The signal recognition particle (SRP) is a ribonucleoprotein
complex that directs the spatially correct synthesis and traffic
of membrane and secreted proteins (Walter and Johnson, 1994;
Walter et al., 2000). In vertebrates, the SRP consists of a ~300
nucleotide-long RNA and six proteins (Walter and Johnson,
1994). The structure and function of the SRP is currently
understood in considerable detail (Keenan et al., 1998; Batey
et al., 2000; Walter et al., 2000; Weichenrieder et al., 2000;
Batey et al., 2001; Beckmann et al., 2001; Keenan et al., 2001;
Weichenrieder et al., 2001; Wild et al., 2001; Hainzl et al.,
2002; Oubridge et al., 2002; Pool et al., 2002; Nagai et al.,
2003), but less is known about how this critically important
ribonucleoprotein particle is assembled in the cell. Studies in
both mammalian cells (Jacobson and Pederson, 1998; Politz et
al., 2000; Politz et al., 2002; Alavian et al., 2004) and yeast
(Ciufo and Brown, 2000; Grosshans et al., 2001) have
implicated the nucleolus as a possible initial site in the
assembly of the SRP. The presence of SRP components in yeast
and mammalian nucleoli constitutes one of several lines of
evidence that the nucleolus has functions beyond its classically
established role in ribosome synthesis (Pederson, 1998; Olson
et al., 2000; Olson et al., 2002; Gerbi et al., 2003).
The oocytes of amphibians and certain other eukaryotic
organisms contain a special class of multiple nucleoli
(Montgomery, 1898; Painter and Taylor, 1942). These multiple
nucleoli are amplified from the genomic ribosomal RNA genes
into hundreds or thousands of extrachromosomal nucleoli that
produce the massive amounts of maternal ribosomes needed to
support early embryonic development (Brown and Dawid,
1968; Gall, 1968; Macgregor, 1972; Gall, 1978). Because these
specialized nucleoli have such a clear developmental purpose,
we were interested to know whether SRP components might
also be present within them. If the biological rationale of the
amplified nucleoli is to produce a maternal stockpile not only
of ribosomes per se, but of translational machinery altogether,
it might be anticipated that they also are committed to SRP
biosynthesis. The findings we now report strongly support this
hypothesis and suggest that at least the initial steps in SRP
ribonucleoprotein assembly, involving SRP RNA and SRP19
protein, occur in these nucleoli.
Materials and Methods
Oocyte microinjection experiments
Female Xenopus laevis at 8-9 cm length were obtained from Blades
Biologicals (Kent, UK) and maintained in an aquarium under optimal
conditions for further growth. Ovary was removed from mature
animals killed under Schedule 1 (Home Office, London, Animals
Scientific Procedures Act 1986) and individual oocytes were released
by stirring the tissue in a solution of 0.2% collagenase (Sigma, Type
I) in OR2 medium minus Ca2+(Evans and Kay, 1991). Mid-
vitellogenic oocytes at stage IV/V (Dumont, 1977) were selected and
maintained in OR2 medium (including 1 mM CaCl2) at 19-20°C.
Fluorescent RNA or protein was microinjected into either the
The signal recognition particle (SRP) is a ribonucleoprotein
machine that controls the translation and intracellular
sorting of membrane and secreted proteins. The SRP
contains a core RNA subunit with which six proteins are
assembled. Recent work in both yeast and mammalian cells
has identified the nucleolus as a possible initial site of SRP
assembly. In the present study, SRP RNA and protein
components were identified in the extrachromosomal,
amplified nucleoli of Xenopus laevis oocytes. Fluorescent
SRP RNA microinjected into the oocyte nucleus became
specifically localized in the nucleoli, and endogenous SRP
RNA was also detected in oocyte nucleoli by RNA in situ
hybridization. An initial step in the assembly of SRP
involves the binding of the SRP19 protein to SRP RNA.
When green fluorescent protein (GFP)-tagged SRP19
protein was injected into the oocyte cytoplasm it was
imported into the nucleus and became concentrated in the
amplified nucleoli. After visiting the amplified nucleoli,
GFP-tagged SRP19 protein was detected in the cytoplasm
in a ribonucleoprotein complex, having a sedimentation
coefficient characteristic of the SRP. These results suggest
that the amplified nucleoli of Xenopus oocytes produce
maternal stores not only of ribosomes, the classical product
of nucleoli, but also of SRP, presumably as a global
developmental strategy for stockpiling translational
machinery for early embryogenesis.
Key words: Immunohistochemistry, In situ hybridization,
Microinjection, Nuclear organization, Nucleolar function
Signal recognition particle assembly in relation to the
function of amplified nucleoli of Xenopus oocytes
John Sommerville1,*, Craig L. Brumwell2,‡, Joan C. Ritland Politz2 and Thoru Pederson2
1Division of Cell and Molecular Biology, School of Biology, University of St Andrews, KY16 9TS, UK
2Department of Biochemistry and Molecular Pharmacology, and Program in Cell Dynamics, University of Massachusetts Medical School,
Worcester, MA 01605, USA
‡Present address: Department of Neuroscience, University of Connecticut Health Center, Farmington, CT 06032, USA
*Author for correspondence (e-mail: firstname.lastname@example.org)
Accepted 13 January 2005
Journal of Cell Science 118, 1299-1307 Published by The Company of Biologists 2005
Journal of Cell Science
cytoplasm or germinal vesicle (nucleus) as previously described in
detail (Ryan et al., 1999). The injection volume was typically 20 nl
for cytoplasmic and 10 nl for nuclear injection. Oocytes were
dissected under oil into germinal vesicles (GVs) and cytoplasm at
various times after microinjection, and RNA or protein distribution
was analysed as detailed below. GV spreads were prepared as
previously described (Callan et al., 1987).
Human SRP RNA, which shares ~87% sequence homology with
Xenopus SRP RNA (Ullu and Tschudi, 1984), was used in this study.
Plasmid phR (Zwieb, 1991; Jacobson and Pederson, 1998) was
linearized by DraI digestion and transcribed with T7 RNA polymerase
in the presence of all four ribonucleoside triphosphates and 5-Alexa
488-UTP (Molecular Probes) present at the same concentration as
UTP, using a Megascript kit (Ambion). A transcript with a sequence
complementary to canine SRP RNA was transcribed with SP6 RNA
polymerase from EcoR1 linearized plasmid pSP7SL (Strub et al.,
1991) with ribonucleoside triphosphates and 5-Alexa 488-UTP as
above. Transcripts were purified using Biogel P-30 spin columns
(BioRad), ethanol precipitated and dissolved at a concentration of
approximately 1 µg/µl in water. Injected specific (sense) and control
(antisense) probes had comparable levels of fluorescence.
Immunoblots and immunocytochemistry
GVs and cytoplasms were isolated and extracted as described
previously (Smillie and Sommerville, 2002). Extracts equivalent to
four GVs and one cytoplasm were denatured in an equal volume of
Laemmli sample buffer (Sigma) and subjected to electrophoresis on
12% SDS-polyacrylamide gels and then electro-transferred onto
nitrocellulose membrane (‘Protran’, Schleicher and Schull). Transfers
were blocked overnight at 4°C in 10% dried nonfat milk dissolved in
phosphate-buffered saline containing 0.1% Tween 20 (PBST)
followed by incubation with a primary antibody diluted in PBST for
1 hour at 20°C, washing through five changes of PBST, and then
incubated with a peroxidase-conjugated secondary antibody diluted in
PBST. Bands were developed using the ECL (Amersham Biosciences)
GV spreads were blocked in 10% fetal calf serum in PBS
(FCS/PBS), incubated with a primary antibody diluted in FCS/PBS
for 1 hour at 20°C, washed through five changes of PBS and then
incubated with a fluorochrome-conjugated secondary antibody diluted
in FCS/PBS). After further washing with PBS, preparations were
mounted in 50% glycerol, 1 mg/ml p-phenylenediamine, pH 8.5, and
viewed in a Leitz Ortholux fluorescence microscope. Controls
omitting a primary antibody incubation showed no secondary
GFP-labelled SRP19 protein
The insert from a fusion plasmid described previously (Politz et al.,
2000) containing the coding sequence for EGFP-SRP19 was cloned
into a bacterial expression plasmid (pET vector, Novagen) containing
a [His]6in-frame coding sequence (Henry et al., 1997) to create a
recombinant plasmid that would express H2N-[His]6-EGFP-SRP19-
COOH. Escherichia coli was transformed with this plasmid and the
expressed GFP-SRP19 protein was recovered and purified by
selection on a Ni NTA agarose column (Qiagen) essentially as
described previously (Henry et al., 1997), except that samples were
concentrated using Biomax-100K filters (Millipore). The purified
protein was diluted in water at a concentration of 1 µg/µl for
microinjection. The localization of GFP-SRP19 protein in the nucleus
after microinjection was analysed by fluorescence microscopy of
spread GV contents. The level of GFP-SRP19 protein in the nuclear
or cytoplasmic fractions was also determined by immunoblotting
using a rabbit antibody to GFP (kindly provided by David Russell,
Washington University School of Medicine) at a dilution of 1/8000
and peroxidase-conjugated anti-rabbit antibody (Sigma) at a dilution
of 1/10,000. In some experiments, oocytes were incubated with
leptomycin B (kindly provided by Minori Yoshida, University of
Tokyo) at a concentration of 50 ng/ml. In other experiments, oocytes
were co-injected in the cytoplasm with both GFP-SRP19 and an
equimolar amount of recombinant human importin-α2(Calbiochem).
Assembly of GFP-SRP19 into a cytoplasmic particle was investigated
by sedimentation of cytoplasmic extracts, prepared as previously
described (Smillie and Sommerville, 2002) on 15-30% linear glycerol
gradients. Some samples were digested before gradient analysis with
ribonuclease A at 1 µg/ml for 30 minutes in the absence of Mg2+
(Walter and Blobel, 1983). Fluorescence in each fraction was read first
in a fluorimeter (VersaFluor™, Biorad) with filters appropriate for
EGFP. Protein was then precipitated from the gradient fractions with
3 volumes of acetone at –20°C and pelleted by centrifugation at
10,000 g for 20 minutes. Dried pellets were dissolved in Laemmli
sample buffer (Sigma) and subjected to immunoblot analysis with
In all, 900, 300, 120, 50, 30 and 25 oocytes at stage I, II, III, IV, V
and VI, respectively, were selected to produce equal volumes of
clarified supernatant after extraction with trichlorotrifluoroethane
(Sigma) and centrifugation at 10,000 g for 2 minutes, followed by
RNA extraction as described previously (Evans and Kay, 1991). RT-
PCR (Ready-to-Go beads, Amersham Biosciences) was run using
primers specific for Xenopus SRP RNA (forward: 5′GCTGTGGCGT-
GTGCCTGTAATCCAG and reverse: 5′GGGTTTTGACCTGCTC-
CGTTTCCGAC) and SRP54 mRNA (forward: 5′CTGGAGGAAA-
TGGCATCTGGCTTGA and reverse: 5′TAGAAGGGTATTCTGGC-
TTTTGTGGC). RT-PCR products amplified after 15, 20 and 25
cycles at 95°C, 48°C and 72°C, respectively, in a MiniCycler (MJ
Research), were separated on 2% agarose gels and 5S RNA was
separated directly from the total RNA sample. Intensity of staining
with ethidium bromide was captured using a GeneSnap system and
measured using GeneTools (Synoptics Ltd).
RNA in situ hybridization
GV spread preparations were incubated with a tetramethylrhodamine-
6-isothiocyanate-labelled peptide nucleic acid probe synthesized
by Applied Biosystems (Politz et al., 2002), complementary to
nucleotides 231-245 of Xenopus SRP RNA. Hybridization was carried
out with 0.3 ng/µl of probe in 40% formamide, 4× SSC, 0.1 M
phosphate, pH 7.2, 0.2 mg/ml tRNA and 0.2 mg/ml denatured
sonicated DNA, for 12 hours at 42°C. Washes were as described
(Politz et al., 2002). As a control, a peptide nucleic acid probe
complementary to nucleotides 192-206 of Schizosaccharomyces
pombe SRP RNA was used.
Detection of endogenous SRP19 protein
To detect endogenous SRP19 protein, extracts were electrophoresed
on 16% polyacrylamide gels and transferred to nitrocellulose as
above. Immunoblotting was with a chicken antibody against the
human SRP19 protein (see below) diluted 1/5000, followed by
peroxidase-conjugated rabbit anti-chicken IgG (Sigma) diluted
GV spreads were immunostained with the SRP19 antibody diluted
1/200, followed by FITC-conjugated rabbit anti-chicken IgG (Sigma)
The antibody to human SRP19 protein was generated in
collaboration with Jos Raats and Walther van Venrooij, University of
Nijmegen, Holland. Human SRP19 protein was expressed in E. coli
Journal of Cell Science 118 (6)
Journal of Cell Science
SRP components in amplified nucleoli
transformed with a (His)6-tagged human SRP19 plasmid and purified
as detailed previously (Henry et al., 1997) and then conjugated to
keyhole limpet haemocyanin. Chickens (Gallus domesticus) were
immunized and sera were screened by ELISA against the immunogen.
An animal displaying a strong immune response (threshold detection
at a serum dilution of 1:12,800) was boosted with another injection.
Immunoglobulin Y was isolated from eggs laid by this animal by
subjecting pooled yolks to lipoprotein depletion and ammonium
sulphate precipitation (Eggcellent IgY Purification Kit, Pierce
Biotechnology), followed by affinity purification on a Sepharose
column to which the recombinant SRP19 protein had been coupled.
Preparations were examined using a Leitz Ortholux Fluorescence
Microscope (100× oil-immersion objective) and photographed on
Kodak Ektachrome P1600 colour reversal film (Kodak Eastman).
Frames were scanned with a Microtek FilmScan 35 using CyberView
interface software (Microtek Europe B.V.) and imported into Adobe
Photoshop. Any recolouring was carried out using the Photoshop
application and merged constrained images were created and
Synthesis of oocyte SRP components coincides with
activity of amplified nucleoli
To establish the occurrence and timing of SRP biosynthesis,
equal masses of oocytes from Xenopus stages I to VI (Dumont,
1972) were examined for the presence of SRP RNA, mRNA
encoding the 54 kDa SRP protein (SRP54, which is the final
protein to be added to the SRP in a reaction that appears to
occur in the cytoplasm) and the 19 kDa SRP protein (SRP19)
RT-PCR was set up with primers specific for Xenopus SRP
RNA and SRP54 mRNA, using total RNA from each oogenic
stage as templates, and reaction products were recorded from
agarose gels after 15, 20 and 25 cycles. Products were detected
at all oogenic stages (Fig. 1A). To compare the levels of SRP
RNA with another RNA polymerase III (pol III) transcript, the
amount of endogenous 5S RNA was recorded from the same
samples (Fig. 1A). Conversion of band densities to amounts
of RNA per oocyte (Fig. 1D) showed that whereas
accumulation of 5S RNA occurs during previtellogenesis
(stages I to II), accumulation of SRP RNA, which is also
transcribed by pol III, occurs throughout oogenesis (including
vitellogenic stages II to VI), although at a slower rate after
stage IV. Thus, the kinetics of SRP RNA synthesis in oocytes
appear to be more akin to those of ribosomal RNA (Scheer,
1973; Scheer et al., 1976) than those of the major pol III
species, 5S RNA and tRNA (Ford, 1971; Mairy and Denis,
1971). On estimating the amounts per oocyte of SRP54
mRNA from RT-PCR (Fig. 1A) and the amounts of SRP19
protein contained within particles sedimenting at ~11S (the
sedimentation rate of mature mammalian SRP) (Walter and
Blobel, 1983) by immunoblotting (Fig. 1B,C), it appears that
the kinetics of synthesis of SRP proteins are similar to those
of SRP RNA (Fig. 1D). Given that accumulation of ribosomes
in oocytes is dependent on the development of active,
amplified nucleoli (Miller and Beatty, 1969), we questioned
whether the observed accumulation of SRP components
through mid-vitellogenesis likewise involves active, amplified
SRP RNA localizes in the amplified nucleoli after
microinjection into the GV
Fluorescent human SRP RNA was injected into the germinal
vesicles (GVs) of stage IV/V oocytes and its distribution
among nuclear components was examined in spread
preparations of isolated GVs (Fig. 2). At 18 hours after
microinjection, distinct, strong fluorescence was observed in
the nucleoli (Fig. 2C). No significant signal was observed in
either the chromosomes or Cajal bodies (Fig. 2A,B), nor in
snurposomes (present throughout the field). All of the
amplified nucleoli examined were labelled. When GVs were
analysed at shorter times after microinjection of SRP RNA,
there was no indication of fluorescence in nuclear structures
other than the nucleoli, suggesting that SRP RNA directly
localizes in the nucleoli after microinjection, rather than first
passing through some other nuclear structures. The nucleolar
localization of SRP RNA was sequence-specific, as shown by
the lack of detectable nucleolar localization of a control RNA
with the antisense sequence to canine SRP RNA (Fig. 2D-F).
Fig. 1. Expression of endogenous SRP RNA and SRP proteins in
Xenopus oocytes. (A) Staged oocytes were collected to give an equal
volume of soluble extract and RNA was extracted for RT-PCR and
quantitation on 2% agarose gels. RT-PCR products were generated
using primers specific for SRP RNA (20 cycles) and SRP54 mRNA
(25 cycles), and 5S RNA was separated directly from the total RNA
sample. (B) Sedimentation analysis of SRP19 protein present in
cytoplasms isolated from stage IV oocytes. Gradient fractions were
immunoblotted using a chicken antibody directed against SRP19
protein. Reactive bands were detected in fractions sedimenting at 10-
12S. Positions of sedimentation mass markers run in a parallel
gradient are indicated by stars (from left: haemoglobin, Mr~67,000;
alcohol dehydrogenase, Mr~180,000; apoferritin, Mr~443,000;
microglobulin, Mr~670,000; immunoglobulin M, Mr~960,000: see
also Fig. 5). Positions of electrophoretic mass markers are shown
(kDa). (C) Immunoblot using anti-SRP19 of the 10-12S fraction
isolated from extracts equivalent to the range of oocyte stages and
oocyte numbers described in (A). (D) Relative amounts of product
per oocyte were calculated by dividing amounts detected from equal
masses of oocytes by the number of oocytes contained in that mass at
each stage. Graph shows: SRP RNA (open circles), SRP54 mRNA
(open squares), 5S RNA (black circles) and SRP19 protein (black
squares). Intensity of staining with ethidium bromide or recorded on
film after ECL was captured using a GeneSnap system and measured
using GeneTools (Synoptics Ltd).
Journal of Cell Science
Endogenous SRP RNA is present in the amplified
The localization of fluorescent SRP RNA in the amplified
nucleoli following its microinjection into the nucleus does not
necessarily mean that endogenous SRP RNA is present there
as well. To address this point, GV spreads were subjected to
RNA hybridization in situ with a peptide nucleic acid (PNA)
probe specific for Xenopus SRP RNA (see Materials and
Methods). As shown in Fig. 3C, hybridization was detected
specifically in the nucleoli and not appreciably in the
chromosomes or other nuclear structures (Fig. 3A). The SRP
RNA hybridization was specific, as shown by the lack of
hybridization when a control probe for yeast SRP RNA
was used (Fig. 3E-G). Fields containing contaminating
autofluorescent yolk platelets are shown to allow comparison
with specific hybridization signals (Fig. 3C,G).
Notably, the localization of SRP RNA signal within the
nucleoli did not appear to be uniform. Nucleoli typically have
a tripartite structure, consisting of small foci called fibrillar
centres (FCs; the sites of the ribosomal genes) situated within
regions called the dense fibrillar component (DFC); these
regions in turn are surrounded by a granular component (GC),
and specific stages of the ribosome biosynthesis pathway have
been assigned to these three domains (Goessens, 1984;
Hernandez-Verdun, 1991; Spector, 1993; Shaw and Jordan,
1995; Scheer and Hock, 1999; Olson et al., 2000). This
classical tripartite organization is also observed in the
amplified nucleoli of amphibian oocytes (Mais and Scheer,
In the nucleolar preparations examined in the current study,
the FCs were identified as the DAPI-staining structures and the
DFC was defined by immunostaining with an antibody to the
Xenopus nucleolar protein, xNop180 (Cairns and McStay,
1995; Schmidt-Zachmann, et al., 1984). In Fig. 3D, the DNA
signal has been computationally coloured green and merged
Journal of Cell Science 118 (6)
Fig. 2. Incorporation of SRP RNA into amplified nucleoli.
Recombinant human SRP RNA (A-C) and control RNA antisense to
canine SRP RNA (D-F) were labelled with Alexa 488 and injected into
the GVs of Xenopus oocytes. After 18 hours, GVs were isolated and
spread preparations were counterstained with DAPI. Fields containing
nucleoli (No) were located by phase-contrast microscopy (A,D) and
generally also contained chromosomes (Ch), Cajal bodies (CB) and B-
snurposomes (smaller particles throughout the field). DNA-rich
regions of chromosomes (chromomeres) and nucleoli (fibrillar centres)
were located by DAPI fluorescence (B,E) and injected RNA was
located by Alexa 488 fluorescence (C,F). SRP RNA was targeted
specifically to nucleoli (C), whereas control antisense RNA failed to
concentrate in any nuclear structures (F), although autofluorescence is
seen from a contaminating yolk platelet (YP).
Fig. 3. In situ hybridization of TRITC-labelled PNA probes with
amplified nucleoli. Probes antisense to Xenopus SRP RNA (A-D)
and sense of yeast SRP RNA (control, E-G) were hybridized to GV
spread preparations. Phase-contrast images (A,E) show nucleoli
(No), chromosomes (Ch) and Cajal bodies (CB). DAPI images (B,F)
show staining of fibrillar centres and chromomeres. TRITC
fluorescence shows that the antisense probe hybridizes to nucleoli,
although not significantly to the fibrillar centres (C). This differential
localization is seen clearly in merged images in which the DAPI
image is computer-coloured green (D). Coincidence of signals
appears as yellow: the small amount of coincidence in nucleoli can
be accounted for by overlapping nucleolar components, whereas
complete coincidence is seen with the wide-spectrum
autofluorescence of a contaminating yolk platelet (YP). The control
probe fails to hybridize to nucleoli, although signal is seen over the
yolk platelets (YP) in (G).
Journal of Cell Science
SRP components in amplified nucleoli
with the in situ hybridization signal (red). In all four of the
nucleoli, the DNA-rich regions and the SRP RNA appear to be
highly segregated, with the probe located largely around the
FCs and extending through much of the DFC and into the GC.
The SRP19 protein is imported into the GV
The findings that microinjected SRP RNA targets the nucleoli
and that the endogenous SRP RNA is localized in them raise
the possibility that the amplified nucleoli are sites of SRP
RNA assembly into nascent SRP particles. The binding of the
SRP19 protein to SRP RNA is the first step in the
biochemically determined assembly pathway of the signal
recognition particle. Therefore, it would be expected that
SRP19 would colocalize with SRP RNA in the nucleoli if
SRP assembly in fact occurs at these sites. To follow SRP19
protein in the oocyte, we used a recombinant GFP-SRP19
fusion protein (see Materials and Methods). We first
investigated whether the GFP-tagged SRP19 protein was
capable of being imported into the nucleus. It was injected
into the cytoplasm of oocytes and its distribution in nuclear
and cytoplasmic fractions was analyzed by immunoblots with
a GFP antibody (Fig. 4). The GFP-SRP19 protein appeared
in the nuclear fraction in increasing amounts over a 10 hour
period (Fig. 4A). The apparent molecular weight of ~47 kDa
is close to that anticipated, as the fusion protein consists of
SRP19 and the ~26kDa GFP, and the persistence of this band
indicates that the nucleus-imported GFP-SRP19 remains
intact. Quantitation of the immunoblot results revealed a
near-linear nuclear import over the 10 hour period of these
experiments (Fig. 4B). By immunoblotting with the SRP19
antibody (see Fig. 1B,C), it was estimated that GFP-SRP19,
recovered from injected oocytes after 10 hours, amounted to
10-20% of endogenous SRP19. In parallel experiments, the
nuclear import factor importin-α was co-injected into the
cytoplasm with GFP-SRP19 protein. This resulted in a
pronounced initial increase in the kinetics of GFP-SRP19
import (Fig. 4B), adding further evidence that the GFP
version of SRP19 was behaving in a physiologically relevant
The SRP19 protein is exported from the GV and is
incorporated into a cytoplasmic RNP particle
To assess further the behaviour of GFP-SRP19 protein in
oocytes, its longer-term distribution between the nucleus and
the cytoplasm was investigated after treatment of oocytes
with leptomycin B, an inhibitor of the Crm1 nuclear export
machinery. Experiments in yeast have established that the
nuclear export of SRP particles is Crm1-mediated (Ciufo and
Brown, 2000; Grosshans et al., 2001), and there is evidence
that this is also the case in mammalian cells (Alavian et al.,
2004). As shown in Fig. 4C, GFP-SRP19 normally began to
leave the nucleus after 10 hours, but treatment with
leptomycinB resulted in an increased level of GFP-SRP19 in
the nuclear fraction at 10 hours of treatment, this effect
becoming more pronounced at 20 hours, suggesting that
GFP-SRP19 export from the oocyte nucleus is Crm1-
We next asked if GFP-SRP19 protein is assembled into a
ribonucleoprotein particle in the oocyte. GFP-SRP19 was
injected into the nucleus and cytoplasmic extracts were
prepared after 48 hours. Glycerol gradient analysis revealed
that the majority of GFP fluorescence sedimented at ~10S
(Fig. 5A), which is close to the sedimentation coefficient
(~11S) of native SRP (Fig. 1B). Treatment of the cytoplasmic
fraction with ribonuclease resulted in a substantial decrease in
the sedimentation velocity of the SRP19 protein to less than
4S (Fig. 5A). In cytoplasms isolated immediately after
injection, the fluorescent signal sedimented close to that
expected for soluble protein monomers (Fig. 5A). The
gradient fractions were further analysed by immunoblotting
with GFP antibody (Fig. 5B), confirming that the fluorescent
signal corresponded to the distribution of intact GFP-SRP19.
Thus, by all these criteria (nuclear import, effect of importin-
α on nuclear import, leptomycinB inhibition of nuclear export
and assembly the exported protein into a ~10S cytoplasmic
particle, the GFP-SRP19 protein behaved in a manner entirely
consistent with the pathway of SRP biosynthesis described in
Fig. 4. Import of GFP-SRP19 into the germinal vesicle and export
back to the cytoplasm. Samples of GFP-SRP19 (20 ng) were injected
into the cytoplasm of oocytes, and GVs and cytoplasms were isolated
under oil at 1, 2, 5 and 10 hours (h) after injection. (A) Protein was
extracted and analysed by immunoblotting using antibodies to GFP.
Bands at 47kDa corresponding to the mass of the fusion protein
(lane S, noninjected protein sample) show a steady increase in
translocation from cytoplasm (C) to GV (N). (B) Ratios of nuclear to
cytoplasmic concentration ([N]/[C]) were calculated from band
density scans and mean values from four different injection
experiments were plotted against time (open circles). In a separate
series of experiments, co-injection of 25ng of the import receptor
importin-α shows an enhanced initial rate of nuclear uptake of GFP-
SRP19 (filled circles). (C) Export of GFP-SRP19 from the germinal
vesicle to the cytoplasm. From 10hours after injection of GFP-
SRP19 into the cytoplasm the nuclear to cytoplasmic concentration
ratio ([N]/[C]) starts to fall (white columns), indicating export from
the GV. This fall is inhibited by incubating the oocytes in the
presence of 50nM leptomycinB (LMB, grey columns).
Journal of Cell Science
The SRP19 protein localizes in the amplified nucleoli
We investigated the nuclear localization of GFP-SRP19 after
import from the cytoplasm. As shown in Fig. 6C, at 1hour after
microinjection into the cytoplasm there was a faint signal in
the nucleoli. After 2-5 hours, the nucleolar signals became
progressively stronger, spreading out from around the DAPI-
staining FCs to encompass the whole nucleolar space (Fig.
6F,I,L). Of the GFP-SRP19 contained in the nucleus at 5hours
postinjection, more than 80% could be pelleted by low-speed
centifugation (3000 g for 5 minutes), indicating that only a
minor component was present in the nucleoplasm. This
observation was confirmed by microscopic examination of
GV contents isolated in the presence of 1 mM Mg2+; the
nucleoplasm, gelled under this condition, exhibiting a much
lower concentration of fluorescence than the embedded
nucleoli (not shown). There was no indication of GFP-SRP19
protein localizing on the chromosomes or Cajal bodies
(compare fluorescence in F,I,L with the phase-contrast images
in D,G,J). All other fields examined failed to show labelling
on structures other than nucleoli. A negative control for
fluorescence is provided by the nuclear behaviour of GFP
itself. As we have shown previously, GFP expressed from
microinjected plasmids equilibrates throughout the oocyte but
does not bind significantly to any nuclear structures (Smillie
and Sommerville, 2002).
When spread GV contents are exposed to low-salt medium
lacking MgCl2,the nucleoli become distended into a necklace-
like array of beaded substructure (Wu and Gall, 1997) due to
a loosening and dissociation of a cortical structure that extends
over the nucleolar surface (Kneissel et al., 2001). When GFP-
SRP19 protein was injected into the cytoplasm and nuclear
spreads were then prepared in a low-salt medium lacking
MgCl2, the signal was observed to be extended in a spatial
configuration that coincided with the distended nucleolar
Journal of Cell Science 118 (6)
Fig. 5. Sedimentation analysis of GFP-SRP19 present in cytoplasm
isolated from injected oocytes. The injected oocytes were either
immediately enucleated and frozen or incubated for 48hours before
enucleation and freezing. Half of the 48hour sample was treated with
ribonuclease A (RNase) for 30minutes in buffer lacking Mg2+.
Clarified cytoplasms from 25 oocytes taken immediately after
injection (open circles), after 48 hours (filled circles) and after 48
hours and treated with RNase (open squares) were loaded on
glycerol gradients and centifuged under the same conditions as
described in Fig.1. Gradient fractions were collected and (A) scanned
at for fluorescence at 510nm (FL, arbitrary units). Fractions from
parallel gradients loaded with the sedimentation markers described in
Fig.1 were scanned at 280nm (Mr, open triangles). The predicted
sedimentation position of SRP (11S, Mr~335,000) is indicated by an
arrow. Fractions were then precipitated with acetone and (B)
analysed by immunoblotting using antibodies to GFP or, in the case
of markers, staining of the gel with Coomassie brilliant blue.
Fig. 6. Incorporation of injected GFP-SRP19 into amplified nucleoli.
At 1 (A-C), 2 (D-F), 3 (G-I) and 5 (J-L) hours after injecting GFP-
SRP19 into the cytoplasm of oocytes, GVs were isolated and spread
preparations were stained with DAPI and examined for fluorescence.
Although nucleoli (No), chromosomes (Ch) and Cajal bodies (CB)
were identified in most fields by phase-contrast (A,D,G,J), and DAPI
staining (B, E, H, K) only nucleoli showed GFP fluorescence
(C,F,I,L). The fluorescent signal is seen to spread with time from
regions around the DAPI-staining fibrillar centres at 2 hours after
injection (E, F) to occupy most of the nucleolar space by 5 hours
Journal of Cell Science
SRP components in amplified nucleoli
substructure (Fig. 7A-C). Moreover, the GFP-SRP19 protein
remained associated with this nucleolar substructure after
storage of preparations in 70% ethanol (Fig. 7D-F), a condition
under which GFP alone is solubilized (J.S., unpublished). This
result establishes that the GFP-SRP19 protein is tightly
associated with nucleolar components.
Endogenous SRP19 is present in the amplified nucleoli
GVs were again spread in low-salt medium lacking Mg2+to
reveal the substructures of the nucleoli at greater spatial
resolution. As shown in Fig. 8C,E, the nucleoli showed distinct
staining with an antibody to SRP19 protein, with no signal
evident in Cajal bodies (Fig. 8A). Very fine foci lacking
immunoreactivity were observed within the nucleolar beads
and these corresponded to the DAPI-stained FCs (Fig. 8B,D).
This suggests that the highest concentration of SRP19 protein
is circumferentially arrayed around the FCs, as was observed
for SRP RNA (Fig. 3). In the merged image (Fig. 8F), the FCs
mostly appeared red, with some immediately adjacent merged
signal (yellow), in turn surrounded by SRP19 alone (green).
This suggests that SRP19 is distributed throughout the nucleoli
except for the FCs, and that some of the protein occupies a
location immediately adjacent to FCs. (It is probable that most
of the merged signal in Fig. 8F represents spatial overlap in the
z-axis.) These results show that the amplified nucleoli are the
major intranuclear sites of the endogenous SRP19 protein in
the oocyte nucleus. Together with the GFP-SRP19 results
and the nucleolar localization of both microinjected and
endogenous SRP RNA, our results strongly support the
hypothesis that the amplified nucleoli of the amphibian oocyte
are involved in SRP biosynthesis.
The initial idea that the nucleolus may have functions other
than ribosome assembly (Pederson, 1998; Pederson, 1999;
Pederson and Politz, 2000) has continued to be borne out by
numerous studies that have revealed the presence in yeast and
mammalian cell nucleoli of various proteins and RNA species
that have no obvious connection with ribosome biosynthesis.
In the present investigation we have identified both the RNA
and a protein component of the signal recognition particle in
the amplified nucleoli of Xenopus oocytes, specifically at a
developmental stage, mid-vitellogenesis, when nucleolar
activity is at a maximum. Previous studies had also identified
SRP RNA and SRP proteins in the nucleoli of somatic
mammalian cells (Jacobson and Pederson, 1998; Politz et al.,
2000; Politz et al., 2002). One interpretation of those findings
was that SRP assembly, at least its initial stages, takes place in
nucleoli. However, another interpretation was that SRP RNA
and SRP proteins remain uncomplexed in nucleoli and serve
some unknown functions unrelated to SRP biosynthesis. This
latter hypothesis can now be viewed as quite implausible as we
show here that SRP RNA and the SRP19 protein are also
present in the oocyte-amplified nucleoli. These nucleoli are
specialized for (at least) ribosome assembly and reside in a
cell-cycle-arrested (meiotic) nucleus. Yet they contain SRP
RNA and an SRP protein, SRP19, which is involved in an early
stage of SRP assembly. Therefore, it seems quite likely that the
presence of the SRP components in oocyte nucleoli reflects an
actual role of these nucleoli in producing a maternal stockpile
of SRP. This interpretation is strongly supported by our
observation that when GFP-SRP19 was injected into the
cytoplasm and localized in the amplified nucleoli, it was
subsequently observed to be exported back to the cytoplasm
where it was identified in a ribonucleoprotein particle having
a sedimentation coefficient in glycerol gradients close to that
of the mammalian SRP particle. The present investigation thus
Fig. 7. Location of GFP-SRP19 in partially dissociated nucleoli. At 5
hours after injecting GFP-SRP19 into the cytoplasm of oocytes, GV
spreads were prepared in the absence of Mg2+and either examined
unfixed (A-C) or after storing the preparations for 18 hours in 70%
ethanol (D-F). Distended nucleolar substructure is evident from the
phase-contrast images (A,D), with globular dense fibrillar component
organized around individual DAPI-staining fibrillar centres (B,E).
The GFP signal remained located at the dense fibrillar component
(C,F) and was absent from chromosomes (D-F) and other nuclear
structures (not shown).
Fig. 8. Immunostaining of amplified nucleoli with antibodies to
SRP19. GV spreads were prepared in low-salt medium without
Mg2+. Nucleoli (No), Cajal bodies (CB) and B-snurposomes were
identified by phase-contrast (A) and DAPI staining (B), but only the
nucleoli were immunostained using chicken anti-SRP19 with FITC-
conjugated anti-chicken IgG as a secondary antibody (C). The
centres of the stained structures are seen to be relatively free of
fluorescence. (D-F) The DAPI staining (D) and the immunostaining
(E) are merged (F), with the DAPI computationally coloured red and
yellow indicating spatial coincidence of the two signals.
Journal of Cell Science
moves the plurifunctional nucleolus concept ahead with respect
to a role in the biosynthesis of the SRP.
The results of this study point to a high mobility of
fluorescent SRP RNA injected into the nucleus of Xenopus
oocytes, since the introduced RNA was observed to traffic
to all of the ~1000 extrachromosomal nucleoli. Similar
observations have been previously made in studies of other
species of RNA injected into the germinal vesicle (Gerbi et al.,
2003; Handwerger et al., 2003). The fact that endogenous SRP
RNA was also found in all of the nucleoli implies that these
molecules traffic extensively from their transcription site
throughout the nucleoplasm to reach all of the amplified
nucleoli. This wide-ranging mobility of RNA in the germinal
vesicle is similar to the diffusive transport of RNA in the
mammalian cell nucleus (Jacobson and Pederson, 1998; Politz
et al., 1998; Politz et al., 1999; Politz et al., 2003).
We were struck by the unique concentration of SRP RNA in
only the amplified nucleoli, and not in other RNA traffic
centres of the Xenopus oocyte nucleus. Similarly, in previous
studies on mammalian cells there was no evidence of SRP
RNA in either of two RNA-rich structures: interchromatin
granule clusters (also known as ‘speckles’) or Cajal bodies
(Jacobson and Pederson, 1998; Wang et al., 2003), and this was
confirmed in the present study in which no association of SRP
RNA with either B-snurposomes (homologous with speckles
in mammalian somatic nuclei) or Cajal bodies was observed in
the Xenopus oocyte nucleus. SRP RNA seems to be distinctive
as one of the few nucleolus-trafficking small RNAs that does
not also have a Cajal body resident stage (Gall, 2000; Gall,
2003; Pederson, 2004).
The present study also provides new information on the
nuclear import of SRP19 protein and its export, studied here
for the first time in amphibian oocytes. Our finding that the
nuclear import of GFP-SRP19 is accelerated by co-
microinjection of importin-α implicates this member of the
importin family in the nuclear import of SRP19 in this
developmentally specialized cell. A previous study of SRP19
protein nuclear import in a detergent-extracted mammalian cell
in vitro system implicated two importins, importin 8 and
transportin, both of which are members of the β-importin
family (Dean et al., 2001). How SRP proteins are imported into
the nucleus of various eukaryotic cells clearly will require
further work, but our results draw attention to importin-α in
Xenopus oocytes. Whether or not SRP19 is actively recruited
to the extrachromosomal nucleoli is not known: a search of the
human SRP19 amino acid sequence did not reveal any regions
corresponding to nucleolus-localizing elements that have been
defined in other nucleolar proteins (S. Yarovoi and T.P.,
unpublished). Our finding that leptomycin B causes an
accumulation of SRP19 protein in the amphibian oocyte
nucleus implicates the Crm1 pathway of nuclear export, which
is the export machinery identified for SRP nuclear export in
yeast (Ciufo and Brown, 2000; Grosshans et al., 2001). We
have found no indication of a nuclear export signal (NES) in
SRP19; indeed, our results with mammalian cells (Alavian et
al., 2004) support an indirect, rather than a direct, export of
nascent SRP by the Crm1 pathway.
Our present findings that both SRP RNA and an SRP protein
are present in the amplified nucleoli of a cell, the amphibian
oocyte, that is designed to stockpile the machinery of
translation, adds considerable weight to the hypothesis that the
nucleolus plays a role in SRP biosynthesis. Moreover, the
present investigation shows that the oocyte is likely to be a
valuable system for investigating further the biosynthesis of the
SRP, both with respect to its nucleolar phase and its overall
developmental timetable. In keeping with numerous studies of
gene expression in this specialized cell, in which features of
transcription, RNA processing and ribonucleoprotein assembly
are quantitatively and cytologically intensified, we are
optimistic that the Xenopus oocyte will be a key system
in continuing studies of both SRP biosynthesis and the
plurifunctional nucleolus concept.
This work was supported by a grant from the Wellcome Trust to
J.S. and U.S. National Institutes of Health grant GM-21595 to T.P. We
are indebted to Jos Raats (University of Nijmegen, The Netherlands)
for his key role in generating the SRP19 antibody. We thank Howard
Fried (University of North Carolina, Chapel Hill, NC) for providing
the SRP19 bacterial expression plasmid, and we acknowledge Serge
Yarovoi in the Pederson laboratory for constructing the GFP fusion
protein. We thank Marion Schmidt-Zachmann (German Cancer
Research Center, Heidelberg) for monoclonal antibody No-114 to
detect xNopp180, David Russell (Washington University School of
Medicine) for the GFP antibody and Minori Yoshida (University of
Tokyo) for leptomycin B. Annemarie Mullin, Katherine Gilchrist and
Ceri Jackson in the Sommerville laboratory are thanked for their help
with the immunoblotting, in situ hybridization and RT-PCR
experiments, and Laura Lewandowski in the Pederson laboratory is
thanked for her skilfull transcription and fluorescent labelling of
Alavian, C. N., Ritland Politz, J. C., Lewandowski, L. B., Powers, C. M.
and Pederson. T. (2004). Nuclear export of signal recognition particle RNA
in mammalian cells. Biochem. Biophys. Res. Comm. 313, 351-355.
Batey, R. T., Rambo, R. P., Lucast, L., Rha, B. and Doudna, J. A. (2000).
Crystal structure of the ribonucleoprotein core of the signal recognition
particle. Science 287, 1232-1239.
Batey, R. T., Sagar, M. B. and Doudna, J. A. (2001). Structural and energetic
analysis of RNA recognition by a universally conserved protein from the
signal recognition particle. J. Mol. Biol. 307, 229-246.
Beckmann, R., Spahn, C. M., Eswar, N., Helmers, J., Penczek, P. J., Sali,
A., Frank, J. and Blobel, J. (2001). Architecture of the protein-conducting
channel associated with the translating 80S ribosome. Cell 107, 361-372.
Brown, D. D. and Dawid, I. (1968). Specific gene amplification in oocytes.
Science 160, 272-280.
Cairns, C. and McStay, B. (1995). Identification and cDNA cloning of a
Xenopus nucleolar phosphoprotein, xNopp180, that is the homolog of the
rat nucleolar protein Nopp140. J. Cell Sci. 108, 3339-3347.
Callan, H. G., Gall, J. G. and Berg, C. A. (1987). The lampbrush
chromosomes of Xenopus laevis: Preparation, identification and distribution
of 5S DNA sequences. Chromosoma 95, 236-250.
Ciufo, L. F. and Brown, J. D. (2000). Nuclear export of yeast signal
recognition particle lacking Srp54p by the Xpo1p/Crm1p NES-dependent
pathway. Curr. Biol. 10, 1256-1264.
Dean, K. A., von Ahsen, O., Gorlich, D. and Fried, H. M. (2001). Signal
recognition particle protein 19 is imported into the nucleus by importin 8
(RanBP8) and transportin. J. Cell Sci. 114, 3479-3485.
Dumont, J. N. (1972). Oogenesis in Xenopus laevis (Daudin). Stages of oocyte
development in laboratory maintained animals. J. Morphol. 136, 153-180.
Evans, J. P. and Kay, B. K. (1991). Biochemical fractionation of oocytes.
Methods Cell Biol. 36, 133-148.
Ford, P. J. (1971). Non-coordinated accumulation and synthesis of 5S
ribonucleic acid by ovaries of Xenopus laevis. Nature 233, 561-564.
Gall, J. G. (1968). Differential synthesis of the genes for rRNA during
amphibian oogenesis. Proc. Natl. Acad. Sci. USA. 60, 553-560.
Gall, J. G. (1978). Early studies on gene amplification. Harvey Lectures Ser.
Gall, J. G. (2000). Cajal bodies: the first 100 years. Ann. Rev. Cell Dev. Biol.
Journal of Cell Science 118 (6)
Journal of Cell Science
1307 Download full-text
SRP components in amplified nucleoli
Gall, J. G. (2003). The centennial of the Cajal body. Nat. Rev. Mol. Cell. Biol.
Gerbi, S. A., Borovjagin, A. V. and Lange, T. S. (2003). The nucleolus: a
site of ribonucleoprotein maturation. Curr. Opin. Cell Biol. 15, 318-325.
Goessens, G. (1984). Nucleolar structure. Int. Rev. Cytol. 87, 107-158.
Grosshans, H., Deinert, K., Hurt, E. and Simos, G. (2001). Biogenesis of
the signal recognition particle (SRP) involves import of SRP proteins into
the nucleolus, assembly with the SRP-RNA, and Xpo1p-mediated export. J.
Cell Biol. 153, 745-762.
Hainzl, T., Huang, S. and Sauer-Eriksson, A. E. (2002). Structure of the
SRP19 RNA complex and implications for signal recognition particle
assembly. Nature 417, 767-771.
Handwerger, K. E., Murphy, C. and Gall, J. G. (2003). Steady-state
dynamics of Cajal body components in the Xenopus germinal vesicle. J. Cell
Biol. 160, 495-504.
Henry, K. A., Zwieb, C. and Fried, H. M. (1997). Purification and
biochemical characterization of the 19-kDa signal recogniiton particle RNA-
binding protein expressed as a hexahistidine-tagged polypeptide in
Escherichia coli. Prot. Expr. Purif. 9, 15-26.
Hernandez-Verdun, D. (1991). The nucleolus today. J. Cell Sci. 99, 475-471.
Jacobson, M. R. and Pederson, T. (1998). Localization of signal recognition
particle RNA in the nucleolus of mammalian cells. Proc. Natl. Acad. Sci.
USA 95, 7981-7986.
Keenan, R. J., Freymann, D. M., Stroud, R. M. and Walter, P. (2001). The
signal recognition particle. Annu. Rev. Biochem. 70, 755-775.
Keenan, R. J., Freymann, D. M., Walter, P. and Stroud, R. M. (1998).
Crystal structure of the signal sequence binding subunit of the signal
recognition particle. Cell 94, 181-191.
Kneissel, S., Franke, W. W., Gall, J. G., Heid, G., Reidenbach, S.,
Schnolzer, M., Spring, H., Zentgraf, H. and Schmidt-Zachmann, M. S.
(2001). A novel karyoskeletal protein: characterization of protein NO145,
the major component of nucleolar cortical skeleton in Xenopus oocytes. Mol.
Biol. Cell 12, 3904-3918.
Macgregor, H. C. (1972). The nucleolus and its genes in amphibian oogenesis.
Biol. Rev. Camb. Phil. Soc. 47, 177-210.
Mairy, M. and Denis, H. (1971). Biochemical studies on oogenesis. 1. RNA
synthesis and accumulation during oogenesis of the South African toad
Xenopus laevis. Dev. Biol. 24, 143-165.
Mais, C. and Scheer, U. (2001). Molecular architecture of the amplified
nucleoli of Xenopus oocytes. J. Cell. Sci. 114, 709-714.
Miller, O. L. and Beatty, B. R. (1969). Extrachromosomal nucleolar genes in
amphibian oocytes. Genetics 61 Suppl., 133-143.
Montgomery, T. H. (1898). Comparative cytological studies, with especial
regard to the morphology of the nucleolus. J. Morphol. 15, 265-582.
Nagai, K., Oubridge, C., Kuglstatter, A., Menichelli, E., Isel, C. and
Jovine, L. (2003). Structure, function and evolution of the signal recognition
particle. EMBO J. 22, 3479-3485.
Olson, M. O., Dundr, M. and Szebeni, A. (2000). The nucleolus: an old
factory with unexpected capabilities. Trends Cell Biol. 10, 189-196.
Olson, M. O., Hingorani, K. and Szebeni, A. (2002). Conventional and
nonconventional roles of the nucleolus. Int. Rev. Cytol. 219, 199-266.
Oubridge, C., Kuglstatter, A., Jovine, L. and Nagai, K. (2002). Crystal
structure of SRP19 in complex with the S domain of SRP RNA and its
implication for the assembly of the signal recognition particle. Mol. Cell. 9,
Painter, T. S. and Taylor. A. N. (1942). Nucleic acid storage in the toad’s egg.
Proc. Natl. Acad. Sci. USA 28, 311-317.
Pederson, T. (1998). The plurifunctional nucleolus. Nucleic Acids Res. 26,
Pederson, T. (1999). Growth factors in the nucleolus? J. Cell Biol. 143, 279-
Pederson, T. and Politz, J. C. (2000). The nucleolus and the four
ribonucleoproteins of translation. J. Cell Biol. 148, 1091-1095.
Pederson, T. (2004). Can telomerase be put in its place? J. Cell Biol. 164, 637-
Politz, J. C., Browne, E. S., Wolf, D. E. and Pederson, T. (1998). Intranuclear
diffusion and hybridization state of oligonucleotides measured by
fluorescence correlation spectroscopy. Proc. Natl. Acad. Sci USA 95, 6043-
Politz, J. C., Yarovoi, S., Kilroy, S. M., Gowda, K., Zwieb, C. and
Pederson, T. (2000). Signal recognition particle components in the
nucleolus. Proc. Natl. Acad. Sci. USA 97, 55-60.
Politz, J. C., Lewandowski, L. B. and Pederson, T. (2002). Signal
recognition particle RNA localization within the nucleolus differs from the
classical sites of ribosome synthesis. J. Cell Biol. 159, 411-418.
Politz, J. C. R., Tuft, R. A. and Pederson, T. (2003). Diffusion-based
transport of nascent ribosomes in the nucleus. Mol. Biol. Cell 14, 4805-4812.
Pool, M. R., Stumm, J., Fulga, T. A., Sinning, I. and Dobberstein, B. (2002).
Distinct modes of signal recognition particle interaction with the ribosome.
Science 297, 1345-1348.
Ryan, J., Llinas, A. J., White, D. A., Turner, B. M. and Sommerville, J.
(1999). Maternal histone deacetylase is accumulated in the nuclei of
Xenopus oocytes as protein complexes with potential enzyme activity. J. Cell
Sci. 112, 2441-2462.
Scheer, U. (1973). Nuclear pore flow rate of ribosomal RNA and chain growth
rate of its precursor during oogenesis of Xenopus laevis. Dev. Biol. 30, 13-
Scheer, U. and Hock, R. (1999). Structure and function of the nucleolus. Curr.
Opin. Cell Biol. 11, 385-390.
Scheer, U., Trendelenburg, M. F. and Franke, W. W. (1976). Regulation of
transcription of genes of ribosomal RNA during amphibian oogenesis: A
biochemical and morphological study. J. Cell Biol. 69, 465-489.
Schmidt-Zachmann, M., Hugle, B., Scheer, U. and Franke. W. W. (1984).
Identification and localization of a novel nucleolar protein of high molecular
weight by a monoclonal antibody. Exp. Cell Res. 153, 327-346.
Shaw, P. J. and Jordan, E. G. (1995). The nucleolus. Ann. Rev. Cell Dev.
Biol. 11, 93-121.
Smillie, D. A. and Sommerville, J. (2002). RNA helicase p54 (DDX6) is a
shuttling protein involved in nuclear assembly of stored mRNP particles. J.
Cell Sci. 115, 395-407.
Spector, D. L. (1993). Macromolecular domains within the cell nucleus. Annu.
Rev. Cell Biol. 9, 265-315.
Strub, K., Moss, J. and Walter, P. (1991). Binding sites of the 9- and 14-
kilodalton heterodimeric protein subunit of the signal recognition particle
(SRP) are contained exclusively in the Alu domain of SRP RNA and contain
a sequence motif that is conserved in evolution. Mol. Cell. Biol. 11, 3949-
Ullu, E. and Tshudi, C. (1984). Alu sequences are processed 7SL RNA genes.
Nature 312, 171-172.
Walter, P. and Blobel, G. (1983). Disassembly and reconstitution of signal
recognition particle. Cell 34, 525-533.
Walter, P. and Johnson, A. E. (1994). Signal sequence recognition and
protein targeting to the endoplasmic reticulum membrane. Ann. Rev. Cell
Biol. 10, 87-119.
Walter, P., Keenan, R. and Schmitz, U. (2000). SRP – where the RNA and
membrane worlds meet. Science 287, 1212-1213.
Wang, C., Politz, J. C., Pederson, T. and Huang, S. (2003). RNA polymerase
III transcripts and the PTB protein are essential for the integrity of the
perinucleolar compartment. Mol. Biol. Cell 14, 2425-2435.
Weichenrieder, O., Wild, K., Strub, K. and Cusack, S. (2000). Structure and
assembly of the Alu domain of the mammalian signal recognition particle.
Nature 408, 167-173.
Weichenrieder, O., Stehlin, C., Kapp, U., Birse, D. E., Timmins, P. A.,
Strub, K. and Cusack, S. (2001). Hierarchical assembly of the Alu domain
of the mammalian signal recognition particle. RNA 7, 731-740.
Wild, K., Sinning, I. and Cusack, S. (2001). Crystal structure of an early
protein-RNA assembly complex of the signal recognition particle. Science
Wu, Z. and Gall, J. G. (1997). “Micronucleoli” in the Xenopus germinal
vesicle. Chromosoma 105, 438-443.
Zwieb, C. (1991). Interaction of protein SRP19 with signal recognition particle
RNA lacking individual RNA-helices. Nucleic Acids Res. 19, 2955-2960.
Journal of Cell Science