From the Department of Cellular and Molecular
Medicine, University of California, San Diego, La Jolla, California
92093-0651 and the ¶ Department of Cell Biology, The Scripps
Research Institute, La Jolla, California 92037
Received for publication, November 18, 2002, and in revised form, March 10, 2003
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ABSTRACT |
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SR proteins and related RS
domain-containing polypeptides are an important class of splicing
regulators in higher eukaryotic cells. The RS domain facilitates
nuclear import of SR proteins and mediates protein-protein interactions
during spliceosome assembly; both functions appear to subject to
regulation by phosphorylation. Previous studies have identified two
nuclear import receptors for SR proteins, transportin-SR1 and
transportin-SR2. Here we show that transportin-SR1 and transportin-SR2
are the alternatively spliced products of the same gene and that
transportin-SR2 is the predominant transcript in most cells and tissues
examined. While both receptors import typical SR proteins in a
phosphorylation-dependent manner, they differentially
import the RS domain-containing splicing regulators hTra2 Small nuclear ribonucleoprotein particles and a large
number of protein factors are required for the assembly of
spliceosomes, where pre-mRNA is processed into mature mRNA (1,
2). SR proteins are a family of non-small nuclear ribonucleoprotein
particles splicing factors that are important for both constitutive and regulated splicing (3). Typical SR proteins contain one or two RNA
recognition motifs, which are responsible for their sequence-specific RNA binding activities, and an RS domain enriched with arginine and
serine repeats in the C terminus (4). Many biochemically and
genetically identified splicing regulators, such as Tra and Tra2 in the
Drosophila sex determination pathway, are related to SR
proteins by the presence of an RS domain (3, 4). Interestingly, two
Tra2 homologues, hTra2 All SR proteins are extensively modified by phosphorylation, which has
been shown to be critical for RS domain-mediated protein-protein interactions important for spliceosome assembly (6-8). Both hypo- and
hyperphosphorylation, however, appear to be inhibitory to the function
of SR proteins in splicing (9), but the precise mechanism for why a
hyperphosphorylated SR is inhibitory remains to be understood,
especially in light of a recent observation that RS repeats in the RS
domain of the SR protein ASF/SF2 can be substituted by RE or RD to
mimic the hyperphosphorylation state in splicing (10). This may be
related to the question whether modulation of SR protein function by
phosphorylation is due to changes in overall charge distribution
brought by phosphorylation in the RS domain, or more specifically, to
the modification at some critical sites, or perhaps, to both.
SR proteins are distributed in a speckled pattern in interphase nuclei
and become dispersed throughout the cell during mitosis as a result of
hyperphosphorylation (11). More recently, hyperphosphorylated SR
proteins were found to be restricted from the nucleus before zygotic
activation of gene expression in nematodes (12). These observations
suggest that phosphorylation may represent an important mechanism to
regulate the activity and localization of SR proteins during the cell
cycle as well as in development.
Although SR proteins are localized in the nucleus at steady state, a
subset of SR proteins is known to shuttle between the nucleus and the
cytoplasm (13). It is thought that SR protein shuttling may reflect a
role in mRNA transport (14) or a way to regulate their function by
localization (15). A functional link between nuclear import and
phosphorylation regulation of SR proteins was first established in
budding yeast where defects in a conserved SR protein kinase resulted
in impaired nuclear import of an SR-like RNA binding protein Npl3p (16,
17). Npl3p is imported by an importin- Here we investigated the relationship between TRN-SR1 and TRN-SR2 and
found that they are actually alternatively spliced products of the same
gene in mammalian cells. We also show that both TRN-SR1 and TRN-SR2
import typical SR proteins in a phosphorylation-dependent manner. Interestingly, TRN-SR1 and TRN-SR2 demonstrate distinct requirements for phosphorylation in mediating nuclear import of RS
domain-containing splicing regulators hTra2 Construction of a TRN-SR1/SR2 Minigene for in Vitro
Splicing--
PCR amplification of the genomic sequences surrounding
TRN-SR1/SR2 exons 10 and 11, as indicated in Fig. 1B, was
carried out using two sets of primers
(5'-gcgaATTCTACTCTGAAAGAAGGCAACC-3' and
5'-ggggtaccAGTGTCATTTTCCTTCCTCTTG-3';
5'-cgggtacCTTTCATTTCTATCTTTTCCTCTTTTTT-3' and
5'-gctctAGAAGTGGCAGAGGAGAGAGA-3'; EcoRI,
KpnI, or XbaI sites for cloning are
underlined, and capital letters are those present in the
TRN-SR1/SR2 genomic sequences). Digested PCR fragments were cloned into
the EcoRI and XbaI sites in pSP72 (Promega). In vitro transcription by Sp6 RNA polymerase and in
vitro splicing using HeLa nuclear extracts were performed under
standard conditions.
RT-PCR Analysis--
Total RNA was extracted from
different human cell lines using TRIzol (Invitrogen). A panel of
human tissue total RNA was purchased from Clontech.
For RT-PCR, 5 µg of total RNA was reverse-transcribed in a 15-µl
reaction using the Moloney murine leukemia virus reverse transcriptase
(Promega), and 1 µl was used for PCR. The forward and reverse primers
for the human TRN-SR1/SR2 gene are 5'-CGCATGAGGGTATCAGACCT-3' and
5'-GAGGCGGACAACTCCTTCTA-3', respectively. The PCR reactions were
carried out using Ampli Tag Gold (Applied Biosystems) for 10 min at
93 °C (activation and denaturation) followed by 35 cycles at
94 °C for 30 s, 60 °C for 30 s, and 72 °C for
30 s. The PCR products were analyzed in 2% agarose gel. Expected
PCR products from cloned TRN-SR1 (312 nucleotides) and TRN-SR2 (210 nucleotides) plasmids were amplified with equal efficiencies, which
served as controls for RT-PCR analysis of total RNA from human tissues and cell lines. No band was detected from isolated total RNA when RT
was omitted.
Preparation of Recombinant Proteins for in Vitro Binding and
Nuclear Import Assay--
Bacterially produced GST-ASF/SF2,
GST-hTra2 In Vitro Modifications, Binding, and Nuclear
Import--
Phosphorylation of GST-ASF/SF2, GST-hTra2
The in vitro import assay was performed as described
previously (26). Briefly, HeLa cells cultured on coverslips with 50% density (about 5 × 104 cells) were permeabilized with
digitonin (Calbiochem), and before each transport reaction, 90-95%
permeabilization was confirmed by staining with trypan blue.
Permeabilized cells were incubated with a 20-µl import reaction mix
at 30 °C for 30 min. Import reactions with cytosol contained 5 µl
of HeLa cytosol, ATP regenerating system (1 mM ATP, 5 mM creatine phosphate, 10 units/ml creatine phosphate
kinase), and 0.5 µg of GFP-tagged transport cargo. Import reactions
with purified components contained the ATP regenerating system, 150 ng
of Ran, 250 ng of NTF2, 0.5 µg of GFP-tagged cargo that was
phosphorylated or mock-phosphorylated by SRPK1, and 0.5 µg of
His-S-TRN-SR or His-S-TRN-SR2. After import, cells were washed in
transport buffer, fixed in 3.7% formaldehyde, and analyzed by
fluorescence microscopy. Control for positive import was an identical
reaction incubated on ice.
TRN-SR1 and TRN-SR2 Are Differentially Spliced
Products--
Sequence comparison reveals that TRN-SR1 and TRN-SR2 are
identical except for two regions, one in the middle and the other at
the C terminus. Alignment of their cDNA sequences indicates that
TRN-SR1 contains an in-frame insertion of 102 nucleotides (34 amino
acids) in the middle and a deletion of a nucleotide (G) near the C
terminus, the latter of which appears to result from a sequencing
error, which has been confirmed by re-sequencing of the cloned
TRN-SR12 and the
corresponding genomic regions in both mice and humans (data not shown).
Thus, TRN-SR1 and TRN-SR2 have the same C termini. The overall sequence
identity at the nucleotide level between TRN-SR1 and TRN-SR2 suggests
that they may result from alternative splicing. Indeed, sequence
alignment between cDNA and genomic sequences in both mice and
humans reveals that the insertion in TRN-SR1 is due to the use of a
proximal 3' splice site relative to the 3' splice site for TRN-SR2 in
exon 11 (Fig. 1).
Sequence comparison with consensus splicing sites indicates that the 3'
splice site for TRN-SR1 contains a poor branchpoint sequence and a
strong polypyrimidine tract, whereas the 3' splice site for TRN-SR2
consists of a strong branchpoint sequence and a poor polypyrimidine
tract (Fig. 1B). In addition, we note another potential
cryptic 3' splice site further upstream the two 3' splice sites (see a
long polypyrimidine tract followed by a bold ag in Fig. 1B). Such a sequence arrangement implies that multiple
3' splice sites in the front of exon 11 may compete with one another in
conjunction with other potential regulatory elements to give rise
alternatively spliced TRN-SR1 and TRN-SR2. To determine whether these
3' splice sites are indeed competent, we isolated by PCR two
TRN-SR1/SR2 genomic fragments surrounding exon 10 and exon 11 and
constructed a minigene for in vitro splicing. As shown in
Fig. 2A, the TRN-SR1/SR2
minigene transcript was inefficiently spliced in HeLa nuclear extracts
relative to the globin (H TRN-SR2 Is Predominantly Expressed in Most Cell Types--
TRN-SR1
was originally isolated from a HeLa cell library (20). However, this
isoform is underrepresented in cDNA and EST databases, indicating
that TRN-SR1 may be a rare isoform. RT-PCR analysis of a panel of human
tissues and cell lines showed that TRN-SR1 is undetectable and TRN-SR2
is the predominant isoform in most cell types (Fig. 2B).
Thus, the cryptic 3' splice site and that for TRN-SR1 appear to be
effectively suppressed in vivo in most cases, even though
the 3' splice site for TRN-SR1 appears to be stronger than that for
TRN-SR2 in in vitro splicing of the minigene (Fig.
2A). It remains to be determined whether TRN-SR1 is only
expressed in some special cell types or induced in response to certain
signaling pathways (see "Discussion").
Both TRN-SR1 and TRN-SR2 Bind and Import SR Proteins in a
Phosphorylation-dependent Manner--
A previous study has
shown that TRN-SR1 is capable of binding and importing GST fused to RS
domains from two prototypical SR proteins, ASF/SF2 and SC35 (20). In
this study, only unphosphorylated RS domain fusion proteins were tested
and potential impact of phosphorylation on TRN-SR1-mediated nuclear
import was not addressed. Subsequently, Tarn and colleagues (21, 23)
reported cloning and characterization of TRN-SR2 (21), which appears to
bind and import phosphorylated ASF/SF2 in a strictly
phosphorylation-dependent fashion. To determine whether the two
isoforms indeed exhibit distinct requirements for phosphorylation in
binding and importing SR proteins, we carried out the in
vitro binding assay using GST-ASF/SF2 and His- or S-tagged TRN-SR1
and TRN-SR2, all produced and purified from bacteria, under identical
conditions. GST-ASF/SF2 was either mock-phosphorylated or
phosphorylated by recombinant SRPK1, a kinase specific for SR proteins
(11). As shown in Fig. 3A,
both TRN-SR1 and TRN-SR2 bound GST-ASF/SF2 in a
phosphorylation-dependent manner. Similar
phosphorylation-dependent binding of GST-ASF-RS and
GST-SC35-RS to TRN-SR1 and TRN-SR2 was also obtained (data not shown).
Thus, both versions of SR protein import receptors appear to behave in
a similar way in their interactions with typical SR proteins. A low
level of specific interaction with unphosphorylated GST-ASF/SF2 by
TRN-SR1 or 2 was detectable, which may explain the reported interaction
between TRN-SR1 and unphosphorylated RS domain fusion proteins,
especially when a large amount of recombinant RS domain fusion proteins
was used in initial binding and import reactions (20).
To verify phosphorylation-dependent nuclear import of SR
proteins by both TRN-SR1 and TRN-SR2, we performed in vitro
nuclear import assays that utilize digitonin-permeabilized HeLa cells (26). As a control, we tested the import of a fluorescein
isothiocyanate-labeled substrate carrying a classical NLS from the SV40
T antigen. This substrate was actively imported in the presence of
cytosol (source of import components) (Fig. 3B). A
GFP-ASF/SF2 fusion protein was similarly imported as demonstrated
previously (20, 23). Recombinant TRN-SR1 and TRN-SR2 were then tested
in this in vitro system for their ability to import mock-
and SRPK1-phosphorylated GFP-ASF/SF2. The results showed that both
receptors actively imported this SR protein in a
phosphorylation-dependent manner (Fig. 3C). Similar phosphorylation-dependent binding and import were
also obtained with GFP-SC35-RS domain fusion protein (data not shown). We therefore conclude that TRN-SR1 and TRN-SR2 function similarly in
binding and importing typical SR proteins tested.
Phosphorylation Is Differentially Required for Nuclear Import of
hTra2 The current study has clarified the structural relationship
between TRN-SR1 and TRN-SR2 and extended previous work on the function
of these two closely related nuclear import receptors for RS
domain-containing splicing factors and regulators. Based on our
expression survey by RT-PCR (Fig. 2B), it is clear that, in
most human tissues and cell lines examined, TRN-SR1 is rare and TRN-SR2
is a predominant isoform. This expression profile is in contrast to
that from in vitro splicing of the TRN-SR1/SR2 minigene
transcript, which was designed to contain all identifiable exonic and
intronic sequence elements including a portion of intronic sequence
downstream exon 11 for potential exon definition effect (29). Further
experiments will thus be required to test additional sequences in
intron 10 to understand why the upstream cryptic splice site and the 3'
splice site for TRN-SR1 are used in in vitro splicing, but
effectively suppressed in most cell types. Our current results also
leave open the possibility that TRN-SR1 might be expressed in some
special tissue and cell types or might be induced by certain signaling
or developmental cues. If the expression of TRN-SR1 can be detected in
future studies, it will serve as an interesting model to investigate
how such tight control of splice site selection is achieved in
mammalian cells.
Although previous studies have documented a role for TRN-SR1 and
TRN-SR2 in mediating nuclear import of SR proteins, our current study
reveals their functional similarities as well as differences in binding
and importing RS domain-containing splicing factors and regulators. In
particular, hTra2 A detailed characterization of how RS domains interact with TRN-SR1 and
TRN-SR2 also remains to be done, which is crucial for understanding the
molecular basis for phosphorylation-dependent and -independent
interactions. Structural and functional studies of the prototypical
receptor importin- From the point of view of splicing regulation, nuclear import of
different RS domain-containing proteins in more than one pathway may be
important for a network control of gene expression at the splicing
level. For splicing factors imported in the
phosphorylation-dependent pathway, the level of such
factors in the nucleus may subject to regulation by the net effects of
kinase and phosphatase activities in the cell, as illustrated in Fig.
5A. Unimported factors may be
directed to a degradation pathway in the cytoplasm, which might be a
mechanism for maintaining homeostasis of some splicing factors and
regulators in specific cell types. In considering the possibility that
the TRN-SR1/SR2 gene might be regulated by alternative splicing in some
special cell types by RS domain-containing proteins, one may speculate
that import of one splicing regulator may alter the ratio of TRN-SR1
and TRN-SR2 to affect import of other RS domain-containing splicing
factors, thereby creating a chain of regulatory events (Fig.
5B). For example, changes in the balance between SR protein
kinase and phosphatase activities might differentially affect nuclear
import and consequently alter the effective concentration of RS
domain-containing splicing regulators in the nucleus. As a result, a
switch from TRN-SR2 to TRN-SR1 might be induced under certain
conditions. The production of TRN-SR1 would then facilitate nuclear
import of some RS domain-containing proteins, such as hTra2 and
hTra2
in different phosphorylation states. We suggest that
differential regulation of nuclear import may serve as a mechanism for
homeostasis of RS domain-containing splicing factors and regulators in
the nucleus and for selective cellular responses to signaling.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and hTra2
, appear to function as general
sequence-specific splicing activators in mammalian cells (5).
-related Ran-binding protein
Mtr10p (18, 19). Likewise, SR proteins can be imported by an Mtr10p homologue identified as transportin-SR
(TRN-SR1)1 in mammalian cells
(20). More recently, a second SR protein import receptor known as
transportin-SR2 (TRN-SR2) and its Drosophila ortholog
(dTRN-SR2) were cloned and characterized (21, 22). Although the
requirement for phosphorylation in TRN-SR1-mediated nuclear import of
SR proteins remains to be investigated, both Mtr10p-mediated nuclear
import of Npl3p in yeast (16) and TRN-SR2-mediated nuclear import of SR
proteins in mammalian cells (23) have been shown to be
phosphorylation-dependent.
and hTra2
. The results have important implications for how SR proteins might be
coordinately regulated in vivo.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, and GST-hTra2
have been described previously (8).
Bacterially produced His-S-TRN-SR1, His-S-TRN-SR2, His-GFP-ASF/SF2,
His-GFP-hTra2
, and His-GFP-hTra2
proteins were purified on nickel
columns. For in vitro import assay, purified proteins were
dialyzed with transport buffer (110 mM KOAc, 20 mM HEPES, pH 7.4, 2 mM Mg(OAc)2, 1 mM dithiothreitol, 1 µg/ml each of leupeptin, pepstatin,
and aprotinin). Purified fluorescein isothiocyanate-bovine serum
albumin-nuclear localization signal (NLS), Ran, and NTF2 were prepared
essentially as described previously (24, 25).
, and
GST-hTra2
by SRPK1 and subsequent in vitro bindings were
performed as described previously (8). Briefly, bacterially expressed
GST fusion proteins were phosphorylated for 6 h, which resulted in
a dramatic mobility shift in SDS gel in all cases. It was estimated by
quantifying incorporated radioactive ATP that 8 to 9 phosphates were
added to GST-ASF/SF2, 15 to 16 to GST-hTra2
, and 17 to 18 to
GST-htra2
. After desalting, 1 µg of GST fusion protein was bound
to glutathione-Sepharose (Amersham Biosciences) and the mixture
was incubated with 1 µg of recombinant His-S-TRN-SR1 or
His-S-TRN-SR2. After binding and extensive washing, bound His-S-tagged
receptor was detected by Western blotting using a His antibody
(Clontech) or the S-tag HRP LumiBlot kit (Novagen).
RESULTS
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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Fig. 1.
TRN-SR1 and TRN-SR2 are products of
alternative splicing. A, diagram of the splicing
pattern of the TRN-SR1/SR2 transcript. The primer set for RT-PCR
analysis is indicated. B, sequences of exon 10 and two
alternative 3' splice sites for exon 11. Upper- and
lowercase letters indicate exonic and intronic sequences,
respectively. Encoded amino acids are indicted by single letter code.
Potential branchpoint sequences for TRN-SR1 and TRN-SR2 based on
comparison with consensus (1) are underlined. The potential
cryptic 3' splice site is indicated by a bold ag
dinucleotide in the intron. Long lines with
arrows, PCR primers used for isolating the genomic
fragments.
) control. Nevertheless, the 5' splice
donor was spliced to all three competing 3' splice sites, but with
descending efficiency. These observations indicate that all potential
splice sites are competent, at least in vitro, but their
selection in vivo may subject to regulation.
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Fig. 2.
Splicing of the TRN-SR1/SR2 minigene in
vitro and TRN-SR/SR2 expression in cell lines and
tissues. A, time course of in vitro splicing
(from 0 to 2.5 h). Splicing of human -globin was carried as a
control (lanes 1-6) and the TRN-SR1/SR2 transcript was
spliced under the same conditions (lanes 7-12). Expected
spliced products for TRN-SR and TRN-SR2 are indicated. Note that three
lariat-exon intermediates migrated above the TRN-SR1/SR2 pre-mRNA,
which is consistent with the use of the 3' splice sites for TRN-SR1 and
TRN-SR2 as well as the cryptic 3' splice site further upstream.
Arrow, spliced product from the use of the upstream cryptic
3' splice site. P, pre-mRNA; M, mRNA;
E, released 5' exon. B, RT-PCR analysis of
TRN-SR1/SR2 expression in human tissues and cell lines.
Arrows on the left indicate the positions of
expected PCR products for TRN-SR1 and TRN-SR2 based on PCR
amplification of corresponding cDNA in the last two lanes.
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Fig. 3.
Phosphorylation-dependent binding
and import of the SR protein ASF/SF2 by TRN-SR1 and TRN-SR2.
A, in vitro pulldown of S- or His-tagged TRN-SR2
and TRN-SR1 by using GST-ASF/SF2, which was either mock-phosphorylated
( P) or phosphorylated by SRPK1 (+P). GST alone
was used as a control. Bound proteins were detected by Western blotting
using anti-S or anti-His antibodies. B, in vitro
import of fluorescein isothiocyanate-labeled bovine serum albumin-NLS
and GFP-ASF/SF2 fusion proteins in the presence of cytosol. Active
nuclear import into digitonin-permeabilized HeLa cells was demonstrated
by incubation of the import reactions on ice or at 30 °C.
C, in vitro nuclear import of mock-phosphorylated
and SRPK1-phosphorylated GFP-ASF/SF2 using recombinant TRN-SR1 and
TRN-SR2.
and hTra2
--
We then extended the analysis to additional
RS domain-containing splicing factors. We were particularly interested
in testing hTra2
and hTra2
, because they can be considered either
as typical SR proteins based on their general splicing enhancer
function in mammalian cells (5) or as RS domain-containing splicing regulators due to their sequence similarity to the splicing regulator Tra2 in the Drosophila sex determination pathway
and their functions in regulated splicing in the neuron (27). In
contrast to phosphorylation-dependent interactions with
ASF/SF2, both TRN-SR1 and TRN-SR2 appeared to bind hTra2
specifically, but phosphorylation did not seem to influence the binding
(Fig. 4A). Titration of
TRN-RS1 and TRN-SR2 indicated that the interaction of these two
receptors with both unphosphorylated and phosphorylated hTra2
was
unlikely due to saturating amounts of the receptors used in the binding
reactions (Fig. 4A). In contrast, while TRN-SR1 exhibited
efficient interaction with hTra2
, regardless its phosphorylation
state, TRN-SR2 bound hTra2
in a manner that was highly dependent on
phosphorylation (Fig. 4A). As expected, the efficiency of
nuclear import paralleled the binding profile. Both TRN-SR1 and TRN-SR2
were capable of importing unphosphorylated and phosphorylated hTra2
with similar efficiencies (Fig. 4B). In contrast, TRN-SR1
was able to import both unphosphorylated and phosphorylated hTra2
,
but TRN-SR2 could only import phosphorylated hTra2
(Fig.
4C). These data indicate that phosphorylation may not be
universally required for nuclear import of all RS domain-containing
splicing factors and regulators. This would allow this class of
splicing factors enter the nucleus via
phosphorylation-dependent and/or
phosphorylation-independent pathways, and thus, the process may be
differentially regulated by phosphorylation in different tissues or
cell types (see "Discussion"). In certain cases, it would not be
surprising that phosphorylation may actually prevent nuclear entrance
of certain splicing suppressors in interphase cells, such as SRp38
described recently (28).
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Fig. 4.
Phosphorylation-dependent and
-independent binding and nuclear import of hTra2
and hTra2
in vitro.
A, binding reactions were the same as described in the
legend to Fig. 3A. GSF-hTra2
and GST-hTra2
were either
mock-phosphorylated or phosphorylated by SRPK1. Beads immobilized with
an equivalent amount of GST-fusion proteins (~1 µg) were used to
pull down increasing amounts (in µl) of recombinant TRN-SR1 and
TRN-SR2 (concentration = 0.1 µg/µl). The first lanes show the
signal for the input (2.5 µl) of TRN-SR1 (upper panel) or
TRN-SR2 (lower panel). B and C,
nuclear import of GSF-hTra2
and GST-hTra2
in different
phosphorylation states by recombinant TRN-SR1 and TRN-SR2.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
seems to be imported by both receptors in a
phosphorylation-independent pathway whereas hTra2
appears to be
differentially imported, depending on its phosphorylation state.
Interestingly, like their Drosophila homologue, both
hTra2
and hTra2
appear to harbor two RS domains, one at the N
terminus and the other at the C terminus. Previous mutagenesis studies
have established the presence of multiple NLS sequences in RS
domain-containing splicing factors and regulators (30, 31), but it is
unclear whether those seemingly redundant NLS sequences in native
proteins function in a parallel or synergistic fashion. In addition,
sequences outside the RS domain of ASF/SF2 may also carry an
independent nuclear targeting signal (10). In the case of hTra2
and
-
, however, preliminary binding studies using deletion mutants
indicate that their import by TRN-SR1 and TRN-SR2 appears to be
mediated by multiple nuclear targeting signals in the RS domains, not
through their RNA recognition
motifs.3 Fine mapping is in
progress to dissect NLS in each RS domain and determine which
NLS-mediated nuclear import depends on phosphorylation and which does not.
show that it can interact with the IBB
motif in the adaptor protein importin-
(32) as well as directly with
a variety of NLS sequences in small nuclear ribonucleoprotein particles
(33), transcription factors (34, 35), cell cycle regulators (36), and
arginine-rich human immunodeficiency virus regulatory proteins (37,
38). Thus, a variety of different types of molecular interactions may
take place in the cargo binding pocket of importin-
. A similar
situation may also hold true for the interaction between TRN-SR1 or
TRN-SR2 and different RS domains, but this will require detailed
structural and functional studies in the future.
, via the
phosphorylation-independent pathway. This potential mechanism would
allow cells to selectively import a subset of RS domain-containing
splicing factors and regulators in response to signaling. This
hypothesis could be tested in the future if cells capable of expressing
TRN-SR1 can be identified.
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Fig. 5.
Model for the potential functional
significance of regulated nuclear import. A,
hypothetical regulation of the interaction between SR proteins and
their nuclear import receptors by kinases and phosphatases.
B, differentially imported RS domain-containing splicing
regulators may in turn alter the splicing pattern of their import
receptors in the nucleus, thereby allowing a subset of splicing factors
to be imported in either phosphorylation-dependent or
-independent pathways.
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ACKNOWLEDGEMENTS |
---|
We thank J. Yeakley and other members of the Fu laboratory for help and suggestions during the course of this project, Jackie Horridge in the Fu laboratory for cloning and sequencing of TRN-SR2 from HeLa total RNA, N. Kataoka and G. Dreyfuss for providing us the TRN-SR1 cDNA, W.-Y. Tarn for close communication and sharing unpublished results, and L. Gerace and T. Hope for critical comments on the manuscripts. We are indebted to L. Gerace in whose laboratory initial in vitro nuclear import assays were carried out.
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FOOTNOTES |
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* This work was supported by Public Health Service Grant GM52872 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Current address: NYU Medical Center, SI 3-10, 540 First Ave., New York, NY 10016.
To whom correspondence should be addressed: Dept. of
Cellular and Molecular Medicine, UCSD, 9500 Gilman Dr., La Jolla, CA 92093-0651. Tel.: 858-534-4937; Fax: 858-534-8549; E-mail:
xdfu@ucsd.edu.
Published, JBC Papers in Press, March 13, 2003, DOI 10.1074/jbc.M211714200
2 N. Kataoka, personal communication.
3 C. Y. Yun, A. L. Velazquez-Dones, and X.-D. Fu, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: TRN, transportin; RT, reverse transcriptase; GST, glutathione S-transferase; NLS, nuclear localization signal.
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