required for the localization of specific components of the nuage and pole plasm, and in particular of Tud.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Details of primers and cloning strategies will be provided
upon request.
GST pull-down assay
Fragments of tud and SmB cDNAs were cloned into pCITE-4
(Novagen). Tud and SmB polypeptides were synthesized using the TNT Coupled
Reticulocyte Lysate System (Promega) in the presence of unlabeled amino acids.
Full-length SmB proteins were synthesized from the LD14049 cDNA
plasmid in the presence of [35S]methionine (Amersham). To
cotranslationally inhibit sDMA synthesis, 0.3 mM S-adenosylhomocysteine
(Sigma) was included in the reaction. GST pull-down assays were performed as
previously described (Anne and Mechler,
2005).
Immunocytochemistry
A GST-Csul fusion protein construct containing a 442-amino-acid peptide
corresponding to amino acid residues 168-609 of Csul was prepared by cloning
the BamHI-XhoI fragment of the csul cDNA in the
pGEX-4T2 GST fusion vector (Pharmacia) and expressed in the bacterial
strain BL21(DE3). Anti-Csul polyclonal antibodies raised in rabbits against
the GST-Csul fusion protein were purified by affinity chromatography on a
protein Aagarose column (Roche) and preadsorbed on proteins extracted from
Escherichia coli.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Homozygous csulP females produce embryos
(csulP embryos) displaying defects in abdominal patterning
and lacking pole cells (Fig.
1B). Flies hatching from csulP larvae are
fully agametic. The severity of the abdominal defects is variable, ranging
from the presence of only one to two abdominal segments
(Fig. 1D) to a fully normal
abdomen. On the basis of the absence of pole cells and the occurrence of
abdominal defects, we classified csulP as belonging to the
`posterior-grandchildless' class of mutations
(Schüpbach and Wieschaus,
1986).
To determine whether csul acts in the posterior-grandchildless
pathway, we investigated two gene transcripts, nanos (nos),
the posterior determinant (Wang and
Lehmann, 1991), and germ cell-less (gcl),
specifically involved in pole cell formation
(Jongens et al., 1992). The
posterior localization of their transcripts in preblastoderm embryos depends
on this pathway. In one half of csulP embryos,
nos mRNA was absent or barely detectable (data not shown), whereas in
the other half it was correctly localized, albeit in much lower amounts than
in wild-type embryos (Fig.
1E,F). Analysis of the Nos protein revealed a Nos gradient
emanating from the posterior pole in only 15 percent of
csulP embryos, whereas Nos remains undetected in the other
embryos (data not shown). In situ hybridization showed that gcl mRNA
is evenly distributed in csulP embryos instead of being
localized to the posterior pole (Fig.
1G,H). From these data we conclude that csul is required
for the posterior localization of at least two transcripts, nos and
gcl.
The posterior-grandchildless group comprises a relatively small number of
genes hierarchically organized with osk orchestrating the activity of
the other genes. As ectopic expression of osk induces pole cell and
abdomen formation at the anterior of the embryo
(Ephrussi and Lehmann, 1992),
we tested whether expression of an osk-bcd3'UTR
transgene would induce pole cells formation in csulP
(Fig. 1I-L). No pole cell could
be detected in csulP; osk-bcd3'UTR
blastoderm embryos. Cuticle examination revealed the absence of duplicated
abdominal structures and a marked atrophy of the head in the majority of the
larvae, indicating a downregulation of osk activity. We therefore
suggest that csul is essential for pole plasm assembly and acts
downstream of osk.
|
). In
contrast to Osk and Vas, no posterior accumulation of Tud could be detected in
csulP, even in very early embryos
(Fig. 1V). From these data we
conclude that csul activity mediates Tud and (to some degree) Vas
accumulation and the maintenance of Osk and Vas at the posterior pole of the
embryo.
csul-dependent pole plasm assembly during oogenesis
As pole plasm assembly occurs during oogenesis we investigated the
distribution of pole plasm components in csulP egg
chambers. osk mRNA and Osk protein distributions appear normal
(Fig. 2A,B; data not shown) and
western blot analysis of ovarian protein extracts revealed similar levels of
Osk proteins and phosphorylation of the short Osk isoform in
csulP and wild-type egg chambers (data not shown). We then
used a GFP-Vas transgene (Sano et
al., 2002) to monitor the distribution of Vas. GFP-Vas is absent
from the pole plasm in the majority of stage 10 csulRM egg
chambers (data not shown). Only a minority of csulRM
oocytes display a small amount of GFP-Vas at the posterior pole
(Fig. 2D). Immunostaining of
csulP egg chambers using anti-Vas antibodies showed a
similar distribution (data not shown). By comparison with early embryos, the
smaller number of stage 10 oocytes displaying a posterior localization of Vas
indicates that Vas may accumulate in the pole plasm during late
csulRM oogenesis to become detectable during early
embryogenesis.
By contrast, we found that Tud fails to concentrate at the posterior pole in csulP oocyte of stage 10 egg chambers (Fig. 2F) indicating that csul activity is essentially required during oogenesis for the localization of Tud, and to a lesser degree for that of Vas, in the pole plasm.
csul-dependent nuage assembly
As a number of pole plasm components localize in the nuage, we analyzed
their pattern of distribution in csul egg chambers. GFP-Vas shows a
normal, albeit weak, nuage localization in csulRM nurse
cells during early oogenesis (Fig.
2H). Subsequently, GFP-Vas progressively fades away and is barely
detected in the nuage of stage 10 egg chambers
(Fig. 2D). By contrast, no Tud
is detected in the nuage in csulP egg chambers, although
Tud accumulates in mutant oocytes (Fig.
2J) and transiently localizes at their anterior margin in stage
6/7 egg chambers (data not shown). In wild-type nurse cells, Mael displays a
punctate perinuclear distribution (Fig.
2K), which is not detected in csulP egg
chambers (Fig. 2L). Hence,
csul is needed for the perinuclear localization of Mael, Tud and Vas,
indicating that csul contributes to the assembly of the nuage.
Molecular characterization of the csul gene
To gain insight into csul function, we isolated the gene by
inverse PCR amplification of a genomic DNA fragment adjacent to the
P-[white+] insertion site. This fragment was used to
isolate DNA segments overlapping the P-element which in turn were
used to recover cDNA clones. Northern blot analysis using genomic DNA probes
from both sides of the P-insert revealed transcripts of 
2kb in
size. Alignment of the cDNA sequences with genomic DNA revealed two types of
transcripts oriented in opposite directions. The P-element was
inserted in one of the transcription units, 51 bp downstream of its 5'
end and 7 nucleotides downstream of the putative translation initiation site.
This transcript was assigned to the csul gene, extending from the
P-insert down to the adjacent Kinesin heavy chain
(Khc) gene. The 3' end of Khc is located 5 nucleotides
downstream of the 3' end of csul. To validate the
identification of the csul gene, we generated a transgene and tested
its ability to complement csul mutations. We found that the fertility
of csulRM females was restored by a single copy of the
XhoI-XhoI P-[csul] transgene
(Fig. 3A). Constructs
containing either the downstream Khc
(Saxton et al., 1991) or the
upstream fidipidine (data not shown) genes failed to restore
fertility to csulRM females.
Alignment of cDNA and genomic DNA sequences revealed that csul is
composed of five exons (Fig.
3B). The csul transcript has a size of about 2 kb
(Fig. 3C) and potentially
encodes a 610 aminoacid protein (accession number: AJ002740). In vitro
translation of a full-length cDNA produced a single polypeptide with an
apparent molecular mass of 67 kDa corresponding to the predicted
molecular mass of Csul (data not shown). Polyclonal antibodies were raised
against the carboxyl-terminal half of bacterially synthesized Csul. On western
blots of ovarian and embryonic protein extracts, these antibodies reacted with
a protein band displaying a molecular mass of 65 kDa, consistent with the
expected mass of Csul (Fig.
3D). No such protein was detected in ovarian extracts of
csulP females. However, long exposure of the immunoblot
revealed a faint band in the 65 kDa range, suggesting that
csulP females may produce a low level of Csul, possibly by
use of an in-frame initiation codon present in the
P-white+ inverted repeat and located 16 nucleotides
upstream of the insertion site (data not shown). This finding suggests that
csulP is a hypomorphic allele.
|
).
To ascertain that Csul acts as a methyltransferase, we investigated whether
amino acid substitutions in the catalytic domain of the enzyme would
inactivate its function. Csul family proteins contain a conserved core region
characterized by motif I [GXGRG], which, together with the Post-I motif, forms
the S-adenosyl-L-methionine binding module
(Ma, 2000). As the GXGRG motif
is highly conserved in the Csul homologs and is required for protein
methylation (Pollack et al.,
1999) we selected this region for mutational analysis,
substituting the conserved residues Gly343 and Arg344
with Ala and Leu residues, respectively.
Wild-type csul and csulG343A;R344L gene
sequences were fused to the tandem affinity purification (TAP)
(Rigaut et al., 1999) tag,
cloned into a transformation vector and transgenic strains were generated.
After introducing the transgenes in a csulRM genetic
background we determined, by western blot, that they both express at similar
level (data not shown) and examined their ability to abolish the
csulRM maternal-effect embryonic phenotype.
csul-TAP and TAP-csul transgenes rescue the development of
csulRM embryos, whereas the
csulG343A;R344L-TAP transgene shows no rescue activity
(data not shown and Fig. 7D).
These results strongly suggest that csul encodes a methyltransferase
whose activity requires an intact catalytic domain.
Sm proteins are substrates of the Csul-Vls complex
We used anti-SYM10 antibodies (
-SYM10), which specifically recognize
proteins containing multiple sDMA-glycine repeats
(Boisvert et al., 2002), to
investigate the pattern of methylated proteins extracted from wild-type,
csul, valois (vls), vas, aub and osk
ovaries. As shown in Fig. 4A,
-SYM10 reacted with five protein bands of a relatively low molecular
mass, ranging from 14 to 30 kDa, in wild-type, osk, vas and
aub protein extracts. In csul and vls the intensity
of four of these bands was strongly reduced. Probing similar protein blots
with the anti-ASYM24 antibodies, which identify proteins containing asymmetric
di-methyl arginyl residues (aDMA) (Boisvert
et al., 2002), we found no change in the pattern of the reactive
protein bands in all extracts. These results corroborate our finding that Csul
and Vls are part of a methylosome complex contributing to sDMA synthesis in
specific proteins (Anne and Mechler,
2005), and indicate that Vls acts as a co-factor of Csul.
Moreover, these data show that Vas, Osk and Aub exert no function in the
Csul-dependent synthesis of sDMA residues.
As the size of the Drosophila proteins recognized by -SYM10
corresponds to that of components required for pre-mRNA splicing in human
cells (Boisvert et al., 2003),
we analyzed whether Sm proteins might be in vivo substrates of Csul. As shown
in Fig. 4B,C, making use of
available mutations in Drosophila genes encoding Sm proteins, we
identified SmB and SmD3 as targets of the Csul-Vls complex. In particular we
found that the amount of an 26 kDa -SYM10 reactive protein was
reduced by half in western blots of heterozygous
SmBBG02775 in comparison to wild-type ovarian extracts. A
similar reduction in intensity was obtained with the Y12 monoclonal antibody
(-Y12) that specifically recognizes methylated SmB
(Paterson et al., 1991).
Similarly, comparison of protein extracts of wild-type and homozygous
SmD3l(2)k131-07 larvae
(Schenkel et al., 2002)
revealed that the -SYM10 signal of a 16 kDa protein is nearly abolished
in the mutant, identifying this protein as SmD3.
To further confirm the Csul-dependent symmetrical methylation of SmB, we
stained ovaries using -Y12. As compared with wild-type ovaries, in
which methylated SmB accumulates in the nuclei of the nurse cells and somatic
follicular cells, -Y12 signal was dramatically reduced in
csulRM egg chambers
(Fig. 4D). Taken together, our
data demonstrate that Csul is required for the symmetrical methylation of SmB
in vivo.
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Interaction between Csul and Tud in vivo is essential for Tud localization
The defective accumulation of Tud in the nuage of csulP
nurse cells led us to evaluate whether Tud might directly interact with Csul.
As shown in Fig. S1 in the supplementary material, we performed pull down
assays, using tagged fragments of Tud
(Golumbeski et al., 1991;
Anne and Mechler, 2005). In
vitro translated fragments of Tud were incubated with immobilized GST-Csul
proteins. After washing, the bound S*Tag-Tud proteins were revealed
by immunodetection. This procedure showed that the JOZ fragment and the 9A1
polypeptide and derivatives display a strong binding, whereas the 3ZS+L
fragments exhibit more moderate binding to GST-Csul whereas GST alone is
unable to bind to Tud fragments. We then used Tud9A1-N to map the binding site
of Tud in Csul.
As shown in Fig. 7A, we found that the 9A1-N fragment of Tud can bind to the N-terminal region of Csul (amino acid residues 1-111). These results suggest that a domain in the N-terminal region of Csul mediates a direct and specific interaction with Tud. To delimit more precisely the N-terminal domain of Csul critical for Tud binding, we generated serial N-terminal truncations of Csul. In this way we identified a critical region for Tud binding between amino acids 60 and 80 (Fig. 7B,C).
We next tested the in vivo relevance of the N-terminal region of Csul with
respect to the localization of Tud in the nuage and the formation of pole
cells. For this purpose we constructed four csul transgenes each
bearing an internal deletion (between amino acids 21-40, 41-60, 61-80 and
81-100, respectively), fused in-frame to the TAP tag, and introduced these
transgenes into the csulRM genetic background.
Immunostaining revealed that the Csul proteins carrying the deletions
21-40 and 41-60 could restore Tud localization in the nuage. By
contrast, deletions 61-80 and 81-100 failed to do so
(Fig. 7D, left panels). Finally
we observed that deletions 21-40 and 41-60 can give rise to the
formation of pole cells and rescue the csulRM
maternal-effect phenotype, whereas deletions 61-80 and 81-100 do
not (Fig. 7D, right panels).
Together with our in vitro interaction results, these findings indicate that
the localization of Tud in the nuage requires interaction with Csul.
sDMA-Sm proteins can bind to Tud in vitro
As sDMA Sm proteins can bind to the Tudor domain of SMN
(Brahms et al., 2001;
Selenko et al., 2001) we
investigated the requirement of sDMA synthesis for Sm binding to Tud. For this
purpose we took advantage of the occurrence of an endogenous type II PRMT
activity in the reticulocyte lysate system
(Brahms et al., 2001). By using
S-adenosyl-homocysteine (SAH), which blocks the activity of protein
methyltransferases (Brahms et al.,
2001), we tested whether SAH could inhibit sDMA synthesis in SmB.
Addition of SAH to the reticulocyte lysate significantly reduced the amount of
-SYM10-immunoreactive SmB polypetides, but did not inhibit SmB
synthesis (see Fig. S2A in the supplementary material). We next incubated
bacterially produced Tud fragments (see Fig. S2B in the supplementary
material) with SmB polypeptides synthesized in the presence or absence of SAH.
As shown in Fig. S2C in the supplementary material, methylation of SmB
promotes its binding to all three tested Tud fragments.
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-SYM10 revealed that all four mutant Csul proteins failed to
symmetrically methylate SmB (Fig.
7E). The finding that the Csul proteins containing the two most
distal deletions (21-40 and 41-60) are still able to restore Tud
targeting to the nuage of csulRM egg chambers indicates
that only the methylation of Sm proteins is impaired. In light of the in vitro
binding results (Fig. 6) we
envisage that the absence of methylation of Sm proteins is primarily due to a
defective binding of SmB to Csul. Whether the two other deletions
(61-80 and 81-100) only affect the binding of Tud to Csul or
structurally compromise other functions of Csul remains an open question but
primarily our data show that we can separate Tud localization in the nuage
from Sm methylation. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Lee et al., 2000;
Branscombe et al., 2001),
homologous to Csul, and the recently identified PMRT7 and PRMT9
(Lee et al., 2005;
Cook et al., 2006) are
responsible for Type II methylation.
By using -SYM10 antibodies that recognize proteins harbouring two
spaced sDMA-glycine motifs (Boisvert et
al., 2002) we identified four major reactive proteins bands as
specific targets of Csul. These proteins are distinct from aDMA-containing
proteins, whose methylation is independent of Csul. Among the sDMA proteins,
we genetically confirmed that the spliceosomal components SmB and SmD3 are
Csul targets. The corresponding mammalian targets have been identified for
PMRT5 (Brahms et al., 2001;
Meister et al., 2001;
Friesen et al., 2001a). As
-SYM10 may only recognize a subset of sDMA proteins methylated by Csul,
further proteomic analysis of ovarian Csul complexes may identify additional
targets of Csul.
The Csul-Vls and human methylosomes interact with sDMA-proteins and Tudor-domain proteins
As indicated by the physical interaction of Csul with Vls
(Anne and Mechler, 2005) and
the size of the native Csul complexes, with a molecular mass of 500 kDa
(data not shown), Csul is part of a large protein complex. In the present work
we show that Vls, the Drosophila homolog of human MEP50, itself a
partner of PRMT5 (Friesen et al.,
2002), is also required in sDMA synthesis on identical target
proteins. However, in the case of pIcln, a component of the human methylosome
of yet unknown function (Friesen et al.,
2001a), we detected no interaction between Drosophila
pIcln and Csul in pull-down assays (data not shown). Furthermore, we found
that both Csul and Vls interact with the N-terminal moiety of SmB. This is in
contrast to PRMT5, which appears to bind to the RG-rich C-terminal domain of
Sm proteins (Friesen et al.,
2001a). Differences in protein interaction and quaternary
structure between the human and Drosophila methylosome may reflect
divergences in the activities of the methylosome between the two species.
Both human (Friesen et al.,
2001a; Meister et al.,
2001) and Drosophila methylosomes lead to sDMA synthesis
on Sm proteins (this study). Similarly to the requirement of sDMA synthesis
for the association of human Sm proteins with the SMN Tudor domain
(Côté and Richard,
2005), we found that Drosophila Sm proteins need to be
symmetrically methylated to bind Drosophila Tud. The binding of
sDMA-Sm to non-overlapping Tud polypeptides indicates that these proteins may
bind to several, if not all Tudor domains in Tud.
The association of human SMN protein with the PMRT5 complex suggests direct
interactions between PMRT5, MEP50 and SMN
(Meister and Fisher, 2002).
Similarly, Drosophila Tud can directly bind to Csul and Vls
(Anne and Mechler, 2005).
However, in contrast to Sm, which binds to multiple sites on Tud, Csul and Vls
more strongly interact with the N-terminal than the C-terminal moiety of Tud,
suggesting a distinct mechanism of association with Tud. Although the specific
binding sites of Csul and Vls on Tud remain to be determined, preliminary
results indicate that each protein binds to a distinct site.
As this work was being completed, another group reported the identification
of the csul gene, termed dart5
(Gonsalvez et al., 2006), and
showed that disruption of this gene (mutant e00797 from the Exelixis
collection) leads to the absence of sDMA synthesis of spliceosomal Sm proteins
without impairing spliceosomal function. This work and ours confirm the
previous finding of Khusial et al.
(Khusial et al., 2005),
indicating that sDMA synthesis on Sm proteins is not required for sRNP
assembly and transport, a critical process for Drosophila
development. In addition, Gonsalvez et al.
(Gonsalvez et al., 2006) also
characterized the maternal requirement of csul for pole cell
formation.
Tud localization in the nuage
In addition to their role in sDMA synthesis, Csul and Vls are required for
Tud localization in the nuage. Our data indicate that csul activity
is also necessary for the proper nuage accumulation of Vas. However, despite
the occurrence of Vas in the nuage of early csul egg chambers, Tud is
absent from this structure, suggesting that the activity of Csul in Tud
localization is independent from that exerted on Vas.
How the Csul/Vls methylosome directs Tud localization in the nuage remains
an open question. The restoration of fertility by mutated csul
transgenes defective in SmB binding, and hence in sDMA synthesis on SmB,
points out the occurrence of a yet unknown protein which should act as a
substrate of Csul and specifically function in germline formation. We favour a
cytoplasmic association of the Csul/Vls methylosome with this substrate and
Tud. Upon methylation the substrate is then transferred to Tud, as indicated
by the preferential binding of sDMA-SmB to Tud polypeptides. In our view, the
interaction between Csul/Vls, the substrate, and Tud may be critical to
position Tud in the vicinity of the site where sDMA synthesis takes place,
thus facilitating the association of Tud with the sDMA-protein. A similar
model has been proposed for the targeting of high-affinity Sm protein
substrates to the SMN complex (Friesen et
al., 2001b). Following the docking of the sDMA protein on Tud, the
Csul/Vls methylosome is released, and the Tud/sDMA protein complex becomes
positioned in the nuage. The docking of the sDMA protein might induce an
allosteric change in Tud, increasing its affinity for a component of the
nuage.
Finally, although Vas is not properly localized at the perinuclear region
of nurse cells in csul and vls mutant egg chambers we notice
that its distribution pattern differs in each mutant. In particular, the level
of Vas in the nuage is comparatively smaller in csul than in
vls mutants (Anne and Mechler,
2005), suggesting that Csul acts independently of Vls in the
localization of Vas to the nuage. Moreover, the finding that Vls specifically
accumulates in the nuage and pole plasm whereas Csul displays a ubiquitous
distribution (data not shown) suggests that both proteins may exert additional
independent functions.
Although the functional relationship between the nuage and pole plasm
remains unresolved, events occurring in the nuage may affect pole plasm
formation. In csul mutant egg chambers, Tud is absent from both the
nuage and the pole plasm and, similarly, a reduced amount of Vas in the nuage
correlates with a decreased level of this protein in the pole plasm. However,
it has been reported recently that a Tud protein containing the Tudor domains
1 and 6-10 could localize to the pole plasm, albeit at a moderate level
compared to full-length Tud, but fail to properly localize to the nuage
(Arkov et al., 2006).
Additional work on the requirement of Csul for Tud localization in the nuage
will be critical for understanding the assembly of this structure, its
dynamical relationship with the pole plasm, and the role of arginine
methylation in protein targeting.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/1/02687/DC1
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ACKNOWLEDGMENTS |
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