* Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724; MRC Human Genetics Unit, Western General
Hospital, Edinburgh, EH4 2XU, United Kingdom; and § Department of Immunology, University of Texas M.D. Anderson
Cancer Research Center, Houston, Texas 77030
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Abstract |
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Expression of most RNA polymerase II transcripts requires the coordinated execution of transcription, splicing, and 3' processing. We have previously shown that upon transcriptional activation of a gene in vivo, pre-mRNA splicing factors are recruited from nuclear speckles, in which they are concentrated, to sites of transcription (Misteli, T., J.F. Cáceres, and D.L. Spector. 1997. Nature. 387:523-527). This recruitment process appears to spatially coordinate transcription and pre-mRNA splicing within the cell nucleus. Here we have investigated the molecular basis for recruitment by analyzing the recruitment properties of mutant splicing factors. We show that multiple protein domains are required for efficient recruitment of SR proteins from nuclear speckles to nascent RNA. The two types of modular domains found in the splicing factor SF2/ ASF exert distinct functions in this process. In living cells, the RS domain functions in the dissociation of the protein from speckles, and phosphorylation of serine residues in the RS domain is a prerequisite for this event. The RNA binding domains play a role in the association of splicing factors with the target RNA. These observations identify a novel in vivo role for the RS domain of SR proteins and suggest a model in which protein phosphorylation is instrumental for the recruitment of these proteins to active sites of transcription in vivo.
Key words: nucleus; phosphorylation; pre-mRNA splicing; recruitment; transcription ![]() |
Introduction |
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EFFICIENT gene expression in vivo requires the coordinated execution of multiple nuclear processes,
including transcription and pre-mRNA splicing.
Upon transcription, heterogeneous nuclear ribonucleoproteins and pre-mRNA splicing factors rapidly associate with the elongating transcript. Excision of introns is then
catalyzed by the assembled spliceosome and in many cases
occurs cotranscriptionally (Sass and Pederson, 1984; Beyer
and Osheim, 1988
; Matunis et al., 1993
; Kiseleva et al.,
1994
). Biochemical evidence for tight coupling of transcription and pre-mRNA splicing includes the physical interaction of the COOH-terminal domain (CTD)1 of the
large subunit of RNA polymerase II (pol II) with pre-mRNA splicing factors and the finding that splicing is severely inhibited in cells that contain a form of the large
subunit of RNA pol II with a reduced CTD (Mortillaro et
al., 1996
; Vincent et al., 1996
; Yuryev et al., 1996
; Corden
and Patturajan, 1997
; Kim et al., 1997
; McCracken et al.,
1997
). Morphological evidence for spatial and temporal
coupling of the two processes is provided by electron and
fluorescence microscopy studies, which have demonstrated
cotranscriptional splicing intermediates and the presence
of spliceosomes on nascent transcripts (Beyer and Osheim, 1988
; Báuren and Wieslander, 1994
; Kiseleva et al.,
1994
).
Virtually all proteins involved in pre-mRNA splicing are
enriched in numerous nuclear compartments, or speckles,
in addition to their diffuse distribution throughout the nucleoplasm (for review see Spector, 1993). Some splicing
factors are also found in the coiled body, a nuclear body of
unknown function (Lamond and Carmo-Fonseca, 1993
).
Morphological examination of speckles by electron microscopy reveals that they consist of two distinct structures: granules 20-25 nm in diameter that are clustered in the interchromatin space and hence are termed interchromatin granule clusters (IGCs), and fibrils ~5 nm in diameter, termed perichromatin fibrils (PFs), often extend from
the periphery of IGCs and are also found dispersed
throughout the nucleoplasm (Fakan, 1994
). Nucleotide
incorporation experiments and gene mapping studies showed that sites of active transcription are found dispersed throughout the nuclear volume and that the majority of actively transcribing genes are found within PFs and
are largely excluded from IGCs (Jackson et al., 1993
, 1998
;
Wansink et al., 1993
; Fakan, 1994
; Xing et al., 1995
). This
is supported by fluorescence microscopy data, which have
placed the position of numerous genes at the periphery of
or between speckles (Huang and Spector, 1991
; Xing et al.,
1995
; Dirks et al., 1997
). These observations indicate that
IGCs are not sites of active transcription and thus not sites of cotranscriptional splicing. It has been proposed that
IGCs are storage/reassembly sites for pre-mRNA splicing
factors and that splicing factors are recruited from these
compartments to sites of active transcription (Jiménez-García and Spector, 1993
; for review see Misteli and Spector, 1998
). Recent observations using time-lapse experiments in living cells support this model. Upon activation of
a gene, pre-mRNA splicing factors were seen to be released from speckles and to be redistributed to sites of active transcription in a recruitment process (Misteli et al., 1997
). This recruitment process might ensure the sufficient
supply of splicing factors to sites of active transcription
and thus serve as a means to spatially coordinate transcription and pre-mRNA splicing in vivo (Jiménez-García and
Spector, 1993
; Huang and Spector, 1996
; Neugebauer and
Roth, 1997
; for review see Misteli and Spector, 1998
). The
molecular basis of splicing factor recruitment to nascent
RNAs has been unknown.
One prominent component of nuclear speckles is the
family of SR proteins (for reviews see Fu, 1995; Manley
and Tacke, 1996
). SR proteins are essential for constitutive splicing in vitro and can influence alternative splice
site selection in a concentration-dependent manner (for
review see Horowitz and Krainer, 1994
). Classical SR proteins contain a COOH-terminal domain rich in ser/arg-dipeptides (RS domain) and either one or two RNA-recognition motifs (RRMs). In addition, a number of SR-like
proteins have been described that contain at least one RS
domain but differ from classical SR proteins in their overall domain structure (for review see Fu, 1995
). The RS domain has been suggested to contain a targeting signal to
mediate localization of these proteins to speckles. In the
Drosophila protein Tra2, the RS domain, and specifically a short peptide sequence within the domain, is necessary
and sufficient for the localization of the protein to speckles
(Li and Bingham, 1991
; Hedley et al., 1995
). In mammalian SR proteins, the RS domain contributes to their localization but is not always necessary, and only in some cases
(for example SRp20) is it sufficient for proper subnuclear
localization (Cáceres et al., 1997
; Gama-Carvalho et al.,
1997
). SR proteins are phosphoproteins, and several kinases that specifically phosphorylate serine residues in the
RS domain and a protein phosphatase 1 (PP1) activity that
dephosphorylates SR proteins have recently been identified (Meyrand et al., 1993
; Gui et al., 1994
; Mermoud et
al., 1994
; Colwill et al., 1996b
; Misteli and Spector, 1996
;
Rossi et al., 1996
; Duncan et al., 1998
; Kuroyanagi et al.,
1998
; Wang et al., 1998
; for review see Misteli and Spector
1998
).
Here we have investigated the molecular mechanism by which SR proteins are recruited from nuclear speckles to active sites of transcription in living cells upon activation of a nearby gene. We find that all modular domains of SR proteins are involved in the efficient recruitment and that different types of domains exert distinct functions in the recruitment process. Specifically, we show that phosphorylation of serine residues in the RS domain is required for the efficient recruitment of SR proteins to a site of active transcription in vivo.
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Materials and Methods |
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Epitope-tagged Expression Plasmids
SF2/ASF, SC35, SRp20, and SRp40 deletion mutants in the pCG-T7 expression vector have been described (Cáceres et al., 1997). RG and GS replacement mutants in the same vector have been described (Cáceres and
Krainer, 1993
). GFP-SF2/ASF-
RS was generated by cutting GFP-SF2/
ASF with ApaI and religation. GFP-SF2/ASF-RG and -GS were generated by replacing a SacII-BamHI fragment in GFP-SF2/ASF-WT with the
corresponding fragment from pCG-T7-SF2/ASF-RG or -GS mutants, respectively.
Cell Lines, Transfections, and Western Blotting
HeLa cells stably expressing the rat homeobox genepem were generated
by cotransfection of pPem-89 containing the tetracycline promoter followed by the genomic pem sequence including the 3' UTR (Maiti et al.,
1996), the plasmid EV-124 containing the tetracycline transactivator, and
the neomycin gene for selection (Gossen and Bujard, 1992
). HeLa cell
clones that exhibited highly repressible expression of pem RNA by addition of 1 µg/ml tetracycline were selected in 1 mg/ml G418, and colonies
were screened by Northern blot analysis. Pem-HeLa cells were routinely
grown in DME (GIBCO BRL, Gaithersburg, MD) supplemented with
10% FCS (Hyclone Labs, Logan, UT) in the presence of 0.4 mg/ml G418
(GIBCO BRL) and transfected as described in Huang and Spector (1996)
.
SR protein mutants and a
-tropomyosin minigene (Helfman et al., 1988
)
were transfected by electroporation of 7 µg DNA and 13 µg of salmon
sperm DNA at 240 V using a Bio-Rad gene pulser (Hercules, CA). For
Western blotting, cell lysates were prepared at 14 h posttransfection and
blotted as described in Misteli and Spector (1996)
. Monoclonal mouse
anti-SF2/ASF antibody recognizing RRM1 of SF2/ASF (Hanamura et al.,
1998
) was used for Western blotting at 1:5. For analysis of the phosphorylation state of proteins, lysates were treated for 30 min at 37°C with 500 U/ml alkaline phosphatase (New England Biolabs, Beverly, MA).
In Situ Hybridization and Indirect Immunofluorescence
HeLa cells carrying the stably integrated rat pem gene were fixed at room
temperature in freshly made 2% paraformaldehyde, 0.5% Triton in PBS
for 5 min, followed by fixation in 2% paraformaldehyde in PBS for 15 min
(Spector et al., 1998). For detection of target DNA, coverslips were
washed twice in PBS for 5 min and incubated for 90 min at 37°C in 50 µl of
100 µg/ml RNAse A (Boehringer Mannheim, Indianapolis, IN) in PBS on
a glass slide. Coverslips were washed three times in PBS for 5 min each,
followed by a rinse in 2× SSC and two 10 min washes in 2× SSC at room
temperature. Immediately before addition of the hybridization cocktail,
cells were denatured in 70% formamide, 2× SSC for 7 min at 78°C. For
detection of RNA, the RNAse step and the denaturation step were omitted. Nick translated probe representing the entire integrated pem sequence, generated as described in Langer et al. (1981)
in 2× SSC, 10%
dextran sulfate, and 1 mg/ml yeast tRNA, was added in a total volume of
20 µl to the coverslip, sealed with rubber cement, and incubated overnight at 37°C. Coverslips were washed in 2× SSC three times for 15 min at room
temperature and once for 10 min in 4× SSC at room temperature. The
probe was detected by incubation for 60 min at room temperature with
avidin-FITC (2.5 µg/ml) in 4× SSC. Coverslips were then washed four
times for 15 min in 4× SSC. After detection of the probe, cells were
washed twice in PBS for 5 min each, and indirect immunofluorescence was performed as described in Misteli and Spector (1996)
without additional fixation or permeabilization steps. Anti-T7 monoclonal antibody
(Novagen, Madison, WI) was used at 1:400 in PBS, anti-SC35 antibody
(Fu and Maniatis, 1990
) at 1:800, anti-SF2/ASF antibody (Hanamura et
al., 1998
) at 1:50. Fluorescently labeled secondary antibodies and avidin-FITC were from Cappel (Durham, NC).
Microscopy
Images were acquired either on a Nikon FXA microscope (Melville, NY)
equipped with a Photometrics SenSys cooled CCD camera (1,320 × 1,035 array; 6.7-µm pixel size) using Oncor Image 2.0.5 (Oncor, Gaithersburg,
MD) or on a Zeiss LSM410 confocal laser scanning microscope (Thornwood, NY) using simultaneous scans to avoid shift between the two optical channels. Time-lapse microscopy of living cells was performed as described (Misteli et al., 1997). In brief, cells at 14 h after transfection were
grown in a FCS2 live-cell microscopy chamber (Bioptechs, Butler, PA) at
37°C and supplied with fresh DMEM medium free of phenol red (GIBCO
BRL, Gaithersburg, MD), supplemented with 10% FCS. The FCS2 chamber was mounted on a Zeiss Axiovert 405M inverted fluorescence microscope equipped with a Photometrics Nu200 cooled CCD camera (1,320 × 1,035 array; 6.7-µm pixel size). For routine observation, a 100×/N.A. 1.3 oil immersion lens was used. Images were acquired using Oncor Image
2.0.5 software. Exposure times were 0.2-0.8 s and a GFP filter (Chroma
Technology, Brattleboro, VT) was used. Images were pseudocolored using the standard Oncor Image 2.0.5 256-level pseudocolor look-up table.
The spectrum of pseudocolors represents the intensities of the fluorescence signal measured for each pixel (gray level 0 in blue, gray level 255 in
red), and speckles appear in red. Line profiles were obtained from unprocessed images with no saturated pixels on a 256-grey value scale using
Oncor Image 2.0.5.
Recruitment Assay in Living Cells
BK virus-transformed hamster fibrosarcoma BKT-1B cells (Moens et al.,
1990) were grown and transcription induced as described (Misteli et al.,
1997
). In situ hybridization using nick translated biotinylated probes for
the full-length BK virus DNA was performed as described (Misteli et al.,
1997
). Images of a particular series were accurately aligned using morphological markers such as shape of nuclei, position of nucleoli, and occasionally cytoplasmic morphological features.
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Results |
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Effect of Deletion Mutations on the Recruitment Behavior of SR Proteins In Vivo
To gain insight into the molecular mechanisms of recruitment of pre-mRNA splicing factors, we tested the ability
of deletion mutants of SR proteins to be recruited to a
newly formed site of transcription in vivo. We used SR
protein mutants, which have previously been characterized with respect to their subnuclear localization and their
function in alternative splice site selection in vitro and in
vivo (Cáceres and Krainer, 1993; Cáceres et al., 1997
).
HeLa cells carrying a stably integrated copy of the rat homeobox gene, pem, were used as an assay system (Maiti et al., 1996
). In this cell line, the pem gene, which contains
four spliced introns, is under the control of the inducible
tetracycline promoter (Gossen and Bujard, 1992
). To assess the recruitment ability of a protein, wild-type or mutant splicing factors tagged with the T7 epitope were expressed for 14 h in the absence of pem transcription. The
pem locus was then activated for 4 h, and pem RNA was
detected by in situ hybridization. Splicing factors were visualized by virtue of the epitope tag. Recruitment of splicing factors to the induced site of transcription was indicated by the colocalization of the in situ hybridization
signal and the immunofluorescence signal.
In the absence of pem expression, the pem locus was not
associated with nuclear speckles and was typically found
between speckles in greater than 95% of cells (Fig. 1).
Upon activation of the pem locus, recruitment of endogenous splicing factors (SF2/ASF, U2-B, SC35) from speckles to the new site of transcription was readily detected
(Fig. 2 A, a-c; data now shown). Recruitment was visualized by the localization of SF2/ASF (Fig. 2 A, a), detected with a specific monoclonal antibody against SF2/ASF (Hanamura et al., 1998
), at the site of the pem RNA signal
(Fig. 2 A, b). This is in agreement with observations by
time-lapse microscopy in living cells that showed that transiently transfected SF2/ASF is efficiently recruited from
speckles to an active transcription site upon activation of
stably integrated genes (Misteli et al., 1997
). Recruitment
is a rapid process, and accumulation of SF2/ASF at the site
of transcription is detectable within 10-20 min after activation of the target genes. Just like endogenous SF2/ASF, transiently expressed full-length SF2/ASF (Fig. 2 A, d-f)
was recruited to the induced site of pem transcription, as
indicated by the overlap of the two signals. Consistent with
the observation that active genes frequently localize to the
periphery of speckles (Huang and Spector, 1991
; Xing et
al., 1995
; Dirks et al., 1997
), in both cases the spots containing the RNA and splicing factor signals were typically
closely associated with the periphery of a larger speckle
(Fig. 2 A, a and d, insets). Identical results were obtained
using conventional wide-field fluorescence or confocal microscopy (data not shown).
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We tested the effect of deletion of either the RS domain or one of the two RRMs of SF2/ASF on the recruitment of the protein to the pem RNA. Deletion of either RRM1 (Fig. 2 A, g-i), RRM2 (Fig. 2 A, j-l), or the RS domain (Fig. 2 A, m-o) prevented accumulation of the mutant protein at the site of induced pem transcription. The pem RNA was now typically found as a distinct spot from speckles and was free of accumulated mutant splicing factor (Fig. 2 A, g, j, and m, insets). The presence or absence of splicing factors at the site of pem RNA was verified by fluorescence intensity measurements along a test-line through the site of pem RNA accumulation and a nearby speckle (Fig. 2 B). In the case of endogenous SF2/ASF and transiently expressed wild-type SF2/ASF, the peaks of the two signals coincided. In contrast, for the deletion mutants the peaks for the pem RNA signal and the mutant splicing factor signal were clearly separated (Fig. 2 B). Quantitation of the number of cells that showed colocalization of splicing factor and the pem RNA demonstrated that endogenous SF2/ASF and wild-type SF2/ASF accumulated in more than 90% of cells at the site of pem RNA, whereas the deletion mutants accumulated in ~30% of cells with pem RNA (Fig. 3). This latter number most likely reflects the random association of the pem transcription site with speckles in the mammalian cell nucleus.
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The RS domains of various members of the SR protein
family all contain multiple arg/ser-dipeptides but are significantly distinct from each other at the amino acid level
(for review see Fu, 1995). We tested whether the RS domain of several SR proteins was required for their recruitment. As for SF2/ASF, deletion of the RS domain in
SRp40, SRp20, or SC35 prevented the mutant proteins
from accumulating at transcription sites (Fig. 3). Furthermore, none of the single RRMs or RS domains was sufficient for recruitment (Fig. 3 and data not shown), indicating that the requirement for recruitment was not strictly
amino acid sequence specific, but might be caused by the
charge distribution within the domain. The lack of recruitment of all mutants tested was not due to the impaired
function of the proteins since they have been shown to be
functionally active in alternative splice site selection in
vivo (Cáceres et al., 1997
).
Evidence for Distinct Roles for the RS Domain and the RRM in the Recruitment Process
Evidence for distinct functions for the RS domain versus
the RRMs of SF2/ASF was obtained in an assay in which
recruitment of SR proteins to a transiently expressed
intron-containing -tropomyosin (
-TM) minigene was
measured. This assay was previously used to demonstrate
that recruitment is dependent on the presence of an intron
in the target RNA (Huang and Spector, 1996
). Similar to the situation during intermediate stages of adenoviral infection, transient overexpression of the
-TM minigene
produces large amounts of pre-mRNA, and as a consequence, the majority of endogenous splicing factors dissociate from speckles and associate with sites of
-TM transcription, resulting in the depletion of splicing factors
from native speckles (Jiménez-García and Spector, 1993
; Pombo et al., 1994
; Huang and Spector, 1996
; Gama-Carvalho et al., 1997
). Under these conditions, transiently expressed full-length SF2/ASF (Fig. 4 A, red) was strongly
recruited to active sites of
-TM expression (Fig. 4 A,
green). In contrast, deletion of either the first or second
RRM in SF2/ASF resulted in the diffuse distribution of
the mutant splicing factor throughout the cell nucleus (Fig.
4 B, red) and prevented significant accumulation of the
mutant protein at sites of
-TM transcription (Fig. 4 B, green). This was most likely due to the weakened interaction of SF2/ASF with
-TM RNA in the absence of either
one of its two RRMs (Cáceres and Krainer, 1993
; Wu and
Maniatis, 1993
; Zuo and Manley, 1993
). Note that the yellow signal in the overlay panel is caused by the diffuse distribution of the mutant protein and does not represent
specific accumulation at transcription sites. In contrast,
SF2/ASF lacking the RS domain was largely retained in
speckles that were frequently (~60%) observed to be distinct from sites of
-TM-transcription (Fig. 4 C, arrows).
The presence of speckles distinct from
-TM transcription
sites indicates that this mutant was less efficiently displaced from endogenous speckles. These observations suggest that in the absence of either one of the RRMs, the
mutant protein was released from speckles but could not
efficiently bind the target RNA, whereas in the absence of the RS domain, the mutant protein was not efficiently released. Thus, we propose that the RS domain and the
RRMs play distinct roles in the recruitment process in
vivo.
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The RS Domain Is Required for Recruitment in Living Cells
To further analyze the role of the RS domain in vivo, we
tested the effect of deletion of the RS domain on recruitment of SF2/ASF in living cells using a previously characterized time-lapse microscopy assay (Misteli et al., 1997).
BKT-1B cells carry stably integrated cAMP-inducible
early genes of BK virus (Moens et al., 1990
), and recruitment to the induced transcription site of BK early genes
can be monitored in individual living cells using SF2/ASF
fused to the green fluorescent protein (GFP) (Misteli et al.,
1997
). As previously demonstrated, upon stimulation of BKV early genes, GFP-SF2/ASF was recruited from speckles and accumulated at the site of transcription (Fig. 5, top,
arrow) (Misteli et al., 1997
). The accumulation is detected
as an area of higher GFP-SF2/ASF concentration (Fig. 5,
red; see figure legend) in the form of a peripheral extension protruding from a nearby speckle (Misteli et al.,
1997
). The extension typically forms gradually upon addition of cAMP after an initial lag period of about 10 min
and peaks at the time of maximal gene expression, in the
case of immediate early genes of BK virus at ~60 min
(Moens et al., 1990
; Misteli et al., 1997
). In contrast, in the
absence of the RS domain, SF2/ASF was not recruited to
the site of transcription (Fig. 5, bottom, arrow). No peripheral extension could be seen at any time during the induction time of immediate early genes of BK virus, and no accumulation of the splicing factor mutant was detected at
the site of transcription (Fig. 5, bottom). This observation
shows that the RS domain is required for the efficient recruitment of splicing factors to transcription sites in living cells.
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Phosphorylation of Serine Residues in the RS Domain Is Required for Efficient Recruitment
A hallmark of the RS domain is its significant posttranslational phosphorylation on serine residues (Roth et al.,
1991; Gui et al., 1994
; Colwill et al., 1996b
). To test
whether phosphorylation of the RS domain is involved in
recruitment to transcription sites, we used mutants of SF2/
ASF in which the RS-dipeptides in the RS domain had
been replaced by either RG- or GS-dipeptides (Fig. 6 A)
(Cáceres and Krainer, 1993
). Western blot analysis of total cell lysates showed that wild-type SF2/ASF was efficiently
phosphorylated in vivo in BHK cells as judged by a shift in
mobility after alkaline phosphatase treatment (Fig. 6 B).
This is in agreement with the recent finding that in vivo
endogenous SF2/ASF is predominantly found in a highly
phosphorylated form (Hanamura et al., 1998
). A similar
shift was observed for SF2/ASF-GS, demonstrating that SF2/ASF-GS was efficiently phosphorylated in vivo (Fig. 6
B). In contrast, deletion of the RS domain prevented all
detectable phosphorylation of the protein (Fig. 6 B) as has
been predicted by phospho-epitope mapping studies which
demonstrated that all phosphorylation sites in SF2/ASF
map to the RS domain (Colwill et al., 1996b
). Similarly,
the RG mutant was not phosphorylated in BHK cells and
migrated identically in the presence or absence of alkaline
phosphatase treatment (Fig. 6 B).
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The ability of these mutants to be recruited to nascent
RNA was then assessed in living cells using the BKT system. The recruitment ability of the mutant proteins in living cells correlated with the phosphorylation state of the
RS domain (Fig. 7). SF2/ASF-GS, like SF2/ASF-WT, was
efficiently recruited to sites of transcription (Fig. 7) as
shown by the formation of a peripheral extension from a
nearby speckle and the accumulation of the mutant protein at the site of transcription. The time course of accumulation of the mutant protein was indistinguishable from
the wild-type protein. In contrast, the RG mutant was retained in speckles after stimulation of transcription of BK
early genes (Fig. 7, bottom) and was not recruited to the
induced genes. This behavior was identical to the RS mutant (see Fig. 5), and no accumulation of splicing factors was observed at any time. These observations demonstrate
that serine phosphorylation of the RS domain is required
for the recruitment of SR proteins from nuclear speckles
to transcription sites in vivo.
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Discussion |
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We have probed the molecular mechanism by which pre-mRNA splicing factors are recruited from nuclear speckles to nascent RNAs in vivo. In a stable, inducible gene expression system, deletion of any single modular domain in
four SR proteins tested prevented efficient recruitment to
the nascent RNA. Furthermore, in a transient transfection
assay in which large amounts of target RNA are produced, we found that the different types of domains play distinct
roles in recruitment. Under these conditions, deletion of a
single RRM in SF2/ASF resulted in the diffuse distribution of the mutant protein in the cell nucleus in the absence of any accumulation at sites of transcription. In contrast, deletion of the RS domain resulted in a protein that
was largely retained in nuclear speckles when challenged
with high levels of -TM RNA. The simplest interpretation of these observations is that recruitment of SR proteins from nuclear speckles to a site of transcription is a
process involving at least two steps, namely the release of
splicing factors from speckles, mediated by the RS domain, followed by their association with the target RNA,
mediated by the RRMs. The role of the RRMs in associating with the target RNA is consistent with the well-documented role of the RRMs in binding to RNA (for review
see Fu, 1995
). The role of the RS domain in the release
from speckles is consistent with the observed effect of deletion of the RS domain on subnuclear distribution of RS
domain-containing proteins. In SRp20 and SC35, both of
which contain only one RRM, the RS domain determines
the subnuclear localization (Cáceres et al., 1997
). The RS
domain is necessary for localization to speckles and is sufficient to target a cytoplasmic reporter protein to nuclear
speckles (Cáceres et al., 1997
). Similarly, in the SR-like
Drosophila proteins Tra and SWAP, the RS domain is sufficient for speckled localization (Li and Bingham, 1991
;
Hedley et al., 1995
). These data argue that the RS domain
can control the association/dissociation properties of SR
proteins with speckles. In support of this idea, we have observed that the RS domain of SRp20 is sufficient for the
release of a reporter protein from speckles upon transient
expression of
-TM RNA (Misteli, T., and D.L. Spector,
unpublished observation). In SF2/ASF, and also SRp40,
association of the protein with nuclear speckles is not dependent on the RS domain alone since deletion of the RS
domain only reduces the level of the protein in speckles but does not completely prevent its association (Cáceres et
al., 1997
). It is likely that for proteins that contain multiple
RRMs, such as SF2/ASF, SRp40, or U2AF65, the speckle
localization is additionally mediated by protein-protein or
protein-RNA interactions occurring through the RRMs,
possibly via snRNPs or other RS domain-containing proteins (Cáceres et al., 1997
; Gama-Carvalho et al., 1997
).
An example of such an indirect localization is the Tra protein lacking its RS domain. This mutant localizes to speckles provided that it can interact with RS domain-containing proteins such as Tra2, but deletion of the interacting
domain displaces the protein from speckles (Hedley et al.,
1995
). Thus, the dissociation and association of RS
domain-containing proteins might be mediated by two
distinct mechanisms (see also Cáceres et al., 1997
). Our
observations suggest that the RS domain functions in recruitment in the context of SR proteins. However, the recruitment function might be a more general feature of the
RS domain in the context of other proteins as well. For example, in U2AF65, an SR-like protein not belonging to the
classical SR family of proteins, the NH2-terminal RS domain is required for recruitment to adenoviral transcripts,
while the RRMs are not required for this event (Gama-Carvalho et al., 1997
).
A trivial explanation for our observations would be that
the recruitment properties of the proteins are the consequence of their functional inactivation as a result of the
deletions. This is unlikely, since no correlation between
function of the protein and recruitment behavior has been
observed. In vivo and in vitro, wild-type SF2/ASF strongly
activates the use of the 13-S 5' splice site in the adenoviral
E1A transcript in a concentration-dependent manner (Cáceres and Krainer, 1993; Zuo and Manley, 1993
; Cáceres et
al., 1997
). The same is true for SF2/ASF lacking the RS
domain or lacking the first RRM. However, in our recruitment assay the wild-type protein was efficiently recruited,
while a mutant lacking the RS domain was not, suggesting
that the recruitment behavior does not simply reflect the
functional properties of the protein. It is not clear at
present how a mutant protein, such as SF2/ASF-
RS, is
able to influence alternative splice site selection, although it is apparently not recruited to the target RNA. One possibility is that a steady-state level of protein below our detection limit is sufficient to affect splice site selection. Alternatively, it is possible that overexpression of the mutant
protein affects the choice of splice sites indirectly.
Because the majority of active transcription sites are localized at the periphery of or outside of speckles (Wansink
et al., 1993; Zhang et al., 1994
; Xing et al., 1995
; Neugebauer and Roth, 1997
), dissociation of splicing factors
from speckles is a requirement for efficient pre-mRNA
splicing in vivo. Our results from living cell experiments
extend observations that imply a function for phosphorylation in the dissociation of SR proteins from nuclear speckles. Several lines of evidence now point to a role of
phosphorylation of serine residues in the RS domain as a
switch for the dissociation of splicing factors from nuclear
speckles. First, we find that in living BHK cells, SF2/ASF
molecules lacking either the RS domain or the serine
phosphorylation sites in the RS domain were not observed
to dissociate from speckles upon transcriptional activation of a nearby gene in contrast to the wild-type protein (Fig.
7) (Misteli et al., 1997
). Second, we observe that in the absence of the RS domain, SF2/ASF was retained in nuclear
speckles distinct from sites of
-TM transcription, whereas
deletion of either RRM resulted in the diffuse nuclear distribution of the mutant protein (Fig. 4). Third, in living interphase cells, the dynamic movements of splicing factors
at the periphery of speckles have been interpreted to represent the continuous movement of splicing factors from speckles to nearby active genes as factors are being recruited (Misteli et al., 1997
). In agreement with a role of
phosphorylation in the release of factors, these movements cease upon addition of protein kinase inhibitors
(Misteli et al., 1997
). Fourth, several kinases, SRPK-1, -2 and Clk-1, -2, -3, and topoisomerase I, which specifically
phosphorylate SR proteins, have recently been identified (Gui et al., 1994
; Colwill et al., 1996b
; Rossi et al., 1996
; Duncan et al., 1997
; Kuroyanagi et al., 1998
; Wang et al.,
1998
). Overexpression of SRPKs or Clks results in the redistribution of splicing factors from a speckled pattern to a
diffuse nuclear pattern and an increase in the solubility of
splicing factors. Fifth, a ser/thr phosphatase I activity has
been identified based on its ability to prevent the association and accumulation of splicing factors in enlarged
speckles upon inhibition of transcription, suggesting that
dephosphorylation of SR proteins facilitates their association with this nuclear compartment (Misteli and Spector, 1996
). Finally, our in vivo results also correlate well with in vitro data on the effect of phosphorylation on splicing factor function. In vitro analyses demonstrate that phosphorylation of SR proteins is a prerequisite for their efficient incorporation into spliceosomes and for their proper
functioning in the splicing process (Tazi et al., 1993
; Mermoud et al., 1994
; Cao et al., 1997
; Xiao and Manley,
1997
). Phosphorylation of SR proteins would thus be expected to occur before the association of SR protein with
the nascent RNA, which is thought to occur outside of nuclear speckles.
Control of the release of splicing factors from nuclear
speckles might be important to ensure the sufficient supply of splicing factors to transcription sites. In addition, it
might also play a role in regulation of alternative splice
site selection. In vitro and in vivo, the choice of splice site
is determined by the ratio of multiple splicing factors (for
review see Horowitz and Krainer, 1994). Controlling the
release of factors from nuclear compartments might be a
way to modify the relative concentrations of multiple factors in the nucleoplasm and thus modulate the choice of
splice site. In support of this interpretation, overexpression of Clk kinases causes a switch of splice sites in several
RNA templates in vivo (Duncan et al., 1997
, 1998
). The
recently identified SR protein kinases are good candidates
to mediate the release of SR proteins from speckles. However, the large number of serine residues in the RS domain
and the overlapping substrate specificities of the identified
kinases makes it difficult to determine which serine residues, or combinations thereof, are important for the proper function and to address the precise roles of the kinases in vivo (Colwill et al., 1996a
; Labourier et al., 1998
;
Wang et al., 1998
).
These observations can be summarized in a working
model for the molecular mechanism of splicing factor recruitment (Misteli and Spector, 1997). In this scenario,
splicing factors reside in nuclear speckles, predominantly
IGCs, and phosphorylation of serine residues in the RS
domain by SR protein kinases has two effects: (a) splicing
factors are displaced from speckles either individually or
complexed with other factors of the RNA-processing machinery (Gui et al., 1994
; Colwill et al., 1996b
; Duncan et al.,
1998
; Wang et al., 1998
) and (b) splicing factors become "active," i.e., they can be incorporated into the spliceosome (Mermoud et al., 1992
, 1994
; Cao et al., 1997
). During or after completion of the splicing reaction, which
takes place in PFs outside of IGCs, the proteins are dephosphorylated (Mermoud et al., 1992
, 1994
; Misteli and
Spector, 1996
), allowing them to return to the IGCs, where
a new round of the phosphorylation cycle starts (see Misteli and Spector, 1998
). We suggest that this arrangement provides a means to control the supply of active splicing
factors at sites of transcription and, thus, is instrumental in
the spatial coordination of transcription and pre-mRNA
splicing within the mammalian cell nucleus.
![]() |
Footnotes |
---|
Received for publication 21 July 1998 and in revised form 4 September 1998.
T. Misteli dedicates this paper to the memory of Thomas Kreis.
T. Misteli was supported by the Human Frontiers Science Program and
the Roche Research Foundation. A.R. Krainer was supported by National
Cancer Institute grant CA13106, M.F. Wilkinson was supported by National Institutes of Health (NIH) grant GM39586 and National Science
Foundation grant MCB-9307963, and D.L. Spector was supported by NIH
grant GM42694.
Address all correspondence to D.L. Spector, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724. Tel.: (516) 367-8456. Fax: (516) 367-8876. E-mail: spector{at}cshl.org
We thank members of the Krainer and Spector laboratories for discussion and critical reading of the manuscript.
![]() |
Abbreviations used in this paper |
---|
-TM,
-tropomyosin;
BKV, BK virus;
CTD, COOH-terminal domain;
GFP, green fluorescent protein;
IGC, interchromatin granule cluster;
PF, perichromatin fibril;
Pol II, RNA polymerase II;
PP1, protein phosphatase 1;
RRM, RNA recognition motif.
![]() |
References |
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