Serine-Arginine-rich Protein p30 Directs Alternative Splicing of Glucocorticoid Receptor Pre-mRNA to Glucocorticoid Receptor
in Neutrophils*
Qing Xu
,
Donald Y. M. Leung
and
Kevin O. Kisich ¶
From the
Division of Pediatric Allergy/Immunology,
National Jewish Medical and Research Center, Department of Pediatrics, Denver,
Colorado 80206 and the ¶Departments of Pediatrics
and Immunology, University of Colorado Health Sciences Center, Denver,
Colorado 80262
Received for publication, January 24, 2003
, and in revised form, April 29, 2003.
 |
ABSTRACT
|
---|
Glucocorticoid (GC) insensitivity is a major clinical challenge in the
treatment of many inflammatory diseases. It has been shown previously that GC
insensitivity, in several inflammatory cell types, is due to an overabundance
of the
isoform of the glucocorticoid receptor (GCR
) relative to
the ligand binding isoform, GCR
. GCR
functions as a dominant
inhibitor of GCR
action. A number of GCR isoforms are created from the
same pre-mRNA transcript via alternative splicing, and the factor or factors
that control alternative splicing of GCR pre-mRNA are of great importance. In
the current study, we have identified the predominant alternative splicing
factor present in human neutrophils, which is known to be exceptionally
GC-insensitive. The predominant alternative splicing factor in neutrophils is
SRp30c, which is one of several highly conserved serine-arginine-rich (SR)
proteins that are involved in both constitutive and alternative splicing in
eukaryotic cells. Inhibition of SRp30c expression with antisense
oligonucleotide strongly inhibited expression of GCR
and stimulated
expression of GCR
. Antisense molecules targeted to other SR proteins
had no effect. Our data indicate that SRp30c is necessary for alternative
splicing of the GCR pre-mRNA to create mRNA encoding GCR
.
 |
INTRODUCTION
|
---|
Glucocorticoid
(GC)1 insensitivity is
a major clinical challenge in the treatment of chronic inflammatory diseases.
The pharmacologic actions of GCs are mediated through intracellular receptors,
the glucocorticoid receptors (GCR). There are two isoforms of GCR in human
cells, GCR
and GCR
, which are generated from a single gene via
alternative splicing of the primary RNA transcript. Several studies indicate
that GC insensitivity has been associated with increased expression of
GCR
(17).
GCR
is truncated at the C terminus, which corresponds to the ligand
binding domain. Thus, GCR
cannot bind GC. In addition, GCR
does
not transactivate GC-sensitive genes and functions as a dominant inhibitor of
GCR
(8). Previous
studies suggest that GCR
:GCR
heterodimer formation may account
for the reduced effectiveness of GC action in cells overexpressing GCR
(8). Recent experiments in our
lab demonstrated that overexpression of human GCR
by mouse hybridoma
cells results in the development of GC insensitivity by these cells
(9).
Regulation of GCR
expression is not well understood. Different cell
types from the same individual can have very different ratios of GCR
to
GCR
. For example, freshly isolated peripheral blood neutrophils are
GC-insensitive. Both the absolute level of GCR
and the ratio of
GCR
to GCR
protein are much higher in neutrophils than in
peripheral blood mononuclear cells (PBMC) from the same individuals
(10). The ratio of GCR
to GCR
can be altered by cytokine stimulation. After stimulation of
neutrophils with IL-8, GCR
mRNA levels increased remarkably, and
GCR
mRNA decreased to undetectable levels. Similarly, exposure of PBMC
to IL-2 and IL-4 resulted in increased GCR
expression and development of
steroid insensitivity (1,
11). Since both GCR
and
GCR
are generated by alternative splicing of the same primary transcript
(1214),
understanding the regulation of this splicing event is of primary importance
for delineating mechanisms of GC insensitivity. Therefore, the goal of this
research has been to identify the factor or factors involved in determining
whether exon 9
is joined to exon 8 to generate mRNA encoding GCR
or whether exon 8 is joined to exon 9
, resulting in mRNA encoding
GCR
.
Pre-mRNA splicing is an essential step in eukaryotic gene expression. It is
a multistep process including the accurate recognition of splice sites,
excision of intronic sequences, and ligation of the 5' and 3' ends
of the resulting fragments
(1519).
The splicing process occurs with the assembly of a multicomponent structure
known as the spliceosome (20).
The major components of the spliceosome are small nuclear ribonucleoprotein
particles U1, U2, and U4/U6, and several proteins including a family of
serine-arginine-rich proteins known as the SR proteins.
SR proteins have a dual role in pre-mRNA splicing in vivo and
in vitro (21). They
are involved in constitutive as well as alternative splicing. SR proteins have
one or two N-terminal RNA recognition motifs and a C terminus rich in
arginine/serine dipeptide repeats (the RS domain)
(22), which mediates protein
interactions with other components of the splicing machinery. In the following
experiments, we have identified the SR protein required for alternative
splicing of the GCR primary RNA transcript into GCR
mRNA
 |
MATERIALS AND METHODS
|
---|
Cell IsolationHuman neutrophils and PBMC were isolated from
normal healthy individuals using a Percoll (Amersham Biosciences)
(23) density gradient. 40 ml
of venous blood was collected in syringes with 4.4 ml of 3.8% sodium citrate
(Fisher). The whole blood was centrifuged at 1200 rpm for 20 min, and then
upper platelet-rich plasma layer was removed and centrifuged at 3000 rpm for
15 min. The platelet-poor plasma supernatant was aspirated from the platelet
pellet. In the meantime, 5 ml of 6% (w/v) dextran (Amersham Biosciences) and
saline was added to 50 ml to the cellular component. Erythrocytes were allowed
to sediment for 30 min at room temperature, and the leukocyte-rich supernatant
was collected. Then the leukocyte-rich supernatant was sedimented at 1000 rpm
for 10 min. The supernatant was discarded, and the white blood cell pellet was
resuspended with 2 ml of platelet-poor plasma. This was underlayed with 42%
(w/v) Percoll followed by 51% (w/v) Percoll. After 5 min, the leukocytes were
centrifuged at 1000 rpm for 10 min. PBMC and neutrophils formed separate bands
at the Percoll density interfaces and were collected. The purity of
neutrophils isolated by this method was >95%, as determined by
Wright-Giemsa staining.
Stimulation of Neutrophils with IL-8 Freshly separated
neutrophils from normal individuals were cultured at a concentration of 1.0
x 106/ml in the presence and absence of IL-8 (0.5 µg/ml)
(R&D systems, Minneapolis, MN) for 2 h. Then cells were harvested, and
proteins were extracted.
Generation of Monoclonal AntibodiesHybridomas CRL-2385
(ATCC, Manassas, VA), which produce monoclonal antibody against a conserved
epitope on a subset of SR proteins, were cultured for 10 days, and then
supernatants were collected
(24).
Western Blot AnalysisCell and nuclear lysates were prepared
from neutrophils, PBMC, and the PLB-985 cell line. Cells were resuspended in
buffer containing 10 mM Tris-HCl (pH 8.3), 1 mM EDTA,
and a mixture of protease inhibitors (0.1 mM phenylmethylsulfonyl
fluoride, 1 µg/ml aprotinin, 1 µM pepstatin, and 1
µM leupeptin). After 30 min, cells were centrifuged at 12,000
rpm for 10 min at 4 °C. Supernatants were collected, and proteins were
resolved by electrophoresis followed by electrophoretic transfer to
polyvinylidene difluoride membranes (Bio-Rad) in Tris-glycine buffer.
Membranes were blocked overnight with blocking buffer (5% milk, 10% 50
mM sodium phosphate, 3% 5 M NaCl, and 0.05% Tween 20)
and then incubated with primary antibody to GCR
(rabbit polyclonal
antibody, ABR Affinity Bioreagents INC, Golden, CO), total GCR (Santa Cruz
Biotechnology, Santa Cruz, CA), and SR proteins (ATCC, CRL-2385) for 2 h,
washed, and then incubated with secondary antibody, horseradish peroxidase
protein A (Amersham Biosciences), for GCR
and total GCR, horseradish
peroxidase-rat anti-mouse IgG1 (Zymed Laboratories Inc., So. San
Francisco, CA) for SR proteins for 1 h. Membranes were washed and developed
with ECL Western blotting detection reagents (Amersham Biosciences).
PLB-985 Cell DifferentiationPLB-985 cells were stimulated
with retinoic acid (Sigma)
(25). 14.9 µl of 6.7
mM retinoic acid were added to 100 ml of medium with 2.0 x
107 cells (final concentration of retinoic acid, 1
µM). Medium was changed on the third day. After 5 days, slides
were made and stained with Diff Quick (Fisher) to evaluate nuclear
morphology.
Transfection of Antisense Oligonucleotides Using
Electroporation PLB-985 cells were washed once in Hanks' balanced
salt solution. 5.0 x 106 cells were resuspended in 500 µl
of RPMI with 10% (v/v) fetal calf serum (without antibiotics) and then
transferred to electroporation cuvettes (Disposable Micro-Electroporation
Chambers, Invitrogen). 10 µg of fluorescein-labeled antisense
oligonucleotides (Midland Certified Reagent Co, Midland, TX) were added to 500
µl of RPMI/fetal calf serum in the cuvettes, mixed gently, and then
incubated at room temperature for 10 min. Cells were permeabilized with a
single pulse using the Invitrogen electroporation apparatus set to 800 ohms to
230 V. These conditions were defined as producing optimal permeabilization
with minimal toxicity in preliminary experiments. The cells were incubated for
2530 min at room temperature in the electroporation cuvettes and then
transferred to flasks containing 25 ml of differentiation medium.
Flow CytometryFlow cytometry was performed on a FACSCaliber
instrument, and analysis used CELL QUEST Software(Becton Dickinson). Uptake
studies were performed using purified oligonucleotides to ensure that only
intact oligonucleotides were studied. 1 ml containing 1.0 x
106 cells was added into 5-ml FALCON tubes (BD Biosciences), and
efficiency of uptake was checked by the flow cytometry. Cells positive for
fluorescein were sorted within 12 h of electroporation and placed into
cultures for differentiation.
RNA Isolation and cDNA PreparationTotal RNA was isolated
from fresh neutrophils, PBMC, and PLB-985 cells by using RNA-Bee (TELTEST,
Friendswood, TX) according to the manufacturer's instructions. Reverse
transcription reactions used
2 µg of total RNA in 20 µl with 200
units of SuperScript II reverse transcriptase (Invitrogen).
Plasmid ConstructionPlasmids used to create standard curves
for real-time PCR were generated as follows: SRp30a cDNA region spanning
nucleotides 111990 GenBankTM (NM_006924
[GenBank]
) was amplified with
forward primer (5'-TTTTCGTCACCGCCATGTC-3') and reverse primer
(5'-CAATTCAACACTTTAGCCCA-3') (all primers were designed using
Macvector 6.5.3) (Accelrys), SRp30b region spanning nucleotides 194630
(GenBankTM NM_003016
[GenBank]
) was amplified with forward primer
(5'-GACCTCCCTCAAGGTGGACAAC-3') and reverse primer
(5'-ACCGAGATCGAGAACGAGTGC-3'), SRp30c cDNA region spanning
nucleotides 312986 (GenBankTM NM_003769
[GenBank]
) was amplified with
forward primer (5'-CCAGGACTTATGGAGGTCGG-3') and reverse primer
(5'-AACCCCACAAAGACAGAACG-3') using cDNA prepared from RNA
extracted from fresh cells. The PCR products were cloned into the PGEM-T
vector (Promega, Madison, WI).
Real-time PCRAn ABI7700 (PerkinElmer Life Sciences) with
Taqman probe sets was used to quantify mRNA levels. Primers were designed to
amplify segments of
200 bp to maximize efficiency. Primer sequences were
as follows: SRp30a, forward primer, 5'-TTGAGTTCGAGGACCCGC-3';
reverse primer, 5'-CGTAATCATAGCCGTCGCG-3'; Taqman probe,
6-fam-CGCGGAAGACGCGGTGTATGG-tam. SRp30b, forward primer,
5'-CGCGGCTTCGCCTTC-3'; reverse primer,
5'-ATGGCATCCATAGCGTCCTC-3'; Taqman probe,
6-fam-TTCGCTTTCACGACAAGCGCGAC-tam. SRp30c, forward primer,
5'-TTTCCGAGTTCTTGTTTCAGGAC-3'; reverse primer,
5'-CTCGCATGTGATCCTTCAGGT-3'; Taqman probe,
6-fam-CCTCCGTCAGGCAGCTGGCAG-tam. Each sample was tested in duplicate, and all
PCR runs were performed three times. Each sequence was quantified relative to
a standard curve of its cognate cloned cDNA sequence.
 |
RESULTS
|
---|
SR Protein Levels in Neutrophils and PBMCTo identify
candidate proteins for the GCR alternative splicing factors, we compared the
complements of SR proteins in cell extracts from human neutrophils, which are
rich in GCR
, with extracts from PBMC, which have low levels of
GCR
. Western blot analysis of cell extracts was performed on 20 µg of
nuclear extracts from neutrophils and PBMC separated from six normal donors.
Both freshly isolated neutrophils and PBMC expressed SR proteins
(Fig. 1). The blots were
developed using chemiluminescence, and the intensity of each band was measured
using a flatbed scanner and NIH Image 1.61. We compared SRp20, SRp30, SRp40,
SRp55, and SRp75 levels in neutrophils and PBMC. However, neutrophils
contained SR proteins predominantly in the 30-kDa range. Levels of SRp30 in
neutrophils were significantly greater than in PBMC when data from the six
individuals were pooled and averaged (3.3 ± 0.97 density units
versus 1.3 ± 0.42 density units (mean ± S.E.);
p = 0.02; t test). Ratios of SRp30 in neutrophils
versus PBMC for the six donors ranged from
1 (donor three) to
6.7 (donor four). To identify the specific SRp30 species present in human
neutrophils, we utilized real-time PCR and specific Taqman probe sets for
SRp30a, SRp30b, and SRp30c. Fig.
2 shows that only mRNAs encoding SRp30a and SRp30c were present in
the neutrophils. Levels of SRp30c mRNA were
23-fold higher (p =
0.03, t test for paired samples) than levels of SRp30a. This suggests
that SRp30c is probably the predominant SR protein present in human
neutrophils.

View larger version (39K):
[in this window]
[in a new window]
|
FIG. 1. Expression of SR proteins in neutrophils and PBMC from six normal
individuals. SRp30 protein levels, analyzed by Western blot, are
significantly higher in neutrophils as compared with PBMC. Data are expressed
as mean ± S.E. analyzed by two-tailed paired t test.
PMN, polymorphonuclear neutrophils.
|
|

View larger version (10K):
[in this window]
[in a new window]
|
FIG. 2. Real-time PCR shows that SRp30c mRNA is significantly higher in
neutrophils from four normal individuals as compared with SRp30a and
SRp30b. Data are expressed as mean ± S.E. and analyzed by
two-tailed paired t test.
|
|
SR Protein Levels in Neutrophils Stimulated with IL-8 We
have demonstrated previously that IL-8 enhances GC insensitivity in
neutrophils by increasing synthesis of GCR
relative to GCR
(10). Therefore, we performed
Western analysis on extracts from neutrophils that had been treated with IL-8
as compared with unstimulated neutrophils
(Fig. 3A). Freshly
isolated neutrophils were treated with IL-8 or medium only for 2 h. 20 µg
of nuclear extracts from neutrophils separated from four normal donors was
separated on SDS-PAGE gels. After transferring to polyvinylidene difluoride
membranes and staining with specific anti-SR monoclonal antibody, the blots
were developed with horseradish peroxidase-conjugated secondary antibody and
chemiluminescence. Densitometry of the films revealed that SRp30 levels
increased significantly in neutrophils treated with IL-8 as compared with
neutrophils incubated in medium only (62 ± 14 density units
versus 32 ± 10 density units; p = 0.04)
(Fig. 3B). Therefore,
conditions that result in increased GCR
levels in neutrophils also
result in increased levels of SRp30 proteins in neutrophils.

View larger version (13K):
[in this window]
[in a new window]
|
FIG. 3. SRp30 levels after IL-8 stimulation in neutrophils. A,
representative Western blot of increased expression of SRp30 proteins in
neutrophils treated for 2 h with IL-8 versus medium only in a normal
individual. B, mean densitometry of SRp30 proteins in neutrophils
from four normal individuals. After 2 h of stimulation with IL-8, SRp30 levels
increased in neutrophils as compared with neutrophils in medium alone. Data
are expressed as mean ± S.E. analyzed by two-tailed paired t
test.
|
|
PLB-985 Cell DifferentiationTo obtain functional evidence
of the identity of the GCR alternative splicing factor, we initially attempted
to specifically inhibit SRp30a and SRp30c expressions in neutrophils using
antisense phosphorothioate oligonucleotides. However, due to the brief
viability of these cells in culture, we were unable to obtain reproducible
results. Therefore, we employed a model of neutrophil-like differentiation
using the PLB-985 cell line
(25). Cells of this line are
capable of granulocytic maturation to become neutrophil-like in the presence
of inducing agents such as retinoic acid. After exposure to retinoic acid, the
cells become neutrophil-like. Fig.
4 illustrates the morphology of PLB-985 cells after 5 days of
exposure to retinoic acid.

View larger version (22K):
[in this window]
[in a new window]
|
FIG. 4. Neutrophilic differentiation of PLB-985 cells. A, PLB-985
cells demonstrating large nuclei and intensely staining cytoplasm before
differentiation. As shown in B, after differentiation, cells exhibit
neutrophil-like morphology, including multilobed nuclei, and lightly staining
cytoplasm.
|
|
GCR
and GCR
Levels in PLB-985 Cells before and after
Neutrophilic DifferentiationTo measure the dynamics of GCR
expression upon differentiation of PLB 985 cells, we performed Western blot
analysis of GCR from lysates of cells prior to and after 5 days of retinoic
acid exposure. Fig. 5 revealed
that GCR
and GCR
levels both increased upon exposure to retinoic
acid relative to undifferentiated PLB-985 cells. However, densitometry
revealed that GCR
levels (5.2 ± 2.4 density units on day 0
versus 143.2 ± 18.2 density units on day five, p =
0.01) increased much more than GCR
(1.82 ± 0.55 density units on
day 0 versus 18.03 ± 4.05 density units on day five,
p = 0.04). This suggests that both synthesis of GCR primary
transcript and alternative splicing are increased upon exposure to retinoic
acid, but the direction of alternative splicing increased toward
GCR
.
SR Protein Levels in Undifferentiated and Differentiated PLB-985
CellsTo examine our hypothesis that enhanced alternative splicing
of GCR pre-mRNA is associated with enhanced expression of SR proteins in the
30-kDa range, we performed Western analysis on cell extracts prior to and
after 5 days of exposure to retinoic acid. Densitometry of the film shown in
Fig. 6A shows that
SRp30 levels significantly increased after differentiation relative to
undifferentiated cells. Fig.
6B shows the density units of SRp30 from three
experiments before and after differentiation (1.08 ± 0.25 density units
versus 77.47 ± 19.30 density units, p = 0.02).
Real-time PCR revealed that upon differentiation, PLB-985 cells expressed
increased levels of SRp30a, SRp30b, and SRp30c. The expression amounts for
SRp30a and SRp30c were much higher than those found in primary neutrophils at
4.3 x 10-5 pmol/ng of 18 S and 3.24 x 10-4
pmol/ng of 18 S, respectively, whereas mRNA for SRp30b was present at
1.2
x 10-5 pmol/ng of 18 S (data not shown). Expression of SRp30b
was in contrast to data obtained from primary neutrophils, which did not
express SRp30b.

View larger version (17K):
[in this window]
[in a new window]
|
FIG. 6. Expression of SR proteins in PLB cells before and after
differentiation. After differentiation, SRp30 proteins selectively
increased in differentiated PLB cells. A representative blot from three
experiments is shown in A, and the mean ± S.E. of three
experiments is shown in B. Data were analyzed by two-tailed paired
t test.
|
|
Inhibition of SRp30c via Antisense Phosphorothioate Oligonucleotides
Inhibits Alternative Splicing of GCR pre-mRNA To determine which
of the three SRp30 proteins present in PLB-985 cells were necessary for
alternative splicing of the GCR pre-mRNA to generate GCR
, each SRp30
protein was specifically targeted with antisense phosphorothioate
oligonucleotides. Electroporation of fluorescein-tagged antisense
oligonucleotides for SRp30a, SRp30b, or SRp30c was performed as described, and
the cells were sorted for the fluorescein-positive population. The transfected
cells were then returned to cultures and exposed to retinoic acid for up to 5
days. Nuclear extracts were then prepared and separated by SDS-PAGE for
Western analysis of GCR
protein levels
(Fig. 7).
Fig. 7A is a
representative Western blot showing that GCR
levels were inhibited for
up to 48 h with antisense oligonucleotide for SRp30c after transfection
relative to control oligonucleotide. Fig.
7B shows the density units of GCR
from three
experiments transfected with antisense oligonucleotide for SRp30a, SRp30b,
SRp30c, and control oligonucleotide. GCR
levels were significantly
inhibited at 24 h after transfection with antisense oligonucleotide for SRp30c
as compared with control oligonucleotide (19.43 ± 11.98 density units
versus 153.1 ± 15.4 density units, p = 0.003). As
shown in Fig. 8, GCR
was
simultaneously elevated following treatment of the cells with anti-SRp30c
relative to cells treated with control oligonucleotide. In the cells
transfected with antisense oligonucleotides for SRp30a or SRp30b, there was no
effect on GCR
levels relative to control oligonucleotide or untreated
cells.
 |
DISCUSSION
|
---|
Pre-mRNA splicing is an essential step in gene expression by eukaryotes. It
is a multistep process including the accurate recognition of splice sites,
excision of intronic sequences, and ligation of the resulting fragments back
into a single molecule. All of these steps are performed by a multimolecular
complex of proteins and RNA known as the spliceosome
(20). Recognition of the
5' and 3' splice sites by the spliceosome requires several small
nuclear ribonucleoprotein particles, including U1, U2, and U4/U6 and the
non-small nuclear ribonucleoprotein particle proteins including the family of
serine/arginine-rich (SR) proteins
(2730).
40% of the human genes may be constitutively spliced
(31). The selection of splice
sites is determined by several parameters including the proximity and strength
of splicing signals (32).
Constitutive splicing can be performed by the minimal spliceosome and involves
ligation of the distal end of the upstream exon with the nearest splice
acceptor, which is at the proximal end of the next exon in the sequence. Among
the different mechanisms of constitutive and alternative splicing, the
possible exclusion of sequences where both the 5' and the 3'
intronic junctions are located within an exon represents a further pattern of
alternative splicing (33). In
alternative splicing, one or more splice acceptors and associated exons are
skipped, resulting in removal of their coding information from the final mRNA.
In the case of GCR, exon 9
is skipped, and exon 9
is ligated
directly to the distal end of exon 8. This results in inclusion of an
alternative C terminus to the GCR. The C terminus encoded by exon 9
included the ligand binding domain, whereas that encoded by exon 9
includes a domain that cannot bind GC. Stimulation of alternative splicing for
other mRNAs such as fibronectin
(3436),
-tropomyosin (37),
apolipoprotein B (apoB) (38),
adenylyl cyclase stimulatory G-protein G
(s)
(39), and survival motor
neuron (40) requires the
coordinated action of specific SR proteins and several classes of cis acting
mRNA elements including purine-rich splicing enhancers known as exonic
splicing elements. These proteins bind and can direct the splicing to
alternative splice sites in a concentration-dependent manner, regulate the
stability of mRNA, and have a general role in mRNA export
(4143).
SR proteins contain one or two N-terminal RNA recognition motifs, which
mediate binding to the exonic splicing elements in the RNA, and a C terminus
rich in arginine/serine dipeptide repeats (the RS domain), which mediates
protein interactions with other components of the spliceosome. Alternative
splicing is a common mechanism to create alternative protein isoforms. A
characteristic of alternative splicing is to introduce stop codons or frame
shifts that can either switch off genes or create proteins with different C
termini.
The strategy used in these studies to identify the factor or factors
involved in selection of the alternative splice site in exon 9
was to
first compare the complement of SR proteins present in cells rich in
GCR
, such as PBMC, with SR proteins present in cells rich in GCR
,
such as neutrophils. This comparison, shown in
Fig. 1, revealed that although
PBMC contain a variety of SR proteins, neutrophils contain predominantly SR
proteins in the 30-kDa range, with minor bands representing SRp75 and SRp20.
The strong band near 30 kDa could contain one or more of SRp30a, SRp30b, or
SRp30c. Of these three candidates, SRp30c has been found to stimulate
alternative splice site selection in CD45 pre-mRNA in leukocytes
(44), whereas SRp30a (SF2/ASF)
has been shown to influence alternative splice site selection in fibronectin
(45,
46),
-tropomyosin
(37), influenza virus
(47), herpes-simplex virus,
and adenovirus E1A pre-mRNAs
(4851).
SRp30b (SC35) has been found to influence splicing of CD45 and implantation
related genes, among others.
To identify which of the SRp30 molecules were present in neutrophils and
could participate in alternative splicing of GCR pre-mRNA, we performed both
reverse transcriptase-PCR and real-time PCR for the mRNAs encoding SRp30a,
SRp30b, and SRp30c. Since SRp30b (SC35) was undetectable by either reverse
transcriptase-PCR or real-time PCR, we concluded that the SRp30a and SRp30c
were the most likely candidates for GCR alternative splicing factors. However,
as SRp30c mRNA was present in neutrophils at much higher levels than SRp30a,
we concluded that the majority of the protein present in neutrophils upon
Western analysis was probably SRp30c.
Conclusive evidence for the identity of the GCR splicing factor or factors
required the ability to specifically inhibit expression of SRp30a and SRp30c
in a cell type that also expressed GCR
. We initially attempted to use
phosphorothioate antisense DNA molecules targeted to SRp30a and SRp30c to
inhibit expression of these proteins in neutrophils, but the short life span
of these cells in culture made it very difficult to obtain reproducible data.
Therefore, we developed a model based of PLB-985 cells, which are from a
promyelocytic leukemia that differentiate into neutrophil-like cells in
response to retinoic acid
(25).
Upon exposure to retinoic acid, PLB-985 cells begin to differentiate, which
takes about 5 days to complete as shown in
Fig. 4. In addition, these
cells express exclusively higher levels of SR proteins in the 30-kDa range
after differentiation; these proteins are undetectable prior to
differentiation. Both GCR
and GCR
increased during
differentiation; however, GCR
increased by
27-fold, whereas
GCR
increased by
10-fold. Therefore, alternative splicing of GCR
pre-mRNA is stimulated during differentiation of PLB-985 cells, coincident
with a dramatic increase in SRp30 proteins. According to real-time PCR,
PLB-985 cells express SRp30a, SRp30b, and SRp30c. Therefore, mRNAs encoding
all three proteins were targeted using specific phosphorothioate antisense DNA
molecules.
Alternative splicing of GCR pre-mRNA to GCR
was not affected when
cells were treated with control phosphorothioate oligonucleotides, nor was it
affected when the oligonucleotides specifically targeted SRp30a or SRp30b.
However, as shown in Fig. 7,
targeting of SRp30c with a specific phosphorothioate antisense molecule
resulted in a dramatic reduction in GCR
production for
24 h after
transfection, whereas GCR
production was stimulated
(Fig. 8). Therefore, SRp30c was
necessary for alternative splicing of GCR pre-mRNA to generate mRNA encoding
GCR
. Since SRp30c represents the major alternative splicing factor
present in neutrophils, we conclude that SRp30c is required for alternative
splicing to generate GCR
mRNA. Since GCR
is intimately involved in
GC insensitivity, and GCR
is dependent on the presence of SRp30c, this
SR protein may make an attractive target for therapeutic intervention.
 |
FOOTNOTES
|
---|
* The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact. 
To whom correspondence should be addressed: National Jewish Medical and
Research Center, 1400 Jackson St., Rm. K926, Denver, CO 80206. Tel.:
303-398-1379; Fax: 303-270-2182; E-mail:
leungd{at}njc.org.
1 The abbreviations used are: GC, glucocorticoid; GCR, glucocorticoid
receptor; apoB, apolipoprotein B; PBMC, peripheral blood mononuclear cells;
SR, serine-arginine-rich. 
 |
REFERENCES
|
---|
- Leung, D. Y., Hamid, Q., Vottero, A., Szefler, S. J., Surs, W.,
Minshall, E., Chrousos, G. P., and Klemm, D. J. (1997)
J. Exp. Med. 186,
1567-1574[Abstract/Free Full Text]
- Leung, D. Y., and Bloom, J. W. (2003) J.
Allergy Clin. Immunol. 111,
1-20[CrossRef]
- Hamilos, D. L., Leung, D. Y., Muro, S., Kahn, A. M., Hamilos, S.
S., Thawley, S. E., and Hamid, Q. A. (2001) J. Allergy
Clin. Immunol. 108,
59-68[CrossRef][Medline]
[Order article via Infotrieve]
- Christodoulopoulos, P., Leung, D. Y., Elliott, M. W., Hogg, J. C.,
Muro, S., Toda, M., Laberge, S., and Hamid, Q. A. (2000)
J. Allergy Clin. Immunol.
106,
479-484[CrossRef][Medline]
[Order article via Infotrieve]
- Sousa, A. R., Lane, S. J., Cidlowski, J. A., Staynov, D. Z., and
Lee, T. H. (2000) J. Allergy Clin.
Immunol. 105,
943-950[CrossRef][Medline]
[Order article via Infotrieve]
- Honda, M., Orii, F., Ayabe, T., Imai, S., Ashida, T., Obara, T.,
and Kohgo, Y. (2000) Gastroenterology
118,
859-866[Medline]
[Order article via Infotrieve]
- Hamid, Q. A., Wenzel, S. E., Hauk, P. J., Tsicopoulos, A.,
Wallaert, B., Lafitte, J. J., Chrousos, G. P., Szefler, S. J., and Leung, D.
Y. (1999) Am. J. Respir. Crit. Care Med.
159,
1600-1604[Abstract/Free Full Text]
- Oakley, R. H., Jewell, C. M., Yudt, M. R., Bofetiado, D. M., and
Cidlowski, J. A. (1999) J. Biol. Chem.
274,
27857-27866[Abstract/Free Full Text]
- Hauk, P. J., Goleva, E., Strickland, I., Vottero, A., Chrousos, G.
P., Kisich, K. O., and Leung, D. Y. (2002) Am. J.
Respir. Cell Mol. Biol. 27,
361-367[Abstract/Free Full Text]
- Strickland, I., Kisich, K., Hauk, P. J., Vottero, A., Chrousos, G.
P., Klemm, D. J., and Leung, D. Y. (2001) J. Exp.
Med. 193,
585-594[Abstract/Free Full Text]
- Leung, D. Y., de Castro, M., Szefler, S. J., and Chrousos, G. P.
(1998) Ann. N. Y. Acad. Sci.
840,
735-746[Abstract/Free Full Text]
- Encío, I. J., and Detera-Wadleigh, S. D. (1991)
J. Biol. Chem. 266,
7182-7188[Abstract/Free Full Text]
- Hollenberg, S. M., Weinberger, C., Ong, E. S., Cerelli, G., Oro,
A., Lebo, R., Thompson, E. B., Rosenfeld, M. G., and Evans, R. M.
(1985) Nature
318,
635-641[Medline]
[Order article via Infotrieve]
- Oakley, R. H., Sar, M., and Cidlowski, J. A. (1996)
J. Biol. Chem. 271,
9550-9559[Abstract/Free Full Text]
- Jamison, S. F., Pasman, Z., Wang, J., Will, C., Luhrmann, R.,
Manley, J. L., and Garcia-Blanco, M. A. (1995) Nucleic
Acids Res. 23,
3260-3267[Abstract]
- Kohtz, J. D., Jamison, S. F., Will, C. L., Zuo, P., Luhrmann, R.,
Garcia-Blanco, M. A., and Manley, J. L. (1994)
Nature 368,
119-124[CrossRef][Medline]
[Order article via Infotrieve]
- Robberson, B. L., Cote, G. J., and Berget, S. M.
(1990) Mol. Cell. Biol.
10, 84-94[Medline]
[Order article via Infotrieve]
- Roscigno, R. F., and Garcia-Blanco, M. A. (1995)
RNA (N. Y.) 1,
692-706[Abstract]
- Tarn, W. Y., and Steitz, J. A. (1995) Proc.
Natl. Acad. Sci. U. S. A. 92,
2504-2508[Abstract]
- Staley, J. P., and Guthrie, C. (1998)
Cell 92,
315-326[Medline]
[Order article via Infotrieve]
- Smith, C. W., and Valcarcel, J. (2000)
Trends Biochem. Sci. 25,
381-388[CrossRef][Medline]
[Order article via Infotrieve]
- Zuo, P., and Maniatis, T. (1996) Genes
Dev. 10,
1356-1368[Abstract]
- Sala, A., Zarini, S., Folco, G., Murphy, R. C., and Henson, P. M.
(1999) J. Biol. Chem.
274,
28264-28269[Abstract/Free Full Text]
- Neugebauer, K. M., Stolk, J. A., and Roth, M. B.
(1995) J. Cell Biol.
129,
899-908[Abstract]
- Tucker, K. A., Lilly, M. B., Heck, L., Jr., and Rado, T. A.
(1987) Blood
70, 372-378[Abstract]
- Deleted in proof
- Guthrie, C. (1991) Science
253,
157-163[Medline]
[Order article via Infotrieve]
- Sharp, P. A. (1994) Cell
77, 805-815[Medline]
[Order article via Infotrieve]
- Manley, J. L., and Tacke, R. (1996) Genes
Dev. 10,
1569-1579[CrossRef][Medline]
[Order article via Infotrieve]
- Graveley, B. R. (2000) RNA (N.
Y.) 6,
1197-1211[Free Full Text]
- Lander, E. S., Linton, L. M., Birren, B., Nusbaum, C., Zody, M. C.,
Baldwin, J., Devon, K., Dewar, K., Doyle, M., FitzHugh, W., Funke, R., Gage,
D., Harris, K., Heaford, A., Howland, J., Kann, L., Lehoczky, J., LeVine, R.,
McEwan, P., McKernan, K., Meldrim, J., Mesirov, J. P., Miranda, C., Morris,
W., Naylor, J., Raymond, C., Rosetti, M., Santos, R., Sheridan, A., Sougnez,
C., Stange-Thomann, N., Stojanovic, N., Subramanian, A., Wyman, D., Rogers,
J., Sulston, J., Ainscough, R., Beck, S., Bentley, D., Burton, J., Clee, C.,
Carter, N., Coulson, A., Deadman, R., Deloukas, P., Dunham, A., Dunham, I.,
Durbin, R., French, L., Grafham, D., Gregory, S., Hubbard, T., Humphray, S.,
Hunt, A., Jones, M., Lloyd, C., McMurray, A., Matthews, L., Mercer, S., Milne,
S., Mullikin, J. C., Mungall, A., Plumb, R., Ross, M., Shownkeen, R., Sims,
S., Waterston, R. H., Wilson, R. K., Hillier, L. W., McPherson, J. D., Marra,
M. A., Mardis, E. R., Fulton, L. A., Chinwalla, A. T., Pepin, K. H., Gish, W.
R., Chissoe, S. L., Wendl, M. C., Delehaunty, K. D., Miner, T. L., Delehaunty,
A., Kramer, J. B., Cook, L. L., Fulton, R. S., Johnson, D. L., Minx, P. J.,
Clifton, S. W., Hawkins, T., Branscomb, E., Predki, P., Richardson, P.,
Wenning, S., Slezak, T., Doggett, N., Cheng, J. F., Olsen, A., Lucas, S.,
Elkin, C., Uberbacher, E., Frazier, M., Gibbs, R. A., Muzny, D. M., Scherer,
S. E., Bouck, J. B., Sodergren, E. J., Worley, K. C., Rives, C. M., Gorrell,
J. H., Metzker, M. L., Naylor, S. L., Kucherlapati, R. S., Nelson, D. L.,
Weinstock, G. M., Sakaki, Y., Fujiyama, A., Hattori, M., Yada, T., Toyoda, A.,
Itoh, T., Kawagoe, C., Watanabe, H., Totoki, Y., Taylor, T., Weissenbach, J.,
Heilig, R., Saurin, W., Artiguenave, F., Brottier, P., Bruls, T., Pelletier,
E., Robert, C., Wincker, P., Smith, D. R., Doucette-Stamm, L., Rubenfield, M.,
Weinstock, K., Lee, H. M., Dubois, J., Rosenthal, A., Platzer, M., Nyakatura,
G., Taudien, S., Rump, A., Yang, H., Yu, J., Wang, J., Huang, G., Gu, J.,
Hood, L., Rowen, L., Madan, A., Qin, S., Davis, R. W., Federspiel, N. A.,
Abola, A. P., Proctor, M. J., Myers, R. M., Schmutz, J., Dickson, M.,
Grimwood, J., Cox, D. R., Olson, M. V., Kaul, R., Shimizu, N., Kawasaki, K.,
Minoshima, S., Evans, G. A., Athanasiou, M., Schultz, R., Roe, B. A., Chen,
F., Pan, H., Ramser, J., Lehrach, H., Reinhardt, R., McCombie, W. R., de la
Bastide, M., Dedhia, N., Blocker, H., Hornischer, K., Nordsiek, G., Agarwala,
R., Aravind, L., Bailey, J. A., Bateman, A., Batzoglou, S., Birney, E., Bork,
P., Brown, D. G., Burge, C. B., Cerutti, L., Chen, H. C., Church, D., Clamp,
M., Copley, R. R., Doerks, T., Eddy, S. R., Eichler, E. E., Furey, T. S.,
Galagan, J., Gilbert, J. G., Harmon, C., Hayashizaki, Y., Haussler, D.,
Hermjakob, H., Hokamp, K., Jang, W., Johnson, L. S., Jones, T. A., Kasif, S.,
Kaspryzk, A., Kennedy, S., Kent, W. J., Kitts, P., Koonin, E. V., Korf, I.,
Kulp, D., Lancet, D., Lowe, T. M., McLysaght, A., Mikkelsen, T., Moran, J. V.,
Mulder, N., Pollara, V. J., Ponting, C. P., Schuler, G., Schultz, J., Slater,
G., Smit, A. F., Stupka, E., Szustakowski, J., Thierry-Mieg, D., Thierry-Mieg,
J., Wagner, L., Wallis, J., Wheeler, R., Williams, A., Wolf, Y. I., Wolfe, K.
H., Yang, S. P., Yeh, R. F., Collins, F., Guyer, M. S., Peterson, J.,
Felsenfeld, A., Wetterstrand, K. A., Patrinos, A., Morgan, M. J., Szustakowki,
J., de Jong, P., Catanese, J. J., Osoegawa, K., Shizuya, H., Choi, S., and
Chen, Y. J. (2001) Nature.
409,
860-921[CrossRef][Medline]
[Order article via Infotrieve]
- Tian, H., and Kole, R. (1995) Mol. Cell.
Biol. 15,
6291-6298[Abstract]
- Bruce, S. R., and Peterson, M. L. (2001)
Nucleic Acids Res. 29,
2292-2302[Abstract/Free Full Text]
- Kuo, B. A., Uporova, T. M., Liang, H., Bennett, V. D., Tuan, R. S.,
and Norton, P. A. (2002) J. Cell.
Biochem. 86,
45-55[CrossRef][Medline]
[Order article via Infotrieve]
- Muro, A. F., Iaconcig, A., and Baralle, F. E. (1998)
FEBS Lett. 437,
137-141[CrossRef][Medline]
[Order article via Infotrieve]
- Du, K., Peng, Y., Greenbaum, L. E., Haber, B. A., and Taub, R.
(1997) Mol. Cell. Biol.
17,
4096-4104[Abstract]
- Gallego, M. E., Gattoni, R., Stevenin, J., Marie, J., and
Expert-Bezancon, A. (1997) EMBO J.
16,
1772-1784[Abstract/Free Full Text]
- Dance, G. S., Sowden, M. P., Cartegni, L., Cooper, E., Krainer, A.
R., and Smith, H. C. (2002) J. Biol.
Chem. 277,
12703-12709[Abstract/Free Full Text]
- Pollard, A. J., Krainer, A. R., Robson, S. C., and Europe-Finner,
G. N. (2002) J. Biol. Chem.
277,
15241-15251[Abstract/Free Full Text]
- Young, P. J., DiDonato, C. J., Hu, D., Kothary, R., Androphy, E.
J., and Lorson, C. L. (2002) Hum. Mol.
Genet. 11,
577-587[CrossRef][Medline]
[Order article via Infotrieve]
- Du, K., Leu, J. I., Peng, Y., and Taub, R. (1998)
J. Biol. Chem. 273,
35208-35215[Abstract/Free Full Text]
- Lemaire, R., Prasad, J., Kashima, T., Gustafson, J., Manley, J. L.,
and Lafyatis, R. (2002) Genes Dev.
16, 594-607[Abstract/Free Full Text]
- Huang, Y., and Steitz, J. A. (2001) Mol.
Cell 7,
899-905[CrossRef][Medline]
[Order article via Infotrieve]
- ten Dam, G. B., Wieringa, B., and Poels, L. G. (1999)
Biochim. Biophys. Acta
1446,
317-333[Medline]
[Order article via Infotrieve]
- Cramer, P., Caceres, J. F., Cazalla, D., Kadener, S., Muro, A. F.,
Baralle, F. E., and Kornblihtt, A. R. (1999) Mol.
Cell 4,
251-258[Medline]
[Order article via Infotrieve]
- Kadener, S., Fededa, J. P., Rosbash, M., and Kornblihtt, A. R.
(2002) Proc. Natl. Acad. Sci. U. S. A.
99,
8185-8190[Abstract/Free Full Text]
- Shih, S. R., and Krug, R. M. (1996) EMBO
J. 15,
5415-5427[Abstract]
- Bruni, R., and Roizman, B. (1996) Proc.
Natl. Acad. Sci. U. S. A. 93,
10423-10427[Abstract/Free Full Text]
- Molin, M., and Akusjarvi, G. (2000) J.
Virol. 74,
9002-9009[Abstract/Free Full Text]
- Himmelspach, M., Cavaloc, Y., Chebli, K., Stevenin, J., and
Gattoni, R. (1995) RNA (N. Y.)
1, 794-806[Abstract]
- Cowper, A. E., Caceres, J. F., Mayeda, A., and Screaton, G. R.
(2001) J. Biol. Chem.
276,
48908-48914[Abstract/Free Full Text]