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First published online 30 January 2008
doi: 10.1242/dev.015438
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Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA.
Author for correspondence (e-mail:
lgavis{at}princeton.edu)
Accepted 18 December 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Drosophila, hnRNP, mRNA localization, Nanos
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Gavis and Lehmann, 1992). Bcd
functions as a transcriptional activator and a translational repressor to
regulate expression of genes required for head and thorax development
(Driever and Nüsslein-Volhard,
1989; Dubnau and Struhl,
1996; Rivera-Pomar et al.,
1996; Struhl et al.,
1989). As a translational regulator, Nos represses synthesis of
the transcriptional repressor Hunchback (Hb) in the posterior of the embryo,
permitting expression of genes required for abdominal development
(Hülskamp and Tautz,
1991; Tautz and Pfeifle,
1989). More recently, additional roles for Nos have been
identified in germline stem cell divisions, germ cell development, and
dendrite morphogenesis (Forbes and
Lehmann, 1998; Kobayashi et
al., 1996; Wang and Lin,
2004; Ye et al.,
2004).
The Bcd and Nos protein asymmetries are generated through the asymmetric
localization and consequent translation of their respective mRNAs
(Berleth et al., 1988;
Driever and Nüsslein-Volhard,
1988; Gavis and Lehmann,
1992; Wang and Lehmann,
1991). Both bcd and nos mRNAs are synthesized
maternally and become localized to opposite poles of the egg during oogenesis.
Whereas anterior localization of bcd depends on active transport
along microtubules (Cha et al.,
2001; Weil et al.,
2006), posterior localization of nos occurs by passive
diffusion and entrapment at the posterior by the specialized posterior
cytoplasm or germ plasm (Forrest and
Gavis, 2003; Weil et al.,
2006). Localization of nos to the germ plasm is essential
to activate nos translation and produce the critical concentration of
Nos required to repress translation of hb mRNA
(Gavis and Lehmann, 1992;
Gavis and Lehmann, 1994).
Thus, when nos localization is abolished by mutations in germ plasm
components such as oskar (osk) or vasa
(vas), Nos is not produced and the resulting embryos lack abdominal
segments. Although nos is highly concentrated at the posterior of the
embryo, its localization is inefficient, such that the majority of
nos mRNA remains unlocalized
(Bergsten and Gavis, 1999). The
obligate linkage of nos mRNA localization and translation prevents
synthesis of Nos in the anterior of the embryo, where it is deleterious to
head and thorax development (Gavis and
Lehmann, 1992; Gavis and
Lehmann, 1994; Wharton and
Struhl, 1989). Recently, posterior localization of nos
has also been shown to be essential for nos function in germ cell
development (Gavis et al.,
2008).
bcd and nos are two of a growing number of mRNAs known to
be localized within Drosophila oocytes and embryos. Since different
mRNAs are targeted to different destinations, specificity for localization
pathways must be encoded in the transcripts and conferred on the cellular
localization machinery by factors that recognize these signals. Cis-acting
sequences that direct intracellular localization have been identified in a
number of localized mRNAs and are usually located in 3' untranslated
regions (3'UTRs) (Gavis et al.,
2007; St Johnston,
2005). Posterior localization of nos mRNA is directed by
a large localization signal within the nos 3'UTR that can be
subdivided into four partially redundant localization elements
(Gavis et al., 1996a).
Individual localization elements exhibit varying degrees of competence to
mediate posterior localization, but at least three contiguous elements are
required to produce wild-type levels of Nos and proper abdominal development
(Bergsten and Gavis, 1999).
Such functional redundancy is characteristic of a variety of mRNA localization
signals including the bcd, Xenopus Vg1 and fatvg, and yeast
ASH1 signals (Chan et al.,
1999; Gautreau et al.,
1997; Gonzalez et al.,
1999; Macdonald and Kerr,
1998).
Previous genetic and biochemical studies suggest that different
nos localization elements are recognized by distinct cytoplasmic
factors that package nos into a ribonucleoprotein (RNP) complex
competent to interact with the germ plasm components
(Bergsten and Gavis, 1999;
Bergsten et al., 2001).
However, the specific localization factors that recognize the nos
localization elements and target nos to the germ plasm have remained
elusive. Although mutations that cause defects in nos localization
have been identified in numerous genetic screens, the effects of these
mutations on nos are most likely to be indirect, as they disrupt
localization or assembly of germ plasm. A recent genetic screen for
nos localization factors identified the chaperone Hsp90 (also known
as Hsp83 - FlyBase) as being important for nos localization, though
its role also appears to be indirect (Song
et al., 2007). Given the redundancy within the nos
localization signal and, consequently, the potential for functional redundancy
among proteins that interact with different nos localization
elements, genetic approaches may fail to identify direct-acting localization
factors.
We have therefore taken a biochemical approach to identify nos
localization factors, starting with an individual nos localization
element. Using a new RNA-affinity purification strategy, we have isolated
Rumpelstiltskin (Rump), a member of the heterogeneous nuclear
ribonucleoprotein (hnRNP) family. Rump appears to be the Drosophila
homolog of hnRNP M (also known as HNRPM in mouse), which has been previously
implicated as a splicing factor in both invertebrates and vertebrates
(Gattoni et al., 1996;
Hase et al., 2006). A
combination of biochemical and genetic data presented here show that Rump is a
direct-acting nos mRNA localization factor. Rump associates with
nos mRNA in vivo and binding of Rump to nos mRNA in vitro
requires a repeated motif within the nos localization signal.
Moreover, binding of Rump in vitro correlates with the ability of this motif
to confer localization function in vivo. Through the analysis of a
rump null mutant, we demonstrate a specific role for rump in
posterior localization of nos. To our knowledge, this is the first
example of an hnRNP M homolog regulating the localization of an mRNA
target.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) were
cloned into the BglII site of pTRAP6 (gift of H. Krause, University
of Toronto, Toronto, Canada) to generate pTRAP6-nos+2'-3X. pTRAP6 and
pTRAP6-nos+2'-3X were linearized with ScaI and
HindIII, respectively, for in vitro transcription by the mMessage
mMachine Kit (Ambion) supplemented with 625 nM [
-32P]UTP.
Radiolabeled capped TRAP+2'-3X (779 nucleotide) and TRAP-ctrl (1261
nucleotide) RNAs were purified by G-50 Sephadex Quick Spin Column (Roche). MCP
was cloned from pCaS-Phsp83-MCP-GFP
(Forrest and Gavis, 2003) into
pGEX-4T-1, fusing MCP to the C-terminus of GST. GST-MCP was purified after
expression in E. coli using glutathione-agarose (Sigma) according to
the manufacturer's instructions.
Extract was prepared from 0- to 2-hour Oregon R embryos in TPB [60 mM HEPES
pH 7.5, 10 mM MgCl2, 150 mM NaCl, 0.1% Triton X-100, 10% glycerol,
1x EDTA-free Complete Protease Inhibitors (Roche)] as described
(Bergsten et al., 2001). All
subsequent purification steps were performed at 4°C. For each sample, 1 ml
of extract (5 mg/ml total protein) was precleared with 40 µl
streptavidin-agarose and 40 µl glutathione-agarose for 30 minutes.
Precleared extract was incubated with 11 µg radiolabeled TRAP-ctrl or
TRAP+2'-3X RNA for 15 minutes, then rotated with 200 µl
streptavidin-agarose in a 1.2 ml RNase-free minicolumn (BioRad) for 2 hours.
The resin was washed with 25 volumes of TPB and bound RNP complexes were
eluted by incubation with 5 mM D-biotin in TPB for 1 hour, with a
second elution for 30 minutes. The eluates were pooled and incubated with 100
µl glutathione-agarose pre-bound with 1 mg GST-MCP for 2 hours with
rotation. The resin was washed with 15 volumes of TPB and proteins were eluted
by treatment with 2 µg RNase A in 200 µl TPB, 2x 30 minutes.
Eluates were pooled, concentrated by TCA precipitation, then resuspended in
boiling sample buffer (Gavis et al.,
1996b). Samples were separated by SDS-PAGE, with the amount of
each sample loaded adjusted for the amount of RNA captured, as determined by
scintillation counting. Proteins were visualized by SYPRO Ruby (Invitrogen)
staining, and the indicated 
70 kDa band was excised and analyzed by mass
spectrometry.
Production of MBP-Rump
The full-length rump coding region was amplified by RT-PCR from 0-
to 2-hour Oregon R embryonic poly(A+) RNA using the primers
5'-AAACTGCAGAGCATGGACGCTAGTAAC-3' and
5'-CCCAAGCTTAAAAGTATGTTAC-3', which introduce PstI and
HindIII sites at the 5' and 3' ends, respectively.
Following digestion with PstI and HindIII, the PCR fragment
was inserted between the PstI and HindIII sites of pMAL-c2
(New England Biolabs), fusing Rump to the C-terminus of MBP. MBP-Rump was
expressed and purified on amylose-agarose
(Kalifa et al., 2006),
exchanged into Storage Buffer (100 mM KCl, 25 mM HEPES pH 7.9, 0.5 mM EDTA,
10% glycerol, 1 mM DTT, 0.5 mM PMSF), and stored at -80°C. For monoclonal
antibody production, the MBP tag was removed using Factor Xa (a gift from F.
Hughson, Princeton University, Princeton, NJ).
UV-crosslinking assay
Radiolabeled RNA probe synthesis and UV-crosslinking were performed as
previously described using 27 µg MBP-Rump per reaction
(Bergsten et al., 2001). The
full-length +2' element [nucleotides 101-186 of the nos
3'UTR (Gavis et al.,
1996a)] was inserted between the SmaI and BamHI
sites of the pBluescript derivative pBS-SK
KP
(Kalifa et al., 2006). The
+2' element deletions (1, nucleotides 101-128; 2, 101-142;
3, 120-162; 4, 128-152; 5, 144-186) were generated by PCR
(1-3, 5) or with annealed oligos (4) and cloned into
pBS-SKKP as above. The +2'(A)G:C, +2'(D)G:C and
+2'(AD)G:C mutants were generated by PCR, introducing G-to-C point
mutations into one or both of the two CGUU motifs (A, nos 3'UTR
nucleotide 120; D, nucleotide 140] in the pBS-SKKPnos+2' plasmid.
Plasmids were linearized with BamHI for probe synthesis. For
immunoprecipitation of crosslinked protein, each RNase-treated reaction was
incubated for 1 hour at 4°C in 200 µl IP buffer (10 mM Tris-HCl pH 7.4,
100 mM NaCl, 2.5 mM MgCl2, 0.5% Triton X-100, 1 mM DTT, 1x
EDTA-free Complete Protease Inhibitors) with 6 µl Protein-G Dynabeads
(Invitrogen) coated with 30 µl antibody. Beads were washed twice with IP
buffer, then resuspended directly in boiling sample buffer and separated by
SDS-PAGE.
Generation of a rump null mutation
The rump1 allele was generated by imprecise excision of
P element P{SUPor-P} CG9373KG02834
(Bellen et al., 2004).
Excisions in trans to Df(3R)by416, which deletes the rump
locus, were screened by single-fly PCR
(Mansfield et al., 2002) using
primer pairs to amplify the genomic regions immediately upstream and
downstream of the P element insertion site. Genomic deletions were
identified by the absence of a PCR product. The rump1
deletion endpoints were determined by PCR amplification with primers flanking
the deletion and sequencing. The nosBN allele
(Wang et al., 1994) was
recombined onto the rump1 and Df(3R)by416
chromosomes to generate rump1 nosBN and
Df(rump) nosBN chromosomes used for the analysis of
nos-tub:nos+2 in Fig.
7.
Transgenes
The nos-tub:nos+2 transgene is described by Gavis et al.
(Gavis et al., 1996a). The
nos-tub:nos+2(A)G:C and nos-tub+2(D)G:C transgenes are
identical to nos-tub:nos+2 except that a G-to-C mutation was
introduced into the first (A) or second (D) CGUU motif within the +2'
sequences, using PCR. The rump rescue transgene contains a 6.5 kb
BanII genomic fragment from BACR19N07 (GenBank AC009350) containing
rump inserted into pCaSpeR4. The rump transgene was
introduced into y w67c23 embryos by P
element-mediated germline transformation
(Spradling, 1986) and multiple
independent lines were isolated.
Northern blotting and immunoblotting
Northern blotting and immunoblotting were performed as previously described
(Kalifa et al., 2006). A
32P-labeled probe for rump was generated by random-hexamer
labeling of the full-length rump EST clone GH11495. For
immunoblotting, anti-Rump 5G4 was used at 1:2000, anti-Snf (gift of P. Schedl,
Princeton University, Princeton, NJ) at 1:20,000. Proteins were detected using
Lumi-Light Western Blot Substrate (Roche).
RNA co-immunoprecipitation
Ovaries from well-fed females were dissected in Schneider's medium
(Invitrogen), rinsed with PBS and frozen in liquid N2.
Approximately 15 mg ovaries or dechorionated embryos per sample were
homogenized on ice in 750 µl RNase-free RNA co-immunoprecipitation buffer
(RCB) [50 mM HEPES pH 7.4, 200 mM NaCl, 2.5 mM MgCl2, 0.1% Triton
X-100, 250 mM sucrose, 1 mM DTT, 1x EDTA-free Complete Protease
Inhibitors, 0.4 mM Pefabloc (Roche)], supplemented with 300 U RNasin
(Promega). Subsequent steps were carried out at 4°C. Soluble extract was
obtained by two sequential 5-minute centrifugations at 16,000
g, then precleared with 40 µl Protein-G Dynabeads for 30
minutes at 4°C. After removal of samples for immunoblotting and
quantitation of RNA input, Rump was immunoprecipitated from 450 µl
precleared extract by incubation for 1 hour with 20 µl Protein-G Dynabeads
bound with anti-Rump 5G4 antibody. Immunoprecipitates were washed eight times
with RCB. Half of the IP was resuspended in RCB, treated with 8 units RQ1
RNase-free DNase (Promega) for 10 minutes at room temperature, then extracted
with phenol:chloroform. RNA was ethanol precipitated with 30 µg tRNA and 5
µg glycogen as carrier and resuspended in DEPC-treated distilled
H2O for RT-PCR. The remainder of the IP was resuspended in 20 µl
boiling sample buffer for immunoblotting.
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Embryonic cuticle preparation, in situ hybridization and immunostaining
Embryonic cuticle preparation and in situ hybridization were performed
according to Gavis and Lehmann (Gavis and
Lehmann, 1992). The statistical significance of embryonic
phenotypes was determined by the 
2 test. Immunostaining of
ovaries was performed as previously described
(Shcherbata et al., 2004).
Embryo immunostaining was performed as described
(Duchow et al., 2005), except
that for anti-Rump immunofluorescence, dechorionated embryos were heat-fixed
in 68 mM NaCl/0.03% Triton X-100, transferred to PBS, and the vitelline
membranes were manually removed using a 30-gauge needle. The following
antibodies and dyes were used: 1:100 anti-Rump 10C3 (immunofluorescence),
1:100 anti-Rump 5G4 (immunohistochemistry), 1:10,000 rabbit anti-Vas (gift of
R. Lehmann, Skirball Institute, New York), 1:1000 Alexa Fluor 568 goat
anti-mouse (Invitrogen/Molecular Probes), 1:1000 Alexa Fluor 488 goat
anti-rabbit (Invitrogen/Molecular Probes), 1:1000 Oregon Green 488 phalloidin
(Invitrogen/Molecular Probes), 1:1000 Hoechst (ovaries), 1:1000 DAPI
(embryos). Images were obtained using standard Nomarksi optics or a Zeiss
LSM510 confocal microscope.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) to
isolate in vitro-assembled RNA-protein complexes containing the 88 nucleotide
nos +2' localization element. We focused on this element
because previous mutational analysis suggested it as a target for multiple
localization factors, and biochemical experiments identified a +2'
element binding activity of 75 kDa (p75) in ovarian and embryonic
extracts (Bergsten et al.,
2001). In vitro-synthesized RNA containing the dual TRAP
purification tags and three tandem copies of the +2' element
(TRAP+2'-3X; Fig. 1A) was
allowed to form RNA-protein complexes with early (0- to 2-hour) embryonic
protein extract. These complexes were isolated first on streptavidin resin,
utilizing the specific interaction between streptavidin and the S1 RNA aptamer
of the TRAP tag. Bound complexes were eluted by the addition of
D-biotin and applied to glutathione resin coated with GST-MS2 coat
protein (GST-MCP), which captures TRAP-tagged RNA through the interaction of
GST-MCP with the six MCP-binding stem-loops present in the tag. This second
purification step served to reduce non-specific background proteins that
interact with the streptavidin-coated agarose beads. Subsequent RNase
digestion allowed specific elution of the protein components of the recaptured
RNP complexes and eluted proteins were separated by SDS-PAGE. To control for
the isolation of non-specific RNA-binding proteins, an RNA of similar length
containing naïve sequence in place of the nos regulatory
sequences (TRAP-ctrl; Fig. 1A)
was used in a parallel purification.
A band of 70 kDa was enriched by the TRAP+2'-3X RNA across
several independent purifications (Fig.
1B and data not shown). In each case, this band was excised and
the protein components were analyzed by mass spectrometry. Sixteen peptides
were recovered spanning the 633 amino acids of a predicted 67 kDa protein
encoded by the gene CG9373. The less intense band of 70 kDa present in
the TRAP-ctrl sample corresponds to the E. coli Hsp70 chaperone, a
contaminant of the GST-MCP protein preparation (data not shown).
CG9373 encodes the Drosophila homolog of the human hnRNP M family
of nucleic acid-binding proteins and is orthologous to the 59 kDa splicing
factor Hrp59 of Chironomus tentans
(Kiesler et al., 2005). Like
other hnRNP M family members, the Drosophila homolog contains three
RNA recognition motifs (RRMs). Based on cross-reactivity of antibodies against
the C. tentans protein with a protein in Drosophila S2
cells, the Drosophila protein has been previously referred to as
Hrp59 (Kiesler et al., 2005).
The phenotypes of a mutation we have generated in CG9373, described below,
have led us to name this gene rumpelstiltskin (rump).
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To identify the binding site for Rump, several overlapping fragments of the
+2' element were assayed by UV-crosslinking to MBP-Rump
(Fig. 2C). Binding of MBP-Rump
to the 5' half of the +2' element (2) was indistinguishable
from binding to the full-length +2' element, whereas no interaction was
detected with the 3' half (5;
Fig. 2C). Two non-overlapping
subregions (1 and 4) were each sufficient for binding by
MBP-Rump, although binding was reduced relative to the overlapping 2
region or the full +2' element (Fig.
2C). This result indicates the presence of multiple binding sites
for Rump in the 5' half of the +2' element.
The 1 and 4 regions each contain a CGUU motif and
linker-scanning mutations that overlap these motifs were previously shown to
disrupt p75 binding and +2' element function
(Bergsten et al., 2001). To
determine whether this sequence is recognized by Rump, a single G-to-C point
mutation was introduced into each CGUU, individually or in combination
[+2'(A)G:C, (D)G:C and (AD)G:C]. Either point mutation significantly
disrupted binding of MBP-Rump to the +2' element, when assayed by
UV-crosslinking (Fig. 2C). By
contrast, a linker-scanning mutation of six residues in the 3' half of
the +2' element [+2'(F)]
(Bergsten et al., 2001) had no
detectable effect on MBP-Rump binding, consistent with the inability of the
5 fragment to bind to MBP-Rump (data not shown). Thus, binding of Rump
to the wild-type +2' element requires the integrity of both CGUU motifs.
The context of these motifs is, however, important for Rump recognition, as
little binding is detected using a synthetic RNA probe consisting solely of
four CGUU repeats (data not shown).
To test whether Rump binding sites are required for localization, we
introduced the (A)G:C and (D)G:C point mutations into the
nos-tub:nos+2 transgene (Fig.
2D). The nos-tub:nos+2 transgene combines the +1 and
+2' elements, which act synergistically to confer substantial, albeit
not wild-type, posterior localization
(Gavis et al., 1996a). Because
only two of the four nos localization elements are present in the
nos-tub:nos+2 transgene, localization signal redundancy is reduced.
Thus, using this transgene permits us to assay the effects of +2'
element mutations on localization function. Both the (A)G:C and (D)G:C
mutation, when tested individually or together, severely compromised posterior
localization in 100% of embryos, consistent with their effect on Rump binding
(Fig. 2E,F and data not shown).
Together, these results suggest that Rump might function in vivo to regulate
the posterior localization of nos mRNA through the +2'
element.
Isolation of a rump null allele
For analysis of Rump function in vivo, we raised monoclonal antibodies to
recombinant Rump protein. In addition, we generated a rump mutation,
rump1, by excision of a P element inserted in the
5'UTR of CG9373 (Bellen et al.,
2004). Imprecise excision of this P element produced a
deletion beginning 426 nucleotides upstream and extending 501 nucleotides
downstream of the insertion site (Fig.
3A). This lesion removes the transcription start site, the entire
5'UTR and the initial 152 codons, and is thus predicted to be a null
mutation of CG9373. Homozygous rump1 flies are viable
through adulthood, facilitating analysis of rump mutant ovaries and
embryos. In contrast to wild-type ovaries, ovaries from
rump1 mutant females lack detectable rump mRNA
(Fig. 3B). Similarly, anti-Rump
monoclonal antibodies fail to detect any Rump protein in mutant ovaries
(Fig. 3C), indicating that
rump1 is a null mutation.
Homozygous rump1 females showed a variable maternal-effect defect in the percentage of progeny that complete embryonic development, ranging from 29% (n=1137) to 65% (n=886). This defect was rescued by a single copy of a genomic transgene containing only the rump transcription unit, with 80% (n=607) to 86% (n=1402) of the corresponding embryos hatching as larvae (P<0.001). Embryos displaying this and other maternal-effect defects will be referred to as rump mutant embryos. In addition to the maternal-effect defect, homozygous rump mutant males produced by parents heterozygous for rump1 exhibited reduced fertility. Further investigation of this phenotype showed that only 10% of males were able to fertilize virgin females within a 10-day period (n=29). Fertility was restored to 100% (n=30) by the rump transgene.
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To determine whether Rump interacts with nos mRNA in vivo as well as in vitro, Rump was immunoprecipitated from wild-type or rump mutant ovary extract and RNA isolated from the immunoprecipitates was analyzed by RT-PCR using primers for nos. Polyadenylated nos mRNA was reproducibly detected in immunoprecipitates from wild-type ovary extract, but not in immunoprecipitates from rump1 ovary extract (Fig. 4A). In addition, a variety of control antibodies failed to immunoprecipitate nos mRNA (Fig. 4A and data not shown). To address whether the interaction between Rump and nos mRNA is maintained in the embryo, a similar co-immunoprecipitation experiment was performed using extract from 0- to 2-hour wild-type or rump1 embryos. RT-PCR showed that nos mRNA is also immunoprecipitated from embryonic extract by anti-Rump antibody in a Rump-dependent manner (Fig. 4B).
Rump protein is detected in ovaries and embryos
Immunoblotting experiments detected Rump in the ovary and early embryo
(Fig. 3C), consistent with a
role in nos mRNA localization. To determine the specific distribution
of Rump, ovaries and embryos were stained with monoclonal anti-Rump
antibodies. From the germarium onward, Rump was detected in the nuclei of the
germline nurse cells and oocyte, as well as in the somatic follicle cell
nuclei (Fig. 5A,B and data not
shown). This distribution is consistent with the previously described nuclear
localization of Rump in Drosophila S2 cells and of its ortholog Hrp59
in the C. tentans Balbiani ring system
(Hase et al., 2006;
Kiesler et al., 2005). We were
unable to detect cytoplasmic staining above background levels in the ovary. In
the newly fertilized embryo, Rump was present throughout the cytoplasm
(Fig. 5C,D). Just prior to
cellularization, Rump could be detected in both the somatic blastoderm nuclei
and their surrounding cytoplasm, as well as in the internal yolk nuclei
(Fig. 5E,F). However, it was
noticeably excluded from the nuclei of the germline precursors, the pole
cells, which are marked by Vas (Fig.
5E',F'). This nuclear exclusion was maintained during
the subsequent migration of the pole cells to the interior of the embryo (data
not shown).
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To complement the above analysis, we partially compromised the nos
localization machinery by using a null mutation in the germ plasm component
tudor (tudtux46)
(Thomson and Lasko, 2004). In
embryos heterozygous for tudtux46, nos levels were
unchanged, but mild abdominal patterning defects were observed in a small
percentage (4%) of embryos (Fig.
6B and data not shown). Elimination of one or both copies of
rump in this sensitized genetic background resulted in an enhancement
of the abdominal segmentation defects in a dosage-dependant manner, with 44%
of embryos showing segmental deletions and fusions in the absence of
rump (Fig. 6B and data
not shown). Similar results were observed using a second tud allele
(tudWC8, data not shown). Once again, the fourth and fifth
abdominal segments were most often affected, consistent with a role for
rump in the regulation of posterior nos activity.
Finally, we reduced nos localization signal redundancy by using
the nos-tub:nos+2 transgene
(Gavis et al., 1996a;
described above), which confers substantial, albeit not wild-type, posterior
localization. Since this transgene includes sequences required for
nos translational control, all of the nos activity that is
produced results from the translation of localized nos-tub:nos+2 mRNA
(Gavis et al., 1996b). A
single copy of the nos-tub:nos+2 transgene partially rescued the
abdominal defects of nos mutant embryos, such that the majority
developed four or more abdominal segments and over 30% were nearly wild-type,
with seven to eight segments (Fig.
7A). In the absence of rump, the ability of the
nos-tub:nos+2 transgene to rescue abdominal segmentation was severely
compromised and more than 90% of embryos developed fewer than four abdominal
segments (Fig. 7A). As a
molecular measure of abdominal segmentation, we monitored expression of the
pair-rule gene even-skipped (eve), which is expressed in
seven stripes corresponding to even-numbered segments
(Macdonald et al., 1986).
nos mutant embryos expressing nos-tub:nos+2 had four to
seven eve stripes, with over 60% of embryos displaying the complete
set of seven stripes (Fig. 7B).
In the absence of rump, this distribution was shifted, such that the
majority of embryos (66%) had fewer than seven stripes
(Fig. 7B). Eve stripes four to
seven, which designate the abdomen, were most sensitive to loss of
rump (Fig. 7C),
consistent with the abdominal segmentation defects of these larvae.
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|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Recombinant and endogenous Rump recognize two CGUU motifs in the +2'
element. A point mutation within either of these motifs is sufficient to
disrupt both interaction with Rump and mRNA localization function. The
demonstration that sequences required for Rump binding are also required for
posterior localization provides strong evidence that Rump plays a direct role
in mediating nos mRNA localization through its interaction with the
nos 3'UTR.
Rump was previously identified in Drosophila S2 cells through its
cross-reactivity with an antibody to the orthologous C. tetans
protein, Hrp59 (Kiesler et al.,
2005). In our experiments, Drosophila ovarian, embryonic
and S2-cell Rump protein migrate with an apparent mobility of 70 kDa,
consistent with its calculated molecular weight of 67 kDa. Mammalian hnRNP M
proteins have been implicated in splicing through their association with
pre-mRNA at an early step in spliceosome assembly
(Kafasla et al., 2002).
Studies in Chironomus have shown that Hrp59 binds to pre-mRNA
co-transcriptionally and remains associated with the RNA until it reaches the
nuclear envelope, and recent analysis of the Drosophila S2 cell
protein shows that it regulates alternative splicing of its own mRNA
(Hase et al., 2006;
Kiesler et al., 2005).
Our results indicate that Rump has multiple functions at various stages of
the Drosophila life cycle. In addition to defects in abdominal
segmentation, many embryos produced by rump mutant females arrest
development prematurely and adult rump mutant males are largely
sterile. Neither the maternal-effect developmental arrest nor the zygotic
sterility phenotypes is characteristic of nos mutants, indicating
that Rump regulates both nos-dependent and nos-independent
processes. This functional pleiotropy is consistent with the isolation of
numerous Rump target mRNAs from S2 cells, the steady state levels of which are
decreased by RNAi knockdown of rump
(Kiesler et al., 2005). Since
nos is not expressed in S2 cells, its behavior in this assay cannot
be determined. However, we detect no alteration in the abundance or splicing
of nos mRNA in rump mutant ovaries or embryos. Instead, our
data demonstrate a specific and previously unidentified role for Rump as an
mRNA localization factor.
|
). The
GGAGG core sequence does not exist within the nos +2' element,
nor within the entire nos 3'UTR. The ability to recognize
different motifs might be conferred by different RRMs within Rump. Similarity
between RRM3 and the human splicing regulator ASF (also known as SF2 and SFRS1
- Human Gene Nomenclature Database) RRM suggests that RRM3 might be
specifically responsible for interaction with splicing targets
(Kiesler et al., 2005),
whereas RRMs 1 and/or 2 might recognize a different set of Rump targets. In
addition, binding of Rump to nos might be cooperative, as mutation of
one or both CGUU motifs is equally deleterious. Consistent with this idea,
Rump self-interaction was detected in a yeast two-hybrid screen
(Giot et al., 2003),
suggesting that Rump might function as a dimer.
Multifunctionality appears to be a common feature of proteins that regulate
mRNA localization and localized translation. For example, Drosophila
Hrp48 (also known as Hrb27C - FlyBase), an hnRNP A/B family member that
participates in localization and translational regulation of osk and
gurken mRNAs (Goodrich et al.,
2004; Huynh et al.,
2004; Norvell et al.,
2005; Norvell et al.,
1999; Yano et al.,
2004), also functions as a regulator of alternative splicing
(Hammond et al., 1997) and
mammalian homologs of Xenopus Vg1RBP60, an hnRNP I protein involved
in Vg1 mRNA localization (Cote et
al., 1999), play various roles in nuclear RNA biogenesis
(Valcarcel and Gebauer, 1997).
Similarly to Rump, activities of two other nos regulators, Glorund
(Glo) and Smaug (Smg), may be distinguished through multiple, distinct binding
specificities. Glo, an hnRNP F/H homolog, represses translation of unlocalized
nos during oogenesis through its interaction with an AU-rich
double-stranded region within the nos 3'UTR
(Kalifa et al., 2006), whereas
its mammalian counterparts regulate alternative splicing through their
interaction with a G-rich motif (Caputi and
Zahler, 2001; Matunis et al.,
1994). Smg represses translation of unlocalized nos in
the embryo through its interaction with Smaug Recognition Elements (SREs) in
the nos 3'UTR (Crucs et al.,
2000; Dahanukar et al.,
1999; Smibert et al.,
1999), but its function in degradation of maternal mRNAs is
SRE-independent (Semotok et al.,
2005).
Elimination of rump function has a more variable effect on
localization of nos-tub:nos+2 RNA than does mutation of Rump binding
sites. Thus, another ovarian protein with a similar recognition motif might be
able to compensate for the loss of Rump. Alternatively, mutation of the Rump
binding site(s) might have a more global effect on the structure of the
localization element, disrupting the function of other localization factors.
The CGUU motifs recognized by Rump flank one of the two SREs recognized by Smg
within the nos 3'UTR and both the (A)G:C and (D)G:C mutations
disrupt the function of the SRE, consistent with the ability of these
mutations to cause pleiotropic effects. By contrast, mutation of the SRE had
only a small effect on p75 binding to the +2' element
(Bergsten et al., 2001),
suggesting that Rump binding is less sensitive to local perturbations.
Finally, several linker-scanning mutations in the +2' element disrupt
the localization function of this element but do not disrupt Rump binding in
vitro (Bergsten et al., 2001)
(our results), suggesting that at least one additional, as yet unknown, factor
mediates localization via the +2' element.
During early and mid-oogenesis, Rump accumulates in the nurse cell nuclei
where nos is synthesized, suggesting that it might first associate
with nos mRNA in the nucleus. In this regard, Rump may resemble
Xenopus Vg1RBP60/hnRNP I, which associates with Vg1 and
VegT mRNAs in the nucleus and travels with the RNAs to the cytoplasm
where the RNP is remodeled (Kress et al.,
2004). Owing to the impenetrability of the late-stage oocyte to
antibodies, we have not been able to determine the distribution of Rump in
late oocytes when nos becomes posteriorly localized. However, Rump
appears cytoplasmic in newly fertilized embryos and is not enriched at the
posterior of the embryo, suggesting that it does not maintain a stable
association with nos once anchored at the germ plasm. Biochemical
evidence for a continued association of Rump with nos during
embryogenesis, together with the uniform distribution of Rump protein,
indicate that in the early embryo Rump is most likely to be associated with
unlocalized, translationally repressed nos mRNA. The close apposition
of Rump and Smg binding sites raises the possibility that binding by these
factors is mutually stabilizing.
Taken together, data presented here suggest a model whereby Rump associates with nos mRNA during oogenesis, possibly in the nucleus, where it participates in the generation or stabilization of localization-competent nos RNP complexes. A small fraction of nos RNPs are entrapped by the germ plasm and this association results in reorganization of the RNP and release of Rump. The majority of nos mRNA remains unlocalized and, consequently, bound to Rump. Whether Rump plays a role in coordinating translational control with nos mRNA localization remains to be determined.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/5/973/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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