1 Department of Embryology, Carnegie Institution of Washington, 115 West
University Parkway, Baltimore, MD 21210, USA
2 Department of Biology, Johns Hopkins University, 3400 North Charles Street,
Baltimore, MD 21218, USA
* These authors contributed equally to this study
Author for correspondence (e-mail:
gall{at}ciwemb.edu
)
Accepted 27 February 2002
![]() |
Summary |
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Key words: Cajal body, Germinal vesicle, Heat shock, Oocyte, Xenopus
![]() |
Introduction |
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Without compelling evidence that CBs originate at specific genetic loci,
emphasis has focused on the possibility that some unique molecule or organelle
might act as the seed or scaffold for their formation. In addition to factors
found elsewhere in the nucleus, CBs are enriched for the marker protein coilin
and the rare U7 snRNA involved in histone pre-mRNA processing (reviewed by
Gall, 2000). Coilin and U7
snRNA have thus been considered as strong candidates for nucleators of CB
formation. Evidence against such a role for coilin is now strong. CBs form in
nuclei assembled in Xenopus egg extract, even when the extract has
been immunodepleted of coilin (Bauer and
Gall, 1997
). Likewise, a mouse knockout for the coilin gene
displays CB-like structures in its nuclei
(Tucker et al., 2001
).
Evidence that U7 snRNA can play a role in CB biogenesis has been presented in
a previous study (Tuma and Roth,
1999
). The study showed that injection of U7 snRNA into the
Xenopus oocyte nucleus (germinal vesicle, GV) was sufficient to
induce formation of small `coiled body-like structures'.
We describe the appearance of mini-CBs (<2 µm) in the GVs of
Xenopus oocytes that have recovered from heat shock. These mini-CBs
are similar in size and number to those described previously
(Tuma and Roth, 1999).
However, an antisense depletion experiment shows that U7 snRNA is not
necessary for their induction by heat shock. Neither transcription of RNA nor
import of newly translated proteins into the GV is required, suggesting that
mini-CBs assemble from pre-existing components in the GV.
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Materials and Methods |
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Fluorescein-U7 snRNA and green fluorescent protein (GFP)-coilin
Fluorescein-U7 snRNA was generated in a 30 µl in vitro transcription
reaction containing 1 µg SalI- linearized #401 plasmid DNA
(Wu et al., 1996), 166 µM
fluorescein-12-UTP (Boehringer Mannheim, Indianapolis, IN), 500 µM ATP and
CTP, 250 µM UTP and GTP, 2.5 mM m7G(5')ppp(5')G cap
(New England Biolabs, Beverly, MA), and 0.4 µM [32P]UTP
(Amersham Life Sciences, Arlington Heights, IL) in a buffer consisting of 50
mM NaCl, 40 mM Tris-HCl (pH 8), 30 mM dithiothreitol, 8 mM MgCl2
and 2 mM spermidine. The total amount of RNA synthesized was estimated by
measuring the efficiency of [32P]UTP incorporation after
trichloroacetic acid precipitation and scintillation counting. The
fluorescein-U7 snRNA product was precipitated twice with ethanol to remove
unincorporated nucleotides and resuspended in diethylpyrocarbonate-treated
H2O at
0.1 µg/µl. In vitro transcripts of a
Xenopus GFP-coilin plasmid were synthesized using SP6 polymerase in a
similar reaction without fluorescein-UTP. The 1.6 kb open reading frame of
Xenopus coilin was obtained by PCR from liver cDNA using primers
based on the Xenopus coilin sequence
(Tuma et al., 1993
). The PCR
product was cloned downstream of GFP in the cloning vector pCS2+GFP
(Huang et al., 1999
).
Transcripts were injected into the cytoplasm of Xenopus oocytes, and
GVs were isolated 18 hours later for a western blot. A band corresponding to
the predicted molecular weight of GFP-coilin was apparent when the western
blot was probed with an antibody against GFP.
Oocyte injections
Needles were pulled from capillary tubing as previously described
(Gall et al., 1999).
Approximately 1 ng fluorescein-U7 snRNA or 1 ng GFP-coilin transcript was
injected into the cytoplasm of oocytes before or after heat shock by means of
a Nanoject Microinjection Apparatus (Drummond Scientific, Broomall, PA). To
deplete oocytes of U7 snRNA, 35 ng of an antisense oligodeoxynucleotide
against bases 1-16 of U7 snRNA (Stefanovic
et al., 1995
) was injected into the oocyte cytoplasm in a volume
of 23 nl. Cells were incubated for a minimum of 12 hours at 18°C before
further experiments were conducted. U7 snRNA was not detectable by northern
blot analysis in nuclei isolated from oligotreated oocytes.
Heat shock
Oocytes were transferred from OR2 saline at 18°C to pre-warmed OR2 at
31.5-33.5°C for a heat shock lasting 2-4 hours. For recovery, the Petri
dish containing heat-shocked oocytes was returned to 18°C for 6-18 hours.
In some experiments, actinomycin D (5-10 µg/ml) or cycloheximide (50
µg/ml) was included in the OR2 medium to inhibit transcription or
translation. Oocytes were pre-incubated in the drug for 2-3 hours at 18°C,
heat shocked while still in the drug, and left in the drug during the recovery
period.
GV spreads
GVs were dissected manually from oocytes and their contents were spread and
attached to microscope slides by centrifugation as previously described
(Gall, 1998). Fixation was in
2% paraformaldehyde in PBS + 1 mM Mg2+ for 1-2 hours.
Immunofluorescence
Immunostaining of GV spreads was carried out as described previously
(Gall et al., 1999). The
following mAbs were used: ARNA-3 against the largest subunit of RNA Pol II
(Krämer et al., 1980
), H1
against Xenopus coilin (Tuma et
al., 1993
), H14 against the C-terminal domain of human RNA Pol II
(Bregman et al., 1995
), K121
against trimethylguanosine (Krainer,
1988
), Y12 against the Sm epitope
(Lerner et al., 1981
),
anti-SC35 against the SR protein SC35 (Fu
and Maniatis, 1990
), 17C12 against fibrillarin
(Pollard et al., 1997
), No 114
against Xenopus Nopp 140
(Schmidt-Zachmann et al.,
1984
), P7-1A4 against Xenopus nucleolin
(Messmer and Dreyer, 1993
) and
4D11 against human hnRNP L
(Piñol-Roma et al.,
1989
). Affinity-purified rabbit polyclonal sera against human
TFIIIA (from R. Roeder) and Xenopus coilin (serum C236) were also
used. Secondary antibodies were Alexa 488- or Alexa 594-labeled goat
anti-mouse IgG (or IgM) or goat anti-rabbit IgG (Molecular Probes, Eugene,
OR). GV spreads were observed with a Zeiss Axioplan fluorescence microscope
(Carl Zeiss, Thornwood, NY) fitted with a filter set for observing fluorescein
or Alexa 488 (excitation filter BP 485/20 and barrier filter BP 520-560) and
Alexa 594 (excitation filter BP 515-560 and barrier filter LP 590). Confocal
laser scanning microscopy was carried out with the Leica TCS NT system (Leica
Microsystems, Exton, PA).
Import assay
Approximately 1 ng fluorescein-U7 snRNA was injected into the cytoplasm of
oocytes. After the cells had been heat shocked at 31.5°C for 4 hours, GVs
were removed in mineral oil (Paine et al.,
1992) and held for a minimum of 4 hours at 18°C. GVs were
transferred in 5 µl of oil to a glass microscope slide and gently squashed
under a 22 mm2 glass coverslip. Typical CBs (2-10 µm) and
mini-CBs (<2 µm) appeared as intensely fluorescent spherical bodies in
the faintly fluorescent nucleoplasm.
[35S]methionine incorporation and protein gels
Oocytes were either held at 18°C (control) or heat shocked at
31.5°C for 1 hour. They were then injected with 23 nl
[35S]-methionine (10 mCi/ml, 1175 Ci/mmol; Amersham Life Science,
Arlington Heights, IL) and returned to 18°C (control) or 31.5°C for 3
hours (heat shock). GVs were removed immediately after heat shock and after
the oocytes had been held at 18°C for a minimum of 12 hours recovery. For
each group, 25 GVs were collected in 30 µl of 5:1 isolation medium (83 mM
KCl, 17 mM NaCl, 6.5 mM Na2HPO4, 3.5 mM KH2PO4, 1 mM
MgCl2, 1 mM dithiothreitol, pH 7.0) and adjusted to a final
concentration of 1x SDS buffer
(Laemmli, 1970). Samples were
split in half and loaded into separate lanes of a 10% discontinuous
SDS-polyacrylamide mini-gel (BioRad Laboratories, Hercules, CA). After
electrophoresis at 30 mA for 1.5 hours, the gel was split in half. One half
was stained with Coomassie Blue and the other half was fixed in 50%
methanol+10% acetic acid for 15 minutes, soaked in 7% methanol, 7% acetic
acid, 1% glycerol for 5 minutes, dried under vacuum at 80°C for 15
minutes, and exposed to X-Omat film (Eastman Kodak, Rochester, NY) at room
temperature for 4 days.
Western blots
Oocytes were either held at 18°C or heat shocked at 31.5°C for 4
hours. Some GVs were removed in 5:1 isolation medium immediately after heat
shock and others after overnight incubation at 18°C. For each sample, 40
GVs were placed in 50 µl of 5:1 isolation medium and adjusted to a final
concentration of 1x SDS loading buffer. Samples were split in half and
loaded into separate lanes of a 10% discontinuous SDS-polyacrylamide mini-gel.
After electrophoresis at 30 mA for 1.5 hours, the gel was split in half. One
half was stained with Coomassie Blue and the other half was transferred at
4°C onto a polyvinylidene fluoride membrane (Immobilon, Millipore,
Bedford, MA) for 1 hour at 100 V. Membranes were briefly immersed in 100%
methanol, rinsed in H2O and blocked overnight at 4°C with 5%
powdered milk in PBS. Primary antibodies were diluted to working
concentrations with 0.025% Tween in PBS. Three mAbs were used: ARNA-3 against
the largest subunit of Drosophila RNA polymerase II
(Krämer et al., 1980), H1
against Xenopus coilin (Tuma et
al., 1993
) and No114 against Xenopus Nopp140
(Schmidt-Zachmann et al.,
1984
). Membranes were incubated with primary antibody for 1 hour
at room temperature, washed for 3x5 minutes with 0.05% Tween-PBS and
incubated with alkaline phosphatase-linked secondary antibodies for 1 hour at
room temperature. Again the membranes were washed for 3x5 minutes with
0.05% Tween-PBS. Finally the blot was exposed to chemiluminescent compound
(Amersham Life Science, Arlington Heights, IL) for 5 minutes at room
temperature and imaged on a Storm 860 scanner (Molecular Dynamics, Sunnyvale,
CA).
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Results |
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Mini-CBs have the same macromolecular composition as larger CBs
Because heat shock can cause aggregation of unfolded proteins
(Morimoto et al., 1994), it
was possible that mini-CBs were simply precipitates of coilin alone or
aggregates of coilin with non-specific proteins. To address this issue, we
carried out immunofluorescence on spread preparations of GV contents after
recovery from heat shock. Specific staining of mini-CBs was observed with
antibodies against the following components: Xenopus coilin (mAb H1
and rabbit serum C236), Sm proteins (mAb Y12), the trimethylguanosine cap
found on several snRNAs (mAb K121), Nopp140 (mAb No114), TFIIIA (rabbit serum
-TFIIIA) and phosphorylated RNA Pol II (mAb H14)
(Fig. 3). Conversely, mini-CBs
failed to stain with antibodies against nucleolin (mAb P7-1A4), the SR protein
SC35 (mAb
-SC35) and hnRNP L (mAb 4D11). Thus, for all antibodies
tested, mini-CBs reacted the same as the larger CBs in the same GV. As
mini-CBs have the same or very similar macromolecular composition as CBs of
control oocytes, it is unlikely that they represent nonspecific aggregates of
unfolded proteins.
|
GFP-coilin and fluorescein-U7 snRNA target to mini CBs
A hallmark of CB activity is their rapid uptake of specific RNAs and
proteins that have been introduced experimentally into the nucleus
(Wu et al., 1994;
Wu et al., 1996
;
Samarsky et al., 1998
;
Narayanan et al., 1999a
;
Narayanan et al., 1999b
;
Sleeman and Lamond, 1999
). To
test if mini-CBs are functionally equivalent to CBs of control cells, we
injected in vitro-synthesized transcripts of GFP-coilin into the oocyte
cytoplasm immediately after heat shock. As in control oocytes, GFP-coilin was
detectable in CBs within 15-30 minutes of injection and became more evident
with time. Mini-CBs formed during overnight recovery from heat shock and they
were similarly labeled (Fig.
4). We also injected fluorescein-U7 snRNA into the oocyte
cytoplasm prior to or immediately after heat shock. GVs were isolated at
various times after injection and the contents examined by fluorescence
microscopy. Fluorescein-U7 was targeted to all CBs within a GV, regardless of
their size (data not shown). Label could be detected in CBs from GVs isolated
only a few minutes after injection, and the intensity of label in CBs
continued to rise over the first few hours after injection. Although
quantitative studies were not carried out, we saw no obvious differences in
the kinetics of uptake between heat-shocked and control oocytes. The
experiments with GFP-coilin and fluorescein-U7 demonstrate that mini-CBs not
only contain specific CB components but actively recruit them from the
nucleoplasm. In this respect they appear to be functionally equivalent to CBs
of typical size.
|
Mini-CB formation does not depend on new protein synthesis
We carried out several experiments to examine the protein composition of
GVs before and after heat shock, looking for changes that might be relevant to
the formation of mini-CBs. Oocytes were heat shocked for 1 hour at 31.5°C,
injected with [35S]methionine, and maintained for an additional 3
hours at the elevated temperature. Some heat shocked oocytes were returned to
18°C for a 12 hour recovery period before GV isolation. GVs were isolated
and newly synthesized proteins were examined by polyacrylamide gel
electrophoresis and autoradiography. The overall protein composition of GVs,
as assessed by Coomassie Blue staining, was similar in all oocytes
(Fig. 5A, lanes 1-3). However,
GVs from heat-shocked oocytes showed a marked deficiency of newly synthesized
proteins relative to control oocytes (Fig.
5B, lanes 4 and 5), consistent with previous studies on whole
oocytes (Bienz and Gurdon,
1982; Horrell et al.,
1987
). During the recovery period
(Fig. 5B, lane 6) the level of
newly synthesized GV proteins returned to normal. Minor differences were
detectable in the lower molecular weight range, where a few bands were more
intense than in the controls and a few less so (stars,
Fig. 5B, lane 6).
|
Although translation was greatly reduced during heat shock, it was not completely abolished, and new proteins were made during the recovery period when mini-CBs were formed. To test whether these newly synthesized proteins were required for mini-CB formation, we used cycloheximide at 50 µg/ml to inhibit all protein synthesis. In control experiments, no incorporation of [35S]methionine was detected in GV proteins from oocytes incubated for 3 hours at 18°C in the drug. Oocytes were then placed in cycloheximide at 18°C for 3 hours, heat shocked for 4 hours, and allowed to recover at 18°C in the presence of the drug. Mini-CBs formed in these cycloheximide-treated oocytes, demonstrating that no new proteins are required for mini-CB formation. Cycloheximide alone, without heat shock, did not induce mini-CBs, indicating that inhibition of protein synthesis by itself does not cause CB formation.
The cycloheximide experiments ruled out the need for newly synthesized proteins. However, formation of mini-CBs might still depend on import of specific stored proteins from the cytoplasm. To exclude this possibility, we isolated GVs from heat-shocked oocytes and allowed them to recover in the absence of cytoplasm. Oocytes were first injected with fluorescein-U7 snRNA, which served as a visible marker for mini-CB formation, and were then heat shocked for 4 hours. GVs from some oocytes were removed under oil, where they were allowed to recover in the absence of cytoplasm. Other oocytes were held intact in an aqueous medium and their GVs removed under oil after the recovery period. Both populations were viewed by direct immunofluorescence after gentle squashing in oil under a coverslip. Mini-CBs were evident in both sets of GVs (data not shown). This result suggests that protein import during heat shock recovery is not required for mini-CB formation. In other words, the GV contains all components necessary for formation of mini-CBs before the process of recovery begins.
Although import of proteins during recovery from heat shock is not needed
for mini-CB formation, modification of pre-existing GV proteins might be
required. To look for gross changes, we examined the patterns of three CB
proteins by western blotting before, during and after heat shock. We saw no
obvious changes in the mobility or amount of coilin, Nopp140, or the largest
subunit of RNA Pol II (Fig. 6).
All three are phosphoproteins (Young,
1991; Meier and Blobel,
1992
; Carmo-Fonseca et al.,
1993
), and differences in mobility might have been seen, if they
had undergone changes in their state of phosphorylation in response to heat
shock.
|
Formation of mini CBs is not dependent on transcription
When transcription is inhibited with actinomycin D or -amanitin, the
lampbrush chromosomes undergo dramatic changes in morphology
(Izawa et al., 1963
;
Schultz et al., 1981
) but CBs
appear essentially unchanged in size and number. Disruption of transcription
by the kinase inhibitor DRB leads to an increase in the number of
B-snurposomes associated with the CBs, but does not noticeably affect the size
or number of CBs themselves (Morgan et
al., 2000
). To test whether heat shock would induce mini-CBs in
transcriptionally inhibited cells, we incubated oocytes in 10 µg/ml
actinomycin D in OR2 saline during heat shock and recovery. As expected, drug
treatment caused contraction of the chromosomes and loss of their
characteristic loops. Because of the continued presence of the inhibitor, the
chromosomes maintained their contracted state during the recovery from heat
shock. Nevertheless, mini-CBs formed in such nuclei, demonstrating that their
formation can proceed in the complete absence of transcription. The
actinomycin results were not entirely unanticipated: we already knew that Pol
II transcription on the chromosomes, which is inhibited by the heat shock
itself, sometimes does not resume during the recovery period when mini-CBs
form. Although heat shock induces mini-CBs in transcriptionally inhibited
oocytes, we emphasize that mini-CB formation is separable from transcriptional
inhibition, since actinomycin treatment in the absence of heat shock does not
cause mini-CBs to form.
Formation of mini-CBs does not depend on U7 snRNA
As in somatic nuclei, U7 snRNA exists at a very low concentration in the
GV. Unlike the splicing snRNAs, which are found in other structures, U7 snRNA
has been demonstrated only in CBs and at a low concentration in the
nucleoplasm (Wu and Gall,
1993; Frey and Matera,
1995
; Wu et al.,
1996
). For this reason U7 snRNA might be limiting for biogenesis
of CBs. Consistent with this possibility, Tuma and Roth have shown that
injection of exogenous U7 snRNA into the Xenopus oocyte nucleus was
sufficient to induce formation of small CB-like structures in the nucleoplasm
(Tuma and Roth, 1999
). Their
experiment left unanswered the question whether U7 snRNA is, in fact,
necessary for formation of CBs. To examine this issue we used an antisense
oligodeoxynucleotide to deplete U7 snRNA from the oocyte. As previously
demonstrated, injection of an antisense oligo into the cytoplasm leads to
rapid destruction of U7 in the GV, presumably by RNase H-mediated digestion of
the RNA-DNA hybrid between endogenous U7 snRNA and the injected oligo
(Stefanovic et al., 1995
;
Bellini and Gall, 1998
). GVs
from U7-depleted oocytes have morphologically normal CBs, demonstrating that
U7 is not required for maintaining the structure of CBs. U7-depleted oocytes
were heat shocked and allowed to recover for a minimum of 18 hours. Mini-CBs
appeared in the GVs of these oocytes as in non-depleted controls
(Fig. 7). All CBs in
U7-depleted oocytes, whether heat-shocked or control, show much reduced
staining with mAb K121 against the trimethylguanosine cap found on several
snRNAs, including U7. We interpret this reduced staining as evidence that U7
is the major capped snRNA in oocyte CBs
(Bellini and Gall, 1998
;
Gall et al., 1999
). The
experiments with U7-depleted oocytes demonstrate that U7 snRNA is not
necessary for maintaining pre-existing CBs or for inducing new CBs after heat
shock.
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Discussion |
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Unlike nucleoli, CBs do not originate on specific DNA sequences
It has been known for many years that some CBs in the amphibian GV are
attached to lampbrush chromosomes at the histone gene loci
(Gall et al., 1981;
Callan et al., 1991
),
suggesting that histone genes might play a role in CB biogenesis comparable
with that of rDNA in the biogenesis of nucleoli. If CBs arise only at histone
gene sites, some additional mechanism must account for the large number of
unattached or free CBs in the GV. Two hypotheses can be considered. Free CBs
might contain amplified copies of the histone genes, similar to the amplified
rDNA in the extrachromosomal nucleoli of the GV
(Brown and Dawid, 1968
;
Gall, 1968
;
Miller and Beatty, 1969
), or
free CBs might form by successive growth and detachment from the histone gene
loci. The first hypothesis, amplification of histone genes, is unlikely, as
histone DNA is not detected in free CBs by in situ hybridization during the
course of experiments on newt lampbrush chromosomes
(Gall et al., 1983
). Moreover,
amplified rDNA is readily detectable in the extrachromosomal nucleoli of the
GV by DAPI staining, whereas CBs are not stained by DAPI. The growth and
detachment model would be reasonable for the slow increase in number of CBs
that takes place during the many weeks of normal oocyte growth. However, it is
more difficult to imagine that hundreds or thousands of mini-CBs arise and
detach from the three known histone loci during the course of several hours in
our heat shock experiments or in the earlier U7 experiments of Tuma and Roth
(Tuma and Roth, 1999
). In
mammalian cultured cells, CBs associate not only with histone genes
(Frey and Matera, 1995
) but
also with several snRNA genes (Frey and
Matera, 1995
; Smith et al.,
1995
; Gao et al.,
1997
; Frey et al.,
1999
; Jacobs et al.,
1999
). Such association with snRNA genes has not been seen in
Xenopus oocytes, but clearly the snRNA genes raise the same issues as
the histone genes. In the case of the oocyte, amplification of the genes for
U1, U2, U4, U5, U6 and U7 has been ruled out by quantitative filter
hybridization of total GV DNA (Phillips et
al., 1992
).
More direct evidence that specific genes are not required as nucleating
sites for CBs comes from nuclei assembled in Xenopus egg extract.
Nuclei with prominent CBs can be assembled not only from sperm chromatin but
also from prokaryotic DNA (Bell et al.,
1992). The latter case effectively rules out the need for any
specific eukaryotic gene as a nucleating site for CBs
(Bauer et al., 1994
;
Gall, 2000
). Even if CB
formation does not require specific DNA sequences, the preferential
association of CBs with histone and snRNA gene loci is indisputable. How and
why CBs reach these loci, if they do not arise there, are questions that must
be addressed by an adequate theory of CB biogenesis
(Abbott et al., 1999
;
Frey et al., 1999
).
No component that is unique to CBs or that might be an essential
nucleating factor has been identified
Among all CB components, the U7 snRNP comes closest to being CB specific.
Roughly 85% of the U7 snRNA in the GV is found in CBs, whereas the rest is in
the nucleoplasm (Wu et al.,
1996). Tuma and Roth have shown that mini-CBs form after injection
of excess U7 snRNA into the Xenopus GV, suggesting that U7 might be a
central nucleating factor for CBs (Tuma
and Roth, 1999
). However, by destroying endogenous U7 snRNA with
an antisense oligodeoxynucleotide before heat shock, we demonstrated that U7
is not essential for mini-CB formation. Furthermore, in these experiments the
removal of U7 has no discernible effect on the morphology of large CBs in
control (or heat-shocked) oocytes, showing that U7 is not required for
maintaining the structural integrity of CBs
(Bellini and Gall, 1998
).
Coilin is specifically enriched in CBs and was originally considered as a
possible nucleating factor. However, CBs are present in nuclei assembled in
coilin-depleted Xenopus egg extract
(Bauer and Gall, 1997). Even
more significant is the fact that coilin knockout mice are viable and exhibit
`residual' CBs in their cells (Tucker et
al., 2001
). In both the in vitro system and the knockout mouse,
CBs contain normal concentrations of fibrillarin and other nucleolar proteins
but fail to recruit Sm snRNPs.
Bell and Scheer (Bell and Scheer,
1997) have examined the role of proteins that are shared by CBs
and nucleoli in the biogenesis of CBs in Xenopus egg extract. They
showed that CBs (which they prefer to call prenucleolar bodies) are formed in
extracts depleted for any of four nucleolar proteins fibrillarin,
nucleolin, Nopp140 and B23/NO38. Their experiments underscore the difficulty
in defining any single component as essential for CB biogenesis or maintenance
(Gall, 2000
).
Four features of CB biogenesis in the oocyte system seem particularly relevant:
These features suggest a model in which mini-CBs originate over a period of hours by accretion of preformed components from the nucleoplasm, without incorporating newly synthesized RNAs or proteins. As the composition of the smallest and largest CBs is the same, growth in size must involve more or less simultaneous addition of all components. One can imagine that mini-CBs, once initiated, increase in size by addition of relatively large macromolecular complexes from the nucleoplasm.
In our heat-shock experiments and in the earlier U7 experiments
(Tuma and Roth, 1999), it was
common to see mini-CBs attached to B-snurposomes. In fact, associations
between B-snurposomes and CBs are a regular feature of Xenopus
oocytes. CBs of all sizes, especially those from younger oocytes, may have one
or more B-snurposomes attached to their surface or embedded in their matrix.
This intimate association between CBs and B-snurposomes suggests that
B-snurposomes may themselves be the elusive nucleating factor. If this is the
case, the search for a single nucleating molecule may be misplaced CBs
may form in association with B-snurposomes under a variety of conditions that
affect the nuclear environment. Several studies demonstrate the CBs are
dynamic structures whose components are in a constant state of flux
(Carmo-Fonseca et al., 1993
;
Wu et al., 1994
;
Wu et al., 1996
;
Samarsky et al., 1998
;
Narayanan et al., 1999b
;
Speckman et al., 1999
;
Morgan et al., 2000
). Ongoing
experiments from our laboratory show that two prominent CB components, U7
snRNA and coilin, equilibrate between the nucleoplasm and CBs with half times
of only a few minutes (K. E. H., unpublished). The numbers and sizes of CBs in
a given nucleus may thus fluctuate depending on subtle changes in the rates of
accumulation and dispersal of their constituent macromolecular complexes. We
suggest that heat shock alters the balance in the oocyte toward accumulation,
and for reasons that are not understood, B-snurposomes serve as preferential
sites for assembly of new CBs. In this model, CBs are an example of the more
general class of self-organizing structures
(Misteli, 2001
).
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Acknowledgments |
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