Exonic Splicing Enhancer-Dependent Splicing of the Gonadotropin-Releasing Hormone Premessenger Ribonucleic Acid Is Mediated by Tra2
, a 40-Kilodalton Serine/Arginine-Rich Protein
Jae Young Seong1,
Jin Han1,
Sungjin Park,
Wolfgang Wuttke,
Hubertus Jarry and
Kyungjin Kim
Hormone Research Center (J.Y.S.), Chonnam National University, Kwangju 500-757, Korea; School of Biological Sciences (J.H., S.P., K.K.), Seoul National University, Seoul 151-742, Korea; and Department of Obstetrics and Gynecology (W.W., H.J.), University of Göttingen, Göttingen 37075, Germany
Address all correspondence and requests for reprints to: Kyungjin Kim, Ph.D., School of Biological Sciences Seoul National University Seoul 151-742, Korea. E-mail: .
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ABSTRACT
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In an earlier study, we found that excision of the first intron (intron A) from the rat GnRH primary transcript is attenuated in non-GnRH-producing cells. This attenuation can be partially relieved by exonic splicing enhancers (ESEs) located in GnRH exons 3 and 4. In the present study, we confirmed that intron A of the mouse GnRH pre-mRNA was not excised in a HeLa nuclear extract (NE) in vitro or in COS-7 cells in vivo. Intron A could, however, be partially removed when exon 3 and/or 4 were linked to exon 2. In the presence of an ESE in exon 4 (ESE4), an addition of GT1 NE further increased the excision rate of intron A, whereas the addition of KK1 (a non-GnRH-producing cell) NE decreased it. To define the GnRH neuron-specific splicing activity, GT1 NE was fractionated by ultracentrifugation and ammonium sulfate precipitation. A 5090% ammonium sulfate pellet (ASP5090) fraction was further precipitated with 20 mM MgCl2 to isolate a serine/arginine-rich (SR) protein fraction. Among the ASP fractions, ASP4050 significantly increased the excision rate of intron A in the presence of HeLa NE or SR protein-rich fraction. However, the ASP4050 fraction alone could not remove intron A. This result suggests the presence of a cofactor protein(s) in the ASP4050 fraction that may mediate the interaction between a 3' spliceosome complex and the ESE4-SR protein complex. UV cross-linking and gel mobility shift analysis revealed that Tra2
but not other SR proteins tested, specifically binds to ESE4. Moreover, Tra2
stimulated intron A excision in a dose-dependent manner. These results imply that Tra2
and a cofactor protein in the ASP4050 fraction are involved in mediating the GnRH neuron-specific excision of intron A from the GnRH primary transcript.
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INTRODUCTION
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GnRH PLAYS A pivotal role in mammalian reproduction and sexual development. The GnRH gene consists of four exons and three introns (1, 2). GnRH transcripts are found in the hypothalamus but also in a variety of extrahypothalamic tissues such as placenta, pituitary, ovary, lymphocytes, and certain brain regions (3, 4, 5, 6, 7, 8). Several lines of evidence, however, indicate that GnRH gene expression and the processing of its primary transcript in extrahypothalamic tissues are different from those in the hypothalamus (3, 8, 9). In these tissues, a greater portion of the GnRH transcripts retains intron A. In addition, an alternatively spliced form that lacks exon 2 is observed in Nb2 cells (immature T lymphocyte cells), Gn11 cells (genetically immortalized GnRH-expressing cells), and mouse hypothalamus (10, 11). Differential processing of the pre-mRNA generally relies on the exon sequences, alternative branch points, pyrimidine content of the 3' splice site, and the secondary structure of the pre-mRNA (12, 13, 14). Intron A retention or exon 2 skipping of GnRH pre-mRNA is most likely due to a suboptimal 3' splice site of intron A (15). Many purine bases reside along the pyrimidine tract of intron A. More importantly, a putative branch point sequence (BPS) is found within the pyrimidine tract, although a BPS is usually found in an upstream region of the pyrimidine tract (16). It is plausible that the suboptimal 3' splice site may hinder spliceosome complex formation, thereby preventing intron A excision. Alternatively, the weakness of the 3' splice site may cause the selection of an alternative 3' splice site of intron B, resulting in exon 2 skipping. Recently, we and other investigators (14, 15) have demonstrated that mutations of the suboptimal 3' splice site toward the consensus greatly increase the intron excision rate.
Exonic splicing enhancers (ESEs) are involved in the facilitatory selection of suboptimal 3' splice sites (13, 17). ESEs mostly consist of purine-rich sequences, but recently nonpurine-rich ESEs have been identified by the systematic evolution of ligands by exponential enrichment method with which many purine- or nonpurine-rich sequences responsible for binding with individual serine/arginine-rich (SR) proteins have been identified (18, 19). In the case of ESE-dependent splicing, SR proteins bind to enhancer sequences or are associated with enhancer complexes, allowing the formation of a spliceosome complex on the weak 3' splice site through the interaction with a U2 small nuclear ribonucleoprotein (RNP) auxiliary factor (20, 21, 22). SR proteins typically contain one or two N-terminal ribonucleoprotein-type RNA binding domains and a C-terminal domain rich in arginine-serine dipeptide repeats (23). At least ten members of this family, including ASF/SF2, SC35, SRp40, SRp55, and SRp75, have been characterized (23). Recently, mammalian homologs of Drosophila transformer-2 (Tra2) have been found to bind sequence specifically to ESE (24) and to be involved in the alternative splicing of certain genes (25, 26). SR proteins also play important roles in ESE-independent splicing (27, 28) as each SR protein is capable of complementing splicing-deficient cytoplasmic S100 extracts that lack SR proteins but contain other factors necessary for constitutive splicing of pre-mRNAs (29). This suggests that SR proteins are essential splicing factors with partially redundant functions.
Using an in vitro splicing system, we recently demonstrated that an ESE located in GnRH exon 4 (ESE4) is crucial for enhanced splicing of GnRH pre-mRNA (15, 30), suggesting the presence of specific factors that interact with ESE4 in GnRH neurons. In this study, we attempted to identify ESE4-recognizing SR proteins and a GnRH neuron-specific cofactor protein(s) that mediates the interaction of SR protein-ESE4 complex and the 3' spliceosome complex.
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RESULTS
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Excision Rate of Mouse GnRH Introns
To examine the excision rate of mouse GnRH introns, each mouse GnRH intron spanned by its neighboring exons was constructed. Unnecessary inner regions of introns were partially truncated (denoted 1
A2, 2
B3, and 3
C4, respectively), as indicated in Fig. 1
. In vitro transcribed RNAs were subjected to an in vitro splicing assay in the presence of HeLa nuclear extract (NE). As shown in Fig. 1
, little excision of intron A was observed, whereas introns B and C were efficiently removed in a time-dependent manner, confirming our previous results (15, 30). The spliced RNAs were identified by RNA size marker and confirmed by RT-PCR and sequencing. Due to their secondary structure, the splicing lariats migrate differentially on different percentage gels, allowing us to identify lariats. Several unexpected bands were cryptic splicing intermediates.

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Figure 1. Excision Rate of Mouse GnRH Introns in Vitro
The 32P-labeled RNA substrates consisting of each GnRH intron and its neighboring exons were synthesized by in vitro transcription. RNAs purified from 6% polyacrylamide gels containing 8 M urea were incubated with HeLa NE as described in Materials and Methods. The products were electrophoresed on 6% polyacrylamide gels containing 8 M urea and then subjected to autoradiography. The RNA substrates are shown at the top of the gels and the time of incubation is specified at the top of each lane. The illustrated RNA structures on the right sides of gels were identified by the RNA size markers and by subjection to higher percentage gels. The spliced RNA (exon-exon) was confirmed by RT-PCR using gel-purified RNAs. Other bands in this and after figures are cryptic splicing intermediate(s), denoted with asterisks. The schematic diagram on the bottom panel shows the restriction map of the GnRH gene and RNA substrates. The sizes of substrates containing each intron and their spliced products are shown in parentheses.
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To examine GnRH pre-mRNA splicing in vivo, RT-PCR was performed with 3 primers (e1-up, iA-up, e2-dn) using deoxyribonuclease (DNase)-treated RNAs derived from GnRH-producing cells (GT1 cells) (31), mouse preoptic area (POA), olfactory bulb, and cerebral cortex. Because e1-up and iA-up primers compete with one another during PCR amplification, the excision rate of intron A of the tissues was obtained by comparing the ratio of the mature mRNA to intron A-containing RNA transcripts. The most efficient excision of intron A was found in GT1 cells and POA, whereas the intron A-containing transcript was abundant in the olfactory bulb and cerebral cortex (Fig. 2A
). This result indicates that intron A retention is highly prevalent in non-GnRH-producing cells, whereas GnRH-producing cells efficiently remove intron A from the primary transcript. Previously we demonstrated, using an in vitro splicing assay system, that incomplete removal of intron A is most likely due to the weakness of the 3' splice site of intron A and that mutations of the 3' splice site toward the consensus completely restored the splicing activity (15). In the present study, an in vivo splicing assay was performed by transfecting intron A-containing DNA constructs into COS-7 cells. A full-length intron A spanned by exons 1 and 2 (denoted 1A2) was constructed. When 1A2 was transfected in COS-7 cells, most of intron A was not removed from the primary transcript (Fig. 2B
). This result is consistent with the in vitro splicing results shown in Fig. 1
. The mutation of the 3' splice site toward the consensus, moving the putative BPS to the upstream region of the pyrimidine tract showed a complete removal of intron A, confirming our previous in vitro finding (15). This result implies that the weakness of the 3' splice site of intron A is likely due to the position of the BPS within the pyrimidine tract of intron A. The attachment of exons 3 and 4 to the end of exon 2 (1A234) partially increased the intron A excision, which may result from the interaction of ESEs in exons 3 and 4 with some splicing factors. Mouse GnRH exons contain two purine-rich sequences: one in the border region overlapping exon 2 and exon 3 (AAGAG/AUGGGCAAGGAGGUGGA) and the other in exon 4 (GAAAGUCUGAU-UGAAGAGGAAGCCAGGCAGAAGAAGA). We prepared NEs from GnRH-producing GT1 cells and non-GnRH-producing mouse granulosa KK1 cells, to compare the ability of these NEs to remove intron A. Addition of GT1 NE increased the intron A excision rate of ESE4-containing pre-mRNAs, whereas KK1 NE rather decreased it (Fig. 3
). GT1 NE did not alter ß-globin pre-mRNA splicing. Together with the previous data (15), it appears that the GT1 NE-specific increase in GnRH pre-mRNA splicing is highly dependent upon ESE4.

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Figure 2. PCR Analysis of GnRH Transcripts
2GnRH mRNA and splicing intermediates in GT1 cells, mouse POA, olfactory bulb, and cerebral cortex were analyzed by RT-PCR using a set of primers (A). GnRH gene constructs under control of the cytomegalovirus promoter were transfected into COS-7 cells. Two days after the transfection, RNA was obtained from the cells and treated with DNase to eliminate possible DNA contaminations. RT-PCR was performed simultaneously using three different primers (B). The primer sequences are indicated in Materials and Methods. Diagram in panel C shows each plasmid construct and primer location. Mutated sequences of the 3' splice site of intron A are also seen (bold/italic; a putative BPS wild-type 3' splice site, bold/underline; mutated sequences, capital/italic; and an artificially created BPS in 1 mA2 construct).
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Figure 3. Splicing Activity of GT1 and KK1 NEs on ESE-Containing RNA Constructs
For the control (CTL), RNA substrates were incubated with HeLa NE (100 µg). To examine the effect of GT1 or KK1 NE, a half volume of HeLa NE (50µg) was replaced by 50 µg of GT1 NE or KK1 NE. The In vitro splicing reaction was carried out for 3 h at 30 C. ß-Globin RNA was used as a control pre-mRNA without putative ESE but with a strong 3' splice site. Arrows indicate the spliced products. Structures of RNA constructs are illustrated in the diagram on the right (E). The ESEs are indicated by gray (ESE3) and black boxes (ESE4). The sizes of pre-mRNA and its spliced products are shown in parentheses. Capital and small characters in ESE sequences are purines and pyrimidines, respectively.
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Ammonium Sulfate-Saturated Pellet (ASP)4050 Contains a Cofactor Protein(s) that Interacts with the ESE4-SR Protein Complex and 3' Spliceosome Complex
The stimulatory effect of GT1 NE on splicing of the GnRH pre-mRNA implicates the presence of one or more certain splicing factors involved in an interaction with ESE4. As a step toward identifying the GT1-specific splicing factors, we fractionated GT1 NE by ultracentrifugation and ammonium sulfate precipitation. To isolate an SR protein-rich fraction, a 5090% ASP (ASP5090) fraction was further precipitated with 20 mM MgCl2 according to the method described previously (29). In the presence of HeLa NE, each ASP fraction showed either a negative or positive effect on the splicing activity. The ASP4050 fraction showed a stimulatory effect on all RNA constructs, having a distinct effect on splicing of 1
A234 (about 2.1-fold increase in splicing rate compared with GT1 addition lane) and 1
Acc34 (4.5-fold increase), whereas it slightly increased splicing of 1
Acs4 (1.2-fold increase) and ß-globin (1.0-fold increase) pre-mRNAs. ASP020 and ASP2030 fractions showed inhibitory effects on 1
A234 and 1
Acc34 splicing (Fig. 4
). The inhibitory effects of ASP020 and ASP2030 fractions on 1
Acs4 and ß-globin pre-mRNA splicing were not as drastic as on 1
A234 and 1
Acc34 splicing. These results indicate that a GT1 NE contains both inhibitory and stimulatory factors for GnRH pre-mRNA splicing. In the presence of an S100 fraction, the cytoplasmic extract lacking SR proteins but containing other splicing-related components, neither ASP020 nor ASP4050 alone exhibited a splicing activity, whereas the SR protein-rich fraction did. The stimulatory splicing activity of ASP4050 fraction was observed in the presence of the SR protein-rich fraction (Fig. 5
).

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Figure 4. Splicing Activity of ASP Fractions of GT1 NE
GT1 NE was fractionated by ultracentrifugation followed by ammonium sulfate precipitation. The ultracentrifuged pellet (UP) was resolved in NE storage buffer and only the soluble fraction was used for splicing reactions. Ultracentrifuged supernatants (US) were further separated by ammonium sulfate precipitation sequentially at 020, 2030, 3040, 4050, and 5070%. US, UP, and each ASP fraction (about 3040 µg) were used in the splicing reaction in the presence of HeLa NE (50 µg). The RNA constructs are the same as shown in Fig. 3 . Arrows indicate spliced products. The splicing rate is shown below the gel. % Splicing, S/(S + P); S, spliced product; P, precursor RNA.
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Figure 5. Splicing Assay in Several Combinations of SR Protein-Rich Fractions with ASP 020, and ASP 4050
Splicing reactions were performed in the presence of cytoplasmic S100 fraction (50 µg) instead of HeLa NE. Left panels show splicing activities of individual fractions of SRp-rich fraction (4 µg), ASP4050 (30 µg), and ASP020 (30 µg). Right panels show the combination of SRp-rich fraction (2 µg) with ASP4050 (15 µg), and ASP020 (15 µg). Arrows indicate spliced products. Lariat structures are indicated on the right side of the gel.
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To characterize the proteins that recognize the ESE4 sequence, ultracentrifuged samples, ASP fractions, and the SR protein-rich fraction were subjected to a UV cross-linking assay using an ESE4 RNA probe that contains ESE4 and its border sequences (Figs. 6
and 7
). Among the GT1 NE proteins, approximately 90-, 60-, and 40-kDa proteins were found to bind to the ESE4 RNA probe. The same pattern was observed in the supernatant but not in the pellet after ultracentrifugation. The 90-kDa and 60-kDa proteins were separated in the ASP020 fraction. The 60-kDa protein was also seen in the ASP2030 fraction. Proteins of approximately 40 kDa were separated in the ASP2030 and ASP3040 fractions. Interestingly, the ASP4050 fraction failed to show the same binding pattern that observed in GT1 NE and had a very weak binding intensity when compared with that seen in other ASP fractions. Because the most efficient splicing activity was found in the ASP4050 fraction, we expected that the ASP4050 fraction contained GT1-specific splicing factors recognizing ESE4. We then attempted to isolate the proteins in ASP4050 that recognize ESE4 using RNA affinity chromatography as performed in a previous study (32). However, the partially purified proteins in the ASP4050 fraction that bound to ESE4 did not increase the GnRH pre-mRNA splicing in the presence of HeLa NE (data not shown). This result indicates that a protein(s) in ASP4050 responsible for enhanced splicing of intron A may not directly recognize ESE4, but that this protein may bind to proteins that already bound to ESE4.

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Figure 6. UV Cross-Linking of ASP Fractions with the ESE4 Probe
Exon 4-containing plasmid (pE4) was digested with BamHI and used for in vitro transcription, resulting in an ESE4 RNA probe. RNA-protein binding reaction was performed for 30 min at 30 C under the same condition, as the splicing reaction except the polyvinyl alcohol was omitted. Yeast tRNA (5 µg) was added as a nonspecific competitor. After the incubation, the samples were UV cross-linked and digested with RNases T1 and A for 10 min at 37 C. Sample buffer (2x) was added to each sample and boiled at 95 C for 510 min. Samples were separated on a 12% SDS-PAGE gel and autoradiographed.
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Figure 7. Western Blot and UV Cross-Linking of SR Protein-Rich Fractions
SR protein-rich fractions were obtained by MgCl2 precipitation of the ASP5090 fraction. ASP5090 fraction was incubated in 20 mM MgCl2 for 1 h on ice, and centrifuged. The pellet was resolved in NE storage buffer. The supernatant (Mg++ supernatant) and pellet (Mg++ pellet) were subjected to Western blot with anti-SC35 antibody to show the presence of SR proteins in the MgCl2-precipitated fraction (A). MgCl2-precipitated fraction was subjected to UV cross- linking assay with the ESE4 RNA probe (B).
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Tra2
, a 40-kDa SR Protein, Binds to ESE4
Several SR proteins are known to regulate the splicing of either weak or strong introns by direct interaction with ESEs (17, 20, 22). The splicing activity in the SR protein-rich fraction allows for a postulation that one of the SR proteins recognizes ESE4 and mediates the GnRH pre-mRNA splicing. Western blots using an anti-SC35 antibody revealed the presence of SR proteins in MgCl2-precipitated proteins (Fig. 7A
) and the UV cross-linking assay showed that among the SR proteins, a 40-kDa protein(s) in size strongly recognized ESE4 (Fig. 7B
). Although we also observed another weakly cross-linked band of 35 kDa (Fig. 7B
), we focused on the more strongly bound 40-kDa protein. These data strongly indicate that the 40-kDa protein(s) might be involved in either constitutive splicing by itself or enhanced splicing of the GnRH pre-mRNA by interacting with certain cofactor protein(s) in the ASP4050 fraction. Based on the protein size, the 40-kDa protein(s) could be SRp40 or Tra2
(24). These two proteins, however, were hardly distinguishable from each other by size fractionation in SDS-PAGE and Western blot using a monoclonal antibody, mAb104 (24).
To determine which protein directly binds to ESE4, we expressed and purified several glutathione-S-transferase (GST)-tagged SR proteins, SRp40, 9G8, SRp20, SRp55, and Tra2
using the baculovirus expression system. UV cross-linking and gel mobility shift analysis revealed that only Tra2
but not other SR proteins examined, was able to bind to the ESE4 RNA probe. The binding of Tra2
was completely blocked by a 200-fold unlabeled ESE4 RNA competitor but not by 200-fold cold mutated ESE4 RNA competitor (Figs. 8A
and 9
). In addition, when human Tra2ß antibody was coincubated with GT1 NE, two distinct supershifted bands were detected (Fig. 8B
). This result demonstrated that Tra2
was able to specifically bind to ESE4. Each recombinant SR protein was able to increase splicing of the ß-globin pre-mRNA in the presence of S100 extract and limited amounts of HeLa NE (Fig. 8C
). SRp40 and SRp55 strongly stimulated ß-globin pre-mRNA splicing, whereas Tra2
slightly increased it. This result is consistent with reports that many SR proteins are functionally redundant in stimulating constitutive splicing (23), and indicates that the failure of the other SR proteins to bind to ESE4 was not simply due to their malfolding or innate inactivity.
To reveal the relationship between the binding affinity of Tra2
to ESE4 and the splicing activity of the GnRH pre-mRNA, the five different mutant ESE4 RNA probes used in a previous study (30) were subjected to gel mobility shift and UV cross-linking assays. Previously, we demonstrated that the GnRH pre-mRNA with an m2 mutation exhibited increased splicing activity, whereas the GnRH pre-mRNA with an m7 mutation, in which two purine-rich domains were severely destroyed, had nearly no splicing activity. The GnRH pre-mRNAs with m10 and m11 mutations exhibited a slight decrease in splicing activity (30). As shown in Fig. 9
, Tra2
bound to wild type, m2, m10, and m11 ESE4 probes, whereas it failed to bind to the m7 ESE4 probe. This result indicates a close relationship between the binding affinity to ESE4 and splicing activity of the GnRH pre-mRNA. To further support this idea, the splicing activity of Tra2
on GnRH pre-mRNA was examined in the presence of an S100 extract and a limited amount of HeLa NE. The addition of Tra2
strongly stimulated the GnRH intron A excision in a dose-dependent manner (Fig. 10A
). In contrast, Tra2
exhibited only a slight activity in ß-globin pre-mRNA splicing (Fig. 10B
). This result suggests that binding of Tra2
to ESE4 is crucial for enhanced splicing of the GnRH pre-mRNA.
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DISCUSSION
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In the present study, we demonstrated that, in GnRH neurons, increased excision of intron A is mediated by ESE4-dependent splicing factors. Tra2
specifically binds to ESE4, exerting a stimulatory effect on GnRH pre-mRNA splicing. Enhanced splicing of GnRH pre-mRNA was also observed when the ASP4050 fraction was added to HeLa NE or the SR protein-rich fraction, indicating the presence of a cofactor protein(s) in ASP4050. These results suggest that Tra2
and a cofactor protein(s) in ASP4050 fraction are responsible for the GnRH neuron-specific enhancement of GnRH pre-mRNA splicing.
Tissue-specific, developmental, and hormonal regulations of pre-mRNA splicing are important mechanisms under which a single gene produces multiple transcripts and proteins with differential ligand binding, cellular localization, or coding capacity (33). Recently, we demonstrated that the intron A excision rate is largely attenuated in the postnatal mouse hypothalamus, and that such an attenuation was relieved during development (34). Moreover, most GnRH transcripts contain intron A in the hypogonadal mice whose GnRH exons 3 and 4 are deleted. Although the biological consequence of the intron A-containing GnRH transcript is poorly understood, our recent finding suggests that the intron A retention in GnRH transcripts is likely to affect the coding capacity of the transcript. Insertion of the intron A sequence into the upstream portion of the luciferase open-reading-frame significantly decreased translation efficiency but not transcription efficiency (34). It is also notable that a GnRH transcript lacking exon 2 cannot produce the GnRH peptide because exon 2 encodes the GnRH peptide (10, 11). Therefore, accurate splicing and increased splicing rate of GnRH pre-mRNA in the POA and GT1 cells are crucial for the efficient production of GnRH.
Efficient removal of intron A may require both ESE4 and a certain splicing factor(s) that recognize ESE4. Considering that most splicing enhancers are active when located within 100 nucleotides from the 3' splice site (12, 20, 35), it is noteworthy that, because ESE4 is located 238 nucleotides downstream from the 3' splice site of intron A, no ESE4-dependent splicing could be predicted. One good example accounting for the ESE4-dependent increase in GnRH pre-mRNA splicing is the Drosophila melanogaster doublesex (dsx) repeat element of the dsx pre-mRNA, that normally functions when located 300 nucleotides downstream of the regulated 3' splice site in the presence of specific regulatory proteins, Tra, Tra2, and a certain SR protein (12, 36). In the absence of Tra/Tra2, these elements are inactive when located 300 nucleotides downstream of the 3' splice site (36). However, in the presence of Tra/Tra2, a very stable enhancer complex is formed, allowing the enhancer to function at this distance (20, 36). Recently, two human homologs of Tra2, Tra2
(37) and Tra2ß (38), were identified. Tra2 protein functions not only in constitutive splicing but also in activated enhancer-dependent splicing (24). Purified Tra2
and ß proteins bind preferentially to RNA sequences containing GAA repeats, a characteristic of many enhancer elements (24). One interesting finding of this study is that a mouse homolog of Drosophila Tra2
specifically recognized ESE4. The involvement of Tra2
in GnRH pre-mRNA splicing is further supported by the fact that Tra2
dose-dependently stimulated the GnRH intron A excision (Fig. 10
). Such a stimulatory effect of Tra2
was greater in GnRH pre-mRNA than in ß-globin pre-mRNA, indicating that Tra2
exerts its action sequence-specifically and may form a stable complex with ESE4 and recruit a splicing factor, allowing ESE4 to function at a long distance from the 3' splice site of intron A.
Although Tra2
is necessary for enhanced GnRH pre-mRNA splicing, we do not exclude the possibility that other factors, such as the particular ratio of one SR protein [and/or heterogeneous nuclear (hn) RNPs] to the other, are also involved in the GnRH neuron-specific splicing events. Indeed, the ratio of hnRNPA1 to particular SR proteins can strongly affect the splicing pattern of certain transcripts (39, 40, 41). Alternatively, the presence of a GnRH neuron-specific splicing factor could also be proposed. An example of tissue-specific splicing factors is the KH-type RNA binding protein Nova-1, which is expressed exclusively in neurons of the central nervous system and activates the inclusion of exons in the glycine receptor and GABAA receptor pre-mRNAs (42). One or more cofactors in the ASP4050 fraction seems to be a GnRH neuron-specific splicing factor as it has an additive effect on splicing of the GnRH pre-mRNA in the presence of either HeLa NE or SR protein-rich fractions. Interestingly, this additive effect is greater in RNA construct in which ESE4 is far from the 3' splice site than those in which ESE4 is very close to the 3' splice site or in the RNA construct that is constitutively active in HeLa NE. This result indicates that ASP4050 is not necessary for constitutive splicing but is required for splicing of pre-mRNA in which ESE is far from the 3' splice site. Thus, it is postulated that a cofactor protein in ASP4050 may bridge the long gap between the 3' spliceosome complex on intron A and Tra2
-ESE4 complex (Fig. 11
). It is well documented that a cooperative association of Tra2 and Tra with members of the SR protein family is required for the enhanced splicing of dsx pre-mRNA (20). A recent study using the yeast two-hybrid system demonstrated that Tra2 proteins are capable of binding a variety of splicing factors (43). Therefore, association of Tra2
with a cofactor protein in the ASP4050 fraction appears to be necessary for the enhanced splicing of GnRH pre-mRNA, although it should be clarified what protein(s) in the ASP4050 fraction is indeed associated with Tra2
.

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Figure 11. A Possible Model for Enhanced Splicing of GnRH pre-mRNA
Efficient splicing of GnRH pre-mRNA may be mediated by the interaction of ESE4 and ESE4-recognizing splicing factors in GnRH neurons. A 40-kDa protein Tra2 specifically binds to ESE4. A cofactor protein (X) in the ASP4050 fraction seems to interact with Tra2 that is already bound to ESE4. This interaction may recruit U2 small nuclear RNP auxiliary factor to recognize suboptimal 3' splice site of intron A, leading to the enhancement of GnRH pre-mRNA splicing.
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It should be noted that there are probably inhibitory regulation mechanisms for GnRH pre-mRNA splicing in GnRH neurons. The decrease in GnRH pre-mRNA splicing by addition of the ASP020 and ASP2030 fractions indicates the presence of inhibitory proteins in GT1 cells. UV cross-linking with the ESE4 RNA probe showed that multiple proteins in these fractions recognized the ESE4 probe. Although the function of these proteins is unknown, it is plausible that the binding of these proteins to ESE4 may interfere with the binding of the SR protein, thereby inhibiting GnRH pre-mRNA splicing. Alternatively, involvement of an inhibitory protein that does not bind to ESE4 should be considered. In the ASP020 and ASP2030 fractions, a polypyrimidine tract binding protein (PTB) was highly detectable by Western blot (data not shown). PTB is well known as an inhibitory splicing regulator that binds to pyrimidine tracts or its authentic PTB-binding sites, which are usually located in the 3' splice site (44). Several putative PTB-binding sites are found in the 3' splice site of intron A. Recently, it was reported that brain PTB, a protein quite homologous to the ubiquitous PTB, binds to the same sequence recognized by PTB and antagonizes the effects of Nova-1 (45). It has also been reported that the cooperative assembly of an hnRNP complex is induced by this tissue-specific homolog of PTB (46). However, the possible inhibitory effect of PTB on the GnRH pre-mRNA splicing should be further elucidated. Nevertheless, the presence of an inhibitory regulator(s) in the ASP020 and ASP2030 fractions suggests that a coordinated regulation by inhibitory and stimulatory factors are involved in the GnRH pre-mRNA splicing in GnRH neurons.
In conclusion, because splicing events directly affect the coding capacity of the transcript, efficient and accurate processing of GnRH pre-mRNA is critical for the efficient production of GnRH, allowing GnRH neurons to specifically maintain their function. GnRH neuron-specific splicing requires the presence of ESE4 and some splicing factors, including Tra2
. Identification of the cofactor protein in the ASP4050 fraction that interacts with Tra2
and 3' spliceosome complex may provide important insight into GnRH gene regulation.
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MATERIALS AND METHODS
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Oligonucleotides
e1-up: 5'-TCACCAGCGGGGAAGACATC-3' [20 oligomer (mer)] - e2-up: 5'-TGATCCTCAAACTGATGGCCGG-3' (22 mer)
- e3-up: 5'-ATGGGCAAGGAGGTGGATCA-3' (20 mer)
- e2-dn: 5'-CTCTTGGAAAGACTCAACC-3' (19 mer)
- e3-dn: 5'-AGAGCTCCTCGCAGATCCCT-3' (20 mer)
- e4-dn: 5'-TGAAATCTACGCTGCTGGG-3' (19 mer)
- iA-up: 5'-TACCTCTGCAGTTTCTGTGA-3' (20 mer)
DNA Constructions
The GnRH DNA fragment containing exon 1, intron A, and exon 2 (1A2) was obtained by PCR with e1-up and e2-dn primers from the mouse liver genomic DNA. StyI and PstI sites in intron A were digested and ligated to remove the inner part of intron A. The ligated DNA was reamplified by PCR using e1-up and e2-dn primers, then cloned into the pGEM-T vector (Promega Corp., Madison, WI). This gene construct was denoted as p1
A2. The DNA fragment containing exon 2, intron B, and exon 3 (2B3) was obtained by PCR using e2-up and e3-dn primers. Digestions of 2B3 with BpuAI and StuI, resulting in p2
B3, removed the inner part of intron B. The PCR product containing exon 3, intron C, and exon 4 (3C4) was digested at the two HincII sites of intron C and ligated to produce p3
C4 (Fig. 1
). GnRH cDNA containing all exons (p1234) was isolated from GT1 cells by RT-PCR. The DNA fragment containing exons 2, 3, and 4 obtained by digestion of BsmI and SalI sites was recloned into p1
A2 to generate p1
A234. To shorten the distance of the ESEs from the 3' splice site of intron A, we created two ClaI sites 3 nucleotides downstream from the 3' splice site and at the first nucleotide of exon 3. These were cut by ClaI and ligated, resulting in the truncation of most of exon 2 (denoted p1
Acc34), which was further digested by ClaI and SacI, and ligated to make p1
Acs4 (Fig. 3
). For in vivo splicing assays, 1A2 was obtained from the mouse genomic DNA by PCR using e1-up and e2-dn primers. Site-directed mutation of the 3' splice site of intron A was performed by PCR with mutated primers (1 mA2). The mutated sequences are indicated in Fig. 2C
. Plasmid 1A234 was cloned by joining of 1A2 and 1234 at a BsmI site in exon 2. The DNA fragments 1A2, 1 mA2, 1A234, and 1234 were recloned into a pEGFP-C1 vector (CLONTECH Laboratories, Inc., Palo Alto, CA) from which the green fluorescent protein sequence was removed, thereby producing cDNAs controlled by the cytomegalovirus promoter. To synthesize the ESE4-containing RNA probe (ESE4 probe), a template DNA (pE4) controlled by the T7 promoter, was cloned by digestion of p1
A234 with ApaI and SacI, which removes the 1
A23 fragment. All of the final products were sequenced by the Sanger dideoxy method (Sequenase 2.0, Amersham Pharmacia Biotech, Little Chalfont, UK).
In Vitro Transcription
Pre-mRNA substrates were synthesized in 25-µl in vitro transcription reactions containing 200 ng of template DNA, 10 U T7 RNA polymerase (Roche Molecular Biochemicals, Nutley, NJ), 0.5 mM diguanosine triphosphate (Roche Molecular Biochemicals), 0.5 mM each of ATP, CTP, uridine triphosphate, 0.05 mM GTP, and 2.5 µl of 32P-GTP (Amersham Pharmacia Biotech). For synthesis of the ESE4 RNA probe, pE4 was linearized with BamHI. In vitro transcription reaction (25 µl for 30 min at 37 C) contained 500 ng of template DNA, 20 U T7 RNA polymerase (Roche Molecular Biochemicals), 0.5 mM each of ATP, CTP, uridine triphosphate, 0.05 mM GTP, and 5 µl of 32P-GTP. The RNAs were purified by electrophoresis on a 6% polyacrylamide gel containing 8 M urea. For the ESE4 RNA probe, a 15% polyacrylamide gel was used.
In Vitro Splicing
In vitro splicing reactions were performed as described previously (15). Briefly, the gel-purified RNA substrates (usually about 20,000 cpm) were subjected to the splicing reaction in a total volume of 25 µl containing 5 mM HEPES (pH 7.9), 20 mM creatine phosphate, 0.4 mM ATP, 0.6% polyvinylalcohol, 3 mM MgCl2, 100 µg HeLa NE, and 20 U RNasin according to Promega Corp.s protocol. The splicing reaction was performed at 30 C for 3 h and stopped by the addition of the 2x stop mix containing 0.5% sodium dodecyl sulfate (SDS), 2 mM EDTA, 3 µg/ml tRNA, 0.03 M sodium acetate, and 0.03 M Tris-HCl (pH 7.4). After phenol/chloroform extraction and subsequent ethanol precipitation, the RNAs were separated on a 6% polyacrylamide gel containing 8 M urea.
Transfection and RT-PCR
COS-7 cells were maintained at 37 C in DMEM plus 10% fetal bovine serum (FBS) under a humidifying atmosphere containing 5% CO2. The cells were transfected with DNA using Lipofectamine (Invitrogen, Carlsbad, CA) according to the manufacturers instructions. Forty-eight hours after transfection, total RNA was prepared from the transfected cells and treated with 10 U DNase (Invitrogen) for 30 min at 37 C to avoid possible DNA contamination. DNase-treated RNAs were boiled for 5 min, extracted with phenol and chloroform, and precipitated with ethanol. The RNA was reverse-transcribed using random hexamers, and the resulting cDNA was amplified by PCR as previously described (47).
GT1 NE Preparation
GT11 cells were maintained in DMEM with 10% FBS under a humidifying atmosphere containing 5% CO2 at 37 C. About 1 x 109 GT11 cells were harvested and the NE was prepared as described by Dignam et al. (48) with a slight modification. A protease inhibitor tablet (Roche Molecular Biochemicals) was added to buffer C (one tablet/10 ml). Instead of buffer D, a nuclear storage buffer containing 40 mM Tris-HCl (pH 7.4), 100 mM KCl, 0.2 mM EDTA, 0.5 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonylfluoride, and 25% (vol/vol) glycerol was used. The final protein concentration was usually 78 mg per ml. Nuclear extractions from KK-1 cells were performed employing the same method.
Ammonium Sulfate Precipitation and MgCl2 Precipitation
About 10 ml of GT1 NE was ultracentrifuged at 360,000 x g for 30 min in a Sovall T-865.1 rotor. The pellet was resuspended in 1 ml NE storage buffer and only the soluble portion was used for splicing and RNA binding assay. The supernatant was transferred to a fresh tube and precipitated sequentially in 020, 2030, 3040, 4050, and 5070% ammonium sulfate. Each ammonium sulfate-precipitated (ASP) fraction was resuspended in an appropriate volume of NE storage buffer to make the final concentration approximately 68 mg protein/ml. The resuspended pellet fractions were dialyzed against 100 vol of NE storage buffer with several changes. GT1 NE precipitated in 5090% ammonium sulfate (ASP5090) was resuspended in a dialysis buffer (65 mM KCl; 15 mM NaCl; 10 mM HEPES, pH 7.6; 1 mM Na2EDTA; 2 mM DTT; 5 mM KF; 5 mM ß-glycerophosphate; and 0.2 mM phenylmethylsulfonylfluoride) and dialyzed against 100 vol of this buffer with several changes. The dialysate was centrifuged for 15 min at 13,000 x g. The supernatant was transferred to clean tubes and MgCl2 was added to 20 mM. After a 1-h incubation on ice, the tubes were centrifuged at 13,000 x g for 30 min. After removal of the supernatants, the pellets were washed with 200 µl of 20 mM MgCl2 dialysis buffer and resuspended in 20 µl of NE storage buffer.
UV Cross-Linking Assay
RNA-protein binding reactions (12.5 µl) contained 5 µl of protein sample (NE, 20 µg; ammonium sulfate precipitation fractions, 20 µg; MgCl2 precipitation, 5 µg; GST-SR proteins, 180 ng), 0.4 mM ATP, 20 mM creatine phosphate, 3 mM MgCl2, 20 U RNasin, 5 µg yeast tRNA, and [
-32P]GTP-labeled RNA probe. All reaction components were mixed and incubated for 30 min at 30 C. UV cross-linking was performed on ice, 4.5 cm away from a UV source at 1.2 J (Stratagene, La Jolla, CA). For competition experiments, a 200 molar excess of unlabeled probe was incubated for 10 min at 30 C before addition of the labeled probe. Each sample was then incubated with 200 U of ribonuclease (RNase) T1 and 200 U of RNase A for 10 min at 37 C. An equal volume (15 µl) of 2x protein loading buffer (100 mM Tris, pH 6.8; 200 mM DTT; 4% SDS; 0.2% BPB; and 10% glycerol) was then added to each reaction, which was then boiled for 5 min and loaded onto an SDS-polyacrylamide gel (5% stacking and 12% separating gels). The gels were dried for autoradiography on x-ray film (Kodak, Rochester, NY).
Immunoblotting
Protein from the ASP5090 fraction and MgCl2-precipitated proteins were separated by 12% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Bio-Rad Laboratories, Inc., Hercules, CA). A 1:2000 dilution of monoclonal antisplicing factor SC-35 (Sigma-Aldrich Corp., St. Louis, MO) or monoclonal antibody mAb104 (ATCC, Manassas, VA) was used as the primary antibody and 1:2500 dilution of horseradish peroxidase conjugated goat antimouse IgG (Promega Corp.) was employed as the secondary antibody. The bands were detected by use of the electrochemiluminescence Western blotting detection kit (ECL Plus, Amersham Pharmacia Biotech).
Protein Purification
GST-tagged SR proteins, SRp40, 9G8, SRp20, SRp55, and Tra2
cloned into Baculovirus transfer vector (CLONTECH Laboratories, Inc.) were used to construct a recombinant baculovirus. The fusion proteins were expressed in Sf9 cells. GST-SR proteins and Tra2
were affinity purified with glutathione-sepharose 4B according to the manufacturers instructions (Amersham Pharmacia Biotech).
EMSA
RNA-protein binding reactions (10 µl) contained 4 µl of protein sample (GST-SR proteins, 120 ng), 0.4 mM ATP, 20 mM creatine phosphate, 3 mM MgCl2, 20 U RNasin, 5 µg yeast tRNA, and [
-32P]GTP-labeled RNA probe. All reaction components were mixed and incubated for 30 min at 30 C. For competition experiments, 200 molar excess of cold competitor was incubated for 10 min at 30 C before addition of the probe. After incubation, 2 or 3 µl of glycerol was then added to each reaction, which was immediately loaded onto a nondenaturing polyacrylamide gel (5%). The gel was dried for autoradiography on x-ray film.
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ACKNOWLEDGMENTS
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We are grateful to S. Stamm for providing human Tra2ß antibody, P. Grabowski for the PTB antibody. We greatly appreciate the services of Biomedical English Editing Service (Portland, OR).
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FOOTNOTES
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The present study was supported by the Ministry of Science and Technology through Korea Brain Science Program and the National Research Laboratory (2000-N-NL-01-C-149). J.Y.S. was a recipient for the fellowship from the Alexander von Humboldt foundation in Germany. J.H. and S.P. are recipients for the scholarship from Brain Korea 21 of the Ministry of Education of Korea.
1 J.Y.S. and J.H. contributed equally to this work. 
Abbreviations: ASP, Ammonium sulfate-saturated pellet; ASP5090, 5090% ASP; BPS, branch point sequence; DNase, deoxyribonuclease; dsx, Drosophila melanogaster doublesex; DTT, dithiothreitol; ESE, exonic splicing enhancers; ESE4, ESE in exon 4; GST, glutathione-S-transferase; hn, heterogeneous nuclear; mer, oligomer; NE, nuclear extract; POA, mouse preoptic area; PTB, polypyrimidine tract binding protein; RNase, ribonuclease; RNP, ribonucleoprotein; SDS, sodium dodecyl sulfate; SR, serine/arginine-rich; SRp, SR protein; Tra2, Drosophila transformer-2.
Received for publication November 14, 2001.
Accepted for publication July 22, 2002.
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