Department of Developmental Genetics, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, Heidelberg 69120, Germany
* Author for correspondence (e-mail: j.anne{at}dkfz.de)
Accepted 2 March 2005
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SUMMARY |
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Key words: Drosophila, Oogenesis, valois, Tudor, Pole plasm assembly
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Introduction |
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Although germline formation and abdominal development represent distinct
developmental processes, molecular and genetic analyses of the
posterior-grandchild-less group of genes indicate that both processes share a
common pathway for producing functional determinants localized at the
posterior pole of the embryo. So far four genes are known to be directly
required in pole plasm function, including osk
(Lehmann and Nüsslein-Volhard,
1986), vasa (vas)
(Schüpbach and Wieschaus,
1986
), tudor (tud)
(Boswell and Mahowald, 1985
)
and valois (vls)
(Schüpbach and Wieschaus,
1986
). Mutants in all four genes are characterized by the absence
or strong reduction of discernible polar granules
(Schüpbach and Wieschaus,
1986
; Boswell and Mahowald,
1985
).
Formation of pole plasm initially depends upon the localization of
osk mRNA (Ephrussi and Lehmann,
1992), which becomes concentrated at the posterior end of the
oocyte in stage 8 egg chambers (Ephrussi
et al., 1991
; Kim-Ha et al.,
1991
). Shortly thereafter, osk mRNA is translated into
two isoforms by use of different initiation codons
(Markussen et al., 1995
;
Rongo et al., 1995
), and the
short form of Osk induces the assembly of polar granules by recruiting
directly the Vas protein, a member of the DEAD-box family of putative RNA
helicases (Lasko and Ashburner,
1988
; Hay et al.,
1988b
) at the posterior pole of the oocyte
(Breitwieser et al., 1996
). Vas
may contribute to the synthesis of specific polar granule components, as
suggested by its requirement for promoting translation of nanos
(nos) (Gavis et al.,
1996
) and gurken
(Styhler et al., 1998
;
Tomancak et al., 1998
). The
other factor involved in pole plasm function is Tud
(Golumbeski et al., 1991
),
which accumulates in polar granules but is also detected in the nuage of nurse
cells and at the periphery of embryonic nuclei
(Bardsley et al., 1993
).
Although the molecular nature of vls remained elusive, this gene
appears to be involved in pole plasm function
(Schüpbach and Wieschaus,
1986
).
Formation of abdomen and pole cells can be mechanistically uncoupled. The
nos gene contributes to abdominal patterning, albeit not to pole cell
formation (Lehmann and
Nüsslein-Volhard, 1991). The localization of nos
mRNA to the posterior pole of the embryo
(Wang and Lehmann, 1991
)
depends upon genes acting in pole plasm and produces a transient gradient of
Nos proteins emanating from the posterior pole
(Wang et al., 1994
). Unlike
the posterior determinant, the determinant involved in pole cell formation is
likely to consist of multiple elements. The gene germ cell-less
(gcl) is one of them (Jongens et
al., 1992
), and ectopic expression of gcl mRNA at the
anterior pole of the embryo produces nuclei with characteristics of pole cell
nuclei with which Gcl protein becomes associated on the inner rim of the
nuclear pores (Jongens et al.,
1994
). Furthermore, mitochondrial ribosomal RNA (mtlrRNA) also
contributes to germ-line determination
(Iida and Kobayashi, 1998
).
Transport of the mtlrRNA from mitochondria to the surface of polar granules is
mediated by Tud protein through a yet unknown mechanism
(Amikura et al., 2001
).
During our analysis of the capsuléen gene (csul; CG3730), we found one major protein partner by using the yeast two-hybrid system. In this report, we show that the protein interacting with Csul is encoded by the vls gene and describe its function in the process of pole cell formation.
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Materials and methods |
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To rescue the vls phenotype a 2.04 kb genomic DNA fragment containing the vls transcription unit and 535 and 195 nucleotides of the 5' and 3' genomic regions, respectively, was cloned into the P-element vector CaSpeR4. The construct was named P-[vls]. Details will be provided upon request. To generate a Vls-HA fusion protein construct, a BglII site was created at the 3' end of the vls-coding sequence of a vls cDNA containing identical 5' and 3' untranslated regions as the genomic transgene. A fragment with 3xHA tags was cut with BglII from plasmid pSM 492 and cloned into the vsl-coding sequence. Details will be provided upon request. A clone containing six copies of HA was selected for microinjection.
Sequencing of mutant alleles
The vls-coding region was amplified by PCR (High Fidelity PCR
Master; Roche) from genomic DNA obtained from single vls1,
vls2 and vls3 flies (primers: sense
5'-gttgctcttccttgctggccgattctc-3', located at position 58
from the translation initiation codon; and antisense,
5'-tgcatttaaactgggctgctgcttcac-3'). The PCR products of 1.46 kb
were cloned into pBluescript. Two clones from independent PCR reactions were
sequenced for each allele using primers covering the coding region. Nucleotide
differences from the wild-type vls sequence were distinguished from
PCR errors by their appearance in both independent clones.
Yeast two-hybrid screen
Standard yeast two-hybrid methods were employed, using the GAL4 system
(Saccharomyces cerevisiae strain Y190). The Csul-coding region was
cloned into the yeast vector pGBT9 to produce a bait construct. This construct
was used to screen the Drosophila embryo MATCHMAKER cDNA library
(Clontech). About 1.2 millions clones were screened.
GST pull-down assay
Full-length vls and vas cDNAs were subcloned into pGEX6P2
(Pharmacia), full-length csul, gus and tud cDNAs into
pCITE-4 (Novagen). Recombinant proteins were synthesized in vitro using the
TNT T7 Coupled Reticulocyte Lysate System (Promega) in the presence of
unlabeled amino acids. GST-fusion proteins expressed in E. coli were
purified with glutathione sepharose (Pharmacia) and washed with the binding
buffer [20 mM HEPES (pH 7.8), 10% glycerol, 300 mM NaCl, 0.1% sodium
deoxycholate, 0.1% NP40 and 0.1% Triton X-100] plus protease inhibitors
(Complete EDTA free from Roche; 1:50 dilution). Recombinant proteins (10% of
the reaction volume) were added to this mixture (in 1 ml) and incubated for 3
hours at room temperature. The beads were washed six times (10 minutes each)
with binding buffer, boiled in loading buffer, and the proteins were separated
by electrophoresis on 8% or 12% SDS-polyacrylamide gels. After transfer to a
polyvinylidene difluoride (PVDF) membrane, the bound proteins were detected by
Western blotting using an S-protein Alkaline Phosphatase conjugate
(Novagen).
Immunoprecipitation
Fly ovaries were homogenized using a plastic pestle in ice-cold IP buffer
[145 mM NaCl, 10% glycerol, 1 mM MgCl2, 1.5 mM
NaH2PO4, 8 mM Na2HPO4 (pH 7.4) and
0.5% NP40] containing protease inhibitors. Lysates were clarified by
centrifugation at 14,000 g for 10 minutes at 4°C. The
supernatants were removed and mixed with 400 units of RNasin (Promega), 30
µl of anti-HA antibody-coupled protein A beads for 5 hours at 4°C on a
rotator. The IP mix was centrifuged at low speed to remove the supernatant.
Protein A-beads were then washed four times with IP buffer for 10 minutes each
and TAP-Csul was detected on a western blot by using alkaline
phosphatase-conjugated IgG (Sigma). TAP-Csul purification was essentially
performed as described by Rigaut et al.
(Rigaut et al., 1999).
Immunocytochemistry
Immunostaining was performed with primary antibodies, including anti-Mael
from rabbit (Findley et al.,
2003), anti-Osk from rabbit (gift of A. Ephrussi), anti-Vas from
rabbit and rat (gift of P. Lasko and A. Nakimura, respectively), anti-Tud from
rabbit (TUD56; gift of S. Kobayashi), and monoclonal anti-HA (clone 16B12;
BAbCO). To reduce background, we incubated first the anti-HA antibodies
(1:400) for 3 hours at room temperature with wild-type ovaries and then
overnight with experimental ovaries. For immunostaining, the ovaries were
fixed for 10 minutes in 4% paraformaldehyde. To visualize components of the
nuage, we used the protocol of Findley et al.
(Findley et al., 2003
).
Chromatin was visualized by staining with Oli-Green (Molecular Probes). Cy3-,
Cy5- and FITC-conjugated secondary antibodies (Jackson ImmunoResearch
Laboratories) were used at 1:200.
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Results |
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Csul and Vls interact in vivo
Among the nearest homologues of Vls we identified in protein databases the
methylosome protein 50 (MEP50) (Fig.
1D). MEP50, the human homologue of Vls, contains six WD repeats
and interacts with PRMT5 (Friesen et al.,
2002), the human homologue of Csul. The finding of an interaction
between Csul and Vls in yeast prompted us to analyze whether such binding
occurs in Drosophila ovaries. To this end, we constructed transgenic
flies bearing both the HA-vls transgene and a Tandem Affinity
Purification (TAP) (Rigaut et al.,
1999
) tagged csul transgene (TAP-csul) shown to
restore csul development (data not shown). Protein extracts of
P{TAP-csul}; vls3; P{HA-vls} ovaries were
immunoprecipitated using rabbit IgG-sepharose beads and the bound
Csul-complexes were released by treatment with recombinant TEV protease. Blots
of released proteins probed with anti-HA antibodies showed an immunoreactive
band of 50 kDa exhibiting the predicted mass of HA-Vls fusion protein
(Fig. 2A). Reciprocally, blots
of ovarian proteins immunoprecipitated with anti-HA antibodies and probed with
IgG antibodies displayed an immunoreactive band of the mass predicted for
TAP-Csul (90 kDa; Fig. 2B).
These results indicate that Csul and Vls can associate in a protein complex in
Drosophila ovaries.
|
Vls is present in nuage and pole plasm
To determine the distribution of Vls during oogenesis, we analyzed the
localization of HA-Vls protein using anti-HA antibodies. Immunostaining of
vls3; P{HA-vls} egg chambers revealed that Vls
accumulates at the posterior pole of the oocyte in stage 10 egg chambers
(Fig. 3A,C) and in the pole
plasm of early syncytial embryos (Fig.
3E,F). We also found Vls in pole cells
(Fig. 3H,I) and in migrating
primordial germ cells (data not shown). During oogenesis, Vls decorates a
region surrounding the nurse cell nuclei
(Fig. 3K), which bears strong
similarity to the nuage (Findley et al.,
2003). By contrast, immunostaining of wild type ovaries using
anti-HA antibodies displayed only an overall residual staining
(Fig. 3B,D,G,J,L). These data
show that Vls is a component of both the nuage and the pole plasm.
|
|
Checking the distribution of Tud during oogenesis revealed that Tud was
absent from the posterior pole of vls mutant oocytes in stage 10 egg
chambers (Fig. 4K), indicating
that Vls may play a role in pole plasm accumulation of Tud. As Tud is also a
component of the nuage encircling nurse cell nuclei
(Bardsley et al., 1993), we
looked at this structure by examining the distribution of three components of
the nuage including Tud, Vas (Hay et al.,
1988a
) and Maelstrom (Mael)
(Findley et al., 2003
). The
use of a transgene expressing a GFP-Vas fusion protein
(Sano et al., 2002
) associated
with the vls chromosome revealed that GFP-Vas was normally localized
in the nuage of vls nurse cells, albeit at a lesser degree compared
with wild type (Fig. 5B)
(Hay et al., 1990
;
Lasko and Ashburner, 1990
).
Examination of Mael showed that this protein is also present in the nuage,
although its distribution diverges from the wild-type pattern. We found that
Mael is concentrated in brighter spots in vls than in wild type at
the periphery of the nurse cell nuclei
(Fig. 5D). By contrast, we were
unable to detect Tud in the nuage of vls nurse cells
(Fig. 5F), although Tud
accumulates in vls oocytes (Fig.
5F) and transiently localizes at their anterior margin (data not
shown). These findings indicate that: (1) vls acts downstream of Vas
for the recruitment of Tud in the nuage; and (2) events occurring in the nuage
are dispensable for the transport of Tud from the nurse cells to the oocyte
but are required for Tud recruitment in the pole plasm.
|
Vls physically interacts with Tud in vitro
The presence in Vls of four WD repeats able to interact with other proteins
(reviewed by Smith et al.,
1999) prompted us to examine whether Vls could physically interact
with proteins localized in the nuage. For this purpose, we performed pull-down
assays using tagged Tud, Vas and Gustavus (Gus)
(Styhler et al., 2002
)
polypeptides.
Five segments of Tud fused to the STag, including JOZ (amino acid
residues 3-273), 9A1 (residues 198-1199), 3ZS+L-N (residues 1198-1981) and
3ZS+L-C (residues 1941-2515) (Golumbeski
et al., 1991), together comprising the complete Tud protein
(Fig. 6A), were in vitro
translated and the synthesized polypeptides were incubated with immobilized
full-length and truncated GST-Vls proteins. Of the five Tud fragments, we
found that only the 9A1 fragment could interact with Vls, whereas JOZ and the
two subfragments of 3ZS+L showed only weak binding
(Fig. 6C). The 9A1 fragment was
further divided into two fragments. The 9A1-N and 9A1-C fragments (residues
198-770 and 751-1199, respectively) displayed a strong binding to Vls
(Fig. 6C).
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Discussion |
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Attempts to isolate additional partners of Vls by using the yeast-two
hybrid system were unsuccessful because of the occurrence of a too large
number of clones growing on selective medium (data not shown), presumably
reflecting the occurrence of WD repeats that are known to mediate interactions
with numerous proteins in eukaryotic cells
(Smith et al., 1999). By using
direct binding assays with proteins involved in pole plasm function, we found
that Vls interacts with Tud.
Vls is a member of the WD-repeat protein superfamily
The feature characterizing the superfamily of WD-repeat proteins is the WD
motif, a 40 amino acid stretch typically containing a GH dipeptide 11-24
residues from its N-terminus and a WD dipeptide at its C terminus, albeit
exhibiting only limited amino acid sequence conservation
(Yu et al., 2000
). When
present in a protein, the WD motif typically occurs in multiple tandem
repeated units. Based on structural analysis, the conformation of the WD
repeat is defined as a series of four-stranded anti-parallel ß sheets,
which fold into a higher-order structure termed a ß-propeller. At least
four repeats are required to constitute a ß-propeller
(Chothia et al., 1997
). We
found that Vls contains four conserved WD repeats and can potentially form a
ß-propeller structure. Interestingly, the nearest mammalian homologue of
Vls, the human MEP50 protein, displays six WD repeats. Although the sequence
of the two additional repeats is conserved overall in Drosophila Vls,
no characteristic GH and WD di-peptides could be found in these repeats
(Fig. 1D).
WD-repeat proteins act as scaffolding/anchoring proteins for a number of
binding partners (Smith et al.,
1999). WD-repeat motifs within one protein can simultaneously bind
several proteins and foster transient interactions with other proteins.
Moreover, WD-repeat proteins occur in relatively stable protein complexes in
which they play a structural role. A similar function can be assigned to Vls
in promoting either permanent or transient interaction with other
proteins.
Evolutionary conserved interaction between Vls and Csul
The human homologue of Vls, the MEP50 protein, was identified through its
association with PRMT5 (Friesen et al.,
2002), the human homologue of Drosophila Csul
methyltransferase. We show that the domain of Vls required for binding to Csul
is small and confined to its C-terminal end. By contrast, the domain of MEP50
specifically involved in the interaction with PRMT5 remains unknown.
MEP50 acts as an adaptor binding to a subset of the Sm proteins and
contributing to their methylation by the PRMT5 methyltransferase
(Friesen et al., 2002).
Biochemical assays indicate that MEP50 is necessary for the methyltransferase
activity of the methylosome, as anti-MEP50 antibodies significantly reduce the
methylation of Sm proteins (Friesen et
al., 2002
). However, the precise role of MEP50 remains elusive.
Its possible functions include a regulation of the enzymatic activity of PMRT5
and the control of the positioning of the substrate for methylation.
The human methylosome complex is involved in the assembly of spliceosomal
U-rich small nuclear ribonucleoproteins (snRNPs) mediated by the survival
motoneuron (SMN) protein, a gene product that is affected in spinal muscular
atrophy (Lefebvre et al.,
1995; Meister et al.,
2002
). SMN is produced ubiquitously and contains a single Tudor
domain that associates with SmB/B', SmD1-D3 and SmE proteins of snRNPs
(Liu et al., 1997
;
Buhler et al., 1999
;
Selenko et al., 2001
). The
assembly of snRNPs mediated by SMN occurs in the cytoplasm and is stimulated
by the PMRT5-methylosome complex that converts specific arginine residues in
the Sm proteins into dimethylarginines
(Meister at al., 2001
;
Friesen et al., 2001a
;
Branscombe et al., 2001
),
facilitating the binding of the Sm proteins to SMN and their association with
snRNA molecules (Friesen et al.,
2001b
; Brahms et al.,
2001
). Ultimately, the assembled snRNPs are released and targeted
to the nucleus, whereas the SMN-PRMT5 complex may dissociate before its
components associate again for a new round of assembly.
In Drosophila, the Tud protein differs from SMN by containing
eight Tudor domains and two Tudor-like domains
(Callebaut and Mornon, 1997),
whereas SMN contains a single Tudor domain
(Pontig, 1997
). It is thus
possible to envisage that Drosophila Tud may bind different
categories of cytoplasmic RNPs through its multiple Tudor domains. However, in
contrast to the PMRT5/MEP50 complex that apparently binds SMN through other
protein(s) present in the complex, we found that Vls can directly bind to Tud
through its first WD repeat. As the C-terminal tail of Vls binds to Csul, it
is possible that the Drosophila Csul/Vls methylosome associates with
Tud through Vls. Thus, we propose that the association between the methylosome
and Tud promotes binding and assembly of specific RNPs on Tud. Further
experiments are needed to unravel the relationship between these proteins, the
targets of Csul methyltransferase activity and the nature of the RNPs
associated to Tud.
vls functions in nuage and pole plasm
vls mutations causing a grandchild-less phenotype are
characterized by agametic larvae exhibiting defects in abdominal patterning
(Schüpbach and Wieschaus,
1986). Eggs laid by homozygous vls females are devoid of
polar granules and consequently the embryos produce no pole cells
(Schüpbach and Wieschaus,
1986
). In these embryos, Tud is absent from the posterior pole
(Bardsley et al., 1993
), and
Vas rapidly disappears from this location during the period of nuclear
cleavage (Hay et al., 1990
;
Lasko and Ashburner, 1990
).
Our analysis reveals that localization of Tud is already absent from the nuage
surrounding the vls nurse cell nuclei. However, the occurrence of Vas
and Mael in the nuage of vls nurse cells indicates that aspects of
this structure can be made independently of Tud.
A pivotal role in the organization of the nuage was assigned to Vas on the
basis of its involvement in localizing specific components such as Aub
(Wilson et al., 1996) and Mael
(Clegg et al., 1997
) in this
structure (Harris and Macdonald,
2001
; Findley et al.,
2003
). To confirm a Vas-dependence of Tud localization in the
nuage, we examined Tud distribution in vas egg chambers and found
that the localization of Tud around nurse cell nuclei is fully abolished (data
not shown). Similarly the absence of Tud in the nuage of vls nurse
cells shows that Tud localization in the nuage is vls dependent.
However, we detected significant amounts of Tud in vls oocytes and at
their anterior border, indicating that Tud accumulation in the nuage is not
required for Tud transport into the oocyte and its anterior margin.
Furthermore, as Vls-HA does not accumulate in early oocytes nor localize at
their anterior margin (data not shown), we conclude that the interaction
between Vls and Tud should be spatiotemporally regulated.
We show that Vls is a component of the nuage and pole plasm. Only a limited
number of proteins display a similar pattern of distribution, including Vas
(Hay et al., 1988a), Aub
(Harris and Macdonald, 2001
)
and Tud (Bardsley et al.,
1993
). The dual location of these proteins indicates that they
either perform distinct functions at each location or exert a function in the
nuage required for their accumulation in the pole plasm. The finding that Vas
absence in the pole plasm correlates with its absence in the nuage supports
the latter possibility (Liang et al.,
1994
).
Inactivation of vls function exerts a further effect on the
production of the short form of Osk protein. As osk mRNA localization
and amount seems normal in vls embryos
(Ephrussi et al., 1991), we
assume that the lower amount of this form detected by immunoblotting in
vls ovaries corresponds to either a defect in Osk synthesis or
stability. We notice, however, that the level of Osk abundance varies
considerably between individual vls oocytes with an apparently normal
level in a small number of oocytes and a markedly reduced level in the
majority of oocytes. The lower amount of Vas detected at the posterior pole of
stage 10 vls or vls2/Df(2L)TW2 oocytes
(Hay et al., 1990
), can be
interpreted as a consequence of the reduced amount of Osk protein, as Vas is
absent from the posterior pole of osk oocytes
(Hay et al., 1990
;
Lasko and Ashburner,
1990
).
The mechanism by which Vls regulates Osk synthesis and/or stability remains
unknown. However, on the basis of Vls localization during oogenesis, we
envisage that vls could regulate the production of the short form of
Osk by two distinct mechanisms. One the one hand, vls can regulate
Osk synthesis by recruiting specific enhancing factors in the pole plasm. On
the other hand, Osk synthesis may be dependent on events mediated by
vls occurring in the nuage. Similarly, efficient osk mRNA
translation in the pole plasm could also be mediated by Aub in the nuage
(Harris and Macdonald, 2001).
Furthermore, recent data point out that the nuage may function in assembling
or reorganizing ribonucleoprotein complexes, particularly those involving
localized or translationally regulated mRNAs
(Snee and Macdonald,
2003
).
The formation of polar granules fully depends upon vls activity
(Schüpbach and Wieshaus,
1986), but only partially upon tud function as polar
granules in reduced number and altered morphology are observed in amorphic
tud pre-blastoderm embryos
(Thomson and Lasko, 2004
).
This raises the question of what the targets of vls function are in
addition to tud and osk. Further experiments will reveal the
components required for vls-dependent formation of polar
granules.
As this work was being completed, another group reported the cloning of
vls by positional mapping (Cavey
et al., 2005). Although Cavey and colleagues identified only two
WD repeats in Vls, they clearly recognized that its nearest homologue is
MEP50. As the human methylosome is formed by MEP50 and PMRT5 homologous to
Drosophila Vls and Csul, respectively, our finding that Vls can
specifically bind to Csul indicates that it is the orthologue of MEP50 and not
a `divergent WD protein'. In contrast to Cavey and colleagues, we found a
restricted and dynamic Vls distribution during oogenesis, first in the nuage
and then at the posterior pole of the growing oocyte. Finally, we observed
that Vls is preferentially incorporated in the forming pole cells. These
findings show that vls may crucially act in the nuage, germ plasm and
pole cells, and are consistent with the vls mutant phenotype. Similar
to Cavey and colleagues, we detected a lower amount of Osk at the posterior
pole of growing vls oocytes, but, in a discrepancy that we cannot
explain, we found that Osk levels were already lower in stage 9 egg chambers,
whereas Cavey et al. only observed a decrement of Osk later during oogenesis
in stage 11 egg chambers.
In conclusion, we demonstrate that Vls can interact physically with at least two proteins, Csul and Tud, which are specifically involved in germ-line determination. Vls, in association with Csul, constitutes the first example of a partner of a dimethylarginine protein methyltransferase whose function has been characterized in vivo. These findings reinforce their cardinal function in a pathway first elucidated through genetic investigations. Our work sets the basis for further investigations on the role of Vls, its dependence upon Csul and its involvement in specific localization of cytoplasmic proteins during the formation of a functional pole plasm.
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ACKNOWLEDGMENTS |
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