* Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois 60208; and DyOGen, INSERM U309, Institut Albert Bonniot, Domaine de la Merci, 38706 La Tronche cedex, France
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Abstract |
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The cell nucleus is organized as discrete domains, often associated with specific events involved in
chromosome organization, replication, and gene expression. We have examined the spatial and functional
relationship between the sites of heat shock gene transcription and the speckles enriched in splicing factors in primary human fibroblasts by combining immunofluorescence and fluorescence in situ hybridization (FISH).
The hsp90 and hsp70 genes are inducibly regulated by
exposure to stress from a low basal level to a high rate
of transcription; additionally the hsp90
gene contains
10 introns whereas the hsp70 gene is intronless. At
37°C, only 30% of hsp90
transcription sites are associated with speckles whereas little association is detected
with the hsp70 gene, whose constitutive expression is
undetectable relative to the hsp90
gene. Upon exposure of cells to heat shock, the heavy metal cadmium, or
the amino acid analogue azetidine, transcription at the
hsp90
and hsp70 gene loci is strongly induced, and
both hsp transcription sites become associated with
speckles in >90% of the cells. These results reveal a
clear disconnection between the presence of intervening sequences at specific gene loci and the association
with splicing factor-rich regions and suggest that subnuclear structures containing splicing factors are associated with sites of transcription.
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Introduction |
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UNDERSTANDING the spatial relationship between
nuclear architecture and genomic function represents a major objective of cell biology. Discrete
subnuclear sites for transcription and splicing have been
described; however, the structure/function relationships of
these organized structures remain unresolved. For example, several studies using labeled precursors for RNA synthesis have shown that the sites of RNA polymerase II
(Pol II)1 transcription are widely distributed in the nucleus
(for reviews see Fakan, 1994; Moen et al., 1995
; Clemson
and Lawrence, 1996
; Huang and Spector, 1996a
; Misteli
and Spector, 1998
). Similarly, active Pol II has been found
randomly distributed in the nucleus (Jiménez-Garcia and
Spector, 1993
; Zeng et al., 1997
), thus suggesting that transcription occurs at multiple sites throughout the nucleus. However, other studies using different reagents have
shown that a subpopulation of the active Pol II is concentrated in specific nuclear regions (Thibodeau and Vincent,
1991
; Bregman et al., 1995
; Blencowe et al., 1996
; Mortillaro et al., 1996
; Patturajan et al., 1998
). A recent study
by Wei et al. (1998)
has shown that individual Pol II transcription sites, detected by BrUTP incorporation, are spatially segregated in the nucleus into ~16 higher order domains, providing support for the compartmentalization of
RNA Pol II transcription within a discrete number of nuclear factories. Consistent with the organization of the
transcription sites into domains, total poly(A) RNAs detected by fluorescence in situ hybridization (FISH) appear
to concentrate in 20-40 nuclear foci (Carter et al., 1991
,
1993
).
The small nuclear ribonucleoproteins (snRNPs) and
various non-snRNP splicing factors such as SC35, U2AF,
U2B, or SF2/ASF detected by immunofluorescence distribute in the nucleus in 20-40 foci of varying size termed
"speckles," in addition to a diffuse nucleoplasmic staining
of lesser intensity (for reviews see Moen et al., 1995
; Clemson and Lawrence, 1996
; Huang and Spector, 1996a
; Lamond and Earnshaw, 1998
; Misteli and Spector, 1998
).
Three classes of nuclear structures enriched in splicing factors have been described by electron microscopy (for reviews see Fakan, 1994
; Puvion and Puvion-Dutilleul,
1996
): (a) the interchromatin granules (IGs), which correspond to the speckles described by immunofluorescence and represent compact structures containing high concentrations of both snRNPs and non-snRNPs splicing factors;
(b) the perichromatin fibrils (PFs), which are less concentrated in splicing factors and may correspond to the diffuse
nucleoplasmic staining; and (c) the coiled bodies (CBs),
which are compact structures highly enriched in snRNPs
but devoid of the essential splicing factor SC35 (for review
see Matera, 1998
).
To what extent do nuclear domains enriched in RNA
Pol II or splicing factors reflect gene expression, sites of
storage, and assembly of macromolecular structures, or recycling centers? In contrast to available methodologies
which have demonstrated the presence of organizing sites
of transcription, it has been difficult to establish the relationship between structure and function for RNA splicing,
specifically to discriminate between actively engaged and
inert splicing complexes. Based on in vitro and in vivo evidence that intron excision is spatially and temporally associated with transcription (Huang and Spector, 1991; Raap
et al., 1991
; Xing et al., 1993
; Wuarin and Schibler, 1994
;
Zhang et al., 1994
), an indirect measure of the role of these
different nuclear structures in transcription and splicing
has thus been to analyze the relative distribution of splicing factors and active sites of transcription. Electron microscopic studies have shown that sites of [3H]uridine incorporation occur preferentially in the vicinity of PFs (for
review see Fakan, 1994
; Puvion and Puvion-Dutilleul, 1996
). Similarly, sites of transcription detected by BrUTP
incorporation reveal little colocalization with the nuclear
speckles (Wansink et al., 1993
; Pombo and Cook, 1996
;
Fay et al., 1997
), and active RNA Pol II is distributed randomly relative to the speckles enriched in splicing factors
(Zeng et al., 1997
). Finally, several viral and endogenous
transcription sites detected by FISH display little or no association relative to the speckles (Zhang et al., 1994
; Lampel et al., 1997
; Smith et al., 1999
). Altogether, these
observations suggest that transcription by RNA Pol II and
splicing would occur in domains scattered throughout the
nucleus but distinct from SC35/snRNP-rich speckles.
In contrast, others have shown that the 20-40 foci enriched in poly(A) RNA colocalize with speckles (Carter et al., 1991, 1993
; Visa et al., 1993
). In addition, several specific transcription sites detected by FISH have
been shown to be spatially associated with these regions
(Huang and Spector, 1991
, 1996a
,b; Wang et al., 1991
;
Lawrence et al., 1993
; Xing et al., 1993
), and a study of
five endogenous genes has shown that inactive genes are
randomly distributed relative to the speckles enriched in
splicing factors, whereas active genes were closely associated with these regions (Xing et al., 1995
). Similarly, the
cytomegalovirus immediate early gene clusters (Dirks et
al., 1997
) as well as the stably transfected homeobox gene
pem (Misteli et al., 1998
) are found associated with nuclear speckles only when they are actively transcribed, thus providing support for a role of the speckles in transcription and splicing.
Three models for the role of speckles in transcription
and splicing have been proposed (Clemson and Lawrence,
1996): (a) speckles represent sites of storage and/or assembly-disassembly of splicing factors; (b) speckles are often
associated with pre-mRNA metabolism of specific genes;
and (c) speckles represent distinct functional entities with
some representing storage sites and corresponding to the
IGs, while others associated with active genes would represent sites of transcription and splicing and more likely
correspond to large accumulations of PFs. In agreement
with the latter model, Huang and Spector (1996a)
have
proposed that upon activation of RNA Pol II transcription, splicing factors would be recruited from their sites
of storage and/or reassembly, the IGs, to the sites of
transcription where nascent transcripts would be spliced. When the transcription rate is very high, a substantial
amount of splicing factors would be recruited to the sites
of transcription, resulting in a granular IG-like appearance
which would be functionally distinct from IGs. This assumption can explain the colocalization observed between
several specific transcripts and the speckles. Consistent
with this proposal, the splicing factor SF2/ASF fused to the
green fluorescent protein is recruited to new sites of viral
transcription upon transcriptional activation in living cells,
thus demonstrating that one function of the speckles is to
supply splicing factors to neighboring active genes (Misteli et al., 1997
). The recruitment of splicing factors to the sites of transcription can be intron-dependent as demonstrated
by Huang and Spector (1996b)
using a set of intron-containing and intronless constructs. However, a caveat of
these studies is that the genes which were studied were either integrated viral genomes or transfected constructs.
To address key issues concerning the involvement of the
nuclear speckles in transcription and splicing activities, we
used a combination of FISH and immunofluorescence to
examine whether the speckles are associated with sites
of transcription of a class of coregulated cellular heat
shock genes including members which are intron-containing (hsp90) (Hickey et al., 1986
) and intronless (hsp70)
(Wu et al., 1985
), and whose expression can be inducibly regulated (for reviews see Morimoto, 1993
; Morimoto et
al., 1996
). This system allows the examination of both the
dynamics of transcription of well studied cellular genes
and the role of introns in association with the speckles.
Our results reveal that both hsp90
and hsp70 transcription sites associate with the speckles upon stress-induced transcriptional activation, independent of the presence of introns.
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Materials and Methods |
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Cell Culture and Stress Induction
Human normal primary fibroblasts were obtained from a skin biopsy performed on a healthy female donor. They were grown in RPMI medium supplemented by 10% fetal calf serum and 100 µg/ml ampicillin. For in situ analysis, cells were grown directly on two-chamber glass slides (Labtek). Heat treatment was performed by immersing the slides or the flasks in a waterbath set up at 42 or 45°C. Cadmium was used at a final concentration of 75 µM for 4 h and azetidine was used at a final concentration of 10 mM for 2 h.
Probes and Antibodies
The pH 2.3 genomic probe covering the entire coding sequence (2.3 kb) of
the human hsp70 gene was used to detect hsp nuclear transcripts (Wu et al.,
1985). The hsp70 gene was detected using the cosmid clone 12HI which
contains a portion of the coding sequence of hsp70 (kindly provided by
Dr. R.D. Campbell, University of Cambridge, Cambridge, UK). cDNA
probes specific for hsp90
(pHS 801) and hsp90
(pHS 811) genes were
obtained from Dr. E. Hickey (University of Nevada, Reno, NV). pHS 801 and 811 probes contain, respectively, 1.3 and 0.9 kb of the coding region
(Hickey et al., 1986
). All probes were labeled by random priming with biotin-14-dATP (GIBCO BRL).
The mouse monoclonal antibody specific for the non-snRNP splicing
factor SC35 (Sigma) was used at a dilution of 1:250 for immunofluorescence (Fu and Maniatis, 1990). The mouse monoclonal Y12 directed
against the Sm protein of snRNPs was obtained from Dr. J.A. Steitz (Yale
University, New Haven, CT) and used at 1:250 (Lerner et al., 1981
). The
mouse monoclonal antibody against the U2B
splicing component (Cappel) was used at 1:50 (Mattaj et al., 1986
). The mouse monoclonal POL3/3
antibody against RNA Pol II was obtained from Dr. E.K. Bautz (University of Heidelberg, Heidelberg, Germany) and used at 1:200 (Krämer et al.,
1980
). The mouse monoclonal CC-3 antibody against RNA Pol II was obtained from Dr. M. Vincent (University of Laval, Quebec, Canada) and
used at 1:500 (Thibodeau and Vincent, 1991
). The mouse monoclonal
MARA3 antibody against RNA Pol II was obtained from Dr. B.M. Sefton
(Salk Institute, La Jolla, CA) and used at 1:200 (Patturajan et al., 1998
).
Combined Immunofluorescence and FISH
Probe Preparation.
100 ng of the cDNA probe or 100 ng of the cosmid
probe was precipitated with 30 µg of salmon sperm DNA. 3 µg of human
Cot I competitor DNA (GIBCO) was also added to the cosmid probes. The pellets were resuspended in 50% formamide/10% dextran sulfate/2× SSC, and denatured for 5 min at 75°C. Cosmid probes were incubated 1 h
at 37°C to allow suppression of repeated sequences (Lichter et al., 1988).
Sample Preparation for Detection of RNA and Proteins.
Immunofluorescence combined to FISH was performed as described previously (Jolly et al.,
1997a). FISH was performed first to enhance the efficiency of hybridization. Briefly, cells were fixed in 4% formaldehyde/PBS. A first permeabilization step was performed by three successive incubations of 5 min each
in 0.5% saponin/0.5% Triton X-100/PBS. After an equilibration step in 20% glycerol/PBS for 20 min, cells were freeze-thawed three times successively by briefly dipping in liquid nitrogen as a second permeabilization
step. Cells were subsequently dehydrated through sequential incubations
in 70%, 90%, and 100% ethanol baths for 5 min each. The denatured
probe was applied to the dry slide and hybridization was allowed to run
overnight. After hybridization, probes were detected using avidin-FITC
(Sigma). After postdetection washes in 4× SSC/0.1% Tween 20, a 45-min
blocking step in 10% FCS/0.3% Triton X-100/PBS was performed, followed by incubation for 90 min at 37°C with the anti-SC35 or the Y12 antibody. Antibody staining was revealed using an anti-mouse-TRITC antibody (Sigma) and nuclei were counterstained with 250 ng/ml DAPI (4',
6-diamidino-2-phenylindole/2 HCl) (Sigma) diluted in an antifading solution consisting in 90% glycerol, 20 mM Tris-HCl, pH 8.0, 2.33% DABCO
(1-4 diazabicyclo [2,2,2] octane) (Sigma).
Sample Preparation for Detection of DNA and Proteins. Immunofluorescence was performed first in this case. After detection with the secondary antibody, cells were rinsed three times with PBS, and subsequently denatured by a 3-min incubation in 70% formamide/2× SSC at 75°C, followed by a 1-min incubation in 50% formamide/2× SSC at the same temperature. The denatured cosmid probe was then applied to the slide. After hybridization, cells were washed three times for 5 min in 50% formamide/2× SSC at 45°C, and three times for 5 min in 0.5× SSC at 60°C. Detection was then performed as described above for nuclear transcript detection.
In Vivo Incorporation of BrUTP
BrUTP incorporation was performed according to a protocol derived
from Wansink et al. (1993). Briefly, cells were rinsed once with PBS for 3 min, and once with a glycerol buffer (20 mM Tris-HCl, pH 7.4, 5 mM
MgCl2, 25% glycerol, 0.5 mM PMSF, 0.5 mM EGTA) for 3 min. Cells
were then permeabilized by incubation for 3 min in the same buffer complemented with 0.05% Triton X-100 and 10 U/ml RNAsin, and subsequently incubated with the transcription cocktail (100 mM KCl, 50 mM
Tris-HCl, pH 7.4, 5 mM MgCl2, 0.5 mM EGTA, 25% glycerol, 1 mM
PMSF, 0.5 mM ATP, 0.5 mM CTP, 0.5 mM GTP, 0.2 mM BrUTP, 160 µM S-adenosyl-methionine, 25 U/ml RNAsin) for 15 min at room temperature. At the end of the reaction, cells were rinsed once in PBS containing
0.05% Triton X-100 and 5 U/ml RNAsin, once in PBS added with 0.5 mM
PMSF and 5 U/ml RNAsin, and subsequently fixed in 4% formaldehyde
in PBS. Incorporation sites were revealed using a mouse anti-BrdU antibody (Sigma) and anti-mouse FITC (Sigma).
Fluorescence Microscopy and Image Analysis
Images were acquired using a confocal laser scanning microscope (Zeiss
LSM 410) using a 63×, 1.25 NA oil immersion objective. Confocal images
were analyzed for the relative distribution of the speckles and hsp transcription sites using software developed at the University of Grenoble
(Monier et al., 1996). Deconvolution was used to revert the distortion of
fluorescent signals due to the point spread function of the microscope
which allowed our ability to define the limits of the speckles. Transcription
sites were defined as associated with a speckle when no pixels were separating the two fluorescent signals. Percentages were determined based on the
analysis of 100 nuclei which corresponds to 200 sites of gene transcription.
RT-PCR Reaction
The RT-PCR reaction was performed as described in Wang et al. (1999).
The reaction was internally controlled by including known amounts of internal control transcripts corresponding to the same genes carrying small
deletions to distinguish from wild-type, thus allowing us to precisely quantify the levels of transcripts (see Wang et al., 1999
for preparation of internal control transcripts).
RNA Extraction.
Total RNAs were extracted using the procedure described by Gough (1988). Briefly, cells at 80% confluency were scraped
and spun down. The cell pellet was resuspended in 200 µl of buffer A (10 mM
Hepes, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, and 2.5 mM
DTT), spun again, and the supernatant was added to 200 µl of ice-cold
buffer B (7 M urea, 0.35 M NaCl, 10 mM EDTA, 10 mM Tris-HCl, pH 7.5, 1% SDS). 400 µl of phenol/chloroform (1:1) was then added, and RNAs
were precipitated as described.
Primers.
Specific sense and antisense primers for hsp70, hsp90, and
hsp90
transcripts (GIBCO BRL) were designed using the MacVector program as follows (the numbers indicate positions on the wild-type transcripts): hsp70 sense: 5'-TTCCGTTTCCAGCCCCCAATC-3'
(nucleotides [nt] 435-455); hsp70 antisense: 5'-CGTTGAGCCCCGCGATCACA-3' (nt 993-974); hsp90
sense: 5'-AAAAGTTGAAAAGGTGGTTG-3' (nt 1803-1822); hsp90
antisense: 5'-TATCCACAGCATCACTTAGTA-3' (nt 2426-2405); hsp90
sense: 5'-AGAAGGTTGAGAAAGGTGACAA-3' (nt 1803-1822); hsp90
antisense: 5'-AAGAAGTTAGAGAGGGAATAAA-3' (nt 2444-2425). The expected sizes
of PCR products are 641, 625, and 558 bp for wild-type hsp90
, hsp90
,
and hsp70 transcripts, respectively, and 531, 499, and 438 bp for in vitro
transcribed hsp90
, hsp90
, and hsp70 transcripts, respectively.
Reverse Transcription. The reaction was performed in a total volume of 20 µl. 2 µg of total RNAs was incubated for 1 h at 37°C with 3.5 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 10 mM DTT, 0.5 mM dNTP, 20 pmol of each antisense primer, 30 pg of each internal control transcript, 400 U RNAsin, and 400 U of Moloney murine leukemia virus reverse transcriptase (Pharmacia).
Polymerase Chain Reaction.
PCR reactions were performed in a final
volume of 50 µl. To the 20 µl of the reverse transcription reaction
were added (to final concentrations): 3.5 mM MgCl2, 50 mM KCl, 10 mM
Tris-HCl, pH 8.3, 20 pmol of each sense primer, 0.5 mM dNTP, 1 µCi
[-32P]dATP, 0.01 µg/µl DNase-free RNAse A, and 50 U Taq polymerase (Pharmacia). The reactions were performed in a Pelletier effect thermal
cycler (MJ Research, PTC100) for 35 cycles (each cycle: 1 min at 92°C, 1 min at 56°C, 1 min at 72°C) with an initial denaturation of 1 min at 94°C
and a final extension at 72°C for 10 min.
Gel. PCR products were analyzed on a 4% acrylamide, 42% (wt/vol) urea denaturing gel.
Quantification.
The levels of wild-type hsp70, hsp90, and hsp90
transcripts were quantified using the PhosphorImager analyzer system (Molecular Dynamics) and normalized by the amount of the corresponding internal control transcripts.
Transcriptional Run-on Assay
Run-on transcription reactions were performed with isolated cell nuclei in
the presence of 50 µCi of [-32P]UTP (Amersham) as described previously (Banerji et al., 1984
). After precipitation, radioactive RNA was hybridized to DNA probes for the human hsp70 gene (pH 2.3), human
hsp90
(pHS 801), pBR322 as a control for nonspecific hybridization, and
the rat gapdh gene (Fort et al., 1985
) as a normalization control for transcription. The intensities of radioactive signals were quantitated using the
PhosphorImager analyzer system (Molecular Dynamics).
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Results |
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Relative Distribution of Splicing Factors and hsp Genes in Unstressed Cells
We investigated the relative distribution of SC35 splicing
factor and sites of hsp70 or hsp90 genes in normal human
fibroblasts. Our rationale for selection of the hsp90
and
hsp70 genes was based on three criteria: (a) both genes are
transcribed at a low basal rate in cells at normal growth
temperatures; (b) the transcription rates of both genes are
induced to high levels upon exposure to heat shock and
other stresses; and (c) the hsp90
gene contains 10 introns
whereas the hsp70 gene is intronless. The relative distribution of hsp70 or hsp90
transcription sites and SC35 splicing factor was analyzed by using a procedure combining
immunofluorescence for the detection of splicing factors
and FISH for the detection of hsp nuclear transcripts
(Jolly et al., 1997a
).
We have demonstrated previously that hsp70 and hsp90 gene expression is induced by heat shock and other
stresses (Watowich and Morimoto, 1988
; Abravaya et al.,
1991
; Shi et al., 1998
; for review see Morimoto et al., 1996
).
At 37°C, hsp90
transcripts are constitutively detected
whereas hsp70 mRNAs were undetectable (Fig. 1 a, lane
a). This corresponded, by transcriptional run-on analysis,
to a very low basal rate of hsp90
gene transcription at
37°C while hsp70 gene transcription was repressed (Fig. 1
b, lane a). Within the nucleus of diploid fibroblasts, hsp
transcripts detected by FISH appear as two foci (Fig. 2).
Because hsp90
transcription rate is low, the foci detected
by FISH may correspond partially to nascent transcripts
which are retained at the site of transcription, as has been
shown for hsp70 transcripts (Jolly et al., 1998
). Codetection of the transcripts and the corresponding gene by
FISH showed a complete overlap of the two hybridization
signals at the level of light microscopy (data not shown).
For the hsp70 gene whose constitutive expression is too
low (Fig. 1), we chose to detect the gene itself by FISH.
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In control cells, SC35 speckles were associated with 30%
of the hsp90 transcription sites, averaging over 200 transcription sites (Fig. 2 a and Table I). In contrast, only 10%
of the signals corresponding to the hsp70 gene were associated with SC35 speckles (Fig. 2 b). No differences in the
size or intensity of associated versus nonassociated transcription sites were observed. Nuclear speckles were found
by quantitative digital imaging analyses to occupy 5-17%
of the nuclear volume (Huang and Spector, 1991
; Carter et al., 1993
) whereas hsp transcripts occupy <1% of the
nuclear volume. The low percentage of association between SC35 speckles and hsp70 transcription sites consequently reflects random distribution, whereas the 30% association with hsp90
transcription sites is significant and
likely reflects the higher basal transcription rate of the
hsp90
gene.
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Relative Distribution of SC35 Speckles and hsp Genes in Heat-shocked Cells
To address whether the distribution of SC35 speckles and
hsp genes is a reflection of introns or of the transcription
rate, we exposed the cells to a heat shock at 42°C or 45°C,
conditions which result in a dramatic elevation of heat
shock gene transcription (Watowich and Morimoto, 1988;
Abravaya et al., 1991
; Shi et al., 1998
; for review see
Morimoto et al., 1996
). The analysis of hsp90
and hsp70
gene transcription rates, by nuclear run-on analysis, revealed that both genes were induced strongly following a
42°C or 45°C heat shock (5- and 12-fold induction for the
hsp90
gene, and 14- and 35-fold induction for the hsp70
gene at 42°C and 45°C, respectively) (Fig. 1 b). Quantification of the mRNA levels by RT-PCR revealed a 11.4-fold
(42°C) and 29.7-fold (45°C) induction of hsp70 mRNA levels (Fig. 1 a, lanes b and c) and a 1.5-fold (42°C) and 2.5-fold (45°C) (Fig. 1 a, lanes b and c) induction of hsp90
mRNA. As expected, the fold-induction of hsp90
transcripts determined by measuring mRNA levels was lower
than for hsp70 due to the higher basal levels of hsp90
transcripts in control cells.
hsp70 or hsp90 transcripts were detected together with
the SC35 splicing factors in heat-shocked cells. As shown
in Fig. 3, 92% of hsp90
transcription sites were observed
to be adjacent to SC35 speckles in the 42°C treated cells
(Fig. 3 a) and 94% in the 45°C treated cells (Fig. 3 c). Likewise, 92% and 93% of the chromosomal sites of hsp70
transcription were associated with a SC35 speckle in cells
exposed to 42°C (Fig. 3 b) and to 45°C (Fig. 3 d), respectively. Identical results were obtained in cells exposed at
42°C or 45°C for only 10 min (data not shown), attesting that the association of splicing factors with transcribing
genes is a very rapid process, directly correlated to the
transcriptional activity of the gene and not due to major
rearrangements of the nuclear architecture as a consequence of heat shock.
|
The high degree of spatial coincidence between SC35
speckles and sites of hsp gene transcription following activation of the heat shock response reveals that a key feature of recruitment of SC35 splicing factors relates to the
dynamics of transcription. A similar spatial association of
the heat-activated hsp90 gene with the speckles has been
reported previously (Lampel et al., 1997
). Our results
demonstrate, however, that the splicing factors do not distinguish between intron-containing and intronless genes. Activated hsp genes were not found to be preferentially
associated with larger speckles as has been observed for fibronectin transcripts (Xing et al., 1993
, 1995
), perhaps reflecting a gene specificity in the pattern of association with
the speckles.
To what extent do our observations reflect features of
SC35 which are not general to splicing complexes? To ensure that our results were not limited to the SC35 splicing
factor or due to the fact that the anti-SC35 antibody only
recognizes a phosphoepitope of the protein, we performed
the same experiments on control and heat-shocked cells
with the Y12 antibody to detect snRNPs (Lerner et al.,
1981) (Fig. 4) or with an anti-U2B''antibody (Mattaj
et al., 1986
) (data not shown). The results obtained for
both antibodies revealed the association of both hsp70
(Fig. 4) and hsp90
transcription sites (data not shown)
with the speckles only in cells exposed to 42°C.
|
Other Stresses Also Induce a Tight Association of Active hsp Genes with SC35 Speckles
To exclude that the redistribution of splicing factors following heat shock was due solely to the effects of elevated
temperatures on nuclear organization, heat shock gene
transcription was activated by exposure to azetidine or
cadmium (Mosser et al., 1988; Williams and Morimoto,
1990
). Both treatments resulted in an increase in hsp70
and hsp90
mRNA levels comparable to those induced by
a 42°C heat shock (i.e., a 10.9- and 12.1-fold increase in
hsp70 mRNA levels, and a 1.4- and 1.5-fold increase in
hsp90
mRNA levels in azetidine- and cadmium-treated
cells, respectively) (Fig. 1 a, lanes d and e). This was corroborated by transcriptional run-on assay showing that
both genes were actively induced in cadmium- and azetidine-treated cells (data not shown). As shown in Fig. 5,
94% and 95% of the hsp90
transcripts were associated
with SC35 speckles in azetidine (Fig. 5 a) and cadmium-treated cells (Fig. 5 c), respectively. Similarly, 90% of the
hsp70 transcription sites were associated with SC35 speckles in azetidine-treated cells (Fig. 5 b) and 93% in cadmium-treated cells (Fig. 5 d). These observations confirm
and extend the results of the heat-induced association of
hsp70 and hsp90
transcription sites with the speckles, and
demonstrate that the dynamic relocalization of splicing
factors is not caused by the thermal effects of heat shock
but is primarily a reflection of the elevated rates of transcription of both gene loci.
|
Effect of Stress on the Transcription and Splicing Activities of the Cells
Human primary fibroblasts are relatively resistant to heat
shock and display a very low percentage of cell death following a 1-h heat shock at 45°C (Jolly et al., 1997b). In addition, general features of nuclear morphology visualized
by light microscopy do not appear to be altered by heat exposure (Jolly et al., 1997a
). To address whether the different inducers of heat shock gene expression employed here
caused a transcriptional arrest, we monitored general transcriptional activity by visualizing the sites of BrUTP incorporation into nascent transcripts (Wansink et al., 1993
). As
shown in Fig. 6, there was no detectable change in the
transcriptional pattern in cells treated with either heat
shock, cadmium, or azetidine when compared to control
cells.
|
We next investigated the distribution of RNA Pol II for
its presence within the speckles and to determine whether
the various stress conditions influenced its distribution.
Since substantial variations in Pol II distribution depending on the cell type and the specific antibody used have
been reported (Thibodeau and Vincent, 1991; Jiménez-Garcia and Spector, 1993
; Bregman et al., 1995
; Blencowe
et al., 1996
; Mortillaro et al., 1996
; Zeng et al., 1997
; Patturajan et al., 1998
), we used three characterized antibodies recognizing different epitopes on the RNA Pol II. As
previously reported for other cell types, the POL3/3 antibody, which recognizes an epitope outside of the COOH-terminal domain (CTD) of Pol II and is independent of
the phosphorylation state of the enzyme, shows a diffuse
nucleoplasmic staining at 37°C (Fig. 7 a) (Krämer et al.,
1980
; Kontermann et al., 1995
). In contrast, the CC-3 antibody, which recognizes a phosphoepitope in the CTD,
stains a subpopulation of Pol II concentrated in speckles
(Fig. 7 b) (Thibodeau and Vincent, 1991
). The MARA3
antibody, which recognizes a phosphoepitope in the CTD
different from CC-3, stains both a diffuse population and a
subpopulation of the RNA Pol II concentrated in the
speckles (Fig. 7 c) (Patturajan et al., 1998
). As previously shown by others, these different patterns correspond to
different subpopulations of RNA Pol II. In our human primary fibroblasts, at least two distinct hyperphosphorylated
forms of RNA Pol II appear to concentrate in the speckles; however, whether these subpopulations represent active forms of the enzyme is still unknown. None of these
patterns were altered by a 42°C (Fig. 7, d-f) or 45°C heat
shock (Fig. 7, g-i), or by cadmium and azetidine treatments (data not shown). These observations showed that
in human fibroblasts at least a subpopulation of RNA Pol
II was localized to the speckles and that the overall distribution of the enzyme was not affected dramatically by
stress.
|
As RNA splicing is affected by heat shock in many organisms (for review see Jolly and Morimoto, 1999), we
chose to examine whether the differential distribution of
hsp genes relative to SC35 speckles during stress was a
consequence of a potential stress-induced arrest in RNA
splicing. In human cells, heat shock results in a redistribution of snRNPs from the speckles to a diffuse nucleoplasmic pattern (Spector et al., 1991
). It has also been shown
that extracts from HeLa cells heat-shocked at 43°C or
higher temperatures are unable to form a functional spliceosome; however, the putative factor(s) inactivated by
heat remains unidentified (Shukla et al., 1990
). To examine whether the association of hsp transcription sites with
speckles was due to a heat-induced retention of unprocessed hsp transcripts at the sites of transcription, we analyzed the transcripts of the two intron-containing hsp90
and hsp90
genes from cells exposed to heat shock at 42°C
or 45°C, cadmium, or azetidine by RT-PCR using primers
surrounding an intron. As shown in Fig. 1 a, the hsp90
and hsp90
transcripts detected under all conditions corresponded only to the expected processed species with no
detection of the predicted precursor species.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
This study is the first to investigate, in mammalian cells,
the relative distribution of splicing factors and endogenous
intronless or intron-containing genes with relation to their
inducible transcriptional activity. At 37°C the inactive
hsp70 gene was associated randomly relative to the distribution of nuclear SC35 speckles, whereas the hsp90 gene
was weakly associated with splicing speckles, consistent
with a low but detectable basal transcription. When the
cells were exposed to various stressors which resulted in the inducible transcription of the hsp genes, both hsp70
and hsp90
genes became associated with the speckles.
The association of splicing factors with the new sites of
transcription is a rapid process, occuring immediately
upon gene activation. In addition, at least two subpopulations of hyperphosphorylated RNA Pol II were found to
concentrate in the speckles, and this distribution was unaffected by stress. Altogether our data demonstrate that the
association of specific genes with splicing factors is a reflection principally of the transcription rate of the endogenous cellular gene and does not depend upon the presence
of introns in the primary transcript. These observations
complement the recent findings by Smith et al. (1999)
that
some intron-containing pre-mRNAs are poorly associated
with increased concentrations of SC35, both demonstrating a disconnection between the presence of introns and
the spatial association with splicing factors.
Is the Association with Splicing Speckles a Measure of Active Gene Transcription?
An important conclusion of our results is that the association of splicing factors with the sites of transcription of endogenous genes is best indicated by the level of transcriptional activity. Under control conditions where heat shock
genes are either repressed or transcribed at low basal levels, they are randomly distributed in regards to splicing
factors or weakly associated with them, whereas upon
gene activation, splicing factors accumulate rapidly at the
sites of abundant nascent transcripts. The significance of
the 30% association of hsp90 transcription sites with the speckles at 37°C is uncertain. Since we did not observe a
significant difference in the fluorescent intensity of associated versus nonassociated transcription sites, this may reflect variation in the relative rate of transcription at the
chromosomal loci. Alternatively, this could reflect variation in the local concentration of splicing factors and the
limitations of light microscopy. Finally, high concentrations
of splicing factors may not be required at the sites of transcription, which may at least in part correspond to sites
of accumulation of full-length mature nascent transcripts rather than growing RNA molecules (Jolly et al., 1998
).
Our results are in good agreement with several previous
studies, showing a redistribution of factors involved in
transcription, pre-mRNA processing, and RNA packaging
in response to changes in gene activity. Indeed, splicing
factors have been shown to relocalize in response to RNA
Pol II inhibition (Carmo-Fonseca et al., 1992; Zeng et al.,
1997
), to viral infection (Martin et al., 1987
; Jiménez-Garcia and Spector, 1993
; Spector et al., 1993
; Pombo et al.,
1994
; Puvion-Dutilleul et al., 1994
; Bridge et al., 1995
), or
to inhibition of pre-mRNA splicing (O'Keefe et al., 1994
).
Moreover, the localization of splicing factors to Balbiani
ring genes occurs in a transcription-dependent manner in
the nuclei of Chironomus tentans (Baurén et al., 1996
), and splicing factors are associated with the loops of lampbrush
chromosomes in amphibian germinal vesicles (Wu et al.,
1991
). In human cells, several in situ studies have shown
that the nuclear speckles are associated predominantly
with transcriptionally active cellular and viral genes (Lawrence et al., 1993
; Xing et al., 1995
; Dirks et al., 1997
; Misteli et al., 1997
, 1998
).
Other observations, however, indicate that certain
highly spliced endogenous pre-mRNAs are poorly associated with increased concentrations of SC35 (Smith et al.,
1999). Similarly, some viral transcripts display little or no
association with speckles (Zhang et al., 1994
; Lampel et al.,
1997
). Altogether, these findings suggest that transcription
is not sufficient for the association of specific genes with
nuclear speckles and reveal a more complex view of the
nuclear compartimentation of transcription and splicing activities. The most likely hypothesis to integrate our observations in the context of other results is the existence of
a gene specificity and/or cell type specificity in the distribution of splicing factors regions (Smith et al., 1999
). Likewise since some active genes predominantly associate with
larger speckles (Xing et al., 1993
, 1995
), we can imagine
that other active genes preferentially associate with very
low amounts of splicing factors which can be missed due to
the limitations of microscopic resolution. The concentration of splicing factors associated with specific transcription sites may be gene-specific and may depend on the
combined effect of several factors such as the size and
complexity of the gene, its transcription rate, and its position in the nucleus and/or its chromosomal environment
which can impose structural constraints, thus limiting the
access of splicing factors to these regions (Moen et al.,
1995
; Xing et al., 1995
; Smith et al., 1999
). Our work now
adds to this by demonstrating that the presence or absence of introns is not a determining factor for the association of active genes with splicing factor-rich regions.
Role of Nuclear Speckles in Transcription and Splicing
The functional significance of splicing factors organized
into subnuclear structures has been uncertain, although
much of the data in the literature are consistent with a role
of splicing factor complexes associated with the transcription and processing of intron-containing genes. The association of splicing factors with intron-dependent sites of
transcription was demonstrated in experiments transiently
or stably expressing intronless and intron-containing genes
in HeLa cells (Huang and Spector, 1996b). The recruitment of splicing factors to sites of transcription based on
a phosphorylation cycle which is tightly correlated with splicing activity also supports this conclusion (Misteli et al., 1998
). Consequently, how do we incorporate the observations of the present study, that splicing factors can also be
associated with sites of transcription independent of introns?
In our analysis, we examined the sites of transcription of
endogenous cellular genes, in contrast to genes reintroduced by transfection or expressed following viral infection (Huang and Spector, 1996b; Misteli et al., 1998
). The
introduction of exogenous nucleic acids, by transfection,
could potentially influence aspects of nuclear organization
(Zhang et al., 1994
) with consequences on endogenous
transcriptional activities and a redistribution of splicing
factors (Huang and Spector, 1996b
). Likewise, the genomic site of integration in stably transfected constructs or integrated viral genomes may also influence their expression (Al-Shawi et al., 1990
).
The association of the intronless hsp70 gene with speckles could be the result of the physical proximity on the
chromosome of a distinct, highly transcribed, intron-containing gene, although this seems unlikely given that our
data show a very high percentage of association between
hsp70 transcription sites and the speckles. In addition, this
association is strictly observed in stressed cells, suggesting
that the putative neighboring gene would need to be
stress-responsive. The only known hsp gene located in the vicinity of the hsp70 gene, which is located in the 6p21.3
region (Harrison et al., 1987), is the hsp90
gene which
maps to the 6p12 locus (Durkin et al., 1993
); the physical
distance between these two genes allows a clear discrimination between the two transcription sites by light microscopy (Jolly et al., 1997c
). Thus, the association observed
between the intronless hsp70 transcription sites and the
speckles is significant.
A more likely explanation for the different interpretations is that the distribution of transcription sites relative
to nuclear speckles varies in a gene-specific manner. Indeed, differential distributions of viral or endogenous
transcripts relative to nuclear speckles have been reported
already, and they may reflect differences in the organization of nuclear RNAs derived from endogenous or from
integrated viral genomes (Lampel et al., 1997). For example, some transcriptionally active genes containing introns
display little or no association with SC35 speckles (Zhang
et al., 1994
; Lampel et al., 1997
; Smith et al., 1999
),
whereas the collagen gene which has numerous introns is
associated with speckles independent of its transcriptional
activity (Xing et al., 1995
). In that respect, we cannot rule
out the possibility of a gene-specific organization of transcription sites with respect to nuclear regions enriched in
splicing factors.
Of potential interest is the observation that active hsp
transcription sites are found adjacent to the speckles
rather than colocalizing with them, suggesting that transcription and splicing activities occur preferentially at the
edges of the speckles where PFs seem to be localized as
described by electron microscopy (for review see Fakan,
1994). A similar observation has been reported for several
other genes (for review see Schul et al., 1998a
). This may
represent a mechanism to accelerate the release of nascent transcripts from the sites of transcription, which would indeed be affected if transcription and splicing were to take
place in the core of the speckle where the concentration in
RNA Pol II and processing factors may be elevated. Alternatively, this situation may simply reflect gene-specific differences in the association with speckles. Whatever the hypothesis, our data show that the distribution of a specific
active gene as adjacent or overlapping with speckles does
not rely on the presence of intronic sequences in the gene.
Why are splicing factors present at sites in the nucleus
where they are apparently not required? A first hypothesis
to explain the recruitment of splicing factors to an intronless gene is that complexes containing splicing factors
could have other functions at the site of transcription in
addition to intron excision. This suggestion is supported by
a recent work in which we have shown that full-length nascent hsp70 transcripts are retained at the site of transcription for a period of <15 min after their completion (Jolly
et al., 1998). Perhaps splicing factors could be involved in a
primary step of the transcription/splicing process to scan nascent transcripts for the presence of introns. An alternative explanation is that active transcription sites would in
fact associate with a subset of active RNA Pol II to which
splicing factors are bound. This is supported by observations that splicing factors and other mRNA processing enzymes interact transiently with the hyperphosphorylated
CTD of RNA Pol II (Mortillaro et al., 1996
; Vincent et al.,
1996
; Yuryev et al., 1996
; Cho et al., 1997
; Du and Warren,
1997
; McCracken et al., 1997a
,b; Schul et al., 1998b
). Even
in mitosis when transcription is arrested, the association between splicing factors and RNA Pol II persists (Kim et al., 1997
). At the cell biological level, at least a subset of RNA Pol II colocalizes with splicing factors within the speckles
(this paper and Thibodeau and Vincent, 1991
; Bregman
et al., 1995
; Blencowe et al., 1996
; Mortillaro et al., 1996
;
Patturajan et al., 1998
), as well as factors involved in the 5'
capping (Cho et al., 1997
; McCracken et al., 1997a
) and in
the 3' mRNA processing (Krause et al., 1994
; Schul et al.,
1998b
). The corecruitment of splicing factors and RNA
Pol II, together with the movement of genes towards speckles, could be beneficial for the cell in several ways.
First, it would accelerate the splicing reaction. Second, by
ensuring a sufficient amount of splicing factors at the site
of transcription, this system would decrease the risk of
producing unspliced transcripts which may generate aberrant and nonfunctional proteins. Third, this system would
provide a feedback control mechanism to arrest transcription if splicing is interrupted (Yuryev et al., 1996
). Fourth,
it would minimize the information quantity required to displace the different factors to the sites of transcription, since the polymerase and splicing factors would all be displaced together as a single complex. This would suggest
the existence of signals both in the transcription and processing apparatus and at the level of the chromosome and/or
within the primary transcript, which determine the recognition between active transcription sites and proteins involved in RNA biogenesis. Such targeting and/or retention
signals, if localized to the nascent transcripts, are not contained within the introns as demonstrated by our results. Identifying these signaling pathways will provide clues to
understand the mechanisms of production and trafficking
of RNAs within the nucleus.
While a growing number of observations support the
idea of a compartimentation of transcription and processing activities in the nucleus, we may still have a simplified
view of a more complex biological situation generated by
the combination of multiple factors varying in a gene-specific and/or cell type-specific manner, therefore proving to
be invalid for certain transcripts. Of particular importance
may be the chromosomal context of the gene, which could dramatically influence the supply of splicing factors to the
gene because of physical constraints (Smith et al., 1999).
We are convinced that further understanding of the complex organization of transcription and splicing activities
within the cell nucleus will come from a large scale analysis of specific endogenous genes, in particular genes with
distinctive transcriptional and splicing characteristics such
as heat shock genes or genes expressed in a tissue-specific manner.
![]() |
Footnotes |
---|
Address correspondence to Caroline Jolly, Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, 2153 North Campus Drive, Evanston, IL 60208. Tel.: (847) 491-3714. Fax: (847) 491-4461. E-mail: c-jolly{at}nwu.edu
Received for publication 23 November 1998 and in revised form 31 March 1999.
C. Jolly was supported by the Association pour la Recherche sur le
Cancer and by the Daniel F. and Ada L. Rice Foundation. R.I. Morimoto
was supported by the National Institutes of Health grant GM38109. M. Robert-Nicoud and C. Vourc'h were supported by the Université Joseph
Fourier and the Ministère de l'Enseignement Supérieur et de la Recherche (ACC-SV No. 5).
We are grateful to Sui Huang, Tom Misteli, Philip T. Moen, and David L. Spector for helpful suggestions; to Li Tai for excellent technical assistance on RT-PCR; to San Ming Wang for providing the RT-PCR method; to Pernette Verschure for help in the BrUTP experiments; to Drs. E.K. Bautz, E. Hickey, B. Sefton, J.A. Steitz, and M. Vincent for providing us with the probes and antibodies used in this study; and to Robert A. Lamb and Robert Holmgren for providing access to the light microscopy facilities.
![]() |
Abbreviations used in this paper |
---|
CTD, COOH-terminal domain; FISH, fluorescence in situ hybridization; IG, interchromatin granule; PF, perichromatin fibril; Pol II, polymerase II; snRNP, small nuclear ribonucleoprotein.
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