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The RNA 3'cleavage factors CstF-64kD and CPSF 100 kDa are concentrated in nuclear
domains closely associated with coiled bodies and newly synthesized RNA
Schul, W.; Groenhout, B.; Koberna, K.; Takagaki, Y.; Jenny, A.; Manders, E.M.M.; Raska, I.;
van Driel, R.; de Jong, L.
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EMBO Journal
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Citation for published version (APA):
Schul, W., Groenhout, B., Koberna, K., Takagaki, Y., Jenny, A., Manders, E. M. M., ... de Jong, L. (1996). The
RNA 3'cleavage factors CstF-64kD and CPSF 100 kDa are concentrated in nuclear domains closely associated
with coiled bodies and newly synthesized RNA. EMBO Journal, 15, 2883-2892.
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Download date: 15 Jun 2017
The EMBO Journal vol.15 no.11 pp.2883-2892, 1996
The RNA 3' cleavage factors CstF 64 kDa and CPSF
100 kDa are concentrated in nuclear domains closely
associated with coiled bodies and newly
synthesized RNA
Wouter Schul, Bastiaan Groenhout1,
Karel Kobernal, Yoshio Takagaki2,
Andreas Jenny3, Erik M.M.Manders4,
Ivan Raska1, Roel van Driel and
Luitzen de Jong5
E.C.Slater Institute, Biocentrum, University of Amsterdam,
Plantage Muidergracht 12, 1018 TV, Amsterdam, The Netherlands,
'Department of Cell Biology, Institute of Experimental Medicine,
Academy of Sciences of the Czech Republic, Albertov 4,
12800 Prague, Czech Republic, 2Department of Biological Sciences,
Colombia University, New York, NY 10027, USA, 3Department of
Cell Biology, Biozentrum, University of Basel, CH-4056 Basel,
Switzerland and 4Department of Physics IV, Royal Institute of
Technology, S-100 44, Stockholm, Sweden
5Corresponding author
The cleavage stimulation factor (CstF), and the cleavage and polyadenylation specificity factor (CPSF) are
necessary for 3-terminal processing of polyadenylated
mRNAs. To study the distribution of 3' cleavage factors
in the nuclei of human T24 cells, monoclonal antibodies
against the CstF 64 kDa subunit and against the CPSF
100 kDa subunit were used for immunofluorescent
labelling. CstF 64 kDa and CPSF 100 kDa were
distributed in a fibrogranular pattern in the nucleoplasm and, in addition, were concentrated in 1-4 bright
foci. Double immunofluorescence labelling experiments
revealed that the foci either overlapped with, or resided
next to, a coiled body. Inhibition of transcription with
a-amanitin or 5,6-dichloro-j-D-ribofuranosyl-benzimidazole (DRB) resulted in the complete co-localization of coiled bodies and foci containing 3' cleavage
factors. Electron microscopy on immunogold doublelabelled cells revealed that the foci represent compact
spherical fibrous structures, we named 'cleavage
bodies', intimately associated with coiled bodies. We
found that .20% of the cleavage bodies contained a
high concentration of newly synthesized RNA, whereas
coiled bodies were devoid of nascent RNA. Our results
suggest that the cleavage bodies that contain RNA are
those that are adjacent to a coiled body. These findings
reveal a dynamic and transcription-dependent interaction between different subnuclear domains, and suggest a relationship between coiled bodies and specific
transcripts.
Keywords: cleavage factors/coiled bodies/nuclear bodies/
PML
Introduction
The nucleus is compartmentalized. By light microscopy
and electron microscopy, many different domains inside
the nucleus can be visualized, which have distinct morpho© Oxford University Press
logies, compositions and probably distinct functions (for
reviews, see Spector, 1993; van Driel et al., 1995). The
most conspicuous example of a nuclear compartment is
the nucleolus (for reviews, see Hemandez-Verdun, 1991;
Raska and Dundr, 1993; Scheer et al., 1993). In this
structure, synthesis and processing of rRNA and assembly
of pre-ribosomal particles takes place. The function of
many other nuclear domains is less clear. Coiled bodies
(Raska et al., 1991), nuclear bodies containing the protein
PML (PML bodies) (Szostecki et al., 1990; Ascoli and
Maul, 1991; Stuurman et al., 1992; Dyck et al., 1994;
Koken et al., 1994; Weis et al., 1994) and clusters of
interchromatin granules (Puvion and Moyne, 1981; Raska
et al., 1990; Spector et al., 1991) have been identified in
nuclei of many cell types. Coiled bodies and clusters of
interchromatin granules contain high concentrations of
splicing factors, suggesting that these domains play some
role in the processing of pre-mRNA.
In addition to splicing, nearly all mammalian mRNAs
are co-transcriptionally or post-transcriptionally modified
by 3' cleavage and polyadenylation (for reviews, see
Wahle, 1992; Wahle and Keller, 1992; Sachs and Wahle,
1993). A number of protein factors are involved in these
reactions. For the cleavage reaction, the cleavage and
polyadenylation specificity factor (CPSF), the cleavage
stimulation factor (CstF), poly(A) polymerase and cleavage factors I and II are required (Christofori and Keller,
1988; Gilmartin and Nevins, 1989; Takagaki et al., 1989).
CPSF and poly(A) polymerase are also essential for
polyadenylation. Little is known about the spatial distribution of the factors involved in these 3' modifications
of mRNA.
CPSF consists of four subunits of 160, 100, 73 and
30 kDa. CstF consists of three subunits of 77, 64 and
50 kDa. The CstF 64 kDa subunit contains a ribonucleoprotein (RNP)-type RNA binding domain (Takagaki et al.,
1992), and can be UV cross-linked to RNA (Takagaki
et al., 1990; Gilmartin and Nevins, 1991). Also the CPSF
160 and 30 kDa subunits can be UV cross-linked to RNA
(Gilmartin and Nevins, 1989; Keller et al., 1991; Jenny
et al., 1994). Monoclonal antibodies (mAbs) against the
50 and 64 kDa subunits of CstF (Takagaki et al., 1990),
and against the 100 and 160 kDa subunits of CPSF (Jenny
et al., 1994) have been used to immunofluorescently
label CstF and CPSF in human cells. These labelling
experiments showed that both factors are localized in the
cell nucleus.
To understand how the nucleus is functionally organized,
we are studying the subnuclear organization and spatial
relationship of the synthesis, 3'-terminal cleavage, polyadenylation and splicing of RNA. Here we investigate
how factors involved in 3'-processing are related spatially
to sites of RNA synthesis, and previously identified nuclear
compartments enriched in other RNA processing factors.
2883
W.Schul et al.
Fig. 1. Immunofluorescent labelling of human T24 bladder carcinoma
cells with mAbs against the CstF 64 kDa subunit (A) and against the
CPSF 100 kDa subunit (B). Note the overall diffuse labelling in the
nucleoplasm, plus a few bright foci.
To this end, immunofluorescence and immunogold
labelling experiments were performed using an anti-CstF
64 kDa mAb and an anti-CPSF 100 kDa mAb, combined
with confocal laser scanning microscopy, three-dimensional image analysis and electron microscopy. We found
that CstF 64 kDa and CPSF 100 kDa are present throughout
the nucleoplasm, except in the nucleolus. In addition, both
factors were concentrated in a few nuclear foci in close
association with two other types of domains, known as
coiled bodies and PML-containing nuclear bodies.
Results
Immunofluorescent labelling of CstF 64 kDa and
CPSF 100 kDa
To study the distribution of the RNA 3'-processing factors,
we used a mAb which specifically recognizes the 64 kDa
subunit of human CstF (Takagaki et al., 1990, 1992) and
a mAb which specifically recognizes the 100 kDa subunit
of human CPSF (Jenny et al., 1994), for immunofluorescence labelling of T24 human bladder carcinoma cells.
As described for HeLa cells (Takagaki et al., 1990; Jenny
et al., 1994; Krause et al., 1994), we found labelling of
both factors throughout the nucleus, except for the nucleoli.
The majority of both labels was found in a homogeneous
fibrogranular pattern in the nucleoplasm. In addition, we
found that CstF 64 kDa (Figure IA) and CPSF 100 kDa
(Figure iB) were concentrated in a few brightly labelled
foci. These foci appeared as small subnuclear domains with
2884
Fig. 2. Optical sections of T24 cells stained for DNA with propidium
iodine (A, C and E) and labelled with anti-CstF 64 kDa (B, D and F).
Cells in metaphase (A and B) or anaphase (C and D) show an overall
CstF 64 kDa labelling in the cell, excluding the chromosomes. No
bright foci, as found in interphase cells, were observed. CstF 64 kDa
begins to accumulate in the nucleus during late telophase when the
nuclear membrane has reformed and the chromosomes start to
decondense (E and F). Bar represents 2 gm.
a distinctly higher fluorescent signal than the surrounding
nucleoplasm. They appeared to be roughly spherical with
a diameter of 0.3-1 gm. Typically, 1-4 CstF foci or CPSF
foci were present per nucleus.
The foci were not observed during mitosis; both cleavage factors were found homogeneously distributed in the
cell, excluding the chromosomes (Figure 2A-D). In late
telophase, when the nuclear membrane has formed and
the chromosomes start to decondense, CstF 64 kDa accumulated again in the nucleus (Figure 2E and F). Apparently,
the foci disintegrate upon entering mitosis and are reassembled after cell division.
Foci of CstF and CPSF are associated with coiled
bodies
Coiled bodies are spherical subnuclear structures, found
in nuclei of plant and animal cells and owe their name to
their distinct appearance in the electron microscope (Raska
et al., 1991). Coiled bodies are highly enriched in the
protein p80-coilin (Andrade et al., 1991) and also contain
high concentrations of splicing factors (Carmo-Fonseca
et al., 1991; Raska et al., 1991; Matera and Ward, 1993),
fibrillarin (Raska et al., 1991), and Nopp140 and NAP57
CstF 64 kDa and CPSF 100 kDa
(Meier and Blobel, 1994). They have proved to be dynamic
structures in the nucleus (Carmo-Fonseca et al., 1992;
Antoniou et al., 1993; Malatesta et al., 1994; for a
review, see Lamond and Carmo-Fonseca, 1993). In the
fluorescence microscope, coiled bodies are visible as bright
nuclear dots, their number, in most cell types, ranging
from one to five per nucleus.
The size, shape and subnuclear distribution of the CstF
and CPSF foci resembled that of coiled bodies. To
investigate whether there is any relationship between these
domains, T24 cells were double labelled with anti-CstF
64 kDa or anti-CPSF 100 kDa, in combination with a
polyclonal antibody against p80-coilin. We used confocal
laser scanning microscopy to compare the two nuclear
domains. Strikingly, the double labelling did not show a
simple co-localization but revealed a complex relationship
between coiled bodies and foci. The foci labelled by antiCstF 64 kDa and by anti-CPSF 100 kDa were found to
overlap either partially or completely with coiled bodies
or were situated adjacent to coiled bodies (Figure 3). The
foci, labelled by the two antibodies against the 3' cleavage
factors, showed an identical distribution relative to coiled
bodies, which made it very likely that both antibodies
labelled the same nuclear structure.
We found coiled bodies and foci labelled by anti-CstF
64 kDa or anti-CPSF 100 kDa paired with a varying
degree of overlap. As shown in Table I, 42% of the foci
labelled by anti-CstF 64 kDa precisely co-localized with
a coiled body. In addition, 51% of the CstF foci were
associated with a coiled body but did not co-localize with
it; 18% could be clearly distinguished as separate round
structures adjacent to coiled bodies (Figure 3A and B).
We found a few single coiled bodies but rarely observed
foci enriched in CstF 64 kDa or CPSF 100 kDa unassociated with a coiled body.
The association and degree of overlap between foci of
3' cleavage factors and coiled bodies was analysed further
quantitatively by measuring red and green signal intensities
along an axis through the foci (Figure 4). The distances
between the peaks in the two signals, representing a coiled
body and a CstF focus, range from zero (Figure 4B) to
-0.5 ,um (Figure 4A). Our findings show that foci of
cleavage factors and coiled bodies are distinct nuclear
domains with a strong spatial and, therefore, possibly
functional relationship.
Foci of 3' cleavage factors appear as distinct
structures in the electron microscope
We used electron microscopy to investigate the structure
of the foci enriched in cleavage factors and their spatial
relationship to coiled bodies in closer detail. Unfortunately,
a post-embedment labelling approach did not provide
sufficient signal for CstF (see Materials and methods).
Pre-embedment labelling, performed according to several
fixation, permeabilization and labelling protocols, only
gave specific CstF 64 kDa labelling under particular
conditions (described in Materials and methods). Coiled
bodies were labelled specifically with p80-coilin, and
often CstF 64 kDa labelling was also found in these
structures. Distinct structures, containing a significantly
stronger CstF labelling than the surrounding material,
could be found in close proximity to a coiled body (Figure
5A and B). These spherical structures had a compact
and fibrous appearance, which differed slightly between
experiments, but was always distinct from a coiled body.
All the available evidence points to the fact that these
structures represent the light microscopically defined foci
of cleavage factors, which we will refer to from now on
as 'cleavage bodies'.
In some cases, the coiled body appeared to contain a
stronger CstF labelling than the adjacent cleavage body.
Interestingly, the coiled body and cleavage body were not
always found in direct contact but also somewhat separated
from each other (Figure 5B). These electron microscopical
data confirm that cleavage bodies are distinct nuclear
structures that have an intimate spatial relationship with
coiled bodies.
Cleavage bodies in various cell types
Foci of RNA 3' cleavage factors were also found in other
cell types, although sometimes they were less frequent or
less distinct than in T24 cells (Figure 6A and B). Human
fibroblasts rarely contained coiled bodies; however, the
few coiled bodies that were found overlapped with a
brightly labelled CstF 64 kDa focus (Figure 6E and F).
HeLa cells often contained a domain associated with
coiled bodies that showed a slightly higher concentration
of CstF 64 kDa (data not shown). CaCo cells were found
to have up to 15 coiled bodies per nucleus. Most coiled
bodies were not associated with a focus of cleavage
factors. However, in 1-5% of the nuclei, bright foci could
be found in or adjacent to coiled bodies (Figure 6C and D).
We have found that foci of RNA 3' cleavage factors
are most prominent in T24 cells and that equivalent
structures can be found in other human cell types.
Some cleavage bodies contain newly synthesized
RNA
To study the relationship between the distribution of the
3' cleavage factors and sites of RNA synthesis, intact
cells were microinjected with 5-bromo-UTP (BrUTP). By
this method, newly synthesized RNA becomes labelled
with 5-bromouridine, which can be detected immunofluorescently, thus visualizing sites of RNA synthesis in the
nucleus (Wansink et al., 1993, 1994). Microinjected cells
double labelled with anti-CstF 64 kDa showed that the
numerous bright spots of newly synthesized RNA did
contain CstF 64 kDa, but that the cleavage factor was not
concentrated specifically at these sites. Cleavage bodies
immunofluorescently labelled with anti-CstF 64 kDa did
sometimes contain a high concentration of newly synthesized RNA (Figure 7); -20% of the cleavage bodies colocalized with a bright spot of labelled RNA. The
remaining cleavage bodies did not appear to contain newly
synthesized RNA. Microinjected cells double labelled with
anti-p80-coilin showed newly synthesized RNA to be
absent from coiled bodies (data not shown). This implies
that only the cleavage bodies that do not overlap with a
coiled body contain newly synthesized RNA.
Cleavage bodies and coiled bodies completely
co-localize after inhibition of RNA polymerase 11
transcription
5,6-Dichloro-,-D-ribofuranosyl-benzimidazole (DRB) and
a-amanitin can selectively inhibit transcription of premRNA by RNA polymerase II (RPII). Upon treatment
2885
W.Schul et aL
Fig. 3. Coiled bodies and foci of 3' cleavage factors are tightly associated dynamic structures. Cells were double labelled with anti-CstF 64 kDa
(A, C and D) (green) or anti-CPSF 100 kDa (B) (green) and anti-p80-coilin (red), and analysed by confocal laser scanning microscopy. Single
optical sections are shown. The cleavage factors are found in a homogeneous fibrogranular pattern throughout the nucleoplasm and concentrated in a
few brightly labelled foci (arrowheads). Coiled bodies and foci of cleavage factors are sometimes found next to each other as separate nuclear
domains (A and B). Treatment of cells with ax-amanitin for 2 h, to inhibit RNA polymerase II transcription, resulted in a complete co-localization
(indicated by yellow) of coiled bodies and foci (C). After 6 h tx-amanitin treatment, CstF 64 kDa was found in large rounded-up speckles often
associated with remnants of coiled bodies (D). Foci of 3' cleavage factors, coiled bodies and some of the PML bodies are found clustered in
ultrastructural trios in the nucleus (E) (see also magnified area). An optical section is shown of a triple labelled cell, labelled with anti-CstF 64 kDa
(green), anti-p80-coilin (red) and an auto-immune serum recognizing PML bodies (blue). Bars represent 2 gm.
with these drugs, splicing factors like snRNPs and U2AF
no longer concentrate in coiled bodies and redistribute in
the nucleus to structures called speckles (Spector et al.,
1991; Carmo-Fonseca et al., 1992). To investigate whether
2886
the association between cleavage bodies and coiled bodies
is similarly transcription dependent, we cultured T24 cells
in the presence of either a-amanitin or DRB for 2 h. The
CstF 64 kDa (Figure 3C) and CPSF 100 kDa (not shown)
CstF 64 kDa and CPSF 100 kDa
Table I. Effect of inhibition of RNA polymerase II on the spatial relationship between coiled bodies and foci of 3' cleavage factors
Treatment
No. of nuclei
No. of CstF foci
Co-localizing
Partially overlapping
Adjacent
Single
-
28
11
6
57
18
11
24 (42%)
17
10
19 (33%)
0
0
10 (18%)
0
0
4 (7%)
1
1
ct-Amanitin
DRB
The number of CstF 64 kDa labelled foci that are co-localizing, partially overlapping and adjacent to coiled bodies is shown. The cells were treated
with a-amanitin or DRB for 3 h to inhibit RNA polymerase II transcription. Percentages in parentheses indicate the number of CstF-foci relative to
the total amount of CstF-foci.
CstF-64k0a
A-
. / .n ~~~p8O coilin
2
o
3
4
5
micrometers
~~~CsTF-64kDa
B
*"/p80 coilin
Q)
C
_
'0..
O
1
2
3
4
5
a
micrometers
Fig. 4. Quantitative analysis of the spatial relationships between foci
of 3' cleavage factors and coiled bodies. The fluorescent signal
intensities of the anti-CstF 64 kDa and anti-p80-coilin labelling are
plotted along an axis through both bodies in one focal plane.
(A) shows two peaks next to each other with little overlap,
representing the coiled body and the focus of cleavage factors in
Figure 3A. The highest intensities in both signals are -0.5 gtm apart,
confirming that both domains are separate nuclear structures. However,
in many cells, the coiled body and the focus of cleavage factors
completely overlap, as shown in (B).
labelling in DRB-treated cells and in the cx-amanitintreated cells showed the same distribution as found in
uninhibited control cells. We still observed coiled bodies
and cleavage bodies, although the numbers of both had
somewhat decreased (0-3 per nucleus).
There was a significant change, however, in the relationship between coiled bodies and the foci of cleavage factors
after x-amanitin or DRB treatment. Both domains showed
an almost complete co-localization (Figure 3C and Table
I). Foci next to bodies or with partial overlap, often seen
in uninhibited cells, were never observed in inhibitortreated cells. Control experiments showed that drugtreated cells had staining patterns typical for transcriptioninhibited nuclei (Carmo-Fonseca et al., 1992), i.e. coiled
bodies no longer contained the U2 snRNP splicing factor,
which almost completely concentrated in 10-20 large
rounded-up speckles, together with the non-snRNP
splicing factor SC-35 (data not shown).
Clearly, CstF 64 kDa and CPSF 100 kDa behaved
differently from the splicing factors mentioned above.
They did not dissociate from coiled bodies but instead
appeared to accumulate in them. These findings show that
Fig. 5. Electron micrographs of thin sections of T24 cells, double
labelled for p80-coilin (small silver particles; arrowheads) and CstF
64 kDa (large silver particles). Coiled bodies, which were identified by
the presence of p80-coilin label (arrowheads), also often exhibited
CstF labelling. A distinct nuclear body (left of the coiled bodies),
containing a higher CstF labelling than the surrounding material but
devoid of p80-coilin labelling, could be observed in close proximity to
the coiled body (B), or in direct contact with the coiled body (A).
These structures, named 'cleavage bodies', most likely represent the
foci of 3' cleavage factors identified by immunofluorescence
microscopy. Bars represent 0.25 ,um.
transcription levels affect the spatial relationship between
coiled bodies and cleavage bodies.
CstF 64 kDa and CPSF 100 kDa redistribute to
rounded-up speckles after prolonged a-amanitin
treatment
After incubation with a-amanitin for 6 h, almost all nuclei
showed a different CstF 64 kDa and CPSF 100 kDa
labelling pattern compared with uninhibited cells or cells
that were inhibited for up to 2 h. After 6 h, the labelling
was concentrated in 10-20 large round structures in
the nucleoplasm (Figure 3D), similar to observations by
Krause et al. (1994) of a-amanitin-treated HeLa cells.
We observed that even after 6 h of a-amanitin treatment
some T24 nuclei still showed small foci rich in CstF
64 kDa or CPSF 100 kDa, which completely co-localized
with foci containing p80-coilin (data not shown). Most
2887
W.Schul et aL
p80-coilin labelling, however, was from disrupted coiled
bodies showing small curved structures in the nucleoplasm
as reported for HeLa cells (Carmo-Fonseca et al., 1992).
Fig. 6. Double labelling experiments with anti-p80 coilin (A, C and E)
and anti-CstF 64 kDa (B, D and F), showed that foci of 3' cleavage
factors associated with coiled bodies occur in various cell types,
although often not as distinct or frequent as in T24 cells (A and B). In
1-5% of the CaCo cells, foci could be found completely or partially
overlapping with a coiled body (C and D). F3035 fibroblasts rarely
contained coiled bodies, but the few that were found always colocalized with a focus enriched with CstF 64 kDa (E and F). Bar
represents 2 ,m.
These remnants of coiled bodies (red structures in Figure
3D) no longer contained high concentrations of CstF
64 kDa or CPSF 100 kDa, and were sometimes found
associated with nucleoli but could also often be found in
close contact with the large rounded-up speckles. In
uninhibited cells, coiled bodies and cleavage bodies never
overlapped with speckles, but no further specific spatial
relationship between the two types of domains was found.
Apparently, initial inhibition of RPII transcription makes
CstF 64 kDa and CPSF 100 kDa become concentrated in
coiled bodies, but prolonged inhibition causes these 3'
cleavage factors and p80-coilin to redistribute into separate
structures. This further demonstrates the dynamic relationship between cleavage bodies, coiled bodies and RPII
activity.
Coiled bodies, cleavage bodies and PML bodies
occur as trios in the nucleus
Besides coiled bodies, many other small subnuclear
domains, often referred to as nuclear bodies, have been
visualized by electron microscopy (de The et al., 1960;
for review, see Brasch and Ochs, 1992). Characterization
of some of these structures has been based on immunolabelling experiments, using a variety of human autoantibodies (Szostecki et al., 1987; Ascoli and Maul, 1991;
Fusconi et al., 1991; Saunders et al., 1991). The use of
mAbs has shown that the proteins PML and SplOO are
highly concentrated in a specific type of nuclear body
(Szostecki et al., 1990; Ascoli and Maul, 1991; Stuurman
et al., 1992; Koken et al., 1994). We will refer to these
structures as PML bodies. Typically, 10-30 PML bodies
are found in the nucleus of a mammalian cell. Their
function is unknown. Experiments have indicated that
coiled bodies very often reside right next to PML bodies,
but do not overlap (Grande et al., submitted).
We have compared the distribution of cleavage bodies
and PML bodies, using the anti-CstF 64 kDa mAb and
the human autoimmune serum SUN3 which recognizes
PML bodies in the nucleus (Szostecki et al., 1987).
Cleavage bodies were often found adjacent to PML bodies,
but they never overlapped (data not shown). Because the
number of PML bodies greatly exceeds the number of
cleavage bodies per nucleus, many PML bodies were not
associated with a cleavage body.
Fig. 7. Some cleavage bodies contained newly synthesized RNA. T24 cells were microinjected with 5-bromo-UTP which is incorporated in RNA.
Sites of newly synthesized RNA are immunofluorescently visualized (A). When double labelled with anti-CstF 64 kDa (B), -20% of the cleavage
bodies contained a high concentration of newly transcribed RNA (compare magnified areas in both images). Single confocal sections are shown. Bar
represents 2
2888
gm.
CstF 64 kDa and CPSF 100 kDa
From this, it seemed likely that coiled bodies, cleavage
bodies and PML bodies could occur grouped together as
trios in the nucleus. To test this, we performed triple
labelling experiments using anti-CstF 64 kDa, anti-p80coilin and SUN3 in combination with three-channel confocal laser scanning microscopy to visualize the three
types of domains simultaneously in one nucleus. As
expected, there was a tight association between coiled
bodies, PML bodies and cleavage bodies in all analysed
nuclei. In cases where the coiled body and cleavage body
did not overlap, three tightly clustered dots could be
observed (Figure 3E). We rarely found a pair of coiled
body and cleavage body that was not associated with a
PML body. The three nuclear domains seemed to form a
tight trio in the nucleoplasm.
Although coiled bodies, PML bodies and cleavage
bodies may be involved in different processes, their
obvious spatial relationship suggests that their functions
are linked.
Discussion
We used specific mAbs against two RNA 3' cleavage
factors to study the distribution of these proteins in the
nucleus of human cells. CPSF comprises four subunits
(160, 100, 73 and 30 kDa) and CstF comprises three
subunits (77, 64 and 50 kDa). Both factors form stable
functional complexes which can withstand various purification procedures (Takagaki et al., 1990; Bienroth et al.,
1991; Jenny et al., 1994). It is, therefore, likely that the
CstF 64 kDa and CPSF 100 kDa labelling represents the
distribution of intact CstF and CPSF, although it cannot
be excluded that some CstF 64 kDa and CPSF 100 kDa
labelling comes from uncomplexed subunits.
The majority of the two 3' cleavage factors was found
distributed in a homogeneous fibrogranular pattern
throughout the nucleoplasm, and not in a distinct punctated
pattern as found for sites of newly synthesized RNA
(compare Figure 7A and B). Nevertheless, our double
labelling experiments showed that CstF 64 kDa was
present at sites of newly synthesized RNA, albeit not in
elevated amounts. This is in good agreement with its
extensively documented role in the processing of premRNA. The CstF 64 kDa labelling outside the sites of
RNA synthesis suggests that only a fraction of the CstF
proteins is actually engaged in the RNA 3' cleavage
process in the nucleus.
The two 3' cleavage factors were also found highly
concentrated in a few foci in the nucleoplasm. Although
we could not compare the CstF foci directly with the
CPSF foci, the identical association with coiled bodies
strongly indicated that CstF 64 kDa and CPSF 100 kDa
are both concentrated in the same nuclear domain. This
nuclear domain was visible in the electron microscope as
a compact spherical fibrous structure, distinct from a
coiled body in appearance and in protein composition.
Based on these observations, we named this novel nuclear
domain 'cleavage body'.
Cleavage bodies, coiled bodies and PML bodies
form a dynamic trio
Our results indicate a tight spatial relationship between
coiled bodies and cleavage bodies, which may indicate a
link between their functions. A cleavage body was sometimes found next to a coiled body, but could also partially
or completely overlap with it. It should be noted that the
observed overlap between coiled and cleavage bodies
could be caused partly by the limited resolution of the
confocal microscope. Two domains next to each other
could often clearly be distinguished; however, two separate
bodies very close together would appear to overlap in the
light microscope. Indeed, electron microscopical observations have indicated that some coiled bodies and cleavage
bodies are so close together that they could not be seen
as separate domains at the light microscopical level.
Moreover, the resolution between subsequent focal planes
in the confocal microscope is much lower than the lateral
resolution and barely above the size of a coiled body and
cleavage body. Therefore, we cannot rule out the possibility
that at least some apparently co-localizing dots were
actually two separate domains above or very close next
to each other, so the percentage of co-localizing and
partially overlapping bodies (Table I) may be an overestimation at the expense of the number of adjacent bodies.
The relationship between coiled bodies and cleavage
bodies is dynamic, since the level of transcription affects
the degree of overlap between the two types of domains:
cleavage bodies and coiled bodies completely co-localized
after inhibition of RPII transcription. Although the light
microscopical data suggest that both structures fuse to
form one domain, there is also the possibility that the
cleavage factors are transferred from the cleavage body
to the coiled body. There are some indications for this
latter possibility from electron microscopical observations,
in which a cleavage body contained less CstF labelling than
the coiled body with which it was associated. However, the
precise mechanism by which both bodies interact is still
unknown. We conclude that cleavage bodies, like coiled
bodies, are dynamic structures that are possibly involved
in the processing of pre-mRNA.
A third nuclear body, rich in the protein PML, was
often found complexed with the pair of coiled body and
cleavage body. The function of PML bodies is unclear.
They never overlap with either coiled bodies or cleavage
bodies and do not seem affected by transcription inhibition
(Grande et al., submitted), but their constitutive presence
in the ultrastructural trios does indicate a role for these
enigmatic nuclear entities in collaboration with coiled
bodies and cleavage bodies.
The role of cleavage bodies in RNA processing
It has been proposed that coiled bodies are compartments
for the assembly or disassembly of multi-snRNP complexes, as part of the splicing cycle (discussed in Lamond
and Carmo-Fonseca, 1993). It is possible that CstF, CPSF
and perhaps other 3'-processing factors similarly undergo
transcription-dependent recycling, assembly or disassembly in cleavage bodies. Parts of the machinery in coiled
bodies for the recycling of splicing factors may also be
used for the recycling of 3' cleavage factors. This could
explain the close association between coiled bodies and
cleavage bodies.
Alternatively or additionally, coiled bodies and cleavage
bodies could play a role in the processing of specific
transcripts. It seems clear that coiled bodies do not
contain newly synthesized RNA, since they become slowly
2889
W.Schul et aL
Fig. 8. Schematic representation of the proposed organisation of a trio
of cleavage body, coiled body and PML body on an active gene. The
cleavage body is in contact with the entire gene or just with the
downstream part containing the cleavage site. The other two bodies do
not contain DNA or newly transcribed RNA, yet are associated with
this specific locus.
labelled with tritiated uridine (Moreno Diaz de la Espina
et al., 1982), do not appear to contain DNA (Thiry, 1994)
and do not contain nascent RNA when labelled with
BrUTP (data not shown; Raska, 1995).
In contrast, we have observed that ~20% of the cleavage
bodies do contain newly synthesized RNA. These RNAcontaining cleavage bodies could not be overlapping with
a coiled body, because coiled bodies never contain newly
synthesized RNA. Thus RNA-containing cleavage bodies
most probably represent those that are located adjacent to
coiled bodies. Remarkably, the percentage of cleavage
bodies found next to a coiled body (18%) is about the
same as the percentage of cleavage bodies found to contain
newly transcribed RNA (-20%). These findings lead us
to propose that this subset of cleavage bodies is associated
with specific sites of RNA synthesis in the nucleus.
Closely associated coiled bodies and PML bodies, although
themselves outside the sites of RNA synthesis, may
be involved in the processing of these specific RNAs
(Figure 8).
Interestingly, recent observations by Frey and Matera
(1995) and Smith et al. (1995) point to a preferential
localization of coiled bodies in the close vicinity of specific
gene loci. They reported that coiled bodies in HeLa cells
are often found associated with the U1 and U2 snRNA
gene clusters and sometimes with the histone gene cluster.
A similar spatial organization has been described for a
specific organelle in the nuclei of amphibian oocytes that
seems closely related to the coiled body: the C-snurposome
(Wu et al., 1991; Wu and Gall, 1993; Gall et al., 1995;
Roth, 1995). C-Snurposomes contain the protein SPH-1,
which is related to p80-coilin (Tuma et al., 1993), and,
like coiled bodies, are found closely associated with the
histone gene loci (Gall et al., 1981; Callan et al., 1991).
Interestingly, both coiled bodies and C-snurposomes are
enriched in U7 snRNA (Wu and Gall, 1993; Frey and
Matera, 1995), necessary for histone 3' maturation
(Marzluff, 1992). We have shown here that coiled bodies
are associated with two other factors involved in the 3'processing of mRNA, namely CstF and CPSF. It is
somewhat puzzling, however, that these last two factors
do not appear to be involved in the 3'-processing of
either the snRNA transcripts, or the histone transcripts.
Apparently, coiled bodies contain elements of different
3'-processing machineries, which could indicate their
2890
involvement in the 3' formation of certain polyadenylated
and non-polyadenylated transcripts.
We have shown that, after inhibtion of RPII transcription, coiled bodies always completely overlap with cleavage bodies. In the proposed model, this may be explained
by the assumption that cleavage body-associated genes
are not transcribed continuously, causing cleavage bodies
and coiled bodies to fuse during the inactive period
and to divide into separate domains when transcription
recommences and the services of the 3' cleavage factors
are required. A cleavage body overlapping with a coiled
body could be associated with an inactive gene, or an
active gene which encodes a non-polyadenylated RNA,
or it could be unassociated with a gene locus at that time.
Our studies have revealed new interesting relationships
between different nuclear domains. Although the precise
role of coiled bodies, PML bodies, cleavage bodies and
other nuclear structures remains unclear, the associations
and interactions we have demonstrat.d may lead to new
insights into their nuclear function.
Materials and methods
Cell culture
T24 cells (from human bladder carcinoma), HeLa cells (from human
cervical carcinoma) and CaCo cells (from human colon carcinoma) were
grown on circular glass coverslips at 37°C under a 10% CO2 atmosphere
in DMEM (Gibco) supplemented with 1% glutamine (Gibco), 10% fetal
calf serum (FCS; Boehringer) and antibiotics [100 IU/ml penicillin and
100 jg/ml streptomycin (Gibco)].
Human skin fibroblasts (Heng89AD and F3035) were grown at 37'C
under a 5% Co2 atmosphere in a 1:1 mixture of Ham's F-10 medium
(Gibco) and DMEM containing the same supplements as for T24, HeLa
and CaCo cells. All cells were used at 50-70% confluency.
For drug treatment, cells were placed in fresh medium containing
50 pig/ml ax-amanitin (Sigma) or 50 ,uM DRB (Sigma) and grown for
another 2 or 6 h before fixation.
Immunofluorescence labelling
Coverslips with attached cells were rinsed once in phosphate-buffered
saline (PBS) and incubated with 2% paraformaldehyde in PBS for
15 min at room temperature. After fixation, cells were rinsed twice with
PBS and permeabilized with 0.5% Triton X-100 (Sigma) in PBS for
5 min at room temperature. Cells subsequently were rinsed twice in
PBS, incubated in PBS containing 100 mM glycine (Sigma) for 10 min
and incubated for 10 min in PBG [PBS containing 0.5% bovine serum
albumin (Sigma) and 0.05% gelatine from cold water fish skin (Sigma)].
For immunolabelling, the following antibodies were used: anti-CstF
64 kDa mAb from mouse (Takagaki et al., 1990), anti-CPSF 100 kDa
mAb from mouse (Jenny et al., 1994), anti-p80-coilin polyclonal
antibody from rabbit (kindly provided by Dr A.I.Lamond), SUN3
human autoimmune serum (kindly provided by Dr L.Bautz) and antiBrdU (Seralab).
Fixed cells were incubated overnight at 4°C with primary antibodies
diluted in PBG. Subsequently, cells were washed four times for 5 min
each time in PBG and incubated with secondary antibodies diluted in
PBG for 1.5 h at room temperature.
For single and double labelling, donkey anti-mouse IgG coupled to
DTAF (Jackson ImmunoResearch Laboratories) and donkey anti-rabbit
IgG coupled to TRITC (Jackson) were used. After labelling, cells were
washed twice for 5 min in PBG and twice for 5 min in PBS, followed
by incubation in PBS containing 0.4 jg/mI Hoechst 33258 (Sigma) for
5 min.
For triple labelling, donkey anti-mouse IgG coupled to DTAF (Jackson)
and donkey anti-rabbit Ig coupled to Texas red (Jackson) were used for
1 h. After this labelling, cells were washed four times for 5 min in PBG
and incubated with mouse anti-human IgG coupled to CyS (Jackson) for
1 h at room temperature. Finally, cells were washed twice for 5 min in
PBG and twice for 5 min in PBS.
All coverslips were mounted in PBS containing 90% glycerol and
1 mg/ml p-phenylenediamine (Sigma).
CstF 64 kDa and CPSF 100 kDa
Microinjection
T24 cells were microinjected with BrUTP as described by Wansink et al.
(1993; 1994). After microinjection, cells were cultured for 10 min at
37°C and subsequently fixed and labelled as described above.
Light microscopy
Images of single-labelled cells were produced on a Leitz Aristoplan
microscope equipped with epifluorescence optics. Pictures were photographed on Kodak Tri-X 400 ASA film. Images of double-labelled cells
were produced on a Leica confocal laser scanning microscope with a
100X/1.35 oil immersion lens. A dual wavelength laser was used to
excite DTAF and TRITC simultaneously at 488 and 514 nm respectively.
The fluorescence signals from both fluorochromes were recorded simultaneously. Optical cross-talk was quantified and subtracted as described
previously (Manders et al., 1992). Image analysis was performed using
SCIL-IMAGE software.
Three-dimensional images of triple-stained nuclei were recorded with
a Sarastro 'Phiobos 1000' confocal microscope (Molecular Dynamics
Inc., Sunnyvale, CA). A 100X/1.3 Planapo oil immersion Zeiss objective
was used. This confocal microscope has been modified for the pixelsimultaneous recording of three different fluorophores. The specimen
was simultaneously illuminated with light of 488, 568 and 648 nm from
an Ar ion and a Kr ion laser. Three photomultipliers were used to record
the different signals. To improve channel separation, the set-up has been
modified for the application of a new technique: intensity-modulated
multiple-beam scanning (IMS) microfluorometry (Carlsson et al., 1994;
Manders et al., 1995). With this technique, optical cross-talk between
different fluorescence signals was reduced substantially (<0.5%).
Immunogold labelling and electron microscopy
Post-embedment immunogold labelling on thin cryosections and thin
lowicryl sections of T24 and HeLa cells was carried out as previously
described (Raska et al., 1995). Whereas p80-coilin labelling was found
satisfactory, this approach did not yield significant labelling for CstF,
despite very gentle fixation conditions such as 30 min fixation in 2%
paraformaldehyde. Only a pre-embedment approach gave a positive
result.
Cells grown on coverslips were washed in PBS, fixed in 2% paraformaldehyde in PBS for 10 s, washed again and permeabilized for 3 min in
1% Triton X-100 in PBS containing 4.5% polyvinylpyrolidone and
0.2 M sucrose at room temperature. Subsequently, the cells were washed
and fixed for 30 min with 2% paraformaldehyde in PBS, washed in PBS
and incubated in 10% FCS in PBS for 10 min. The cells were incubated
with primary antibody anti-CstF 64 kDa in 5% FCS for 3 h, washed in
PBS, and incubated with anti-mouse 1 nm gold adducts (Aurion,
Belgium) in 5% FCS in PBS, overnight at 4°C. The cells were further
fixed for 20 min in 2% paraformaldehyde in PBS. Silver intensification
was carried out with the Aurion intensification kit in two consecutive
30 min intervals. For immunogold double labelling, the cells additionally
were exposed to anti-p80-coilin (polyclonal antibody from rabbit, kindly
provided by Dr E.Chan) in 5% FCS for 3 h, washed in PBS, and
incubated with anti-rabbit 1 nm gold adducts in 5% FCS in PBS and a
second round of intensification (2 X 30 min) was performed. After
fixation, the cells were embedded in epon. A diamond knife from
Diatome (Suisse) and a Reichert (Leica) Ultracut E ultramicrotome were
used for sectioning. The sections were stained with 5% uranyl acetate
in water for 60 min. The sections were observed in an Opton 109
electron microscope equipped with transfibre optics.
Acknowledgements
The authors thank Dr R.W.Dirks for his help with triple labelling
experiments, and Drs C.M.van Drunen and M.A.Grande for helpful
discussions. The electron microscopic work was supported by grant no.
304/93/0594 of the Grant Agency of Czech Republic and grant no.
539401 of the Grant Agency of the Academy of Sciences of the
Czech Republic.
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Received on May 18, 1995; revised on December 12, 1995