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First published online July 11, 2006
doi: 10.1242/10.1242/dev.02456
,
1 European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg,
Germany.
2 Biological Research Center of the Hungarian Academy of Sciences, Institute of
Genetics, H-6701 Szeged, POB 521, Temesvari krt. 62, Hungary.
3 The Wellcome Trust/Cancer Research UK Gurdon Institute & the Department of
Genetics, University of Cambridge, Tennis Court Rd, Cambridge CB2 1QN,
UK.
¶ Authors for correspondence (e-mail: ephrussi{at}embl.de; erdelyim{at}brc.hu)
Accepted 23 May 2006
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: oskar, Non-coding RNA, Polarity, Oogenesis, Drosophila
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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During Drosophila oogenesis, 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, oskar RNA is exported from the nurse cells into the
oocyte cytoplasm, where the RNA accumulates as a translationally silent
transcript. During mid-oogenesis (stage 8), oskar mRNA is transported
towards the posterior pole, leading to its asymmetric localization
(Ephrussi et al., 1991;
Kim-Ha et al., 1991).
oskar mRNA 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 hunchback mRNA 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
oskar alleles cause a different, stronger oskar phenotype:
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 mRNA.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Although the mutations were isolated in EMS and P element mutagenesis,
molecular analysis reveals that the oskA87 and
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 osk187
homozygous 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)pXT103 genomic 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 oskar sequence 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 oskA87 mutant 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)pXT103 and nosA10/Df(3R)pXT103 females 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'-AACGAGCAAGAAGACGACGCTACAGTCTTG-3') and bcd_rt (5'-GCGAATAGCGTATTGCAGGGAAAGTATAGA-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/Df(3R)pXT103, pCogGal4:VP16/UAS oskWT;; oskA87,NanosGal4: VP16Df(3R)pXT103, and pCogGal4:VP16/UAS osk3'UTR;; oskA87, NanosGal4:VP16/Df(3R)pXT103 females using SYBR Green1 chemistry (Molecular Probes) on an ABI PRISM 7900HT real-time PCR apparatus. oskK10 mRNA was amplified using primers oskK10_for (5'-CTCCTGTCTAATCAACGAAAGG-3') and K10_rev (5'-TTGACCATGGGTTTAGGTATAATG-3'), and primers A87RT and circ osk 3' were used to amplify total osk mRNA. For normalization, bcd mRNA 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
Laboratories).
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'-AGCGAGAACAACGGTACCATCATCGAG-3'; ATG
GGT) and osk54Hind
(5'-AAGAAAGCTTTCAAACATAAAGCTACTCACTCCTACTCACTGATGCTCGATATCGTGATT-3')
was used as primer for a second round of PCR together with M1BssHII
(ATGCGC). The product was then reamplified with the outside primers
M1BssHII
(5'-TAGGATCCAAGAATATTGGATCACTTTCCTCCAAGCGCGCGCCGCAGTCACA-3') 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)pXT103 and
P(transgene)/SM6B;
oskA87/Df(3R)pXT103 females [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)pXT103
background 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/UAS oski(1,2,3);;
oskA87,Nanos-Gal4:VP16/Df(3R)pXT103;
pCog-Gal4:VP16/UAS osk3'UTR;; oskA87,Nanos-Gal4:VP16/Df(3R)pXT103;
pCog-Gal4:VP16/w1118; UAS osk-K10/+; oskA87,Nanos-Gal4:VP16/Df(3R)pXT103.
Test females were collected as virgins and mated with Oregon-R (Fig. 3B), or w1118 males (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 normalized values.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Lehmann and Nüsslein-Volhard,
1986), both oskA87 and
osk187 produce 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 in osk187 and
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
osk187 egg 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)pXT103 egg 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 (Fig. 1C,D).
|
;
Rongo and Lehmann, 1996),
females carrying only an oskA87 or
osk187 allele
[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)pXT103 oocytes
(Fig. 2B), indicating that
Staufen accumulation in the oocyte is mediated by oskar mRNA. 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 oskar mRNA
(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
(Fig. 3B).
oskar transcript but not Oskar protein is required for completion of oogenesis
The surprising observation that the osk54,
osk84 and osk346 nonsense 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 oskA87 and
osk187 is 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
osk54 nonsense 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 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)pXT103 and
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.
|
). 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)pXT103 females, 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
oski(1,2,3), in which the three oskar
introns were deleted (Fig.
3C). oskA87/Df(3R)pXT103
females expressing oski(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)pXT103 females 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 oskar 3'UTR to
rescue the oskA87/Df(3R)pXT103 early oogenesis
arrest. Remarkably, expression of the oskar 3'UTR alone from
the UAS osk3'UTR 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
oskar RNA, independent of Oskar protein, and demonstrates that the
oskar 3'UTR is sufficient to perform this function.
|
). 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 midoogenesis. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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|
). 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 oskar 3'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 oskar RNA 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.
|
ACKNOWLEDGMENTS |
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|
Footnotes |
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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
|
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