Serine-Arginine-rich Protein p30 Directs Alternative Splicing of Glucocorticoid Receptor Pre-mRNA to Glucocorticoid Receptor {beta} in Neutrophils*

Qing Xu {ddagger}, Donald Y. M. Leung {ddagger} § and Kevin O. Kisich ¶

From the {ddagger}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
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 {beta} isoform of the glucocorticoid receptor (GCR{beta}) relative to the ligand binding isoform, GCR{alpha}. GCR{beta} functions as a dominant inhibitor of GCR{alpha} 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{beta} and stimulated expression of GCR{alpha}. 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{beta}.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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{alpha} and GCR{beta}, 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{beta} (17). GCR{beta} is truncated at the C terminus, which corresponds to the ligand binding domain. Thus, GCR{beta} cannot bind GC. In addition, GCR{beta} does not transactivate GC-sensitive genes and functions as a dominant inhibitor of GCR{alpha} (8). Previous studies suggest that GCR{alpha}:GCR{beta} heterodimer formation may account for the reduced effectiveness of GC action in cells overexpressing GCR{beta} (8). Recent experiments in our lab demonstrated that overexpression of human GCR{beta} by mouse hybridoma cells results in the development of GC insensitivity by these cells (9).

Regulation of GCR{beta} expression is not well understood. Different cell types from the same individual can have very different ratios of GCR{alpha} to GCR{beta}. For example, freshly isolated peripheral blood neutrophils are GC-insensitive. Both the absolute level of GCR{beta} and the ratio of GCR{beta} to GCR{alpha} protein are much higher in neutrophils than in peripheral blood mononuclear cells (PBMC) from the same individuals (10). The ratio of GCR{beta} to GCR{alpha} can be altered by cytokine stimulation. After stimulation of neutrophils with IL-8, GCR{beta} mRNA levels increased remarkably, and GCR{alpha} mRNA decreased to undetectable levels. Similarly, exposure of PBMC to IL-2 and IL-4 resulted in increased GCR{beta} expression and development of steroid insensitivity (1, 11). Since both GCR{alpha} and GCR{beta} 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{alpha} is joined to exon 8 to generate mRNA encoding GCR{alpha} or whether exon 8 is joined to exon 9{beta}, resulting in mRNA encoding GCR{beta}.

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{beta} mRNA


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell Isolation—Human 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 Antibodies—Hybridomas 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 Analysis—Cell 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{beta} (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{beta} 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 Differentiation—PLB-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 25–30 min at room temperature in the electroporation cuvettes and then transferred to flasks containing 25 ml of differentiation medium.

Flow Cytometry—Flow 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 Preparation—Total 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 Construction—Plasmids used to create standard curves for real-time PCR were generated as follows: SRp30a cDNA region spanning nucleotides 111–990 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 194–630 (GenBankTM NM_003016 [GenBank] ) was amplified with forward primer (5'-GACCTCCCTCAAGGTGGACAAC-3') and reverse primer (5'-ACCGAGATCGAGAACGAGTGC-3'), SRp30c cDNA region spanning nucleotides 312–986 (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 PCR—An 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
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SR Protein Levels in Neutrophils and PBMC—To 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{beta}, with extracts from PBMC, which have low levels of GCR{beta}. 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.



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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.

 


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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{beta} relative to GCR{alpha} (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{beta} levels in neutrophils also result in increased levels of SRp30 proteins in neutrophils.



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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 Differentiation—To 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.



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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{alpha} and GCR{beta} Levels in PLB-985 Cells before and after Neutrophilic Differentiation—To 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{alpha} and GCR{beta} levels both increased upon exposure to retinoic acid relative to undifferentiated PLB-985 cells. However, densitometry revealed that GCR{beta} 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{alpha} (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{beta}.



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FIG. 5.
Western blot of total GCR in PLB cells before and after differentiation. After differentiation, expression of GCR{alpha} and GCR{beta} increased. However, GCR{beta} level increased significantly more as compared with GCR{alpha} level. A representative blot from three experiments is shown in A, and the mean ± S.E. of three experiments is shown in B analyzed by two-tailed paired t test.

 

SR Protein Levels in Undifferentiated and Differentiated PLB-985 Cells—To 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.



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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{beta}, 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{beta} protein levels (Fig. 7). Fig. 7A is a representative Western blot showing that GCR{beta} 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{beta} from three experiments transfected with antisense oligonucleotide for SRp30a, SRp30b, SRp30c, and control oligonucleotide. GCR{beta} 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{alpha} 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{beta} levels relative to control oligonucleotide or untreated cells.



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FIG. 7.
Expression of GCR{beta} in differentiated PLB cells transfected with control oligonucleotide and antisense oligonucleotides for SRp30a, SRp30b, and SRp30c. Proteins were extracted from days 1, 2, and 3 after differentiation and before differentiation. SRp30c antisense oligonucleotide, but not SRp30a or SRp30b or control oligonucleotides, significantly inhibited the expression of GCR{beta} at 24 h after transfection. A representative blot from three experiments is shown in A, and the mean ± S.E. of three experiments is shown in B. *, GCR{beta} levels are significantly inhibited in cells on day 1, with antisense oligonucleotide for SRp30c compared with control oligonucleotide. p = 0.003. Data were analyzed by two-tailed unpaired t test.

 


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FIG. 8.
Expression of GCR{alpha} in differentiated PLB cells transfected with control oligonucleotide and antisense oligonucleotide for SRp30c. Proteins were extracted from day 0 (prior to differentiation), day 1, day 2, and day 3 after differentiation. SRp30c antisense oligonucleotide significantly stimulated the expression of GCR{alpha} relative to control oligonucleotide. A representative blot from three experiments is shown in A, and the mean ± S.E. of three experiments is shown in B. *, GCR{alpha} levels are significantly stimulated in cells following treatment with antisense oligonucleotide for SRp30c compared with control oligonucleotide. p < 0.01. Analyzed by two-tailed unpaired t test.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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{alpha} is skipped, and exon 9{beta} 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{alpha} included the ligand binding domain, whereas that encoded by exon 9{beta} includes a domain that cannot bind GC. Stimulation of alternative splicing for other mRNAs such as fibronectin (3436), {beta}-tropomyosin (37), apolipoprotein B (apoB) (38), adenylyl cyclase stimulatory G-protein G {alpha}(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{beta} was to first compare the complement of SR proteins present in cells rich in GCR{alpha}, such as PBMC, with SR proteins present in cells rich in GCR{beta}, 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), {beta}-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{beta}. 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{alpha} and GCR{beta} increased during differentiation; however, GCR{beta} increased by ~27-fold, whereas GCR{alpha} 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{beta} 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{beta} production for ~24 h after transfection, whereas GCR{alpha} production was stimulated (Fig. 8). Therefore, SRp30c was necessary for alternative splicing of GCR pre-mRNA to generate mRNA encoding GCR{beta}. Since SRp30c represents the major alternative splicing factor present in neutrophils, we conclude that SRp30c is required for alternative splicing to generate GCR{beta} mRNA. Since GCR{beta} is intimately involved in GC insensitivity, and GCR{beta} 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. Back

§ 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. Back



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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