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Endocrinology Vol. 142, No. 10 4454-4461
Copyright © 2001 by The Endocrine Society

ARTICLES

First Intron Excision of GnRH Pre-mRNA During Postnatal Development of Normal Mice and Adult Hypogonadal Mice

Jae Young Seong, Bong Won Kim, Sungjin Park, Gi Hoon Son and Kyungjin Kim

Hormone Research Center (J.Y.S.), Chonnam National University, Kwangju 500-757, Korea; and School of Biological Sciences (B.W.K., S.P., G.H.S., K.K.), Seoul National University, Seoul 151-742, Korea

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: kyungjin{at}snu.ac.kr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Previously, we demonstrated that excision of the GnRH first intron (intron A) was largely attenuated in non-GnRH-producing tissues but accelerated in GnRH neurons. In the present study, we examined the splicing rate of GnRH pre-mRNA in developing normal mice and adult hypogonadal mice. The preoptic area and cerebral cortex were removed from mice at ages 1–7 wk. GnRH pre-mRNA splicing was examined by competitive RT-PCR using a variety of primer sets. The ratio of mature mRNA to intron A-containing RNA species in the preoptic area was lowest in 1- and 2-wk-old mice, significantly augmented in 3-wk-old mice, and further increased until adulthood. In contrast, the ratio of mRNA to intron A-containing RNA in the cerebral cortex was extremely low, drastically decreased in 3-wk-old mice, and remained at low levels until adulthood. These data indicate a preoptic area-specific increase in intron A excision during development. Intron B or C excision in the preoptic area was not significantly changed during development. To elucidate the possible involvement of the exonic splicing enhancers located in GnRH exons 3 and 4 in the developmental increase in intron A excision, we examined the splicing rate of GnRH pre-mRNA in hypogonadal mice whose GnRH exons 3 and 4 were truncated. The intron A excision in the preoptic area of hypogonadal mice was significantly lower than that of normal mice but similar to that in other tissues, such as cerebral cortex and olfactory bulb. To support the functional relevance of intron A-containing RNA species, we examined the translation efficiency of intron A-containing RNA. Insertion of intron A sequence into the upstream portion of the luciferase open reading frame significantly decreased translation efficiency. The present study demonstrates that intron A excision in the preoptic area is developmentally regulated in normal mice but largely attenuated in hypogonadal mice, indicating the functional importance of intron A excision in GnRH pre-mRNA splicing.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GnRH IS A HYPOTHALAMIC decapeptide that plays a pivotal role in the neuroendocrine regulation of pituitary functions. The majority of GnRH-synthesizing cell bodies and GnRH mRNA is concentrated in the preoptic area (POA) of the brain (1, 2). However, in addition to the POA, several endocrine tissues and extrahypothalamic regions of the brain express GnRH gene transcripts, although their levels are very low (3, 4, 5). Studies on the GnRH gene structure have indicated that the GnRH gene is composed of four short exons and three long introns and that the open reading frame for GnRH preprohormone initiates at exon 2 (6). Interestingly, the first GnRH cDNA clone from the human placenta contained the first intron (intron A), so this cDNA has a long 5' untranslated region (UTR) (7). Subsequent studies demonstrated that intron A is largely retained in a variety of extrahypothalamic tissues (8, 9). RNase protection assays and sensitive RT-PCR with rat and mouse GnRH pre-mRNAs revealed that introns B and C were rapidly excised but that intron A was excised slowly (10, 11, 12). Our recent study using an in vitro splicing system demonstrated that introns B and C were efficiently excised, whereas intron A was not (13). These results suggest that GnRH pre-mRNA processing begins with the splicing of introns B and/or C from the primary gene transcript. Hence, intron A excision appears to be a rate-limiting step of GnRH pre-mRNA processing.

The attenuation of intron A excision is most likely attributable to a suboptimal 3' splice site of intron A. Mutations in such suboptimal 3' splice sites toward consensus sequences significantly increased the splicing rate in vitro (13). In addition, purine-rich sequences in GnRH exon 3 and/or exon 4 are proposed to constitute another parameter regulating intron A excision. In the presence of the purine-rich sequences, intron A could be partially excised, indicating that the purine-rich sequences in exons 3 and 4 may serve as exonic splicing enhancers (ESEs) (13). Moreover, the addition of nuclear extracts from GnRH neuronal cells (GT1 cells) further increased the intron A excision rate in the presence of ESEs. These results suggested the presence of a putative GnRH neuron-specific splicing factor(s) that interacts with ESEs located in GnRH exons 3 and 4. Such an interaction appears to represent a fine-tuning mechanism underlying the efficient production of mature GnRH mRNA.

In addition to the GnRH neuron-specific mechanism of GnRH pre-mRNA splicing, one may postulate the presence of a possible regulation mechanism by which the splicing efficiency in GnRH neurons can be altered under certain conditions. Recently, Roberts and colleagues (14) found an uncoupling of the transcriptional rate and the posttranscriptional process of GnRH transcripts during mouse embryonic and postnatal development. In their study, alterations in GnRH primary transcript levels were not accompanied by similar changes in GnRH mRNA. Although it is currently unclear how this posttranscriptional regulation participates in GnRH gene regulation, it is likely that changes in the splicing rate of GnRH pre-mRNA may contribute to this process. To address this possibility, in the present study, we examined the excision of intron A (ratio of mature mRNA to intron A-containing RNA) in the POA and cerebral cortex (CTX; a non-GnRH-producing region) of developing normal mice. To elucidate the functional relevance of ESEs in GnRH exons 3 and 4 in intron A excision in vivo, intron A excision was examined in the POA of adult hypogonadal (hpg) mice in which GnRH exons 3 and 4 were deleted (15). Moreover, to determine the functional importance of intron A-retained RNA species, its translation efficiency was examined.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and tissue preparation
Female ICR mice at ages 1–7 wk were obtained from Seoul National University Animal Breeding Center. The hpg mice (HPG/Bm, Gnrhhpg/+) were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were housed in a temperature-controlled chamber under a 14-h light/10-h dark photocycle (light on at 0600 h) with food and water supplied ad libitum. Female mice were killed by cervical dislocation between 1300 and 1500 h. The POA and other brain regions such as the CTX and olfactory bulb (OB) were sampled as previously described (13). Animal experiments were conducted in accordance with the Guidelines for the Care and Use of Experimental Animals at Seoul National University.

RT-PCR analyses
RNAs were isolated with the acid guanidinium thiocyanate-phenol-chloroform method (16). To eliminate possible DNA contamination, 5-µg RNA samples were further treated with 10 U of DNase I (Life Technologies, Inc., Gaithersburg, MD) for 20 min at 37 C in the presence of 10 U of RNase inhibitor (Promega Corp., Madison, WI) followed by a phenol/chloroform extraction and ethanol precipitation. DNase-treated RNAs were subjected to RT-PCR as previously described (13). Sets of primers were purchased from Sigma-Genosys (Woodlands, TX). The primers used in this study were as follows: e1-up, 5'-GGAAGACATCAGTGTCCCAGA-3'; e2-up, 5'-AAACACTGAACACTTGGTTGAG-3'; e2-dn, 5'-CTCTTGGAAAAGAATCAACCAATG-3'; iA-up, 5'-TACCTCTGCAGTTTCTGTGA-3'; e3-dn, 5'-AGAGCTCCTCGCAGATCCCTA-3'; iB-dn, 5'-GTCCAAAGTGCAAGGAGGAA-3'; iB-up, 5'-AAGCAACCATCACAACTCCA-3'; e3-up, 5'-ATGGGCAAGGAGGTGGATCA-3'; iC-up, 5'-CAACAGCTCCACAAGATGCT-3'; and e4-dn, 5'-TGAAATCTACGCTGCTGGGT-3'. The primer locations and the expected sizes of PCR products are shown in Fig. 1Go. DNA fragments 123 (containing exons 1, 2, and 3) and A23 (intron A, exons 2 and 3) were obtained by RT-PCR and used for the titration of competitive PCR. To determine the actual amount of GnRH mRNA, a competitive RT-PCR method was used as previously described (17, 18). A deletion mutant GnRH cDNA was generated by cleaving wild-type GnRH cDNA with ScaI and AsuII restriction enzymes and was used as an internal control for determining GnRH mRNA levels (18). A 1-µg RNA sample was applied to the RT reaction mixture containing 200 U of RNaseH- Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.), 50 pmol of random hexamer, 20 U of RNase inhibitor (Promega Corp.), 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 5 mM MgCl2, and 1 mM deoxynucleotide triphosphate. The RT mixture was overlaid by 50 µl of light mineral oil. The RT reaction was carried out at 37 C for 50 min followed by a 5-min denaturation period at 99 C. Two microliters of RT sample was amplified in 40 µl of the PCR mixture containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2 mM MgCl2, 20 pmol of upstream and downstream primers, and 1 U of Taq DNA polymerase (Perkin-Elmer Cetus, Norwalk, CT). Forty cycles of PCR amplification were carried out under conditions of denaturation at 94 C for 1 min, primer annealing at 60 C for 1 min, and primer extension at 72 C for 1 min. Ten-microliter aliquots of PCR products were electrophoresed on a 1.5% agarose gel in Tris-acetate-EDTA buffer, stained with ethidium bromide, and photographed under UV illumination with Polaroid 667 films (Polaroid, Cambridge, MA). To determine the levels of the intron A-containing RNA species, conventional RT-PCR was performed with 28 PCR cycles followed by Southern blot analysis.



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  		Figure 1. GnRH gene structure and primer locations. All of the primers used in this study are shown in the GnRH gene structure (top). The PCR products and the expected sizes are shown in the diagram (bottom).

 
Southern blot hybridization
After rinsing with double distilled water, the gel was soaked in 0.5 M NaOH, 1 M NaCl solution for denaturation of double stranded DNA followed by resoaking in 0.5 M Tris-HCl (pH 7.4), 1.5 M NaCl solution for neutralization. The DNA was then blotted onto Nytran membrane (Schleicher & Schuell, Inc., Dachen, Germany) by capillary transfer method. The Nytran membrane was prehybridized in 50% formamide, 10x Denhardt’s solution, 6x standard sodium phosphate EDTA buffer (SSPE), 1% SDS, and 50 µg/ml salmon sperm DNA. Hybridization was carried out overnight at 42 C in the same buffer including 32P-labeled probes. The membrane was washed twice in 6x SSPE, 0.5% SDS at room temperature for 15 min and twice in 1x SSPE, 1% SDS at 42 C for 15 min. The washed filter was exposed to x-ray film at -70 C for 6 h (13).

Plasmid constructions

Mouse GnRH gene fragments containing exon 1 and/or intron A were amplified from mouse genomic DNA by PCR. All upper primers contained HindIII restriction sequence at their 5' ends, and all lower primers contained the NcoI site. PCR products were cloned into pGEM-T Easy vector (Promega Corp.), and sequence identities were confirmed by chain termination sequencing methods. Luciferase reporter plasmids were constructed by insertion of each fragment into pGL3-control vector (Promega Corp.) using HindIII/NcoI sites, which reside between the simian virus 40 minimal promoter and the luciferase coding sequence. To avoid the excision of the intron A region in the cells, the last G base of intron A was omitted. The primers used for cloning are as follows: E1 H3 up, 5'-AAGCTTAACTGTGCTCACCAGCGGGGA-3'; IA H3 up, 5'-AAGCTTGTACAGTTCTTTGTTGTTCT-3'; E1 N1 dn, 5'-CCATGGCTGTTTGGATGTGAAAGTCA-3'; IA N1 dn, 5'-CCATGGTAAGGGACATCAAGACACAGA-3'; IA 810 up, 5'-GCGAAGCTTATTGACTTGGAGGAACT-3'; and IA 810 dn, 5'-GCGCCATGGAGTTCCTCCAAGTCAAT-3'.

Cell culture and transient transfection
Chinese hamster ovarian (CHO-K1) cells were maintained in DMEM supplemented with 4 mM glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin/streptomycin, and 10% FBS under a humidifying atmosphere containing 5% CO2 at 37C. For transfection experiments, cells were plated in 60-mm dishes and grown to 40–60% confluence for 1–2 d. Cells were washed twice with Dulbecco’s PBS, and medium was changed to serum- and antibiotic-free DMEM before transfection. Plasmids for transfection experiments were purified with QIAGEN columns (QIAGEN GmbH, Hilden, Germany) according to the manufacturer’s instructions and dissolved in TE buffer (1 mM Tris, pH 8.0, 0.1 mM EDTA). One microgram of plasmid DNA was transfected using the LipofectAMINE PLUS reagent (Life Technologies, Inc., Grand Island, NY). Excess DNA complexes were washed out with Dulbecco’s PBS the next day. After more than 24 or 48 h of incubation, cells were harvested and subjected to luciferase assay or Northern blot hybridization.

Luciferase assay
For luciferase assay, luciferase reporter constructs and cytomegaloviral promoter-driven ß-galactosidase constructs were cotransfected. Cell extracts were prepared by incubating cells in 0.3 ml of reporter lysis buffer (Promega Corp.) for 15 min at room temperature. After brief centrifugation, supernatants were obtained and kept at -70 C until assay. ß-Galactosidase and luciferase assays were performed using commercial enzyme assay kits (Promega Corp.). Cytomegaloviral promoter-driven ß-galactosidase activity was used to normalize the transfection efficiency.

Northern blot analysis
Total RNAs were isolated by a single step acid guanidinium thiocyanate-phenol-chloroform method as previously described (16). For Northern blot hybridization, 30 µg of each RNAs was resolved on a 1.2% formaldehyde agarose gel and transferred to a Nytran filter (pore size, 0.45 µm; Schleicher & Schuell, Inc.) for 18 h by diffusion blotting. The GnRH or luciferase complementary RNA probe was prepared using a commercial in vitro transcription system (Promega Corp.) in the presence of [32P]UTP. Hybridization procedures were performed as previously described (13).

Data analysis
The intensity of PCR bands was determined using Bio1D image analysis software (Vilber-Lourmat, Marne-La-Vallee, France). Intron A excision and GnRH mRNA levels were calculated on the basis of the standard curve. Data were statistically evaluated using one-way ANOVA followed by Fisher’s least significant difference test for a post hoc comparison. Statistical significance was set at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Changes in intron A excision during postnatal development of normal mice
Competitive RT-PCR has been shown to be a useful method that circumvents PCR variation or artifacts, allowing investigators to measure the amount of target mRNA (17, 18). To examine splicing efficiency, we compared the mRNA levels of intron-containing RNA species by simultaneous amplification of both cDNA fragments in the same tubes. The primer locations and expected sizes of the PCR products are indicated in Fig. 1Go. A titration of the competitive PCR was performed with three primers, e1-up, iA-up, and e3-dn, in the presence of a constant amount (1 fg) of A23 DNA fragment and a serial dilution (0.1–10 fg) of 123 DNA fragment, because the amount of GnRH mRNA ranges from 0.1 to 10 fg/µg total RNA. The standard curve was constructed based on the observed ratio of 123 to A23 PCR products as a function of a given ratio of 123 to A23 cDNAs, showing a linear regression coefficiency of 0.98 (Fig. 2AGo). To compare expression levels of the mature mRNA and intron A-containing RNA, 123 and A23 cDNAs were coamplified in the same tube. The ratio of 123 to A23 was calculated on the basis of the standard curve shown in Fig. 2AGo. The ratio of 123 to A23 in the POA was lowest in 1- and 2-wk-old mice (2.08 ± 0.21 and 3.15 ± 0.38, respectively), began to increase significantly in 3-wk-old mice (6.37 ± 0.66; P < 0.01 compared with those of 1- and 2-wk-old mice), and further increased until adulthood in 7-wk-old mice (9.56 ± 1.19). In contrast, the ratio of 123 to A123 in the CTX (0.32 ± 0.03 in 1-wk-old mice) was substantially lower than that in the POA, decreased in 3-wk-old mice (0.12 ± 0.04), and remained unchanged until adulthood (Fig. 2BGo). This result suggests that intron A excision (as defined by assessing the ratio of 123 to A23) in the POA is developmentally regulated. To examine whether such an increase in intron A excision corresponds with an increase in GnRH mRNA levels, we performed competitive RT-PCR with an exogenous internal control that was generated by a truncation of an internal part of GnRH cDNA (18). GnRH mRNA levels were calculated on the basis of a standard curve as previously described (17, 18). Compared with GnRH mRNA levels in the POA of 1-wk-old mice (1.58 ± 0.42 fg/µg total RNA), GnRH mRNA levels gradually increased during development. Specifically, a significant increase was first observed in 3-wk-old mice (5.50 ± 0.86 fg/µg total RNA; P < 0.01) and was further augmented until adulthood (13.39 ± 2.06 fg/µg total RNA) (Fig. 3AGo). This result is consistent with a recent finding reported by Gore et al. (14). GnRH mRNA levels in the CTX, however, were undetectable in the presence of 0.2 fg of mutant GnRH cDNA at all ages sampled (data not shown). Levels of the 1A23 RNA species in both the POA and CTX were determined under an optimized PCR condition followed by Southern blot analysis (Fig. 3BGo). To optimize the PCR condition, PCR was performed with different numbers of cycles and different initial inputs of reverse transcribed RNA (data not shown). 1A23 levels in the POA were not significantly changed, but 1A23 levels in the CTX were decreased by 22% in 4-wk-old mice. It is noteworthy that the alteration in GnRH mRNA levels during development roughly parallel the changes in intron A excision shown in Fig. 2BGo.



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  		Figure 2. Intron A excision. A, Titration and standard curve of competitive PCR for the quantitation of 123 and A23 was performed in the presence of 1 fg of the A23 DNA fragment and a serial dilution of the 123 DNA fragment. Total RNA was extracted from the POA and CTX of developing mice at the age of 1–7 wk. B, RT-PCR was performed in the presence of three primers, e1-up, iA-up, and e3-dn, allowing the simultaneous amplification of the mature mRNA (123) and intron A-containing RNA species (A23). Intron A excision was calculated by the ratio of 123 to A23 on the basis of a standard curve as shown in A. Closed circles represent the mean ± SEM of the POA (n = 5), and open circles represent the mean of duplication of the CTX. *, P < 0.01 vs. POA from 1-wk-old mice.

 


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  		Figure 3. GnRH mRNA and 1A23 RNA levels during postnatal development. A, RT-PCR was performed with primers e1-up and e3-dn in the presence of 0.2 fg of mutant cDNA as an internal control. B, 1A23 RNA levels were determined by 28 PCR cycles with primers iA-up and e3-dn followed by Southern blot analysis. Closed circles and closed triangles represent the mean ± SEM (n = 5) of RNA levels in the POA and CTX, respectively. *, P < 0.01 vs. 1-wk-old mice.

 
Introns B and C excision during development
Intron B2B) with the 23 transcript using the primer set e2-up, iB-dn, and e3-dn, showed no significant changes during development (Fig. 4AGo). Intron C excision during development was also examined by comparing RNA species containing exons 3 and 4 (34) and intron C and exon 4 (C4). Both RNA species 34 and C4 were continuously increased during development, with no significant changes in the ratio of 34 to C4 (Fig. 4BGo). This result indicates no developmental control of introns B and C excision. However, compared with intron A-containing RNA (A23), the intron B-containing RNA species (B3 or A2B) was gradually increased during development (Fig. 4Go, C–E), which was very similar to that shown in Fig. 2BGo. This result indicates that the GnRH gene transcriptional rate (represented by the intron B-containing RNA species B3 and A2B) was increased during development, with a coincident increase in intron A excision, thereby increasing GnRH mRNA levels during development.



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  		Figure 4. Introns B and C excision during development. RNA samples from the POA were subjected to RT-PCR. A, To determine intron B excision, e2-up, iB-dn, and e3-dn primers were used. B, Intron C excision was examined by simultaneous amplification of 34 and C4 RNA species using primers e3-up, iC-up, and e4-dn. C and D, To compare the primary transcript with the intron A-containing RNA, primers iA-up, iB-up, and e3-dn or primers iA-up, iB-dn, and e3-dn were used, respectively. E, Observed ratios of PCR products. The ratios were obtained from A, B, C, and D, respectively. Closed circles, open circles, closed triangles, and open triangles indicate the ratios of 23 to 2B, 34 to C4, B3 to A23, and A2B to A23, respectively.

 
Intron A excision of hpg mice
hpg mice do not undergo reproductive development and show extremely low levels of pituitary gonadotropins because they lack the ability to synthesize the GnRH peptide as a result of a deletion of two exons (exons 3 and 4) in the GnRH gene (15). Previously, we demonstrated that ESEs in GnRH exons 3 and 4 were involved in the enhanced excision of intron A (13). In the in vitro splicing system, pre-mRNA containing intron A with its neighboring exons (1A2) could not be spliced. However, when exons 3 and 4 were linked up with exon 2 (1A234), intron A could be excised, indicating that exons 3 and 4 serve as ESEs. To gain insight into the functional significance of the ESEs in vivo, we examined intron A excision in hpg mice. Using the primer set e1-up, iA-up, and e2-dn, spliced RNA species (12) and unspliced RNA species (A2) were simultaneously amplified with RNA samples from the POA, OB, and CTX of the adult hpg (hpg/hpg), heterozygous (hpg/+), and normal (+/+) mice. The levels of unspliced RNA species (A2) were not significantly different among the tissues from either hpg or normal mice. However, strong signals of spliced RNA species (12) were observed only in the POA of heterozygous and normal mice (Fig. 5Go). The ratio of 12 to A2 RNA species clearly showed that intron A excision in the POA of hpg mice was significantly lower than that of normal and heterozygous mice. This result suggests that GnRH transcripts in the POA of hpg mice mostly remain unspliced, probably because of the lack of ESEs in the GnRH exons 3 and 4, whereas GnRH transcript in the POA of normal mice undergo further processing with the help of ESEs in exons 3 and 4, resulting in the production of mature mRNA.



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  		Figure 5. Intron A excision of hpg mice. Total RNAs were obtained from the POA, OB, and CTX of adult hpg homozygous (hpg/hpg), heterozygous (hpg/+), and normal (+/+) mice. RNA was amplified by RT-PCR in the presence of three primer, e1-up, iA-up, and e2-dn, allowing the simultaneous amplification of the mature mRNA species (12) and the intron A-containing RNA species (A2). Intron A excision was obtained by determining the ratio of 12 to A2 cDNAs. Bars represent the mean ± SEM (n = 5). *, P < 0.01 vs. POA of hpg/hpg mice.

 
Inhibitory effect of the intron A sequence on the translation of the downstream open reading frame
To demonstrate the functional relevance of intron A-retained RNA species, we examined the effect of the intron A sequence on translation efficiency. Because the intron A-retained transcript has a very long 5' UTR (the coding region for GnRH prohormone starts at the second exon), one may assume that such a long 5' UTR may affect translation efficiency. To address this possibility, we used fusion constructions of the 5' UTR of the mouse GnRH gene and the luciferase coding region that were transfected into CHO-K1 cells (Fig. 6AGo). Whereas E1-Luc (exon 1-luciferase) gene constructs elicited slightly decreased luciferase activity compared with that of control reporter plasmid, intron A-containing gene constructs, such as IA-Luc (intron A-luciferase) and E1IA-Luc (exon1-intron A-luciferase), exhibited a drastic reduction in luciferase activity (Fig. 6BGo). However, similar amounts of luciferase mRNA were observed among the experimental groups, as revealed by Northern blot analysis (Fig. 6CGo).



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  		Figure 6. Effects of retained intron A on the translational efficiency of downstream luciferase reporter. A, Scheme of GnRH-luciferase fusion gene constructs. Mouse GnRH exon 1 (E1) and/or intron A (IA) DNA fragments were fused to luciferase open reading frame. All fusion constructs were under the control of the simian virus 40 minimal promoter. B, Measurement of luciferase activity. The fusion constructs and the control luciferase vectors were transfected into CHO-K1 cells. Luciferase activities were measured 24 h after transfection. Data are shown as mean ± SE (n = 4–12). ß-Galactosidase activities were measured for normalizing transfection efficiency. C, Northern blot analysis of luciferase mRNA. Total RNAs were isolated from transfected CHO-K1 cells. Thirty micrograms of each RNA sample was resolved on a 1.2% formaldehyde gel and transferred to a Nytran filter. The hybridization was performed using 32P-labeled luciferase cRNA probe.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we demonstrate for the first time that the ratio of GnRH mRNA to intron A-containing RNA in the POA was altered during mouse postnatal development. The ratio of mRNA to intron A-containing RNA in the POA was significantly increased in 3-wk-old mice and further augmented until adulthood, which was accompanied by a simultaneous increase in mature mRNA levels during development. Interestingly, intron A excision was largely attenuated in the POA of adult hpg mice in which GnRH exons 3 and 4 were deleted. This may provide evidence that ESEs in GnRH exons 3 and 4 may be involved in the developmental increase in intron A excision in the POA of normal mice. Moreover, we found a decreased translation efficiency of intron A-retained RNA species. These data clearly indicate that GnRH expression across development is finely regulated at multiple levels such as transcription, posttranslation, and translation, providing further evidence that enhanced intron A excision is a prerequisite for GnRH expression in GnRH neurons.

The significant increases in the ratio of mRNA to intron A-containing RNA, which occur as a function of the age of the mouse, seem to be attributable to enhanced intron A excision during development. However, one may also argue that the mRNA level is increased because of the enhanced stability of the mRNA in the absence of any change in excision over time. Indeed, several lines of evidence indicate that GnRH mRNA levels are altered by changes in mRNA stability. Using GT1-7 cells, Roberts and colleagues (19, 20) demonstrated that phorbol ester or calcium ionophores can decrease the GnRH mRNA half-life by shortening the poly(A) tail length. Using the in vivo system, Roberts and colleagues (12, 21, 22) also proposed the possible involvement of GnRH mRNA stability in the regulation of GnRH gene expression in response to steroid or neural inputs by comparing the primary transcript with mRNA levels. Thus, GnRH mRNA stability could increase as a function of neural inputs (e.g. glutamate) that may be present during development. However, the increasing ratio of mRNA to intron A-containing RNA with age shown in Fig. 2BGo is most likely attributable to increasing intron A excision based on the following reasons. First, as illustrated in Fig. 4AGo, there was no significant difference among the ratios of intron B-containing RNA (probably a primary transcript) to 23 RNA (probably mRNA) at each time point examined. The absence of a change in the ratio of intron B-containing RNA to mRNA in the presence of a gradual increase in GnRH mRNA levels (Fig. 3AGo) indicates that transcription rate appears to increase with age. Thus, the increases in mRNA levels shown in Figs. 2BGo and 3AGo are most likely caused by an increase in transcription rate rather than the increase in mRNA stability.

Second, as shown in Fig. 4Go, B and C, the level of intron B-containing RNA over intron A-containing RNA increases with age, which is very similar to the ratio pattern of mRNA to intron A-containing RNA shown in Fig. 2BGo. Such a response profile indicates that intron A excision is accelerated during mouse development. Third, although an age-related increase in mRNA stability with no change or decrease in intron A-containing RNA stability could be interpreted as producing a differential regulation of RNA stability between mRNA and intron A-containing RNA, it would require a differential regulation of poly(A) tail length and degradation of RNA, which is mostly dependent on the sequence or structure of the 3' UTR (23). It should be noted that GnRH mRNA and intron A-containing RNA share the same 3' UTR, so both RNA species should be controlled by the same regulatory mechanism. This raises the possibility that there is a differential regulation of RNA stability between mRNA and intron A-containing RNA through the intron A sequence itself, because the presence of intron A is the only difference between mRNA and intron A-containing RNA species (Fig. 5). If that is the case, the ratio of 12 to 1A2 RNA species in hpg mice should be higher than that shown in Fig. 5AGo, because 1A2 RNA species would be more rapidly degraded compared with 12 RNA. However, the data in Fig. 5AGo clearly show that the ratio of 12 to 1A2 in hpg mice was very low compared with that in normal mice, indicating that the differential regulation of RNA degradation through the intron A sequence seems not to be the cause of these changes. Thus, we favor the idea that enhanced intron A excision with age appears to better explain the data than an increase in mRNA stability.

Previously, we demonstrated that the intron A retention observed in non-GnRH-producing tissues is attributable to a suboptimal 3' splice site of intron A. The 3' splice consensus contains a polypyrimidine tract followed by CAG (YnCAG) (24). The branch point sequence located in the upstream region of the pyrimidine tract is also regarded as a part of the 3' consensus (25). However, the pyrimidine tract of the 3' splice site of GnRH intron A contains many purine bases. Moreover, a putative branch point sequence resides within the pyrimidine tract but not upstream of the pyrimidine tract. On such a suboptimal splice site, the 3' spliceosome complex may not be formed; thus, intron A is retained. Mutation of this 3' splice site toward a consensus sequence greatly increased the splicing rate, indicating an intrinsic weakness of the 3' splice site of intron A (13). Recently, a GnRH transcript lacking exon 2 was found in Gn11 cells and immune cells (26, 27). Thus, all of the three RNA species (intron A retention, exon 2 skipping, and mature mRNA) could be produced from a single GnRH gene. Exon 2 skipping from GnRH pre-mRNA would be explained by the same context as intron A retention. The 3' spliceosome complex may not be formed in the 3' splice site of intron A, but a 3' spliceosome complex formed in the 3' splice site of intron B may interact with the 5' spliceosome complex formed in the 5' splice site of intron A. Thus, exon 2 could be removed together with intron A and intron B.

Another parameter determining 3' splice site selection is the ESE sequence that is usually found downstream of the regulated 3' splice site (28, 29). We previously found that intron A could be partially excised in the presence of ESEs located in GnRH exons 3 and 4 (13). Moreover, intron A could be more efficiently excised by the addition of GT1 nuclear extract in the presence of GnRH exons 3 and 4. It is assumed that in the POA of normal mice, introns B and/or C are first excised from the GnRH primary transcript, producing the 1A234 RNA species. Then, the majority of the 1A234 RNA species undergoes further processing of intron A excision with the help of ESEs located in exons 3 and 4, generating the mature mRNA. Therefore, the truncated exons 3 and 4 present in hpg mice may contribute to a diminished capacity for intron A to be excised in hpg mice (Fig. 7Go). Together, these results clearly indicate that ESEs in GnRH exons 3 and 4 are required for the enhanced excision of intron A from the primary transcript and that there is a GnRH neuron-specific splicing factor(s) interacting with those ESEs.



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  		Figure 7. A scheme of intron A excision in normal and hpg mice. In the POA of normal mice, the GnRH primary transcript undergoes RNA processing beginning with introns B and/or C excision, producing the 1A234 RNA species. The majority of 1A234 RNA undergoes further processing of intron A excision with the help of ESEs located in exons 3 and 4, generating the mature mRNA predominantly, as indicated by a thick arrow. The mature mRNA is a good template for the production of large amounts of GnRH peptide. However, in the POA of hpg mice, because of the lack of ESEs, a majority of RNA transcripts remain unspliced, as indicated by the thick arrow, whereas intron A is barely excised, as indicated by the dashed arrow. Because of the lower production of intron A-excised RNA transcript, hpg mice would produce extremely low amounts of GnRH peptide. Gray boxes in exons 3 and 4 indicate ESEs.

 
It should be noted that a coding sequence for GnRH preprohormone initiates at exon 2 (6). The first exon contains the 5' UTR. Exon 2 codes for the signal peptide, the GnRH decapeptide, and the N-terminal 11 amino acids of the GnRH-associated peptide. Exons 3 and 4 code for the next 45 GnRH-associated peptide residues and the termination codon (2). Thus, the intron A-retained RNA species in normal and hpg mice contains a long 5' UTR. Because exon 2 is intact in hpg mice, despite the lack of exons 3 and 4, it could be postulated that hpg mice could produce the GnRH peptide like normal mice. However, little GnRH is detectable in hpg mice (15). Our findings raise the possibility that the attenuated production of the GnRH peptide in hpg mice may result not only from a low transcription rate but also from a low level of intron A excision. Although intron A-retained RNA transcripts are found in a variety of tissues, their biological consequence is poorly understood. One could suggest that a long 5' UTR of intron A is likely to affect translational efficiency. In fact, sequence analysis reveals that intron A provides many AUGs and at least five conserved Kozak sequences (30), suggesting the existence of multiple short open reading frames. The presence of multiple AUGs or Kozak sequences in intron A may, in part, interfere with the translational efficiency. This possibility can be supported by recent work in which several long 5' UTRs of neuronal nitric oxide synthase mRNA profoundly inhibited translation (31). Indeed, we have attempted to examine the issue specifically with regard to the translational efficacy of intron A-retained RNA. This was accomplished by transfecting CHO-K1 cells with a eukaryote expression vector in which the intron A sequence was inserted into the upstream region of the luciferase gene. There were no alterations in luciferase mRNA levels between the control and the intron A-inserted group, as revealed by Northern blot analysis. However, a drastic decrease in luciferase activity was observed in the intron A-inserted groups (Fig. 6Go), suggesting that intron A can strongly inhibit the translational efficacy of the downstream gene. Therefore, it appears that an intron A-retained RNA transcript cannot be translated successfully into hpg mice. In normal mice, the increased splicing of GnRH pre-mRNA in the POA during development is important for the efficient production of the GnRH peptide, which is in good agreement with the gradual increase in GnRH release from the hypothalamus that occurs during postnatal development (32).

Previously, we suggested the presence of a GnRH neuron-specific splicing factor(s) that may interact with ESEs located in GnRH exons 3 and 4 to facilitate intron A excision (13). The present study indicates that a developmental maturation of the splicing machinery may occur to increase the splicing rate of GnRH pre-mRNA in GnRH neurons. Although it is unknown what factor(s) is involved in this process, the present data suggest that developmental changes in this factor(s) may play a role in intron A excision.


   Footnotes
 
This study was supported in part by the Ministry of Science and Technology through the Korea Brain Science Program and a National Research Laboratory (NRL) grant (to K.K.). B.W.K., S.P., and G.H.S. were supported by a Brain Korea 21 research fellowship from the Korea Ministry of Education.

Abbreviations: CHO-K1 cells, Chinese hamster ovarian cells; CTX, cerebral cortex; ESEs, exonic splicing enhancers; hpg, hypogonadal; OB, olfactory bulb; POA, preoptic area; SSPE, standard sodium phosphate EDTA buffer; UTR, untranslated region.

Received March 9, 2001.

Accepted for publication June 29, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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