* Division of Cellular and Molecular Medicine, Department and School of Medicine, University of California, San Diego, La
Jolla, California 92093-0651; and Department of Molecular and Cell Biology, Harvard University, Cambridge, Massachusetts
02138
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The SR superfamily of splicing factors and
regulators is characterized by arginine/serine (RS)-rich
domains, which are extensively modified by phosphorylation in cells. In vitro binding studies revealed that RS
domain-mediated protein interactions can be differentially affected by phosphorylation. Taking advantage of
the single nonessential SR protein-specific kinase
Sky1p in Saccharomyces cerevisiae, we investigated RS
domain interactions in vivo using the two-hybrid assay.
Strikingly, all RS domain-mediated interactions were
abolished by SKY1 deletion and were rescuable by
yeast or mammalian SR protein-specific kinases, indicating that phosphorylation has a far greater impact on
RS domain interactions in vivo than in vitro. To understand this dramatic effect, we examined the localization
of SR proteins and found that SC35 was shifted to the
cytoplasm in sky1 yeast, although this phenomenon
was not obvious with ASF/SF2, indicating that nuclear
import of SR proteins may be differentially regulated by phosphorylation. Using a transcriptional repression
assay, we further showed that most LexA-SR fusion
proteins depend on Sky1p to efficiently recognize the
LexA binding site in a reporter, suggesting that molecular targeting of RS domain-containing proteins within
the nucleus was also affected. Together, these results reveal multiple phosphorylation-dependent steps for SR
proteins to interact with one another efficiently and
specifically, which may ultimately determine the splicing activity and specificity of these factors in mammalian cells.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
PRE-mRNA splicing takes place in an RNA machine
known as the spliceosome, which consists of small
nuclear ribonucleoprotein particles (snRNPs)1 and
non-snRNP protein factors. The RNA components in the
spliceosome form the catalytic core through a series of dynamic RNA-RNA interactions which are likely to be mediated and/or stabilized by non-snRNP protein factors (for
recent reviews see Nilsen, 1998; Staley and Guthrie, 1998
).
Among the best characterized non-snRNP splicing factors
are SR proteins which contain one or two RNA recognition motifs and a signature RS domain containing multiple
serine and arginine repeats (for reviews see Fu, 1995
;
Manley and Tacke, 1996
). The RNA recognition motifs
bind to RNA sequences in a coordinated fashion to determine splicing specificity (Chandler et al., 1997
) and commit pre-mRNA substrates to the splicing pathway (Fu,
1993
), whereas the RS domains mediate specific protein-
protein interactions in a number of spliceosomal assembly
steps (Wu and Maniatis, 1993
; Kohtz et al., 1994
; Roscigno
and Garcia-Blanco, 1995
; Tronchère et al., 1997
). In fact,
the RS domain is present in a large number of proteins,
many of which are proven splicing factors and/or regulators (for review see Fu, 1995
).
SR proteins are especially interesting because their
functions in splice site selection and spliceosome assembly
appear to be regulated by phosphorylation. In vitro, phosphorylation of RS domains can enhance the interactions
between RS domain-containing proteins (Xiao and Manley, 1997; Wang et al., 1998
) and prevent their nonspecific
binding to RNA (Tacke et al., 1997
), which may explain
the requirement of phosphorylated SR proteins to initiate spliceosome assembly at early steps (Mermoud et al., 1994
;
Cao et al., 1997
). On the other hand, dephosphorylation
appears to be required for the resolution of the spliceosome to produce spliced products (Tazi et al., 1993
; Mermoud et al., 1994
; Cao et al., 1997
). Thus, the phosphorylation-dephosphorylation cycle of RS domains appears to
accompany, and likely regulate, the splicing cycle.
The impact of phosphorylation on SR protein function
in vivo is further manifested by the localization and intracellular trafficking of splicing factors in mammalian cells.
Most splicing factors are unevenly distributed in the nucleus, giving rise to a speckled appearance in all somatic
animal cells examined to date (for review see Spector,
1993). Although nuclear speckles contain polyadenylated RNA (Xing et al., 1993
) and are associated with some actively transcribing genes (Xing et al., 1995
), most splicing
reactions appear to take place cotranscriptionally in the
nucleoplasm and, therefore, splicing factors need to be recruited to sites of transcription (Misteli et al., 1997
, 1998
).
Importantly, this intranuclear movement of splicing factors appears to be mediated by RS domain phosphorylation (Gui et al., 1994a
; Colwill et al., 1996a
; Cáceres et al.,
1997
; Misteli et al., 1997
, 1998
; Duncan et al., 1998
). Because SR proteins are known to affect splice site selection
in a concentration-dependent manner, such phosphorylation-dependent localization of splicing factors among different nuclear pools may be a mechanism to regulate alternative splicing. Indeed, it was demonstrated recently that alternative splicing in vivo can be modulated by phosphorylation, presumably through modifying SR proteins
(Duncan et al., 1997
; Kanopka et al., 1998
). These observations illustrate the importance of understanding the
function and regulation of SR proteins by specific kinases
in cells.
Mammalian cells express several SR protein-specific kinases, of which the best characterized are the SRPK and
Clk/Sty families of kinases (Gui et al., 1994a; Colwill et al.,
1996a
b; Duncan et al., 1998
; Kuroyanagi et al., 1998
; Wang
et al., 1998
). These two kinase families are structurally distinct, exhibit different substrate specificities, and are differentially expressed (Gui et al., 1994b
; Colwill et al.,
1996b
; Wang et al., 1998
). The SRPK and Clk/Sty kinases
are also differentially localized in cells: SRPKs are present
in both the cytoplasm and the nucleus (Wang et al., 1998
),
whereas Clk/Sty kinases appear to localize exclusively in
the nucleus (Colwill et al., 1996a
; Duncan et al., 1998
). The
existence of multiple SR protein kinases with distinct substrate specificity and localization in mammalian cells suggests that RS domain-mediated interactions may be subject to coordinate regulation by phosphorylation in a
temporal- and spatial-specific manner. Therefore, it is important to study phosphorylation regulation of pre-mRNA
splicing at both the biochemical and cellular levels.
In this study, we established a novel model system using
Saccharomyces cerevisiae to investigate the importance of
phosphorylation in RS domain-mediated protein-protein
interactions in vivo. Unlike mammalian cells that express
many SR protein kinases, budding yeast express only a single conserved SR protein-specific kinase, Sky1p, which
was cloned and characterized recently in our lab (Siebel et
al., 1999). Structural and functional studies demonstrate that Sky1p is a SRPK family member that has the same
high activity and specificity for mammalian SR proteins as
SRPK1 and SRPK2. Moreover, increasing evidence suggests that budding yeast express proteins that are structurally and functionally related to mammalian RS domain-
containing proteins. Therefore, yeast provide a powerful genetic system to study SR protein function in vivo, which
will provide clues to the function and regulation of SR
proteins in mammalian cells. Here, we took advantage of
the fact that SKY1 is not essential for vegetative growth in
yeast. Therefore, we were able to express mammalian RS
domain-containing proteins and study their interactions in
cells with and without SRPK-mediated phosphorylation,
an approach that was not possible in mammalian cells. Our
results demonstrate that RS domain-mediated protein- protein interactions in vivo are entirely dependent on the
activity of Sky1p, which can be substituted by SRPK1 or
Clk/Sty from mammalian cells. Further examination of RS
domain interactions in this system reveal that, in addition
to the modulation of protein affinities, SRPKs appear to
play an important role in mediating nuclear import of SR
proteins as well as in finding their targets within the nucleus.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In Vitro Binding
GST pull-down assays were performed as described (Wang et al., 1998).
Bacterially expressed GST-ASF/SF2 (100 µg) was phosphorylated in vitro
using baculovirus-expressed GST-SRPK1 (10 U, see Gui et al., 1994b
) for
6 h at 30°C with or without 1 mM ATP. Proteins were desalted on G-50
columns (Pharmacia) and rebound to glutathione beads before binding to
TNT (Promega) translated U1-70K or U2AF35. Empty beads or GST-loaded beads were used as controls.
Two-Hybrid Interactions
Two-hybrid assays were performed as described using the LexA system
(Gyuris et al., 1993; Wu and Maniatis, 1993
). Strain EGY48 was deleted of
the SKY1 gene by recombination with a kanamycin resistance expression
unit flanked by SKY1 genomic sequences (Wach et al., 1994
). The deletion encompassed 316 bp 5' of the initiation codon to 541 bp 5' of the termination codon, and was confirmed by PCR. Bait and prey plasmids were
gifts from J. Wu (Wu and Maniatis, 1993
) or were constructed in pEG202
and pJG4-5 with modified polylinkers (Wang et al., 1997
). Expressed proteins were verified by Western blotting with a monoclonal anti-LexA antibody (Clontech) using the enhanced chemiluminescent detection system
(Pierce) in conjunction with HRP-labeled goat anti-mouse antibodies. Kinase expression plasmids were constructed in p415GAL1 (Mumberg et al.,
1994
) for selecting Leu+ phenotype (Wu and Maniatis, 1993
). Deletions of
the RS domains in U2AF65 (residues 1-65) and U2AF35 (residues 191-
240) were made by PCR.
-Galactosidase activity is expressed as Miller
units: OD420 × 1,000/OD600 × time (min) × culture volume assayed
(ml), determined at 30°C.
Immunolocalization
Freshly streaked yeast clones bearing prey plasmids (HA tagged) were inoculated in 5 ml YPGal/Raff and grown for 3 h at 30°C. Yeast were fixed
with 0.7 ml 37% formaldehyde and processed for immunofluorescence as
described (McConnell et al., 1990), using the 12CA5 antibody at 1:100.
GFP-ASF/SF2 Expression
Humanized GFP (pGreenLantern-1; Life Technologies) was fused in
frame to the NH2 terminus of ASF/SF2 wild-type cDNA or the KS substitution mutant (Cáceres and Krainer, 1993). The fusion protein was expressed from pcDNA3 (Invitrogen) in HeLa cells for 48 h before visualization of live cells. Whole cell extracts (200 µg) were treated with 500 U
CIP (Boehringer Mannheim; Misteli et al., 1998
) and subjected to Western analysis using an anti-GFP antibody (Chemicon).
Transcriptional Repression
Both the parent EGY48 and corresponding sky1 strains were grown on
5-FOA plates to eliminate the pSH18-24 two-hybrid reporter (lacZ) plasmid. The resulting strains were cotransformed with pEG202-based bait
plasmids and pJK101, a transcriptional repression reporter (Golemis and
Brent, 1992
), then assayed for
-galactosidase activity. Maximal transcription was determined using a modified bait plasmid lacking LexA.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Differential Modulation of RS Domain Interactions In Vitro
Phosphorylation of the SR protein ASF/SF2 by SRPK1,
SRPK2, or Clk/Sty has been shown previously to enhance
its interaction in vitro with the U1 snRNP-specific 70-kD
protein (U1-70K) in a GST pull-down assay (Xiao and
Manley, 1997; Wang et al., 1998
; Fig. 1). This enhancement
may result from the neutralization of charges, allowing RS
domains to interact efficiently (Xiao and Manley, 1997
).
To test the generality of this principle, we examined the
effect of SRPK1-mediated phosphorylation on ASF/SF2
binding to another RS domain-containing splicing factor,
U2AF35, an interaction proposed for pairing the 5' and
3' splice sites (Wu and Maniatis, 1993
). Unexpectedly,
binding of unphosphorylated ASF/SF2 to U2AF35 was
readily detectable, and this interaction was largely unaffected by SRPK1-mediated phosphorylation (Fig. 1).
These results indicate that phosphorylation differentially
modulates the affinity between RS domain-containing
splicing factors, depending on the pairs examined. A similar observation was also made independently by Xiao
and Manley (1998)
.
|
Detection of Phosphorylation-dependent Interactions In Vivo
To examine the role of phosphorylation in protein-protein
interactions under physiological conditions, we took advantage of the single conserved SR protein-specific kinase
in S. cerevisiae to investigate RS domain interactions in the
presence or absence of phosphorylation. Mammalian SR
proteins expressed in both wild-type and SKY1 deletion
(sky1) yeast strains were soluble, but differed in their
phosphorylation states, as determined by mobility shift on
SDS-PAGE as well as Western blotting with mAb 104, a monoclonal antibody specific for the phosphoepitope
present in all SR proteins (data not shown; Siebel et al.,
1999
). As shown in Fig. 2 a, LexA-ASF/SF2 and LexA-SC35 expressed in wild-type yeast migrated more slowly than those from sky1
yeast (left four lanes), indicative of
a change in phosphorylation states (see Xiao and Manley,
1997
). Reexpression of Sky1p, but not an ATP binding site
mutant (Sky1p K187M), restored the phosphorylated form
of ASF/SF2 (right two lanes). Therefore, Sky1p appears to
be the only endogenous kinase capable of efficiently phosphorylating mammalian SR proteins expressed in yeast.
This system offers a unique opportunity to test the phosphorylation dependence of RS domain interactions in vivo
using the two-hybrid strategy.
|
The results of two-hybrid assays are shown in Fig. 2 b.
As a control, the interaction between the nuclear receptor
transcription factor T3R and its corepressor NCoR, which
are not SRPK1 substrates (data not shown), was unaffected by the SKY1 deletion. In contrast, the interactions
between RS domain-containing splicing factors, all SRPK
substrates, were largely eliminated in sky1 yeast. Growth assays of these interactions agreed with the quantitative
assays (data not shown). In particular, RS domain-mediated interactions between U1-70K and either ASF/SF2 or
SC35 were completely abolished in sky1
yeast, consistent
with the in vitro binding data. As expected, the interaction
between ASF/SF2 or SC35 with U2AF35 was detectable in
wild-type yeast, but we never detected any interaction between these three RS domain-containing splicing factors
in parallel experiments in sky1
yeast. Surprisingly, the
two-hybrid interaction between U2AF65 and U2AF35
was also severely affected by the SKY1 deletion, despite
the fact that their interaction takes place outside their RS
domains (Zhang et al., 1992
; see below). These results
clearly indicate that phosphorylation has a far greater impact on RS domain interactions in vivo than in vitro, reflecting additional phosphorylation-dependent events for
the interaction between RS domain proteins in cells.
Functional Rescue by Yeast and Mammalian SR Protein Kinases
To further demonstrate that Sky1p-mediated phosphorylation is critical for RS domain-containing proteins to interact with one another, we conducted kinase rescue experiments. As shown in Table I, Sky1p, but not its ATP
binding site mutant, was able to restore the two-hybrid interactions examined, proving that Sky1p kinase activity is
required. Because mammalian cells express more than one
kinase family for SR proteins, the yeast model system provides an opportunity to evaluate functional similarities between different mammalian SR protein-specific kinases.
Therefore, we extended the rescue experiments to SRPK1
and Clk/Sty from mammalian cells. As shown in Table I,
both SRPK1 and Clk/Sty, but not corresponding ATP
binding site mutants, were able to restore the interactions of U1-70K with both ASF/SF2 and SC35. In contrast, an
unrelated kinase, the catalytic subunit of protein kinase A,
did not rescue these interactions. Therefore, kinases from
both the SRPK and Clk/Sty families appear to be functionally equivalent in this assay. The restoration of these
two-hybrid interactions by different SR protein kinases
strongly argues against the possibility that the lack of RS
domain-mediated interactions in sky1 yeast is due to a
general defect resulting from the SKY1 deletion. Furthermore, the ability of Clk/Sty to restore the two-hybrid interactions in sky1
yeast implies that there may be no endogenous kinase with the same activity and specificity as Clk/
Sty in S. cerevisiae, whereas mammalian cells have evolved
multiple SR protein kinases to regulate RS domain-containing splicing factors.
|
Nuclear Localization of SC35 Affected by SKY1 Deletion
The absolute dependence of RS domain-mediated interactions in vivo on phosphorylation is in sharp contrast to
the affinity changes modulated by phosphorylation in
vitro. This difference raises the possibility that other aspects of SR protein function may be affected by phosphorylation in vivo and, therefore, offers an opportunity to investigate additional phosphorylation-dependent events.
For instance, hypophosphorylated SR proteins may be sequestered in the nucleus due to nonspecific binding to
other proteins or nucleic acids, as suggested by in vitro
RNA binding studies (Tacke et al., 1997; Xiao and Manley, 1997
). Therefore, we examined the localization of expressed SR proteins in yeast by immunohistochemistry. As
shown in Fig. 3, SC35 expressed from the prey vector was
largely localized in the nucleus in wild-type yeast, but dispersed uniformly in both the cytoplasm and the nucleus in
sky1
yeast. In other experiments using a prey vector lacking an exogenous nuclear localization signal, SC35 was
uniformly distributed in wild-type cells, but excluded from
the nucleus in sky1
yeast, leaving a "hole" in the immunofluorescence image (data not shown). These unanticipated findings suggest that Sky1p-mediated phosphorylation may regulate nuclear import of SC35 expressed in
yeast. However, the localization of ASF/SF2, U1-70K, and
U2AF35 seemed mostly unaffected by the SKY1 deletion
as they were generally uniformly distributed in both the
nucleus and cytoplasm of wild-type and sky1
yeast (Fig. 3
and data not shown). These observations suggest for the
first time that SRPK-mediated phosphorylation plays an
important role in nuclear import of SR proteins, although
not all SR proteins are affected in the same way.
|
To obtain further evidence that SRPK-mediated phosphorylation plays a role in nuclear localization of RS domain proteins in mammalian cells, we took advantage of
the KS substitution mutant of ASF/SF2 in which arginines
in the RS domain are replaced by lysines (Cáceres and
Krainer, 1993). We have shown previously that ASF/SF2-KS is not a substrate for the SRPK family of kinases, but
can be phosphorylated by Clk/Sty (Colwill et al., 1996b
). Consistent with the observation that there appears to be
no Clk/Sty-like kinase activity in yeast, ASF/SF2-KS did
not interact with U1-70K in the two-hybrid assay even in
wild-type yeast (data not shown). We expressed wild-type
ASF/SF2 and ASF/SF2-KS as GFP fusion proteins in
HeLa cells, and expressed proteins were examined for
phosphorylation-dependent mobility shifts on SDS-PAGE
(Fig. 4 a). Treatment with alkaline phosphatase caused an
increase in the mobility of wild-type ASF/SF2, but had no
effect on that of the KS mutant, suggesting that GFP-ASF/ SF2-KS expressed in mammalian cells is not phosphorylated. The localization of these proteins in HeLa cells is
shown in Fig. 4 b. Wild-type GFP-ASF/SF2 localized in
the nucleus in a speckled pattern characteristic of endogenous splicing factors. In contrast, GFP-ASF/SF2-KS
showed significant accumulation in the cytoplasm. ASF/
SF2-KS was also localized in the nucleus in enlarged
speckles, which is consistent with a role of SRPKs in dissociating splicing factors from nuclear speckles, as suggested
earlier (Gui et al., 1994a
). These observations reinforce
the idea that SRPK-mediated phosphorylation plays an
important role in both nuclear import and intranuclear localization of SR proteins.
|
Evidence for Phosphorylation-dependent Molecular Targeting in the Nucleus
Although inefficient nuclear localization may be a contributing factor for impaired protein-protein interactions
involving SC35, it cannot explain the absolute phosphorylation dependence of all the RS domain-mediated interactions we tested in sky1 yeast. Furthermore, the two-hybrid
interaction between U2AF65 and U2AF35 in sky1
yeast
was also substantially reduced (Fig. 2 b) despite the fact
that this interaction is not RS domain-mediated (Zhang et
al., 1992
). When the RS domains of U2AF65 and U2AF35 were deleted, their two-hybrid interaction became highly
efficient in both wild-type and sky1
yeast, even surpassing the interaction between full-length proteins in wild-type yeast (Table II). These observations suggest that the
phosphorylation state of their RS domains may influence the U2AF65-35 interaction in eukaryotic cells, even
though this interaction can take place in bacteria (Rudner
et al., 1998
). In an attempt to correlate inefficient two-hybrid interactions with a defect in heterodimer formation between U2AF65 and U2AF35, we conducted gel filtration and coimmunoprecipitation experiments and detected similar amounts of U2AF65 and 35 in large protein
complexes from both wild-type and sky1
yeast (data not shown). Because these complexes may contain additional
proteins, we cannot determine whether phosphorylation
directly affects the efficiency of heterodimer formation.
Alternatively, Sky1p may indirectly affect the outcome of
the two-hybrid interaction assay by regulating molecular
targeting of its substrates to each other and/or to the reporter gene in the nucleus.
|
To demonstrate that SRPKs play a direct role in assisting RS domain-containing proteins to locate their specific
protein or RNA targets, we adapted the following transcriptional repression assay (Golemis and Brent, 1992),
which allowed a quantitative measure of phosphorylation-dependent molecular targeting of RS domain proteins in
vivo. As diagrammed in Fig. 5, LexA operators were inserted between a GAL1 upstream activating sequence
(UAS) and the transcriptional start site for lacZ such that
LexA or LexA fusion proteins bind to the operators and
repress lacZ expression. In both wild-type and sky1
yeast, lacZ expression was constitutive in the absence of
LexA, and repressed in its presence. The ability of a control LexA-T3R fusion protein to repress lacZ expression was not affected by the SKY1 deletion. In contrast, the targeting of LexA-SR fusion proteins was dramatically, although not equally, affected by the SKY1 deletion in this
assay (Fig. 5). In particular, the LexA-SC35 fusion protein
fully repressed lacZ expression in wild-type yeast, but
showed little repression in sky1
yeast, probably reflecting
the significant impact of Sky1p-mediated phosphorylation on the fusion protein at both nuclear localization and intranuclear targeting steps. A similar SKY1-dependent effect was also seen, although less pronounced, with ASF/
SF2, U1-70K, and U2AF65, but was not evident with
U2AF35. These observations provide an explanation for
the decreased interaction between U2AF65 and U2AF35
in sky1
yeast in the two-hybrid assay. The U2AF65 fusion protein expressed from the bait vector in sky1
yeast
could not efficiently locate its target and activate transcription of the reporter gene, even though targeting of
U2AF35 was unaffected by phosphorylation. Based on
these results, we conclude that phosphorylation plays a direct role in the molecular targeting of RS domain-containing proteins within the nucleus.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this report, we have taken advantage of the fact that apparently only one SR protein-specific kinase is conserved
in the relatively simple eukaryote S. cerevisiae. Therefore,
we were able to address the functional requirements of SR
protein-specific kinases in mediating SR protein-protein
interactions in vivo. An equivalent kinase deletion experiment cannot be done in mammalian cells due to the presence of at least four identified kinase activities capable of
phosphorylating RS domain-containing proteins (for reviews see Fu, 1995; Misteli and Spector, 1997
). The yeast
model system is especially attractive due to the fact that
many aspects of RNA metabolism and fundamental mechanisms of nuclear import/export are well conserved between mammalian cells and yeast. For example, this model
organism was used successfully to address HIV rev function in mediating specific RNA transport in yeast, although yeast is not a natural host for this virus (Stutz and
Rosbash, 1994
; Stutz et al., 1995
). Furthermore, the two-hybrid protein-protein interaction and transcriptional repression assays have been used widely to address the function of mammalian proteins in yeast, and often accurately
reflect interactions that occur normally in mammalian cells. Our current findings illustrate multiple steps at
which the SR superfamily of splicing factors may be regulated by phosphorylation and our data are consistent with
published observations on the effects of phosphorylation
and dephosphorylation on SR proteins in mammalian cells
(see below). Although a phosphorylation-dependent nuclear localization defect was observed using mammalian
SC35 expressed in yeast, a similar phenomenon also occurs with an endogenous Sky1p substrate, Npl3p (Yun,
C.Y., and X.-D. Fu, manuscript submitted for publication),
indicating that conserved regulatory mechanisms may operate in both yeast and mammalian cells. Most advantageously, the use of yeast has made it possible to provide
quantitative measures of intracellular trafficking of SR
proteins, which have been largely descriptive in previous studies.
Nuclear Import of SR Proteins
Our localization studies showed increased cytoplasmic accumulation of SR proteins in the absence of SRPK-mediated phosphorylation. The nuclear localization signal for
SC35 appears to be confined to its RS domain (Cáceres et al.,
1997), and, therefore, the effect of SRPK-mediated phosphorylation on SC35 localization may be readily detectable. In contrast, sequences both inside and outside of the
RS domain of ASF/SF2 seem to be required for the protein to properly localize in the nucleus (Cáceres et al., 1997
), and, as a result, nuclear import of ASF/SF2 may be
less efficient but not abolished without SRPK-mediated
phosphorylation. This possibility is consistent with the observations that ASF/SF2 mutants, including ASF/SF2-KS
and RS domain-deleted ASF/SF2 (Misteli et al., 1998
),
were localized in both the cytoplasm and the nucleus of
transfected HeLa cells. Together, these results suggest
that the localization of different SR proteins may be differentially facilitated by phosphorylation, which may reflect distinct mechanisms for their nuclear import.
Increased cytoplasmic localization of hypophosphorylated SR proteins may be due to inefficient nuclear import
or accelerated export. Recently, it was reported that overexpression of Clk/Sty or SRPK2 in mammalian cells increases cytoplasmic localization of ASF/SF2 (Cáceres et al.,
1998; Kuroyanagi et al., 1998
; our unpublished observations), indicating that SR protein-specific kinases may
play an active role in facilitating nuclear export of SR proteins. Alternatively, the dissolution of nuclear speckles by
these kinases elevates the nucleoplasmic pool of SR proteins, which may indirectly lead to an increase in detectable cytoplasmic protein levels, as previously suggested
(Cáceres et al., 1998
). In either case, the observation that
over-phosphorylation appears to stimulate SR protein export is incompatible with the idea that the lack of phosphorylation also accelerates nuclear export of SC35 in
sky1
yeast. Therefore, we favor the interpretation that SKY1-mediated phosphorylation functions at the nuclear
import step, which provides a rationale for our earlier
findings that all SRPK family members from yeast to humans are largely, but not exclusively, localized in the cytoplasm (Takeuchi and Yanagida, 1993
; Wang et al., 1998
;
Siebel et al., 1999
). Together, these observations suggest
that SRPKs may catalyze phosphorylation of their substrates in the cytoplasm and facilitate their nuclear import.
Molecular Targeting to Nascent Transcripts
In yeast, we observed that unphosphorylated SR proteins
appear to distribute uniformly in the nucleoplasm, yet
their efficient targeting to appropriate molecular targets
(in this case, the LexA binding site) requires phosphorylation mediated by Sky1p. These data are consistent with the
recent report on phosphorylation-dependent recruitment
of SR proteins to nascent transcripts (Misteli et al., 1998).
In addition, the transcriptional repression assay provided a
quantitative measure of recruitment, and, therefore, allowed the comparison of different RS domain-containing
splicing factors. For example, we observed that targeting
of the SC35 fusion protein is more dependent on phosphorylation than that of ASF/SF2 and other RS domain-containing splicing factors, which may result from the phosphorylation dependence of SC35 for both efficient nuclear
import and intranuclear movement. The mechanism for
phosphorylation-dependent targeting is not entirely clear,
but may be explained by the possibility that an unphosphorylated RS domain may interact with other nuclear constituents. For example, it was suggested, based on in
vitro experiments, that phosphorylation is necessary for
sequence-specific binding of SR proteins to their RNA
targets (Tacke et al., 1997
; Xiao and Manley, 1997
).
In addition to increasing protein-nucleic acid binding
specificity, phosphorylation can modulate protein-protein
interactions among RS domains in vitro (Xiao and Manley, 1997; Wang et al., 1998
). Our finding that phosphorylation increases ASF/SF2-U1-70K interactions, but has little effect on ASF/SF2-U2AF35 interactions, suggests that
phosphorylation can differentially modulate protein-protein interactions depending on the protein pairs. Therefore, phosphorylation may regulate molecular targeting of
RS domain-containing proteins to their appropriate protein partners after being recruited to actively transcribing
regions in the nucleus. This phosphorylation-dependent selectivity may be crucial for an orderly assembly of splicing factors on specific transcripts at specific stages of the
splicing reaction.
Dynamic Distribution of Splicing Factors in the Nucleus
In mammalian cells, splicing factors are believed to be
"stored" in nuclear speckles (for reviews, see Spector,
1993; Singer and Green, 1997
) and recruited to nascent
transcripts to carry out the splicing reaction. The exchange
of SR proteins between the nucleoplasm and nuclear
speckles is highly dynamic, as illustrated by monitoring the
movement of GFP-ASF/SF2 in living cells (Misteli et al.,
1997
, 1998
). Earlier studies have shown that increased phosphorylation releases SR proteins to the nucleoplasm
(Gui et al., 1994a
; Colwill et al., 1996a
; Misteli et al., 1997
;
Duncan et al., 1998
; Wang et al., 1998
), making them generally available to be recruited to the sites of transcription
and splicing, whereas dephosphorylation appears to be essential for the resolution of spliceosomes (Mermoud et al.,
1994
), and as a result, contributes to the accumulation of
SR proteins in nuclear speckles (Misteli and Spector,
1997
). In the current study, SR proteins in sky1
yeast remained soluble, suggesting that unphosphorylated SR proteins may not form nonspecific aggregates resembling inclusion bodies in yeast, yet they do not interact with each
other efficiently. Interestingly, we observed that removal
of the RS domains from both U2AF65 and U2AF35 allowed them to interact very efficiently in both wild-type
and sky1
yeast, even better than the interaction between
the full-length proteins in wild-type yeast. This result implies that even in wild-type cells, some proteins may not be sufficiently phosphorylated for efficient interaction. Because we detect U2AF65/35 in large complexes in both
wild-type and sky1
yeast, it is possible that phosphorylated and unphosphorylated proteins may interact with
different sets of proteins.
In fact, interactions involving hypophosphorylated RS
domain-containing splicing factors may resemble those in
nuclear speckles where a series of rearranged protein-protein and protein-RNA interactions have been triggered by
phosphatases during splicing. To some extent, these dephosphorylation-induced rearrangements may cause the
coalescence of splicing complexes on nascent transcripts to
give rise to the appearance of speckles in the nucleus.
Therefore, one might imagine that nuclear speckles are
postsplicing structures for recycling of splicing factors,
rather than storage sites where splicing factors are randomly accumulated. This scenario may best explain the
observations that nuclear speckles contain essentially all splicing factors as well as poly (A)+ mRNA (Xing et al.,
1993, 1995
), but are not sites for the majority of nascent
transcripts detected by BrUTP pulse labeling (Jackson et
al., 1993
).
A Model for Phosphorylation-dependent Movement of Splicing Factors in Cells
A synthesis of our results with published reports is presented in a model (Fig. 6), depicting the dependence of SR
protein trafficking on phosphorylation. In this model,
nuclear import of newly translated or shuttling RS domain-containing proteins is facilitated by phosphorylation. Newly imported SR proteins are then partitioned between speckles and the nucleoplasm based in part on the
extent of phosphorylation. A large body of evidence suggests that mRNA splicing takes place cotranscriptionally
in the nucleoplasm whereas nuclear speckles have been
described as "storage sites" for splicing factors. Release of
SR proteins from speckles is facilitated by phosphorylation (Gui et al., 1994a; Colwill et al., 1996a
), which is a prerequisite for their subsequent recruitment to nascent transcripts (Misteli et al., 1998
). We show in the current study
that specific targeting of nucleoplasmic SR proteins to
their appropriate RNA and protein partners are also affected by phosphorylation. Once assembled on nascent
transcripts, the resolution of splicing complexes during the
splicing reaction appears to require dephosphorylation of
SR proteins (Mermoud et al., 1994
), which may accompany a series of rearrangements involving both RNAs and
proteins in the spliceosome. As a result, hypophosphorylated SR proteins may be engaged in interactions with a
distinct set of proteins, causing them to remain in speckles,
as mature transcripts proceed to nuclear export.
|
In summary, the novel system developed in this study enabled us to dissect this complex process in a relatively simple genetic background. The data presented in this report illustrate that SR protein kinases can affect translocation of RS domain-containing proteins from the cytoplasm to the nucleus as well as molecular targeting within the nucleus, in addition to modulation of RS domain affinities observed in vitro. Defects in combinations of these processes provide a probable explanation for the critical dependence of RS domain interactions on SRPK-mediated phosphorylation in cells. These phosphorylation-dependent steps provide multiple avenues for pre-mRNA splicing regulation in response to internal or external signaling.
![]() |
Footnotes |
---|
Address correspondence to Xiang-Dong Fu, CMM Room 239, University of California, Mail Code 0651, 9500 Gilman Dr., La Jolla, CA 92093-0651. Tel.: (619) 534-4937. Fax: (619) 534-8549. E-mail: xdfu{at}ucsd.edu
Received for publication 9 November 1998 and in revised form 17 March 1999.
J.M. Yeakley is the recipient of a National Institutes of Health (NIH)
postdoctoral fellowship. H. Tronchère was supported by the Human Frontiers Science Program. J.A. Dyck is funded by the Damon Runyon-Walter
Winchell Cancer Research Foundation. X.-D. Fu is a Leukemia Scholar.
This work was supported by an NIH grant to X.-D. Fu.
We thank Mira Guzijan, Harold Fisk, and Jane Wu for assistance and expert advice; and Lana Feng, Jiwu Wang, Chi Yun, Brent Gravely, and Tom Maniatis for critical comments. T3R and NCoR control plasmids were kindly provided by M.G. Rosenfeld.
![]() |
Abbreviation used in this paper |
---|
snRNPs, small nuclear ribonucleoprotein particles.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Cáceres, J.F., and A.R. Krainer. 1993. Functional analysis of pre-mRNA splicing factor ASF/SF2 structural domains. EMBO (Eur. Mol. Biol. Organ.) J. 12: 4715-4726 [Abstract]. |
2. |
Cáceres, J.F.,
T. Misteli,
G.R. Screaton,
D.L. Spector, and
A.R. Krainer.
1997.
Role of the modular domains of SR proteins in subnuclear localization and
alternative splicing specificity.
J. Cell Biol.
138:
225-238
|
3. |
Cáceres, J.F.,
G.R. Screaton, and
A.R. Krainer.
1998.
A specific subset of SR
proteins shuttles continuously between the nucleus and the cytoplasm.
Genes Dev.
12:
55-66
|
4. | Cao, W., S.F. Jamison, and M.A. Garcia-Blanco. 1997. Both phosphorylation and dephosphorylation of ASF/SF2 are required for pre-mRNA splicing in vitro. RNA. 3: 1456-1467 [Abstract]. |
5. |
Chandler, S.D.,
A. Mayeda,
J.M. Yeakley,
A.R. Krainer, and
X.-D. Fu.
1997.
RNA splicing specificity determined by the coordinated action of RNA recognition motifs in SR proteins.
Proc. Natl. Acad. Sci. USA.
94:
3596-3601
|
6. | Colwill, K., T. Pawson, B. Andrews, J. Prasad, J.L. Manley, J.C. Bell, and P.I. Duncan. 1996a. The Clk/Sty protein kinase phosphorylates SR splicing factors and regulates their intracellular distribution. EMBO (Eur. Mol. Biol. Organ.) J. 15: 265-275 [Abstract]. |
7. |
Colwill, K.,
L. Feng,
J.M. Yeakley,
G.D. Gish,
J.F. Cáceres,
T. Pawson, and
X.-D. Fu.
1996b.
SRPK1 and Clk/Sty protein kinases show distinct substrate
specificities for serine/arginine-rich splicing factors.
J. Biol. Chem.
271:
24569-24575
|
8. | Duncan, P.I., D.F. Stojdl, R.M. Marius, and J.C. Bell. 1997. In vivo regulation of alternative pre-mRNA splicing by the Clk1 protein kinase. Mol. Cell. Biol. 17: 5996-6001 [Abstract]. |
9. | Duncan, P.I., D.F. Stojdl, R.M. Marius, and J.C. Bell. 1998. The Clk2 and Clk3 dual-specificity protein kinases regulate the intranuclear distribution of SR proteins and influence pre-mRNA splicing. Exp. Cell Res. 241: 300-308 |
10. | Fu, X-D.. 1993. Specific commitment of different pre-mRNAs to splicing by single SR proteins. Nature. 365: 82-85 |
11. | Fu, X.-D.. 1995. The superfamily of arginine/serine-rich splicing factors. RNA. 1: 663-680 |
12. | Golemis, E.A., and R. Brent. 1992. Fused protein domains inhibit DNA binding by LexA. Mol. Cell. Biol. 12: 3006-3014 [Abstract]. |
13. | Gui, J.F., W.S. Lane, and X.-D. Fu. 1994a. A serine kinase regulates intracellular localization of splicing factors in the cell cycle. Nature. 369: 678-682 |
14. |
Gui, J.-F.,
H. Tronchère,
S.D. Chandler, and
X.-D. Fu.
1994b.
Purification and
characterization of a kinase specific for the serine and arginine-rich pre-mRNA splicing factors.
Proc. Natl. Acad. Sci. USA.
91:
10824-10828
|
15. | Gyuris, J., E. Golemis, H. Chertkov, and R. Brent. 1993. Cdi1, a human G1 and S phase protein phosphatase that associates with Cdk2. Cell. 75: 791-803 |
16. | Jackson, D.A., A.B. Hassan, R.J. Errington, and P.R. Cook. 1993. Visualization of focal sites of transcription within human nuclei. EMBO (Eur. Mol. Biol. Organ.) J. 12: 1059-1065 [Abstract]. |
17. | Kanopka, A., O. Muhlemann, S. Petersen-Mahrt, C. Estmer, C. Ohrmalm, and G. Akusjarvi. 1998. Regulation of adenovirus alternative RNA splicing by dephosphorylation of SR proteins. Nature. 393: 185-187 |
18. | Kohtz, J.D., S.F. Jamison, C.L. Will, P. Zuo, R. Lührmann, M.A. Garcia, Blanco, and J.L. Manley. 1994. Protein-protein interactions and 5'-splice-site recognition in mammalian mRNA precursors. Nature. 368: 119-124 |
19. | Kuroyanagi, N., H. Onogi, T. Wakabayashi, and M. Hagiwara. 1998. Novel SR-protein-specific kinase, SRPK2, disassembles nuclear speckles. Biochem. Biophys. Res. Commun. 242: 357-364 |
20. | Manley, J.L., and R. Tacke. 1996. SR proteins and splicing control. Genes Dev. 10: 1569-1579 |
21. | McConnell, S.J., L.C. Stewert, A. Talin, and M.P. Yaffe. 1990. Temperature-sensitive yeast mutants defective in mitochondrial inheritance. J. Cell Biol. 111: 967-976 [Abstract]. |
22. | Mermoud, J.E., P.T. Cohen, and A.E. Lamond. 1994. Regulation of mammalian spliceosome assembly by a protein phosphorylation mechanism. EMBO (Eur. Mol. Biol. Organ.) J. 13: 5679-5688 [Abstract]. |
23. | Misteli, T., and D.L. Spector. 1997. Protein phosphorylation and the nuclear organization of pre-mRNA splicing. Trends Cell Biol. 7: 135-138 . |
24. | Misteli, T., J.F. Cáceres, and D.L. Spector. 1997. The dynamics of a pre-mRNA splicing factor in living cells. Nature. 387: 523-527 |
25. |
Misteli, T.,
J.F. Cáceres,
J.Q. Clement,
A.R. Krainer,
M.F. Wilkinson, and
D.L. Spector.
1998.
Serine phosphorylation of SR proteins is required for their recruitment to sites of transcription in vivo.
J. Cell Biol.
143:
297-307
|
26. | Mumberg, D., R. Muller, and M. Funk. 1994. Regulatable promoters of Saccharomyces cerevisiae: comparison of transcriptional activity and their use for heterologous expression. Nucl. Acids Res. 22: 5767-5768 |
27. | Nilsen, T.W. 1998. RNA-RNA interactions in nuclear pre-mRNA splicing. In RNA Structure and Function. R. Simons and M. Grunberg-Manago, editors. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 279-307. |
28. | Roscigno, R.F., and M.A. Garcia-Blanco. 1995. SR proteins escort the U4/ U6.U5 tri-snRNP to the spliceosome. RNA. 1: 692-706 [Abstract]. |
29. |
Rudner, D.Z.,
R. Kanaar,
K.S. Breger, and
D.C. Rio.
1998.
Interaction between
subunits of heterodimeric splicing factor U2AF is essential in vivo.
Mol.
Cell. Biol.
18:
1765-1773
|
30. | Siebel, C., L. Feng, C. Guthrie, and X.-D. Fu. 1999. Conservation of yeast protein kinase specific for the SR family of splicing factors. Proc. Natl. Acad. Sci. USA. In press. |
31. | Singer, R.H., and M.R. Green. 1997. Compartmentalization of eukaryotic gene expression: causes and effects. Cell. 91: 291-294 |
32. | Spector, D.L.. 1993. Macromolecular domains within the cell nucleus. Annu. Rev. Cell Biol. 9: 265-315 . |
33. | Staley, J.P., and C. Guthrie. 1998. Mechanical devices of the spliceosome: motors, clocks, springs, and things. Cell. 92: 315-326 |
34. | Stutz, F., and M. Rosbash. 1994. A functional interaction between Rev and yeast pre-mRNA is related to splicing complex formation. EMBO (Eur. Mol. Biol. Organ.) J. 13: 4096-4104 [Abstract]. |
35. | Stutz, F., M. Neville, and M. Rosbash. 1995. Identification of a novel nuclear pore-associated protein as a functional target of the HIV-1 Rev protein in yeast. Cell. 82: 495-506 |
36. |
Tacke, R.,
Y. Chen, and
J.L. Manley.
1997.
Sequence-specific RNA binding by
an SR protein requires RS domain phosphorylation: creation of an SRp40-specific splicing enhancer.
Proc. Natl. Acad. Sci. USA.
94:
1148-1153
|
37. | Takeuchi, M., and M. Yanagida. 1993. A mitotic role for a novel fission yeast protein kinase dsk1 with cell cycle. Mol. Biol. Cell. 4: 247-260 [Abstract]. |
38. | Tazi, J., U. Kornstadt, F. Rossi, P. Jeanteur, G. Cathala, C. Brunel, and R. Lührmann. 1993. Thiophosphorylation of U1-70K protein inhibits pre-mRNA splicing. Nature. 363: 283-286 |
39. | Tronchère, H., J. Wang, and X.-D. Fu. 1997. A protein related to splicing factor U2AF35 that interacts with U2AF65 and SR proteins in splicing of pre-mRNA. Nature. 388: 397-400 |
40. | Wach, A., A. Brachat, R. Pohlmann, and P. Philippsen. 1994. New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast. 10: 1793-1808 |
41. |
Wang, H.-Y.,
W. Lin,
J.A. Dyck,
J.M. Yeakley,
Z. Songyang,
L.C. Cantley, and
X.-D. Fu.
1998.
SRPK2: a differentially expressed SR protein-specific kinase
involved in mediating the interaction and localization of pre-mRNA splicing
in mammalian cells.
J. Cell Biol.
140:
737-750
|
42. |
Wang, J.,
Z. Dong, and
L.R. Bell.
1997.
Sex-lethal interactions with protein and
RNA. Roles of glycine-rich and RNA binding domains.
J. Biol. Chem.
272:
22227-22235
|
43. | Wu, J.Y., and T. Maniatis. 1993. Specific interactions between proteins implicated in splice site selection and regulated alternative splicing. Cell. 75: 1061-1070 |
44. | Xiao, S.-H., and J.L. Manley. 1997. Phosphorylation of the ASF/SF2 RS domain affects both protein-protein and protein-RNA interactions and is necessary for splicing. Genes Dev. 11: 334-344 [Abstract]. |
45. |
Xiao, S.-H., and
J.L. Manley.
1998.
Phosphorylation-dephosphorylation differentially affects activities of splicing factor ASF/SF2.
EMBO (Eur. Mol. Biol.
Organ.) J.
17:
6359-6367
|
46. | Xing, Y., C.V. Johnson, P.R. Dobner, and J.B. Lawrence. 1993. Higher level organization of individual gene transcription and RNA splicing. Science. 259: 1326-1330 |
47. | Xing, Y., C.V. Johnson, P.T. Moen, J.A. McNeil, and J.B. Lawrence. 1995. Nonrandom gene organization: structural arrangements of specific pre-mRNA transcription and splicing with SC35 domain. J. Cell Biol. 131: 1635-1647 [Abstract]. |
48. | Zhang, M., P.D. Zamore, M. Carmo-Fonseca, A.I. Lamond, and M.R. Green. 1992. Cloning and intracellular localization of the U2 small nuclear ribonucleoprotein auxiliary factor small subunit. Proc. Natl. Acad. Sci. USA. 89: 8769-8773 [Abstract]. |