Department of Genetics, Center for Human Genetics and Program in Cell
Biology, Case Western Reserve University and University Hospitals of
Cleveland, Cleveland, OH 44106-4955, USA
Present address: Department of Biochemistry, University of Mississippi Medical
Center, Jackson, MS 39216-4505, USA
* Author for correspondence (e-mail: gxm26{at}po.cwru.edu)
Accepted 6 October 2002
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Summary |
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Key words: RNA processing, Nuclear bodies, Phosphorylation, SMN, snRNA, snoRNA
![]() |
Introduction |
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In addition to their ability to move throughout the nucleus, another
fascinating feature of nuclear bodies is their capacity to form within the
nuclear milieu without apparent support structures
(Platani et al., 2002).
Several nuclear domains, such as Sam68 bodies, PML bodies, Cajal bodies and
gems contain `signature' proteins (Sam68, PML, coilin and SMN, respectively)
that are used as markers for each domain. Characterization of these marker
proteins has revealed that they each share the ability to self-associate
(Chen et al., 1997
;
Hebert and Matera, 2000
;
Lorson et al., 1998
;
Perez et al., 1993
). Indeed,
self-association may be a common theme utilized by nuclear body marker
proteins to provide a scaffold upon which other components of the respective
domain can then coalesce (Hebert and
Matera, 2000
; Misteli,
2001
). Despite elucidation of marker proteins and characterization
of a myriad of other protein and RNA constituents in nuclear bodies,
understanding of mechanisms that regulate the size and number of these nuclear
domains is lacking. Such mechanisms must exist and are probably propagated
either through intrinsic properties of the marker proteins (e.g. structure or
expression level) or extrinsic factors that affect protein-protein
interaction. Interestingly, Lamond and co-workers recently showed that
transient upregulation of SmB protein in cells that normally do not display
Cajal bodies promotes the nucleation of these structures
(Sleeman et al., 2001
). This
finding supports the idea that Cajal bodies play a role in some aspect of
snRNP metabolism and suggests that snRNP expression levels can affect the
localization of coilin.
Post-translational modification of marker proteins may trigger
conformational changes that facilitate self- or other protein interactions
crucial for nuclear body assembly. PML bodies, for example, require the SUMO
modification of the PML protein in order to properly form
(Ishov et al., 1999;
Kamitani et al., 1998
;
Muller et al., 1998
).
Additionally, the phosphorylation state of Sam68 affects its ability to
selfoligomerize (Chen et al.,
1997
) and thus may affect its capacity to form nuclear bodies.
Likewise, coilin hyperphosphorylation, which occurs during mitosis
(Carmo-Fonseca et al., 1993
),
results in a reduction in self-association
(Hebert and Matera, 2000
).
Cajal bodies disassemble during mitosis and reform at early- to mid-G1 phase
(Andrade et al., 1993
;
Carmo-Fonseca et al., 1993
).
Thus, the phosphorylation status of coilin might influence Cajal body
formation. We have also shown that phosphorylation can control localization by
exposing or sequestering a putative nucleolar localization signal
(Hebert and Matera, 2000
).
This idea has precedence, as the function of the retinoblastoma protein is
thought to be controlled by a similar process of sequential phosphorylation
events (Harbour et al.,
1999
).
We have recently shown that symmetrical dimethylation of arginines within
the coilin RG box motif is vital for the in vivo incorporation of the SMN
complex into CBs (Hebert et al.,
2002). Without this modification, the SMN complex fails to
localize in CBs and forms gems. Therefore, coilin methylation, like
phosphorylation, can affect nuclear body formation and composition.
We are interested in understanding how nuclear body size and number are regulated. In this report, we set out to identify regions of coilin, if any, that are responsible for controlling these processes. Surprisingly, we found that coilin proteins from human, mouse and frog displayed different localization patterns when expressed in both HeLa cells and mouse embryonic fibroblasts (MEFs). Additional experiments using MEFs derived from coilin-knockout embryos showed that specific residues within the C-terminus of coilin are important for the proper regulation of CB numbers. Finally, although the coilin self-association domain resides in the N-terminus of the protein, point mutations and small truncations in the distal C-terminus affect its ability to form or target to CBs. We conclude that coilin contains an intrinsic nuclear body formation potential, shared among other nuclear body marker proteins, but is subject to increasing layers of regulation from frog, to mouse, to human.
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Materials and Methods |
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Cell culture, transfection and immunofluorescence
The mouse embryonic fibroblast cell line was established from
coilinknockout (-/-) mice as described previously
(Tucker et al., 2001). The
coilin-knockout MEF or HeLa cells were grown in DMEM (GIBCO BRL), supplemented
with 10% FBS (GIBCO BRL). Cells were grown to subconfluency on chambered
slides (Nunc) and transfected for 24 hours. MEF cells were transfected using
LippofectAMINE (GIBCO BRL); HeLa cells were transfected using SuperFect
(Qiagen) as directed. Cells were fixed in 4% paraformaldehyde, extracted in
0.5% Triton X-100 and processed for microscopy as previously described
(Frey and Matera, 1995
).
Immunofluorescence was carried out with myc (1:40; Santa Cruz Biotechnology),
R288 [1:100 (Andrade et al.,
1993
)] and R508 [1:200 (Chan et
al., 1994
)] antibodies.
![]() |
Results |
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|
The C-terminus of coilin regulates Cajal body number
In addition to finding that coilin is a self-associating protein and that
this domain affects C-terminal epitope recognition, we also have shown that
fusion of GFP to the coilin C-terminus results in formation of numerous foci
(Fig. 2)
(Hebert and Matera, 2000).
Normal coilin localization (diffuse nucleoplasmic plus bright foci) is
obtained upon fusion of GFP to its N-terminus
(Fig. 2). However, fusion of
the GFP moiety to the C-terminus results not only in numerous coilin foci but
also in a reduction in the nucleoplasmic pool of protein
(Fig. 2). Given the results in
Fig. 1, it seemed plausible
that fusion of GFP to the C-terminus of coilin produced an aberrantly folded
protein, culminating in a large increase in the number of coilin foci. In
other words, the coilin C-terminus regulates CB number. This hypothesis is
supported by a truncation mutant, coilin(1-481), that produces the same
phenotype observed for the coilin-GFP fusion
(Fig. 2). Furthermore, larger
C-terminal truncations of coilin result in varied patterns of localization,
from nucleolar accumulations to large nucleoplasmic inclusions, called
pseudo-CBs (Bohmann et al.,
1995
; Hebert and Matera,
2000
). These results suggest that, in vivo, the coilin C-terminus
masks the N-terminal region and that fusion of this domain to GFP or outright
truncation of C-terminal residues unmasks the self-association domain.
|
In order to test this idea further, we wanted to determine whether
formation of coilin foci could take place in the cytoplasm or if it were
restricted to the nucleus. If foci formation were limited to the nucleus, it
would imply that important interactions or modifications (e.g.
phosphorylation) are localized within this cellular compartment.
Alternatively, formation of foci in the cytoplasm would indicate either that
coilin modifiers exist in the cytoplasm or that foci formation is an intrinsic
property of the coilin N-terminus. The nuclear localization signals (NLSs)
reside within the loosely conserved internal region that links the more highly
conserved N- and C-terminal domains
(Bohmann et al., 1995). We
therefore created internal deletions spanning coilin residues 106-234. In the
GFP-coilin background, the
NLS protein shows exclusively cytoplasmic
localization, lacking foci, in HeLa cells
(Fig. 2, lower left panel).
Surprisingly, endogenous coilin localization was not altered in cells
expressing the GFP-coilin
NLS (data not shown). Thus, it was possible
that deletion of the NLS region could affect the self-association activity of
coilin. To test this, and to verify that coilin foci formation is limited to
the nucleus, we took advantage of the coilin construct that generates a
plethora of dots, coilin-GFP. HeLa cells transfected with coilin
NLS-GFP
typically displayed two to five foci in the nucleus, with additional bright
staining throughout the cytoplasm (Fig.
2, lower middle panel). Thus despite the lack of an NLS, a small
amount of coilin
NLS-GFP protein was imported into the nucleus, perhaps
by binding to endogenous coilin molecules. In order to test this idea, we
transfected coilin
NLS-GFP into coilin-knockout cells
(Tucker et al., 2001
) and
found that, indeed, the construct was completely restricted to the cytoplasm
in all cells (Fig. 2, lower
right panel). It should be noted that HeLa cells expressing
coilin
NLS-GFP displayed a markedly reduced number of foci compared with
the parental construct (Fig. 2,
compare upper middle and lower middle panels). Nonetheless, the ability to
form bodies in the nucleus and not in the cytoplasm verifies the idea that the
subcellular localization of coilin is important for their formation.
Coilins from different species demonstrate specific nuclear body
regulatory potentials
When transiently expressed in HeLa cells at low to medium levels, human
GFP-coilin properly localizes to the nucleoplasm and the CB. However, high
levels of expression result in reorganization of ectopic coilin, endogenous
coilin, and other constituents of the CB, such as SMN, to the nucleoplasm
(Hebert and Matera, 2000).
High levels of overexpression do not alter the overall nuclear architecture
since other nuclear structures, such as PML bodies, are unaffected
(Hebert and Matera, 2000
).
Furthermore, the GFP moiety is not responsible for this phenotype because
coilin constructs with a myc-tag display the same effect (K.B.S., M.D.H. and
A.G.M. unpublished). In contrast to the results using the human coilin
constructs in HeLa cells, high levels of GFP-mouse coilin overexpression do
not abolish foci formation. Instead, cells expressing high levels of mouse
GFP-coilin displayed the unregulated pattern of coilin foci observed for human
coilin(1-481) or coilin-GFP (Figs
2 and
3). Similarly, frog YFP-coilin
also displayed a multitude of foci when expressed in HeLa cells
(Fig. 3). Surprisingly,
transfection of these three coilins into a murine embryonic fibroblast (MEF)
cell line lacking endogenous coilin
(Tucker et al., 2001
) revealed
additional species-specific regulatory differences. For example, human coilin
failed to form CBs in most of these MEFs, even in those with low expression
levels (Fig. 3). By contrast,
mouse coilin readily formed foci in MEF cells when expressed at low levels.
MEF cells expressing high levels of mouse coilin showed only nucleoplasmic
staining. Thus expression of heterologous coilins in human cells resulted in
dysregulation of CB numbers. When mouse coilin, for example, was expressed in
murine cells, the number of coilin foci did not increase. However, the number
of coilin foci was completely unregulated when frog coilin was expressed at
high levels in both cell types (Fig.
3). This was most easily observed upon expression of frog coilin
in the knockout MEFs. In this background, large coilin aggregates were
observed, even in the cytoplasm. Furthermore, the pool of nucleoplasmic frog
coilin in MEF cells was virtually non-existent.
|
In summary, human coilin is able to effectively target to pre-existing CBs in HeLa cells when expressed at low levels but is unable to target, and indeed disrupts, CBs at high expression levels. In MEF cells lacking endogenous coilin, the human protein is ineffective at nucleating CBs (Fig. 3). Mouse coilin is able to effectively form CBs in mouse cells at low expression levels but, like human coilin in HeLa cells, becomes diffusely nucleoplasmic at higher levels of expression. In the HeLa background, however, mouse coilin displayed numerous foci. Similar findings were observed for frog coilin in both HeLa and MEF cells. At least for mouse and frog coilin proteins, the expression level was an important determinant for nuclear body formation. Human coilin, however, appears to have additional constraints regarding its ability to form nuclear bodies.
Chimeric coilin constructs delineate the Cajal body regulatory
region
We next set out to better define the region within coilin that governs CB
formation and number. Given that truncation or fusion of the C-terminus
resulted in formation of numerous coilin foci
(Fig. 2), we speculated that
this region might play an important regulatory role. These findings, coupled
with the observation that, compared with the human protein, mouse coilin has
an increased potential for nuclear foci formation in HeLa cells
(Fig. 3), led us to postulate
that the C-terminus of mouse coilin imparts differential regulatory
constraints. To test this idea, we generated chimeric constructs of human and
mouse coilin by swapping the C-terminal 96 amino acids
(Fig. 4). Following expression
in coilin-knockout MEFs, we assessed the capacity of the mutants to form
nuclear foci (Fig. 4). As
previously observed (Tucker et al.,
2001), cells expressing moderate levels of mouse coilin typically
display foci (87%), whereas those expressing human coilin rarely display foci
(7%; Fig. 4). Furthermore, the
number of foci per cell was greatly reduced (0.09 for human versus 2.84 for
mouse). Strikingly, replacement of the mouse C-terminus with the corresponding
human portion of coilin reduced both the frequency of cells displaying foci
and the numbers of foci per cell (Fig.
4, compare 87% for mouse with 1% for the mouse/human chimera).
Conversely, replacement of the human C-terminus with the mouse region
increased the number of cells that displayed coilin foci by 10-fold
(Fig. 4, 7% for human versus
70% for human/mouse). We conclude that the C-terminal 96 amino acids of human
coilin downregulates the capacity for nuclear body formation, whereas the
corresponding region in mouse coilin increases this capability.
|
Critical serine residues in the C-terminus of coilin control nuclear
body formation
Despite the apparent phenotypic differences in nuclear-body-forming
potential, an alignment of the various coilin C-termini demonstrates a high
level of identity in this region (Fig.
5A). Interestingly, frog coilin shows several residues that are
substantially changed compared with the corresponding amino acids in human and
mouse (e.g. frog contains DEE at 499-501 compared with NGA in human). These
differences, although slight, may account for the observed localization
phenotypes. Curiously, the biggest differences between human and mouse are in
the very C-terminal, serine-rich, portion. Given that human coilin
phosphorylation occurs exclusively on serine residues
(Carmo-Fonseca et al., 1993)
and that several of the serines in its C-terminus are predicted to be
phosphorylated (NetPhos, Technical University of Denmark;
http://genome.cbs.dtu.dk/services/NetPhos/),
we decided to systematically generate mutations in this region and assess
their nuclear-body-forming potentials. The mutations were each produced in
GFP-coilin backgrounds, and the constructs were subsequently transfected into
HeLa cells or coilin knockout MEFs. A summary of the mutations and their
proclivities to form nuclear bodies in MEF cells is shown in
Fig. 5B. The mutation of
arginine 561 in human coilin to a stop codon resulted in a variable phenotype.
In HeLa cells, human coilin(1-560) was mostly nucleoplasmic with nuclear
aggregates, although a small percentage of cells (<10%) appeared normal
(data not shown). In coilin-knockout MEFs, human coilin(1-560) mirrors that
observed in HeLa, with the exception that no cells displayed coilin foci.
|
The corresponding truncation mutation in mouse coilin (arginine 557 to
stop) showed a dramatic change in localization when expressed in HeLa cells
compared with full-length mouse coilin
(Fig. 5C). Most HeLa cells
transfected with mouse coilin(1-556) displayed primarily nucleoplasmic
staining, even at lower expression levels. Additionally, high expression
levels of mouse coilin(1-556) resulted in nucleoplasmic staining and
corresponding disassembly of endogenous CBs
(Fig. 5C, arrow). The
overexpression of mouse coilin(1-556) in HeLa cells is reminiscent of the
pattern seen for the high expression of human coilin in HeLa cells, which
likewise results in nucleoplasmic staining and subsequent disruption of CBs
(Hebert and Matera, 2000).
Curiously, overexpression of wild-type mouse coilin in HeLa cells also reduced
the amount of endogenous coilin in CBs, despite the observed foci present for
mouse coilin (Fig. 5C, top
panels, arrow). Thus we conclude that removal of the C-terminal 13 amino acids
of mouse coilin effectively makes it behave like the human protein. In support
of this idea, MEF cells transfected with mouse coilin(1-556) no longer
displayed CBs, but showed nucleoplasmic staining
(Fig. 5B). Therefore, these 13
amino acids affect not only the ability of coilin to target to preformed CBs,
but the capacity to form foci in the absence of endogenous coilin.
Larger C-terminal truncations of both mouse (1-477) and human (1-481) coilin resulted in formation of multiple foci in MEF cells (Fig. 5B). However, smaller C-terminal deletions (human: 1-516 and 1-560; mouse: 1-512, 1-535 and 1-556) lost the ability to form coilin foci when transfected into MEF cells. Curiously, a construct lacking the C-terminal five residues, mouse(1-564), was capable of generating nuclear bodies whereas mouse(1-556) did not, thus narrowing a region that may structurally inhibit nuclear body formation to residues 478-563 in mouse coilin (Fig. 5B).
Given the serine-rich composition of the C-terminal 13 residues in mouse coilin (5 out of 13), we speculated that the phosphorylation states of these residues might play a role in the ability to form foci. In particular, we were interested in the last two serines of mouse coilin (S567 and S568), which are conserved in human coilin (Fig. 5A). Mutation of these serines, along with the C-terminal threonine in mouse coilin SST to AGA, resulted in a remarkable mislocalization both in MEF and HeLa cells (Fig. 5B,C). Indeed, mouse coilin (AGA) and (1-556) showed virtually identical overexpression localization patterns in HeLa (Fig. 5C). These findings led us to speculate that S567 and S568 in the mouse protein need to be phosphorylated to properly form or target CBs. When the corresponding serines were mutated to aspartates (SST to DDT), mimicking a constitutively phosphorylated state, the ability to form foci in the absence of endogenous coilin and to produce numerous coilin foci in HeLa cells was retained (Fig. 5B,C). However, mutation of mouse residues S567 or S568 (to either D or A) individually had little effect on the localization pattern (Fig. 5B, data not shown). Point mutagenesis of other residues suspected to affect the folding of the C-terminus also failed to produce notable changes in coilin sublocalization compared with the parental construct (Fig. 5B). Thus mutation of both serines 567 and 568 together had an effect whereas no effect was observed when mutated singly.
We speculated that the mutants that cannot form nuclear bodies (mouse 1-556 and SST to AGA) were somehow deficient in coilin self-association. Coimmunoprecipitation assays using these constructs, however, failed to show any differences in the abilities of the mutated coilins to interact with coilin or other components of the CB, such as SMN (data not shown). Therefore, it is possible that the mutations in the mouse coilin background specifically affect the ability to target or form CBs. Our inability to identify human coilin constructs, other than coilin-GFP and GFP-coilin (1-481), that form foci in MEF cells (Fig. 5B) suggests the existence of additional regulatory controls for human coilin.
Other nuclear body proteins display unregulated foci formation
To generalize the idea that, apart from coilin, proteins considered markers
for different nuclear bodies can induce foci formation upon overexpression, we
surveyed the literature. Notably, various mutants of Sam68, the marker protein
for Sam68 nuclear bodies, appear to have lost the ability to regulate nuclear
body formation (Chen et al.,
1999). Cells transfected with wild-type Sam68 typically display
two to five nuclear bodies, along with a diffuse nucleoplasmic staining.
However, mutants such as Sam68
L1 produce numerous foci (up to 30) along
with a concurrent loss of nucleoplasmic staining
(Chen et al., 1999
). Several
human coilin mutants also display a similar phenotype
(Fig. 2). PML, which is
considered to be the marker protein for PML bodies, also generates more PML
bodies when overexpressed in HeLa cells and larger (but fewer) PML bodies when
overexpressed in knockout MEFs (Ishov et
al., 1999
; Mu et al.,
1997
).
On the other hand, overexpression of wild-type SMN, which is vital for gem
formation, reportedly does not change the number of gems in transfected cells
(Pellizzoni et al., 1998). We
have found, however, that high expression of myc-tagged SMN, both in HeLa
cells and coilin knockout MEFs, results in formation of numerous foci
(Fig. 6). The additional foci
are present both in the nucleus and the cytoplasm. The phenotype is similar to
that observed upon ectopic expression of the SMN
N27 mutant protein
(Pellizzoni et al., 1998
). The
main difference is that the enlarged nuclear foci seen with SMN
N27
(Pellizzoni et al., 1998
) were
not observed in cells overexpressing the wild-type protein. The large
cytoplasmic blobs (Fig. 6A,
bottom) were observed only at the higher expression levels. In HeLa cells, SMN
overexpression produces nuclear foci which sometimes contain coilin and can be
considered CBs (Fig. 6A, arrow)
and other foci that lack coilin and thus can be considered gems
(Fig. 6A, arrowheads). In
summary, we conclude that the overall level of SMN expression
(Fig. 6) and the extent of
coilin arginine dimethylation (Hebert et
al., 2002
) are major determinants in gem formation.
|
![]() |
Discussion |
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Interestingly, frog coilin does not have a serine-rich C-terminus unlike
the mouse and human proteins (Fig.
5A). Furthermore, unlike the human protein
(Carmo-Fonseca et al., 1993),
Xenopus coilin is not highly phosphorylated (M. Bellini, personal
communication). It is possible, therefore, that frog coilin is `locked' in a
conformation that renders it competent for nuclear body formation and thus is
insensitive to phosphorylation. Consequently, frog coilin would be expected to
readily form nuclear foci (Fig.
3). Human and mouse coilin appear to have greater regulatory
constraints with regard to their ability to form nuclear bodies. This idea is
supported by findings using the human coilin
NLS mutants
(Fig. 2), which are incapable
of forming cytoplasmic foci. These results suggest that factors responsible
for phosphorylating coilin are located in the nucleus.
Given that alterations in coilin phosphorylation levels can affect both
self-association and nucleolar localization
(Hebert and Matera, 2000;
Lyon et al., 1997
;
Sleeman et al., 1998
), it is
likely that this modification plays an important role in CB targeting as well.
We showed that, unlike human coilin, a chimeric human/mouse construct can
readily form foci in murine cells lacking endogenous coilin. A reciprocal
construct containing mouse coilin with the human tail displayed a phenotype
similar to that of the human protein expressed in the mouse cells
(Fig. 4). Considering that
mutation of the coilin N-terminal self-association domain alters C-terminal
epitope recognition by the anti-coilin Ab R508, we speculated that
self-association would be regulated via the C-terminal tail.
Coimmunoprecipitation experiments, however, failed to show any change in
coilin self-association among the various mutants. Furthermore, the
observation that mouse coilin(1-556) and coilin(AGA) disrupt the endogenous
coilin in HeLa cells supports the notion that self-interaction is not affected
(Fig. 5C). Although it is
conceivable that self-interaction is required for nuclear body formation,
additional cellular factors [e.g. high levels of Sm proteins
(Sleeman et al., 2001
)] and/or
modifications of coilin may be necessary. Since mutation of the mouse coilin
C-terminal residues to mimic constitutively phosphorylated serines (SST to
DDT) results in wild-type levels of foci formation, we conclude that these
sites are normally phosphorylated. Since kinase recognition sites are often
dependent on previous phosphorylation events, the phosphorylation of these
residues could facilitate additional serine phosphorylation elsewhere within
the coilin protein. The fact that mutation of individual S567 or S568 serines
to mimic an unphosphorylated state had no effect on mouse coilin localization
emphasizes the complexity of coilin phosphorylation. Furthermore, the
inability of human coilin to form foci at high expression levels in HeLa cells
(or to form any foci at all in MEF cells) may be due to titration of the
putative coilin kinases. We have shown that coilin is a substrate for the
kinases CDK2-cyclinE (Liu et al.,
2000
) and casein kinase II
(Hebert and Matera, 2000
);
however, phosphatases that modulate the phosphorylation state of coilin have
not been described. Current studies are focused on identification of residues
within coilin that are post-translationally modified and thus alter its
conformation, localization and function.
As an alternative model, it is possible that mutation of the coilin
C-terminus may not affect phosphorylation, but rather, alter a binding site
between coilin and other proteins. The C-terminus may be needed to interact
with another protein essential for CB formation, and overexpression of coilin
may titrate out this integral component thus dispersing CBs. Another such
example exists whereby overexpression of the immediate-early protein IE1 leads
to a disruption in PML body formation by interacting with the major structural
factor PML (Ahn et al., 1998).
Additionally, coilin itself may encode the structural integrity of the CB, and
the mouse coilin C-terminal mutants deficient in CB formation may simply yield
an alteration in coilin structure rather than phosphorylation, leading to CB
dispersal. Future experiments will need to address whether the implicated
residues are indeed phosphorylated in vivo and if differences in
phosphorylation levels can account for CB number differences between cell
lines.
In conclusion, various nuclear body marker proteins share a common capacity
for self-association and nuclear body formation. However, simply making more
protein is not always sufficient to generate additional nuclear foci,
especially in the case of CBs. Interestingly CBs can be induced to form in
cell lines that do not typically possess them by overexpression of Sm proteins
(Sleeman et al., 2001). Given
that post-translational modification of coilin plays a vital role in Cajal
body composition (Hebert et al.,
2002
) and formation (this work), we are especially interested in
assessing the modification status of coilin in the nucleoplasmic versus CB
compartments. Recently developed methods for CB purification
(Lam et al., 2002
) should
greatly aid such an endeavor.
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Acknowledgments |
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