In many organisms, determination of body axis relies on
molecular asymmetries established during oogenesis or
early embryogenesis. In Drosophila melanogaster, maternally
provided mRNAs and proteins are transported into the growing
oocyte and stored until they are required at later stages of
During Drosophilaoogenesis, a germline stem cell located at the
anterior tip of the germarium divides to generate a new stem cell and
a sibling cystoblast (reviewed by Gilboa and Lehmann, 2004; Huynh
and St Johnston, 2004). The cystoblast undergoes four rounds of
divisions with incomplete cytokinesis, giving rise to a 16-cell cyst
consisting of 15 nurse cells and an oocyte interconnected by
cytoplasmic bridges called ring canals. The nurse cells provide the
oocyte with most of the mRNAs and proteins required for its
development, and for the development of the future embryo until the
onset of zygotic transcription (reviewed by Lawrence, 1992;
Spradling, 1993). One of these mRNAs encodes the posterior
determinant Oskar (Ephrussi et al., 1991; Kim-Ha et al., 1991).
During early oogenesis, oskarRNA is exported from the nurse cells
into the oocyte cytoplasm, where the RNA accumulates as a
translationally silent transcript. During mid-oogenesis (stage 8),
oskarmRNA is transported towards the posterior pole, leading to its
asymmetric localization (Ephrussi et al., 1991; Kim-Ha et al., 1991).
oskarmRNA is exclusively translated at the posterior pole, where it
initiates assembly of the pole plasm (Kim-Ha et al., 1991;
Markussen, 1995; Rongo et al., 1995).
oskar mRNA produces two Oskar isoforms, Long Oskar and
Short Oskar, generated by the use of two alternative start codons,
called M1 and M2 (Markussen et al., 1995; Rongo et al., 1995).
Together, the two Oskar proteins induce posterior pole plasm
assembly and localization by recruiting the additional factors
necessary for abdomen and germline formation in the future embryo
(Ephrussi and Lehmann, 1992). Embryos from classical oskar
mutant mothers fail to form posterior structures and lack germ cells
(Lehmann and Nüsslein-Volhard, 1986), because they fail to recruit
Vasa protein and nanos (nos) mRNA to the posterior pole (Ephrussi
et al., 1991; Hay et al., 1990; Lasko and Ashburner, 1990). Vasa is a
highly conserved component of the germline, and is required for
abdomen and germline formation in Drosophila (Schüpbach and
Wieschaus, 1986). Nanos, the abdominal determinant, acts by
repressing translation of maternal hunchbackmRNA in the posterior
region of the embryo, allowing posterior activation of gap genes and,
thus, formation of posterior structures (Gavis and Lehmann, 1992;
Wang and Lehmann, 1991).
Classical oskar mutants were isolated in screens for maternal
effect genes required for anteroposterior patterning of the embryo
(Lehmann and Nüsslein-Volhard, 1986). These classical alleles all
express a significant amount of oskar mRNA, but lack functional
Oskar proteins and thus produce embryos lacking germ cells and
abdomen (Ephrussi et al., 1991; Kim-Ha et al., 1991). Here, we
describe two new oskar mutant alleles showing a strong reduction
or complete absence of oskar mRNA, respectively. Intriguingly,
these new oskaralleles cause a different, stronger oskarphenotype:
the early arrest of oogenesis leading to a complete failure in egg
production. Using a rescue approach with a number of transgenes
unable to produce Oskar protein, we show that the oskar mRNA
transcript, but not the protein, is required for early oskar function.
In particular, its 3?UTR is sufficient to overcome the early
oogenesis arrest, thus revealing an unexpected function for oskar
A translation-independent role of oskar RNA in early
Andreas Jenny1,*,†, Olivier Hachet1,†,‡, Péter Závorszky1,2, Anna Cyrklaff1, Matthew D. J. Weston3,§,
Daniel St Johnston3, Miklós Erdélyi2,¶and Anne Ephrussi1,¶
The Drosophila maternal effect gene oskar encodes the posterior determinant responsible for the formation of the posterior pole
plasm in the egg, and thus of the abdomen and germline of the future fly. Previously identified oskar mutants give rise to offspring
that lack both abdominal segments and a germline, thus defining the ‘posterior group phenotype’. Common to these classical oskar
alleles is that they all produce significant amounts of oskar mRNA. By contrast, two new oskar mutants in which oskar RNA levels
are strongly reduced or undetectable are sterile, because of an early arrest of oogenesis. This egg-less phenotype is complemented
by oskar nonsense mutant alleles, as well as by oskar transgenes, the protein-coding capacities of which have been annulled.
Moreover, we show that expression of the oskar 3? untranslated region (3?UTR) is sufficient to rescue the egg-less defect of the RNA
null mutant. Our analysis thus reveals an unexpected role for oskar RNA during early oogenesis, independent of Oskar protein.
These findings indicate that oskar RNA acts as a scaffold or regulatory RNA essential for development of the oocyte.
KEY WORDS: oskar, Non-coding RNA, Polarity, Oogenesis, Drosophila
Development 133, 2827-2833 (2006) doi:10.1242/dev.02456
1European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg,
Germany. 2Biological Research Center of the Hungarian Academy of Sciences,
Institute of Genetics, H-6701 Szeged, POB 521, Temesvari krt. 62, Hungary. 3The
Wellcome Trust/Cancer Research UK Gurdon Institute & the Department of Genetics,
University of Cambridge, Tennis Court Rd, Cambridge CB2 1QN, UK.
*Present address: Mount Sinai School of Medicine, Brookdale Department of
Molecular, Cell and Developmental Cell Biology, Annenberg Bldg. 18-92, Box 1020,
One Gustave L. Levy Place, New York, NY 10029, USA
†These authors contributed equally to this work
‡Present address: Swiss Institute for Experimental Cancer Research, Chemin des
Boveresses 155, CH-1066 Epalinges, Switzerland
§Present address: Pharmaceuticals Research, Lehman Brothers, 1 Broadgate, London,
EC2M 7HA, UK
¶Authors for correspondence (e-mail: email@example.com; firstname.lastname@example.org)
Accepted 23 May 2006
MATERIALS AND METHODS
Isolation and molecular characterization of the new oskar alleles
All Drosophila strains used in this study that have been previously reported
are described in FlyBase. oskA87was isolated on a ru st e ca chromosome in
an EMS mutagenesis screen using a sensitized genetic background (D. St J.,
unpublished). osk187was identified in a P element mutagenesis screen
(Erdélyi et al., 1995). Although the mutations were isolated in EMS and P
element mutagenesis, molecular analysis reveals that the oskA87and osk187
mutations were induced by a ZAM (Leblanc et al., 1997) retrotransposon
and an I element (Crozatier et al., 1988), respectively. Inverse PCR on a
circularized SspI digest of genomic DNA purified from osk187homozygous
flies was performed by amplification with primers U8 (5?-TTGCGCCTGC
TCTTTCGCCTTC-3?) and L5 (5?-TTTGTTCCCATTCGCCCACCAT-3?).
The amplification product was cloned using a pCRScript kit (Stratagene)
and an I element was identified in the sequence, which was obtained by
sequencing using vector primers. oskA87/Df(3R)pXT103genomic DNA was
digested with SnaBI. Inverse PCR using L5 and oskSeq1 primers (5?-
CGAAAAGCACCGTAAGTCTC-3?) was performed on the circularized
template. The PCR product was cloned into Topo T/A plasmid (Invitrogen).
Sequence analysis of the fragment revealed the insertion of a ZAM
retrotransposon (Accession Number, AJ000387) in reverse orientation with
respect to oskar transcription. Using ZAM element and the oskar-specific
primers ZAM5?rev (5?-GTATGCGTTGTTCTGTCTGAG-3?) and circ osk
3?(5?-TAACTGCAGTTGGTCTTTTCATCCGTT-3?), the 5? end of the
ZAM element and the flanking oskarsequence were amplified. The insertion
site of the ZAM element was precisely determined by cloning into Topo T/A
vector and sequencing the PCR product.
Southern analysis of the oskA87mutant DNA revealed a band whose size
precisely reflected the insertion of a ZAM retrotransposon (data not shown).
Northern blot analyses were performed using poly(A)+selected RNA and
radiolabeled antisense oskar and rp49 RNA probes (Roche RNA labeling
kit), according to standard protocols.
For RT-PCR analysis, total RNA from the abdomen of three to six females
was extracted using the Absolutely RNA RT-PCR Miniprep kit (Stratagene)
including a DNAse digest. Oligo-dT primed cDNA was synthesized with
Superscript II (Invitrogen) according to standard procedures. To show the
absence of osk transcript in oskA87(Fig. 1C), RT-PCR was performed on
cDNAs from oskA87/Df(3R)pXT103and nosA10/Df(3R)pXT103females
with primers A87RT (5?-TTGCTGAGCCACGCCCAGAA-3?) and
Bi_osk_control (5?-ACATTGGGAATGGTCAGCAG GAAATC-3?) for 40
cycles (annealing temperature 55°C). Primers bcd_up (5?-AACGAGCAAG-
AAGACGACGCTACAGTCTTG-3?) and bcd_rt (5?-GCGAATAGCG-
TATTGCAGGGAAAGTATAGA-3?) were used as positive control.
Quantitative real-time RT-PCR (Fig. 4) was performed on cDNAs from
pCogGal4:VP16/w1118; UAS osk-K10/+; oskA87,NanosGal4:VP16/
VP16Df(3R)pXT103, and pCogGal4:VP16/UAS osk3?UTR;; oskA87,
NanosGal4:VP16/Df(3R)pXT103females using SYBR Green1 chemistry
(Molecular Probes) on an ABI PRISM 7900HT real-time PCR apparatus.
oskK10 mRNA was amplified using primers oskK10_for (5?-CTCC-
TGTCTAATCAACGAAAGG-3?) and K10_rev (5?-TTGACCATGGGT-
TTAGGTATAATG-3?), and primers A87RT and circ osk 3? were used to
amplify total oskmRNA. For normalization, bcdmRNA was amplified using
primers bcd_up (5?-AACGAGCAAGAAGACGACGCTACAGTCTTG-3?)
and bcd_rt (5?-GCGAATAGCGTATTGCAGGGAAAGTATAGA-3?).
Amplification efficiencies were comparable as determined using serial 10-
fold dilutions of an initial osk PCR product as substrate.
In situ hybridization and immunohistochemistry
Whole-mount antibody staining and in situ hybridization using fluorescent
RNA probes were performed as previously described (Hachet and Ephrussi,
2004; Tomancak et al., 1998). For whole-mount antibody staining, antigens
were detected using the following primary antibodies: mouse anti-BicD (a
mix of monoclonals 1B11 and 4C2, 1:10 dilution; gift of Beat Suter), rabbit
anti-Staufen (pre-adsorbed, 1:2000 final dilution), rat anti-Bruno (1:5000
dilution) (Filardo and Ephrussi, 2003), mouse anti-Orb (a mix of 48H and
6H4 monoclonals, 1:20 dilution; Developmental Studies Hybridoma Bank)
and rabbit anti-Par-1 (1:40 dilution) (Tomancak et al., 2000). For fluorescent
detection, Rhodamine- or FITC-coupled goat anti-mouse, rabbit or rat
secondary antibodies were used (1:500; Jackson Immuno Research
Construction of transgenes
P(mM1 mM2stop) was made in a two-step PCR reaction. The product of a
first round of PCR, generated using primers linkerM2mut (5?-AGC-
GAGAACAACGGTACCATCATCGAG-3?; ATGrGGT) and osk54Hind
ATGCTCGATATCGTGATT-3?) was used as primer for a second round of
PCR together with M1BssHII (ATGrCGC). The product was then
reamplified with the outside primers M1BssHII (5?-TAGGATCCAA-
and osk54Hind, then cloned into pBluescript (pBSNTL) and sequenced. The
fragment was then used to replace the wild-type BamHI (oskar
promoter)–HindIII (first intron) fragment of pGem11go6.45, a pGEM11
vector with a 6.45kb XhoI-ApaI genomic DNA fragment encompassing the
oskar locus. The mutated oskar gene was then transferred into pCasper4
(Pirrotta, 1988) as an XhoI-NotI fragment (pCaspNTL).
To generate P(mM1SLmM2), the hairpin HP7, which blocks scanning by
small ribosomal subunits (Kozak, 1989a), was cloned as a blunt-ended
BamHI-HindIII fragment into the blunt-ended SphI site of pBSNTL
(pBSNTL HP7; orientation: reconstituted BamHI site proximal to the oskar
transcription start site). The BamHI-XcmI fragment of pGem11go6.45 was
then replaced by the corresponding fragment of pBSNTL HP7 and the BstXI
site of the resulting plasmid was destroyed by cutting, filling and religation.
This resulted in an additional frame shift (CCACTGG instead of
CCACCTGG; sequence not canonical owing to fill-in artefact). The mutated
oskar transgene was then transferred as an XhoI-NotI fragment into
pCasper4, resulting in P(mM1SLmM2).
pUASp oskWT and pUASp osk?i(1,2,3) were constructed by cloning
genomic and cDNA versions of oskar as BamHI/NsiI fragments of pGem
g.osk and pGem g/c.osk, respectively, into pUASp Casper (Rorth, 1998)
digested with BamHI/PstI. pGem g.osk was constructed by subcloning a 6.45
kb XhoI-ApaI fragment of oskar genomic DNA into pGem11Zf (Promega).
pGem g/c.osk was constructed by replacing a 2425 bp BssHII-SacII fragment
of pGem g.osk containing all of the oskar introns with the equivalent 2024
bp fragment of the oskar cDNA of Blue-osk (Ephrussi et al., 1991).
Complementation and rescue analysis
Trans-heterozygous oskar mutant females (A87/54, A87/84, A87/346,
187/54, 187/84 and 187/346) were produced, as well as P(transgene)/SM6B;
osk187/Df(3R)pXT103and P(transgene)/SM6B; oskA87/Df(3R)pXT103females
[in which P(transgene) represents the various rescue constructs under osk
promoter]. Rescue analysis using the UAS yeast inducible promoter was
performed in the oskA87/Df(3R)pXT103background using pCog-Gal4:VP16
and Nanos-Gal4:VP16 drivers simultaneously.
Flies of the following genotypes were analyzed:
pCog-Gal4:VP16/UAS oskWT;; oskA87,Nanos-Gal4:VP16/Df(3R)pXT103;
pCog-Gal4:VP16/w1118; UAS osk-K10/+; oskA87,Nanos-Gal4:VP16/
Test females were collected as virgins and mated with Oregon-R (Fig.
3B), or w1118males (Fig. 3D). The egg-laying capacity of at least 30
individual test females from each experiment was monitored over four days,
in egg-laying blocks on apple-juice agar plates, at 25°C. Values (including
the wild-type controls) were normalized to the average of eggs laid per day
per Oregon-R or w1118 female. The standard deviation was calculated from
oskar RNA null mutants fail to complete oogenesis
Two new oskar (osk) mutants, oskA87and osk187, were isolated in
independent mutagenesis experiments. When transheterozygous
with the oskar nonsense alleles osk54, osk84and osk346 (Kim-Ha et
Development 133 (15)
al., 1991; Lehmann and Nüsslein-Volhard, 1986), both oskA87and
osk187produce embryos that display the classical ‘posterior group
phenotype’ (lack of an abdomen and germline; data not shown; see
also Fig. 3A,B) and thus fail to complement previously known oskar
alleles. Molecular analysis revealed the presence of I (Crozatier et
al., 1988) and ZAM (Leblanc et al., 1997) transposable elements in
the upstream regulatory sequences or 1st exon of oskar inosk187and
oskA87, respectively (Fig. 1A). The position of these mutagenic
elements suggested that they might affect the production or stability
of oskar transcripts. Consistent with this hypothesis, northern
analysis, in situ hybridization and RT-PCR of mutant osk187egg
chambers confirmed that only residual amounts of oskar RNA are
present, which are even further reduced in hemizygous animals (Fig.
1B,D; data not shown). In the case of oskA87/Df(3R)pXT103egg
chambers, we detected no oskar mRNA either by RT-PCR or by in
situ hybridization. oskA87 therefore is a true RNA null allele of oskar
Surprisingly, and in contrast to females carrying the previously
known nonsense alleles of oskar, osk54, osk84and osk346, which do
not express Oskar protein (as judged by western blots) and are thus
considered strong loss of function alleles (Markussen et al., 1995;
Rongo and Lehmann, 1996), females carrying only an oskA87or
osk187allele [oskA87/Df(3R)pXT103, osk187/Df(3R)pXT103] fail to lay
eggs and are sterile, owing to an early arrest of oogenesis. In both
mutants, oocytes are determined and begin to develop, as indicated
by the accumulation of BicD (Suter and Steward, 1991), Orb (Lantz
et al., 1994), Bruno (Webster et al., 1997) and Par1 (Vaccari and
Ephrussi, 2002) in the oocyte (Fig. 2D,F,H,J). Only Staufen, a RNA-
binding protein (St Johnston, 1992), the localization of which within
the oocyte is interdependent with that of oskar mRNA (St Johnston
et al., 1991), is not detected in oskA87/Df(3R)pXT103oocytes (Fig.
2B), indicating that Staufen accumulation in the oocyte is mediated
by oskarmRNA. Defects in oskA87/Df(3R)pXT103 egg chambers first
become evident at stage 2, when fragmentation of the normally
compact karyosome is observed (Fig. 2L). Mutant egg chambers
continue to develop until stage 7, when they begin to degenerate.
To further prove that the oogenesis defects are due to mutations
in oskar, we performed genetic rescue experiments using three oskar
transgenes, encoding either both of the Oskar isoforms (Markussen
et al., 1995; Rongo et al., 1995), or each isoform individually (Fig.
3A). The first transgene, P(osk+) (Markussen et al., 1995), consists
of a genomic DNA fragment that encompasses the oskar locus and
encodes both Oskar isoforms. P(M1L) contains a mutation in M1,
the first translation initiation site in oskar mRNA, and therefore
produces only Short Oskar, the isoform responsible for pole plasm
formation (Markussen et al., 1995). Conversely, P(M139L)produces
only Long Oskar, owing to a mutation in M2, the second translation
initiation site in oskarmRNA (Markussen et al., 1995). Long Oskar
is essential for the cortical anchoring of oskar RNA and thus for
correct localization of the pole plasm, but fails to rescue the
abdominal and germ cell defects of the oskar protein null mutants
(Markussen et al., 1995; Rongo et al., 1995; Vanzo and Ephrussi,
2002). All three transgenes, P(osk+), P(M1L) and P(M139L), fully
complement the egg-less phenotype of oskA87/Df(3R)pXT103 and
osk187/Df(3R)pXT103, indicating that these are indeed oskar alleles
oskar transcript but not Oskar protein is required
for completion of oogenesis
The surprising observation that the osk54, osk84and osk346nonsense
mutant alleles (Kim-Ha et al., 1991) rescue the early oogenesis
defect of the new oskar mutants (giving rise to the ‘posterior group
phenotype’; see also Fig. 3A,B), suggested that the early function
of oskar might be mediated by oskar RNA, rather than by Oskar
protein. To rule out the possibility that a truncated, unstable and
thus undetectable Oskar peptide that is responsible for rescue of the
oogenesis defects of oskA87and osk187is produced by the nonsense
alleles, we constructed two translationally incapacitated, protein
null oskar alleles, and tested their ability to rescue the new alleles.
The first construct, P(mM1 mM2stop), consists of an oskar gene
identical to the osk54nonsense allele, but whose capacity to produce
a short peptide initiating from M1 or M2 was abolished by mutation
of M1 and M2 to CGC and GGT, respectively (Fig. 3A). The
second construct, P(mM1SLmM2), also containing a mutated M1
and M2, was additionally designed to prevent the initiation of
translation by blocking scanning small ribosomal subunits, as well
Translation-independent role of oskar RNA
Fig. 1. Characterization of the oskA87and osk187alleles.
(A) Schematic representation of the oskar locus and the insertion points
of the transposable elements in the new oskar alleles. E1 through E4
represent the oskar exons. The first exon contains a 15 nucleotide
untranslated region. In oskA87, a ZAM retrotransposable element is
inserted 51 bp upstream of the first intron, in reverse orientation
relative to oskar. In osk187, an I element is inserted 534 bp upstream of
the oskar transcription initiation site, in the same orientation as oskar.
(B,C) Northern blot and RT-PCR analysis of oskar mRNA in the new
oskar mutants. (B) Northern blot: WT1 sample is RNA from wild-type
(Oregon R) egg chambers of stages 1 to 14, and WT2 from wild-type
egg chambers of stages 1 to 7-8. The eglRC12sample represents ovaries
whose development arrested during oogenesis at stages similar to the
new oskar mutants. osk54and osk84are nonsense alleles of oskar (Kim-
Ha et al., 1991). Only trace amounts of oskar mRNA are detected in
homozygous osk187egg chambers and no oskar mRNA is detected in
osk187/Df(3R)pXT103egg chambers. (C) RT-PCR: in contrast to bcd (upper
panel), no oskar mRNA is detected by RT-PCR in oskA87/Df(3R)pXT103
egg chambers (lower panel, lane 2) while a 126 bp band is detected in
a control amplification from nosA10/Df(3R)pXT103(lower panel, lane 1),
an allele of nanos originating from the same genetic background as
oskA87. Lanes 3: negative controls without addition of template.
(D) oskar RNA in situ hybridization in osk187and oskA87. Fluorescently-
labeled antisense oskar RNA probe was used to visualize the presence
of endogenous oskar RNA in stage 3-4 wild-type, osk187/Df(3R)pXT103
and oskA87/Df(3R)pXT103mutant egg chambers. Compared with wild
type, considerably less or no oskar RNA is detected in
osk187/Df(3R)pXT103and in oskA87/Df(3R)pXT103egg chambers,
as to abolish putative translation of Oskar peptides that might
initiate from in-frame methionine codons elsewhere in the oskar
transcript. To this end, a sequence predicted to adopt a stable hairpin
structure and that has been shown to block translation by stalling
scanning ribosomes (Kozak, 1989b) was inserted between mutated
M1 and M2, and a frame-shift was introduced downstream of M2
(Fig. 3A). Both P(mM1 mM2stop) and P(mM1SLmM2) fully
complement the egg-less phenotype of oskA87/Df(3R)pXT103and
osk187/Df(3R)pXT103, to the same extent as the original P(osk+)
transgene (Fig. 3B). However, the embryos produced lack an
abdomen, confirming the absence of Oskar protein. These results
demonstrate that no feature of Oskar protein is required for early
oogenesis, indicating that this function of oskar is mediated by
another aspect of the gene.
The 3? ?UTR of oskar is sufficient to rescue the
oogenesis arrest phenotype of oskA87
All constructs that rescue the oogenesis arrest phenotype of the
oskar RNA null alleles (see above) contain the endogenous oskar
promoter, the coding region, the introns and the oskar 3?UTR. We
therefore wanted to address which region of the oskar locus is
required for its function in early oogenesis. To exclude the
promoter region, we made use of a set of transgenes in which the
oskar promoter was replaced by the yeast UAS promoter, thus
placing the oskar gene under Gal4 transcriptional control (Brand
and Perrimon, 1993). We first analyzed the UAS oskWT transgene
which only differs from the P(osk+) transgene by the replacement
of the oskar promoter region by the UAS promoter (Fig. 3C).
When driven by a combination of pCogGal4:VP16 and
NosGal4:VP16 drivers, UAS oskWT rescues the early oogenesis
arrest of oskA87/Df(3R)pXT103females, as well as the posterior
group phenotype of the progeny (Fig. 3D; data not shown). This
rules out an involvement of the promoter region in oskar rescue
activity and demonstrates that these drivers are sufficient to drive
expression of a rescuing transgene. To assess a possible role of
oskar intronic sequences in early oskar function, we tested the
rescue ability of a UAS oskar transgene called UAS osk?i(1,2,3),
in which the three oskar introns were deleted (Fig. 3C).
oskA87/Df(3R)pXT103females expressing osk?i(1,2,3) produce a
normal number of eggs (Fig. 3D), indicating that the oskar introns
are not essential for early oskar function.
We then tested whether the oskar-coding region or the 3?UTR
are required to rescue the early oogenesis phenotype. Expression
of a UAS osk-K10 transgene in which the oskar 3?UTR was
replaced by that of K10 (Fig. 3C) (Riechmann et al., 2002) under
the control of the same Gal4 drivers successfully used above, is
unable to rescue the early oogenesis defect of the new oskar
alleles, even when the flies are raised at 29°C in order to increase
the expression level of the transgene (Fig. 3D and not shown). To
confirm the functionality of this transgene, we tested whether
oskA87/osk+heterozygous flies overexpressing UAS osk-K10
produce delocalized Oskar activity during embryogenesis. Indeed,
83% of embryos laid by such females are bicaudal, showing that
the UAS osk-K10 transgene is functional. Furthermore, real-time
PCR on cDNA of ovaries from oskA87/Df(3R)pXT103females
expressing the UAS osk-K10 transgene under control of both Gal4
drivers confirmed that the UAS osk-K10 transgene is expressed at
early stages of oogenesis (before stage 7 when oogenesis arrests;
Fig. 4C; see 4A and 4B for schematic and exact genotypes). The
transcript is, however, present at lower levels than the rescuing
oskWT transcript, probably because of the degeneration of the
ovaries (note that in wild-type background, the transcript levels of
UAS oskWT and UAS oskK10 are similar; data not shown).
Nevertheless, antibody staining showed that the UAS osk-K10
RNA is translated well before oogenesis arrests (Fig. 4D). The fact
that UAS osk-K10 fails to rescue early oogenesis confirms our
observation that Oskar protein does not provide early oskar
function. In addition, it indicates that the oskar 3?UTR might
provide the early oogenesis function of oskar.
We, thus, directly tested the capacity of the oskar3?UTR to rescue
the oskA87/Df(3R)pXT103early oogenesis arrest. Remarkably,
expression of the oskar 3?UTR alone from the UAS osk3?UTR
Development 133 (15)
Fig. 2. An oocyte is specified in oskA87/Df(3R)pXT103. (A,B) Whole
mount antibody staining of oskA87/Df(3R)pXT103ovaries (B) shows that
Staufen is not enriched in the oskA87mRNA null oocytes compared with
wild type (A). (C-J) In contrast, the oocyte markers BicD (Suter and
Steward, 1991), Orb (Lantz et al., 1994), Bruno (Webster et al., 1997),
and Par-1 (Shulman et al., 2000; Tomancak et al., 2000) accumulate in
a single, posterior cell in both wild-type (C,E,G,I) and
oskA87/Df(3R)pXT103(D,F,H,J) egg chambers, indicating that early steps in
oocyte specification occur normally in the mutant. (K,L) DAPI staining
of chromatin in stage 4 wild-type (K) and oskA87/Df(3R)pXT103(L) egg
chambers. The compact structure of the karyosome (arrow) is detected
within the wild-type oocyte nucleus. oskA87/Df(3R)pXT103mutant
oocytes show a fragmented karyosome structure (arrow).
transgene (Fig. 3C) (Filardo and Ephrussi, 2003) driven by the
combination pCogGal4:VP16 and NosGal4:VP16 is sufficient to
rescue the early oogenesis and egg-less phenotypes (Fig. 3D). As
expected, the 3?UTR also rescues the karyosome defect (Fig. 5D),
but does not rescue the late oskar phenotype of the resulting
embryos, which display the ‘posterior group phenotype’ (data not
shown). This demonstrates that the function of oskar during early
oogenesis is mediated by oskarRNA, independent of Oskar protein,
and demonstrates that the oskar 3?UTR is sufficient to perform this
We also analyzed the localization of Staufen in the ovaries of
oskA87/Df(3R)pXT103females expressing either the UAS osk3?UTR
or the UAS osk-K10 transgene. In the case of UAS osk-K10, no
Staufen protein was detected in the oocyte, indicating that Staufen
fails to associate with osk-K10 RNA (Fig. 5B). By contrast,
expression of the oskar 3?UTR alone was sufficient to restore
Staufen accumulation in the oocyte, showing that Staufen
associates with oskar RNA though its 3?UTR (Fig. 5C). However,
during late oogenesis, Staufen and oskar 3?UTR RNA fail to
localize at the posterior of oskA87/Df(3R)pXT103oocytes [compare
Translation-independent role of oskar RNA
E1 E2E3 E4
E1E2 E3 E4
A87 / Df
187 / Df
Eggs per day (norm. to wild-type)Eggs per day (norm. to wild-type)
Fig. 3. The oskar 3? ?UTR is sufficient to rescue the oogenesis arrest phenotype of oskar RNA null alleles. (A,C) Schematic of oskar alleles
and transgenes. Solid black bars represent the oskar promoter and 5?UTR (left) and the 3?UTR (right). M1 and M2 are the two translation initiation
sites of oskar. E1 through E4 indicate the oskar exons. Black dots in osk54, osk84, osk346and P(mM1 mM2stop) transcripts show the positions of the
stop codons in the nonsense alleles and the transgene. P(osk+) is a wild-type oskar transgene that encodes both the long and short Oskar isoforms
and fully rescues the oskar strong loss of function alleles (Markussen et al., 1995). Black dots at M1 and M2 in P(oskM1L), P(oskM139L), P(mM1
mM2stop) and P(mM1SLmM2) indicate the mutated translation initiation sites. HP7 represents the hairpin loop sequence (Kozak, 1989a) inserted
between M1 and M2, and the grey dot shows the frame-shift mutation inserted in P(mM1SLmM2). (C) Light grey bars represent the UAS promoter.
UAS oskWT expresses the oskar gene (including introns) under the control of the yeast UAS promoter. UAS osk?i(1,2,3) expresses an oskar RNA
whose three introns have been deleted (dark grey bar), under control of the UAS promoter. Note that all UAS-driven transcripts contain an intron
derived from the pUASp vector. Dashed line in UAS osk-K10 indicates the K10 3?UTR. UAS osk3?UTR expresses only the 3?UTR of oskar without any
oskar coding region under control of UAS. To ensure continued high-level expression throughout oogenesis, expression of all UAS transgenes was
driven by pCOG:Gal4VP16 and nanos:Gal4VP16 simultaneously. Flies were grown at 25oC. In the case of the UAS osk-K10 transgene, rescue was
also tested at 29oC, at which Gal4-induced expression is maximal. (B,D) Average number and standard deviation produced daily by females of the
genotype indicated underneath (normalized to the average number of eggs laid by wild-type females). See text for details. (B) Grey bar: Oregon-R
control. Black and white bars: alleles or transgenes in oskA87/Df(3R)pXT103and osk187/Df(3R)pXT103background, respectively. (D) Grey bar: w1118
control. Black bars: transgenes in oskA87/Df(3R)pXT103 background.
Fig. 5F,H with 5E,G (UAS osk WT)]. These observations confirm
our previous results showing that the oskar 3?UTR is not sufficient
for oskar RNA localization at the posterior of the oocyte (Hachet
and Ephrussi, 2004). It therefore appears that the function supplied
by the oskar 3?UTR for oocyte progression past early oogenesis
is independent of the assembly of the oskar RNP localization
complex that mediates its transport to the oocyte posterior in mid-
The new oskar alleles oskA87and osk187reveal that oskar plays a
crucial role during early oogenesis. This early function of oskar is
unusual, as it is mediated by oskar RNA, and not by Oskar protein.
The low but detectable amount of oskar RNA observed in osk187
mutants indicates that a threshold exists, below which egg chambers
fail to develop. Our findings raise the possibility that many protein-
coding genes may fulfill additional non-coding functions and thus
increase the spectrum of gene function.
What might the function of oskar RNA be during early
oogenesis? Oskar protein serves as a scaffold for the assembly of
cytoplasmic structures essential for germline development, the polar
granules, at the posterior pole of the oocyte and embryo. During
early oogenesis, oskar RNA might provide a similar scaffold
function for assembly of cytoplasmic complexes essential for the
progression of oocyte development. RNAs are associated with
proteins in ribonucleoprotein complexes during most of their
existence, from their emergence as nascent transcripts in the nucleus,
during nucleo-cytoplamic transport, to their final cytoplasmic
localization, translation or degradation (Shyu and Wilkinson, 2000).
Development 133 (15)
Fig. 4. UAS osk-K10 produces Oskar protein before oogenesis
arrest in oskA87/Df(3R)pXT103. (A) Scheme showing transgene
structure and priming sites of PCR primers used for Real-time PCR.
Large blue boxes represent oskar coding region. Grey and orange thin
boxes represent oskar and K10 UTRs, respectively. Thin lines correspond
to introns. Oskar specific primers are in red, K10 specific primer is in
green. (B) Genotypes of ovaries analyzed. Numbers correspond to
numbers in panels A, C. (C) Two percent agarose gel showing end
products of Real-time PCR reactions performed in triplicate using cDNA
of early stage ovaries of genotype numbered on top. bicoid (bcd) was
used for normalization. UAS oskK10 is about 15 fold less abundant
than UAS osk WT. NTC: non-template control. (D) Antibody staining of
non-rescued ovaries from females expressing UAS oskK10 (genotype
#1) show that Oskar protein (green) is expressed as early as in germaria.
F-actin and DNA are in red and blue, respectively.
Fig. 5. The oskar 3? ?UTR is sufficient for
Staufen transport to the oocyte, but not for
oskar RNA localization at the posterior pole.
(A-C) Staufen revealed by immunofluorescence in
stage 4 oskA87/Df(3R)pXT103 egg chambers
UAS oskWT (A), UAS oskK10 (B), and UAS
osk3?UTR (C). (D) The karyosome defect is
rescued in egg chambers expressing
osk3?UTR. White arrow marks an intact
karyosome revealed by DAPI stain. (E-H) Top and
bottom panels show stage 10 oskA87/Df(3R)pXT103
egg chambers expressing UAS-driven oskWT and
osk3?UTR, respectively. (E,F) Staufen detected by
immunofluorescence is shown in green; DAPI
staining is in red. (G,H): oskar RNA detected by
fluorescent in situ hybridization is shown in red;
DAPI staining is in blue.
Staufen protein requires oskar RNA for its transport from the nurse
cells into the oocyte (Fig. 2B). In addition, we have shown that it is
the oskar 3?UTR that mediates accumulation of Staufen protein in
the oocyte (Fig. 5). This reveals that the well-known mutual
interdependence of Staufen and oskar mRNA in their localization
during oogenesis is mediated by interaction of Staufen with the
oskar3?UTR. Of the candidate proteins we examined, only Staufen
showed a clearly altered distribution in the oskar RNA null mutant,
yet Staufen itself does not appear to play a role during early
oogenesis (St Johnston et al., 1991). It is therefore reasonable to
assume that oskar RNA acts as a structural partner for the transport
into the oocyte of additional, so far unidentified proteins or RNAs
essential for its development. In this regard it is interesting to note
that both VgT RNA and the non-coding Xlsirts RNA have been
shown to mediate anchoring of several RNAs at the vegetal pole of
the Xenopus oocyte (Heasman et al., 2001; Kloc and Etkin, 1994;
Kloc et al., 2005).
Alternative functions of the oskar 3?UTR are also plausible. In
particular, the oskar 3?UTR might bind and sequester a negative
regulator that, in its free form (i.e. in an oskar RNA null background),
inhibits early oogenesis. One candidate that has been shown to bind
to the oskar 3?UTR is the translational regulator Bruno (Kim-Ha et
al., 1995). However, overexpression of Bruno – at least in the
presence of wild-type levels of oskar mRNA – does not cause a
phenotype similar to that of the oskarRNA null mutant (Filardo and
Ephrussi, 2003). Thus, to fully understand the mechanism of
oogenesis arrest resulting from absence of oskar mRNA, it will be
important to identify other proteins and RNAs binding to the oskar
3?UTR that are required for egg chamber development.
We thank Elisa Wurmbach for help with QRT-PCR and Beat Suter for anti-BicD
antibodies. The monoclonal antibodies orb6H4 and orb4H8 developed by Paul
Schedl were obtained from the Developmental Studies Hybridoma Bank
developed under the auspices of the NICHD and maintained by The University
of Iowa, Department of Biological Sciences, Iowa City, IA 52242, USA. This
work was supported by grant T048393 from the Hungarian National Science
Foundation (OTKA) to M.E. A.J. was supported by fellowships from the Swiss
National Fonds and EMBO and O.H. by a fellowship from a predoctoral
‘Allocation de Recherche’ from the French government. A.J. and P.Z. also
received support from a Human Frontier Science Program grant to A.E.
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Translation-independent role of oskar RNA