Establishment and maintenance of the germline is an essential
process for all sexually reproducing organisms. Germ cells, which
carry the genetic information to the next generation, are
simultaneously totipotent and highly specialized (Wylie, 1999). 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
Arginine methyltransferase Capsuléen is essential for
methylation of spliceosomal Sm proteins and germ cell
formation in Drosophila
Joël Anne1,2,*, Roger Ollo2, Anne Ephrussi3and Bernard M. Mechler1
Although arginine modification has been implicated in a number of cellular processes, the in vivo requirement of protein arginine
methyltransferases (PRMTs) in specific biological processes remain to be clarified. In this study we characterize the Drosophila PRMT
Capsuléen, homologous to human PRMT5. During Drosophila oogenesis, catalytic activity of Capsuléen is necessary for both the
assembly of the nuage surrounding nurse cell nuclei and the formation of the pole plasm at the posterior end of the oocyte. In
particular, we show that the nuage and pole plasm localization of Tudor, an essential component for germ cell formation, are
abolished in csul mutant germ cells. We identify the spliceosomal Sm proteins as in vivo substrates of Capsuléen and demonstrate
that Capsuléen, together with its associated protein Valois, is essential for the synthesis of symmetric di-methylated arginyl residues
in Sm proteins. Finally, we show that Tudor can be targeted to the nuage in the absence of Sm methylation by Capsuléen, indicating
that Tudor localization and Sm methylation are separate processes. Our results thus reveal the role of a PRMT in protein localization
in germ cells.
KEY WORDS: Drosophila oogenesis, Protein arginine methyltransferase, Pole plasm, Nuage, capsuléen
Development 134, 137-146 (2007) doi:10.1242/dev.02687
1Department of Developmental Genetics, Deutsches Krebsforschungszentrum, Im
Neuenheimer Feld 280, D-69120 Heidelberg, Germany. 2Laboratoire de Biologie
Moléculaire de la Drosophile, Département de Biologie Moléculaire, Institut Pasteur,
Paris F-75015, France. 3Developmental Biology Unit, European Molecular Biology
Laboratory, Heidelberg D-69117, Germany.
*Author for correspondence (e-mail: firstname.lastname@example.org)
Accepted 10 October 2006
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.
MATERIALS AND METHODS
Plasmid constructs were generated by PCR amplification of the relevant
DNA segments (High Fidelity PRC Master; Roche), which were subcloned
into appropriate vectors. vas, tud, nos, gcl and SmD3 cDNA plasmids were
kindly provided by P. Lasko, R. Boswell, R. Lehmann, T. Jongens, and H.
Schenkel, respectively. The plasmids pBS1479 (C-TAP) and pBS1761 (N-
TAP) were obtained from Cellzome (Heidelberg). SmB and SmD1 cDNAs
(BDGP EST project clones LD14049 and RE39488, respectively) were
provided by the Drosophila Genomics Resource Center (DGRC),
Bloomington, IN, USA. Interstitial deletions in csul were prepared using a
QuickChange site-directed mutagenesis kit (Stratagene) with the
modifications of Makarova et al. (Makarova et al., 2000). 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 LD14049cDNA
plasmid in the presence of [35S]methionine (Amersham). To co-
translationally 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).
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 csulcDNA in the pGEX-4T2GST
fusion vector (Pharmacia) and expressed in the bacterial strain BL21(DE3).
Development 134 (1)
Fig. 1. Embryonic phenotype of csulP. (A-D) The csul gene belongs
to the ‘posterior-grandchildless’ group. Immunohistochemical detection
(horseradish peroxidase) of Vas protein (A,B) and cuticle preparations of
first instar larvae (C,D) in wild-type (A,C) and csulP(B,D). Anterior is to
left. Pole cell formation and abdominal patterning are defective in csulP.
(E-H) csul activity is required for the localization of maternal
determinants. Distribution of nos (E,F) and gcl (G,H) transcripts
detected by whole-mount in situ hybridization in wild-type (E,G) and
csulP(F,H) embryos. In csulPthe accumulation of nos mRNA is reduced
at the posterior pole (F), whereas gcl mRNA is evenly distributed (H). (I-
L) csulPsuppresses the bicaudal phenotype induced by osk-bcd3?UTR.
Immunohistochemical staining of Vas protein (I,J) and cuticle
preparations of first instar larvae (K,L) in osk-bcd3?UTR (I,K) and csulP;
osk-bcd3?UTR (J,L). Although the cuticle has a normal polarity in csulP;
osk-bcd3?UTR, the head is malformed, indicating that the embryo
contains a residual amount of nos activity at the anterior pole. (M-P)
Distribution of Osk protein in wild-type (M,O) and csulP(N,P) embryos.
Immunofluorescent detection of Osk (red). DNA detected using Oli-
Green (green). After nuclear cycle 6, the level of Osk is strongly reduced
at the posterior pole in csulPembryos (P). In older csulPembryos (insert
in P) trace amounts of Osk can be detected at the posterior pole. (Q-T)
Distribution of Vas protein in wild-type (Q,S) and csulP(R,T) embryos.
Immunofluorescent detection of Vas (red); DNA (green). Similar to Osk,
Vas staining is strongly reduced in the pole plasm of csulPembryos (T)
and is rarely detected at the posterior pole of syncytial embryos (insert
in T). (U,V) Distribution of Tud protein in wild-type (U) and csulP(V)
embryos. Immunofluorescent detection of Tud (red); DNA (green). No
localized Tud staining is observed in csulPembryos.
Anti-Csul polyclonal antibodies raised in rabbits against the GST-Csul
fusion protein were purified by affinity chromatography on a protein A-
agarose column (Roche) and preadsorbed on proteins extracted from
For immunostaining, primary antibodies were anti-Osk from rabbit, anti-
Vas from rabbit and rat (gifts from P. Lasko and A. Nakamura, respectively),
anti-Tud from rabbit (TUD56; a gift from S. Kobayashi), anti-Mael from
rabbit (a gift from S. Findley), anti-Nos from rat (a gift from R. Wharton),
mouse monoclonal anti-HA (clone 16B12; BAbCO), anti-Sm (clone Y12;
NeoMarkers), and anti-dimethyl-arginine (SYM10 and ASYM24) from
rabbit (Upstate). DNA was visualized by staining with Oli-Green (Molecular
Probes). Biotinylated and Cy3-conjugated secondary antibodies (Jackson
ImmunoResearch Laboratories) were used at 1:200.
Maternal-effect phenotype of csul
The csulPmutation was identified in a screen for maternal-effect
mutations induced by a P-[white+] transposon. The P-element
insertion was mapped to 52F on chromosome 2 (data not shown).
Following P-element excision, we recovered numerous (92/150)
fertile white flies producing fully viable and fertile progeny,
indicating that the P-insert was the cause of the maternal-effect
phenotype (see below). Moreover, we isolated a series of viable
white females producing embryos similar to those laid by csulP
females. These alleles resulted from a partial excision of the P-
[white+] transposon and are named csulRM.
Homozygous csulPfemales produce embryos (csulPembryos)
displaying defects in abdominal patterning and lacking pole cells
(Fig. 1B). Flies hatching from csulPlarvae 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 csulPas belonging to
the ‘posterior-grandchildless’ class of mutations (Schüpbach and
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
csulPembryos, nosmRNA 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 csulPembryos, 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 oskorchestrating the
activity of the other genes. As ectopic expression of oskinduces 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.
Distribution of pole plasm components in early
We then determined whether csul activity is needed for the
localization of Osk and Vas (Fig. 1M-T). We found that Osk and
Vas are present at the posterior pole of early syncytial csulP
embryos, albeit at a reduced level for Vas. However, starting from
nuclear cycle 6, Osk and Vas signals significantly decrease and are
nearly completely absent from the posterior pole of late syncytial
Role of PRMT Capsuléen in germ cell formation
Fig. 2. csul activity contributes to nuage and pole plasm
assembly during oogenesis. Distribution of Osk (A,B), GFP-Vas
(C,D,G,H), Tud (E,F,I,J), and Mael (K,L) proteins in stage 10 egg
chambers (A-F) and previtellogenic egg chambers (G-L). Left and right
columns show wild-type and csulPegg chambers, respectively. (A,B) Osk
immunostaining (red) and DNA (green). Osk is correctly synthesized and
positioned at the posterior pole of csulPoocytes. (C,D,G,H) GFP-Vas
(green) and DNA staining (red). GFP-Vas is only detected in the nuage
of previtellogenic csulPegg chambers and disappears in later stages of
egg chamber development. In comparison with wild type, the amount
of GFP-Vas is markedly reduced in the pole plasm of csulPstage 10
oocytes. (E,F,I,J) Tud immunostaining (red) and DNA (green). Although
Tud is synthesized in csulPegg chambers, it is absent both from nuage
and pole plasm. (K,L) Mael immunostaining. Mael localization in the
nuage is abolished in csulPegg chambers. o, oocyte; n, nurse cell
Development 134 (1)
Fig. 3. See next page for legend.
csulPembryos. In a few pre-blastoderm embryos we detected Osk-
and Vas-stained particles closely associated with nuclei at the
posterior pole (inserts in Fig. 1P,T). These structures may represent
remnants of polar granules, similar to those detected in
hypomorphic tud mutant embryos (Hay et al., 1990). 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
As pole plasm assembly occurs during oogenesis we investigated the
distribution of pole plasm components in csulPegg 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 csulPand 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 csulRMegg chambers (data not
shown). Only a minority of csulRMoocytes 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 csulRMoogenesis to become detectable during
By contrast, we found that Tud fails to concentrate at the posterior
pole in csulPoocyte 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
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 csulPegg 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
csulgene, 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 csulRMfemales.
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 amino-
acid 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 csulPfemales. However, long exposure of the
immunoblot revealed a faint band in the 65 kDa range, suggesting
that csulPfemales may produce a low level of Csul, possibly by use
of an in-frame initiation codon present in the P-white+inverted
Role of PRMT Capsuléen in germ cell formation
Fig. 3. Molecular analysis of the csul locus. (A) Restriction map of
the genomic DNA covering the csul locus with the location of the P
element insertion in csulP. Below the map are shown three transcription
units in this locus and their orientation, as well as the three genomic
fragments used for constructing transgenes. Transcription orientation of
the fidipidine (?), csul (?), and Kinesin heavy chain (Khc) genes is
indicated by arrows. The analysis of complementation of the csulP
mutation by transgenes shown on the right demonstrates that the ?
gene corresponds to csul. (B) Restriction map of the csul gene, with
alignment of the csul transcript. Exons are drawn as boxes and the
putative open reading frame is indicated in black. (B) BamHI; (P) PstI; (R)
EcoRI; (Xb) XbaI; (Xh) XhoI. (C) Northern blot showing a single ovarian
csul transcript of ~2 kb. RNA marker sizes (in kb) are indicated on the
left. (D) Western blot detection of Csul in wild-type and csulPovarian
protein extracts using anti-Csul antibodies. A band of ~65 kDa (arrow)
is detected in wild-type but not in csulP. Protein load in each well was
normalized by using anti-actin antibodies (data not shown). The anti-
Csul antibodies recognize another protein of ~38 kDa unrelated to
Csul. (E) Alignment of the amino acid sequences of Csul and
homologous proteins performed using the Pileup program of the
Wisconsin Package (Genetics Computer Group) reveals that csul
encodes a protein-arginine methyltransferase similar to human PMRT5.
Gaps in the amino acid sequence, indicated by dots, were introduced
for optimal alignment. At each position of the alignment, residues
identical in all sequences are background-shaded blue, and functionally
conserved (i.e. more than half of the amino acids of a column) residues
with strong or weak similarities are background-shaded red and
orange, respectively. The asterisks over the positions 243 and 244
indicate the substitutions made in the csulG343A;R344Ltransgene.
GenBank accession number sequences are as follows: CP4855_AG:
EAA14767; PRMT5_HS: AF167572; zC14A17.2_DR: CAD60861;
F6E21.40_AT: T10666; Skb1_SP: U59684; YLB5_CE: U10402;
B208.200_NC: T49572; Hsl7p_SC: U65920.
repeat and located 16 nucleotides upstream of the insertion site
(data not shown). This finding suggests that csulPis a hypomorphic
Csul acts as a protein methyltransferase
Csul protein sequence was found to exhibit significant similarity to
protein-arginine methyltransferases (PRMTs), which catalyse the
formation of symmetric di-methyl arginyl (sDMA) residues (Fig.
3E). Csul homologues can be identified in organisms ranging from
the budding yeast Saccharomyces cerevisiaeto humans (Ma, 2000).
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 Gly343and
Arg344with Ala and Leu residues, respectively.
Wild-type csul and csulG343A;R344Lgene 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 csulRMgenetic
background we determined, by western blot, that they both express
at similar level (data not shown) and examined their ability to abolish
the csulRMmaternal-effect embryonic phenotype. csul-TAP and
TAP-csul transgenes rescue the development of csulRMembryos,
whereas the csulG343A;R344L–TAPtransgene shows no rescue activity
(data not shown and Fig. 7D). These results strongly suggest that
csul encodes a methyltransferase whose activity requires an intact
Sm proteins are substrates of the Csul-Vls
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 SmBBG02775in 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)k131-07larvae
(Schenkel et al., 2002) revealed that the ?-SYM10 signal of a 16
kDa protein is nearly abolished in the mutant, identifying this protein
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
Development 134 (1)
Fig. 4. Symmetrical methylation of SmB and SmD3 proteins is
dependent on Csul and Vls. (A) Csul and Vls are required for sDMA
synthesis. Protein extracts of wild-type (WT), csulRM, vls1, osk84/pXT103,
vasQ7, vasO11and aubHN2/aubN11ovaries were separated by SDS-PAGE,
blotted and probed with ?-SYM10 (left panel) and ?-ASYM24
antibodies (right panel). Anti-ribosomal p40 antibodies (?-p40) were
used as a loading control (left panel, lower blot). The intensity of four
SYM10-reactive protein bands in the range of 15 to 26 kDa was
strongly reduced in csulRMand vls1protein extracts. No change of the
intensity of the ASYM24-reactive bands was observed in any mutant
background. [B] and [C] indicate the position of protein bands shown in
B and C, respectively. (B) SmB is symmetrically dimethylated in vivo.
Ovarian protein extracts of wild-type and SmBBG02775/CyO females were
separated by SDS-PAGE, blotted and probed with ?-SYM10 and ?-Y12
antibodies. By comparison to wild type, the intensity of the
SYM10/Y12-reactive protein band is significantly reduced in
SmBBG02775/CyO. (C) SmD3 is symmetrically dimethylated in vivo. Protein
extracts of wild-type and homozygous SmD3l(2)k131-07larvae were
separated by SDS-PAGE, blotted and probed with ?-SYM10 antibodies.
Arrow indicates the position of SmD3. (D) SmB immunostaining using
?-Y12 antibodies. sDMA-SmB localizes in the nuclei of the nurse cells
and somatic follicular cells of wild-type egg chambers but is undetected
in csulRMegg chambers.
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
Csul and Vls interact with Sm proteins in vitro
As Sm proteins can be methylated by the Csul-Vls methylosome,
we investigated by binding assays whether these proteins can
physically interact together. We found that Csul and Vls can bind
to SmB (Fig. 5), SmD1, and SmD3 (data not shown). We then
determined the reciprocal binding domains between Csul and
SmB. First we synthesized 35S-labeled S-tag SmB1-199(full-
length), SmB1-106(N-terminal moiety with Sm domains) and
SmB83-199(C-terminal moiety with RG repeats) polypeptides and
incubated them with GST-Csul, GST-Vls, or GST alone. We
found that the deletion of the RG domain had no effect on SmB
binding to Csul or Vls, indicating that the N-terminal moiety
containing the Sm domains mediates SmB interaction with Csul
or Vls. Then, by using a similar approach we assigned the SmB
binding domain of Csul to the N-terminal part (amino acid
residues 1-200) (Fig. 6). Further analysis showed that both N- or
C-terminal truncations in this domain (fragments 20-610 and 1-
111, respectively) prevent SmB binding to Csul (Fig. 6 and data
not shown), indicating that the N-terminal part of Csul contains at
least two domains necessary for interaction with SmB.
Interaction between Csul and Tud in vivo is
essential for Tud localization
The defective accumulation of Tud in the nuage of csulPnurse 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 immuno-
detection. 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 csulRMgenetic
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 csulRMmaternal-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
Role of PRMT Capsuléen in germ cell formation
Fig. 5. SmB binds to Csul and Vls. Left panel: GST-Csul and GST-Vls
proteins stained with Coomassie Blue. Middle panel: [35S]S•Tag-SmB
fragments were separated by SDS-PAGE and detected by fluorography.
Right panel: Following incubation with GST-Csul or GST-Vls, the bound
[35S]S•Tag-SmB fragments were separated and detected as described
above. No binding was detected with GST alone (data not shown).
Below these panels the binding results of SmB and derivatives as well as
those of SmD1 and SmD3 to Csul and Vls are summarized graphically.
Size and structure of the SmB, SmD1 and SmD3 proteins are depicted
with the two Sm domains and the RG dipeptides shown as orange
boxes and circles, respectively. N- and C-terminal amino acid residues of
the Sm proteins and fragments are indicated.
Fig. 6. SmB-binding domain in Csul. (Upper panel) Full-length GST-
Csul or derivatives were purified from bacteria and incubated with
[35S]S•Tag-SmB. Bound [35S]S•Tag-SmB proteins were separated by
SDS-PAGE and detected by fluorography. Amino acid numbers are
given across the top. ‘Input’ was one tenth of the synthesized
[35S]S•Tag-SmB proteins. The smallest fragment of Csul showing
binding to SmB encompasses amino acids 1-200. (Lower panel) The
amount of GST-Csul polypeptides was visualized by Coomassie Blue
staining. (Right) Representation of the GST-Csul fragments used for the
mapping and the summary of the results, with the identified domain
required for SmB-binding in Csul shown in blue.
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.
Tud localization and Sm protein methylation may
be unrelated processes, both driven by Csul
To further explore the relationship between Sm methylation and pole
plasm function, we investigated the methylation status of Sm proteins
in extracts of transgenic csulRMovaries producing Csul proteins with
interstitial deletions in the N-terminal region. Western blot analysis
using ?-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 csulRMegg
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.
Csul is a Type II PRMT
csul encodes a Type II PRMT, which transfers methyl groups from
S-adenosyl-L-methionine to the guanidinium group of arginyl
residues. PRMTs can be divided into two major categories,
catalyzing the synthesis of aDMA (Type I) or sDMA (Type II)
residues, respectively. The mammalian PRMT5 (Pollack et al.,
1999; 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
Development 134 (1)
Fig. 7. A domain in the N-terminal region of Csul is essential for
the methylation of Sm proteins and localization of Tud.
(A-C) Identification of the Tud9A1-N-binding domain in Csul. (A) Full-
length GST-Csul or derivatives were purified from bacteria and
incubated with S•Tag-Tud9A1-N. Bound S•Tag-Tud9A1-N was detected
as described in Fig. S1 of the supplementary material (upper panel). The
amount of GST-Csul proteins was visualized by Coomassie Blue staining
(lower panel). Amino acid numbers are given across the top. ‘Input’
was one tenth of the S•Tag-Tud9A1-N extract. The smallest fragment
of Csul showing binding to Tud9A1-N encompasses the first 111 amino
acids of Csul and may thus be distinct from the SmB binding site.
(B) Delimitation of the Tud-binding domain using N-terminal truncation
of Csul. Detection of S•Tag-Tud9A1-N bound to the N-terminal
truncated Csul polypeptides revealed that the sequence following
amino acid residue 60 is required for Tud9A1-N binding. (C)
Representation of the GST-Csul fragments used for the mapping and
the summary of the binding results are indicated on the right. The
identified domain of Csul necessary for Tud-binding is shown in blue.
(D,E) Use of interstitial deletions in the N terminus region of Csul to
determine the domain necessary for Tud localization in the nuage and
sDMA methylation of SmB. (D) The Tud9A1-N-binding domain of Csul
is required for Tud localization in the nuage and pole cell formation.
Egg chambers and embryos from homozygous csulRMfemales
expressing the different transgenes were stained using anti-Tud (red;
left column) and anti-Vas (red; right column) antibodies, respectively.
DNA staining is in green. The deletions uncovering residues 21-40 and
41-60 can properly target Tud in the nuage and rescue the csul
phenotype. (E) The N-terminal region of Csul is necessary for sDMA
synthesis on SmB protein. Transgenes expressing full-length or modified
Csul proteins were introduced into the csulRMbackground and ovarian
extracts were prepared from the homozygous females. Western blot
analysis using ?-SYM10 antibodies indicates that none of the deletion
transgenes are able to rescue the methylation of the SmB protein
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 Drosophilamethylosome 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 csulgene, 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 thatcsul
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
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
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
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.
J.A. dedicates this work to the memory of his grandparents, André and
Henriette Costentin. We are particularly grateful to Jean-René Martin, Susan
Sather, and Anna Raibaud in the initial phase of this work and to Seth Findley,
Thomas Jongens, Satoru Kobayashi, Paul Lasko, Ruth Lehmann, Peter Tolias
and Robin Wharton for providing reagents. We would like to thank Christof
Niehrs and the members of the DKFZ group, Gunter Merdes, Istvan Török,
Joachim Marhold, Dennis Strand, Rolf Schmitt, and Ioannis “PAOK Olé”
Illiopoulos, for their constant help. This study was supported by grants to
B.M.M. from the European Commission (BMH1-CT94-1572 and QLRI-CT2000-
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