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Institute of Reproductive Medicine (J.G., L.L., M.G., T.M., C.M., M.S.), University Hospital, D-48129 Münster, Germany; and Institute of Biochemistry (S.S.), 91054 Erlangen, Germany
Address all correspondence and requests for reprints to: Jörg Gromoll, Ph.D., Institute of Reproductive Medicine, Domagkstraße 11, D-48129 Münster, Germany. E-mail: Joerg.Gromoll{at}ukmuenster.de.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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A hallmark of the LHR gene and the closely related FSH receptor (FSHR) and TSH receptor (TSHR) genes is a marked alternative splicing of the primary transcript (3). Several alternative splicing mechanisms have been identified, the most common type being exon skipping (removal of one or several complete exons), followed by exon inclusion, alternative acceptor, or donor splice sites (4). Whereas skipping of one or several exons results in in-frame deletion of amino acids without changing the open reading frame, exon inclusion or alternative acceptor or donor splice sites results in truncated receptors due to the presence of premature termination codons (5, 6).
Studies in sheep and rats, as well as other species, have shown that splicing of the LHR is regulated in a temporal and spatial manner during follicular or testicular development and that the abundance of the different variants varies significantly (7, 8, 9). Functional studies demonstrated that some of them are translated and, in principle, are capable of hormone binding. However, nearly all of them do not reach the membrane and remain trapped within the cell (10, 11). Recently, a LHR splicing variant lacking exon 9 was shown to interact with the full-length LHR when expressed in vitro and to modulate the expression of the wild-type LHR (12). Common to all studies is the lack of any information concerning the mechanism underlying splicing in the LHR gene. This also holds true for the TSHR and FSHR gene.
After the original observation that the wild-type LHR mRNA lacks exon 10 in the New World monkey (NWM) Callithrix jacchus (13), we proposed that this receptor belongs to a new class of LHR, which we named type 2. LHR type 2 represents the wild-type LHR in the whole NWM lineage (14). Interestingly, exon 10 is only missing at the mRNA and at the protein level because Southern blot experiments indicated the presence of a putative exon 10 at the genomic level (our unpublished data and Ref. 13). Additional studies in several primate lineages indicated that exon 10 is completely lacking in the LHR cDNA of all NWMs, whereas in humans and other primates exon 10 is constitutively included in the mRNA (14, 15). This indicates tight functional constraints for constitutive splicing of exon 10, suggesting its essential role for LHR function in these species. Exon 10 encodes a part of the hinge region, linking the extracellular to the transmembrane domain. Removal of exon 10 from the human LHR results in different responsiveness to human CG and LH, with a significantly enhanced preference toward human CG (16, 17). Therefore, the part of the LHR protein encoded by exon 10 might be instrumental for receptor activation in species possessing CG.
Alternative splicing is regulated by a tightly regulated balance of trans- and cis-acting factors. Detailed studies on alternative splicing of the CFTR and SMN2 genes that are involved in human diseases indicate that single exonic or intronic point mutations can cause exon skipping (18). The fact that exon 10 is completely absent in LHR type 2 could therefore be due to mutations disrupting splice sites or affecting binding of splicing factors or, possibly, to species-specific splicing factors. Thus, LHR type 2, aside from its unique biochemical properties, represents an interesting model to investigate the molecular mechanisms underlying splicing in glycoprotein hormone receptors. In this study we delineate, for the first time, the molecular mechanism underlying constitutive exon 10 inclusion in the human and exon 10 skipping in the marmoset LHR.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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) (EMBL accession no. AM181324).
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The putative marmoset exon 10 consists of 81 bp and displays 86% nucleotide identity to exon 10 of the human and mouse LHR (Fig. 2). Interestingly the splice sites are conserved, with AG at the 3' and GT at the 5' end (Fig. 2). A splice site score calculator program revealed high scores for a normal splicing mechanism of the putative exon 10, which perfectly maintains splicing phase 2 with CT at the 3'-end encoding for leucine (CTN), as in all other exons of the LHR including exon 10 from other species (14). The deduced amino acid sequence of the putative marmoset exon 10 revealed 77% identity compared with human and mouse exon 10. This homology is similar to the overall homology between the marmoset and the human LHR (13). Thus, no obvious sequence alterations were identified that could explain the absence of exon 10 usage in the marmoset.
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). For the marmoset minigene only a smaller amplicon lacking exon 10 could be detected. No alternative splicing was observed for any minigene. This corroborates original findings in ovaries and testes (14, 15) showing no alternative splicing of exon 10 and strengthens the view of a very tightly regulated mechanism controlling exon 10 processing.
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, m2–m4). This strongly suggests that no repressing factor/activity was binding to the marmoset-specific intronic sequences.
Exon 10 Usage Requires Both Intronic and Exonic Sequences
To circumscribe and to identify the intronic sequences adjacent to exon 10 responsible for its usage, genomic sequences encompassing exon 10 from human or marmoset DNA and including 450, 300, 150, or 45 bp 5' and 3' of exon 10, respectively, were cloned into the exon trap vector pSPL3, followed by transient transfection of COS-7 cells and RT-PCR. The typical pattern of exon 10 usage of the human and marmoset construct, respectively, was completely maintained when using the 450-bp or the 300-bp construct (data not shown) as well as when using the 150-bp or the 45-bp construct (Fig. 4, upper and middle panel). This indicates that sequences responsible for exon 10 usage must be located within the region encompassing exon 10 and 45 bp upstream and downstream of it.
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). Transfection and RT-PCR of these chimeric, exon trap constructs showed alternative splicing. In particular, the marmoset-human-marmoset construct, consisting (from 5' to 3') of the 5'-flanking intron of the marmoset exon 10 (45 bp), the human exon 10 (81 bp), and the 3'-flanking intron of the marmoset exon 10 (45 bp) showed a shift from constitutive skipping to 51% of exon 10 inclusion. Conversely, the human-marmoset-human construct, consisting of the 5'-flanking intron of the human exon 10 (45 bp), the marmoset exon 10 (81 bp), and the 3'-flanking intron of the human exon 10 (45 bp), showed an even higher extent (76%) of exon 10 usage. These experiments indicated that exon 10, along with one or both intronic flanking regions within a distance of 45 bp, contributes to exon 10 splicing or skipping and that the human intronic regions are presumably more important than the exonic sequences for the constitutive use of exon 10.
Essentially the same results were obtained with the human granulosa cell line COV 434 (Fig. 4, lower panel), serving as a cell type naturally expressing the LHR. Therefore, no species-specific splicing factors seem to be involved.
Comparative Genomics of Exon 10 and Its Flanking Regions Identifies Candidate Nucleotides Responsible for Exon 10 Usage
We previously showed that exon 10 is lacking in all NWMs whereas it is present in all other primate lineages (14). Assuming a common mechanism underlying exon 10 skipping in all NWMs, we reasoned that comparing the corresponding sequences from different primates could help to identify candidate nucleotides. We therefore cloned and sequenced the 171-bp genomic region comprising exon 10 and the 45 bp upstream and downstream of it from four additional NWMs (Saimiri sciureus, Saguinus fuscicollis, Lagothirx lagothricha, Pithezia pitheria) as well as from the Old World monkey Macaca fascicularis, the lemur Eulemur coronatus, and the chimpanzee (Pan paniscus). Primers were designed based on sequence homology of human, marmoset, and mouse LHR. Inspection of the sequences obtained revealed the presence of only a few nucleotide changes specifically present in the NWM (Fig. 2, gray boxes). We pinpointed unique nucleotide substitutions at position –19 within the 3'-intronic sequence upstream of exon 10, in which a T present in all other species is replaced by an A in M. fascicularis and in the NWM, and at position +26 within the coding region of exon 10 where a G is substituted by an A. In addition, a highly conserved T at position –10 is replaced by a C only in C. jacchus.
Sequence analysis using the ESE (exon splicing enhancer) software program yielded several important splicing motifs. First, two polypyrimidine (Y = C or T) stretches (n = 7 and n = 12), important cis-acting sequence elements directing intron removal in pre-mRNA splicing, are directly adjacent to the 3'-splice site of human exon 10 (Fig. 2, underlined). In the marmoset the first poly-Y stretch is disrupted by the purine A at position –19 (Fig. 2, gray box) and in the second stretch the pyrimidine T at position –10 is replaced by the pyrimidine C (gray square). Second, within the coding sequence of human exon 10 a sequence stretch displayed some homology to an SC35 binding site signature (consensus sequence GRYYcSYR; human sequence CATTTCTG), which was lacking in the marmoset and in all other NWMs due to the A substitution at position +26. Other than these differences, binding sites for other splicing factors such as SRp40, SF2/AF, and SRp55 were identical both in human and marmoset exon 10 (data not shown).
Three Nucleotides within a 45-bp Stretch at the Intron 9-Exon 10 Boundary Specifically Regulate Exon 10 Usage
Through oligonucleotide site-directed mutagenesis, the nucleotides at positions –19 and +26 in the marmoset sequence were changed into the corresponding human nucleotides and vice versa (Fig. 5, upper part). When the intronic marmoset –19 A was mutated into human T, a small increase in exon 10 inclusion (11% compared with < 1%) was observed, whereas no change in splicing was observed in the corresponding human construct (Fig. 5, m –19 and h –19). However, when the human sequence was restored in the marmoset by mutating the nucleotide A at position +26 into a G, significantly increased splicing (60%) could be observed (Fig. 5, lane m 26). Conversely, by replacing the human G at position +26 by an A, only a slight decrease in exon 10 splicing efficiency could be detected (93%; Fig. 5, lane m26 and h 26). Surprisingly, combining both exonic and intronic changes at position –19 and +26 in both species resulted in a slightly more pronounced exon 10 usage, but neither complete exon 10 skipping in the human nor complete splicing in the marmoset LHR (data not shown) was achieved. We therefore considered the unique exchange at position –10 present only in the marmoset sequence (Fig. 2). Replacing the C at position –10 of the marmoset by a T resulted in a drastic effect on exon 10 inclusion in the marmoset sequence (56%), whereas in the corresponding human construct a drastic reduction of exon 10 splicing (61%) was observed (Fig. 5, lane m –10 and h –10). When both intronic nucleotide changes at position –19 and –10 were introduced, exon 10 inclusion further increased to 91% for the marmoset, whereas in the human exon 10 splicing was lowered to 60% (Fig. 5, lane m –19/–10 and h –19/–10). A similar high inclusion rate was obtained for marmoset exon 10 by changing the two nucleotides at position –19 and +26 (80%), which reached 100% inclusion when the nucleotides at position –10 and +26 were changed (Fig. 5, lane m –19/26 and m –10/26). In the final experiments all three nucleotide changes at position –19, –10, and +26 were combined within one construct This almost completely reversed the wild-type pattern of human exon 10 splicing (only 5% residual inclusion) (Fig. 5, lane h –19/–10/26) and of marmoset exon 10 skipping (100% inclusion) (Fig. 5, lane m –19/–10/26). Thus, the nucleotides at position –10 and +26 and, to a lesser extent at position –19, represent key sites in regulating exon 10 skipping or splicing of the LHR. To study further whether SC35 interacts with the identified putative binding site at position +26 we performed RNA interference (RNAi) experiments. Although SC35 protein expression, as revealed by Western blots, could be significantly reduced in COS-7 cells transiently transfected with RNAi specific for SC35 by at least two of the three RNAis tested (356 and 589: Fig. 6A), an effect on exon 10 usage could be detected neither for the wild-type human LHR nor for the marmoset LHR mutated at the three positions –19, –10, and +26 (Fig. 6B). Thus, although the nucleotide sequence displays a putative SC35 binding site signature, this is not involved in the regulation of exon 10 usage.
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, the secondary RNA structure of human (H) and marmoset (M) wild-type exon 10 and its adjacent intronic sequences are markedly divergent. Whereas for the human exon 10 the 3'-acceptor splice site (uucAG) is located in a short stem (see inset H), in the marmoset the acceptor site is positioned at the edge of a loop (inset M). In the human RNA the putative SC35 sequence stretch (ranging from nucleotide 23–28; Fig. 2) is the RNA stretch. Thus, both the 3'-intronic sequence and part of exon 10 form a short stem, obviously necessary for the recognition of the 3'-splice site. In the marmoset, by contrast, only a loop with no complementary sequences can be observed. In addition, the overall secondary structure and the number of stems and loops are different between the two species. Introducing sequentially point mutations at position –19, –10, and +26 or combinations thereof significantly changes the secondary structure predictions [supplemental Fig. 1, a–h, for human exon 10 and supplemental Fig. 2, a–h (published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org). For the human exon 10 this results in transformation of the 3'-acceptor site to a loop structure (H' and inset), whereas a stem transformation is found in the marmoset (M' and inset) (Fig. 7). Interestingly, the stem formed resembles exactly the human exon 10 wild-type conformation with a complementary strand consisting of nucleotide position 23–28 (see inset) of the putative SC35 binding motif. Moreover the overall secondary structure including number of loops and stems is very similar to that of the human RNA (compare H and M'). Single mutations (–19, –10, and +26) resulted in an intermediate configuration between H and H' and between M and M' (supplemental Figs. 1 and 2); however, no association between free energy values and extent of exon splicing was found. Thus, the formation of stems covering the 3'-acceptor splice site of exon 10 is associated with exon 10 usage.
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, significant stimulation could be obtained with all constructs and hormone preparations used, suggesting that the insertion of exon 10, either of marmoset or human origin, does not change the functional response of the marmoset LHR. Because only two hormone concentrations were used, the previously reported right shift of the LH dose-response curve with the LHR lacking exon 10 (17) cannot be appreciated in this experiment.
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DISCUSSION |
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Inspection of the putative marmoset exon 10 sequence and its genomic organization revealed two interesting features. First, there is a significant expansion of the intronic regions 9 and 10 due to the presence of LINE-1 elements; second, exon 10 sequence is highly conserved when compared with other LHRs and the splice sites are intact. The LINE-1 elements, making up approximately 7500 bp of the total intronic regions 9 and 10, are considered a driving force in evolution, reshaping genome content by their ability to retrotranspose. Random integration of LINE-1 elements may cause gene or exon silencing or can generate additional exons and even new genes (21, 22, 23). However, most LINE-1 elements are transcriptionally silent. The LINE-1 elements found in the analyzed region of the marmoset LHR gene lack an open reading frame and are inactive. In addition, our experiments demonstrated that they do not influence exon inclusion (Fig. 3). Thus, it seems likely that the retrotransposition of LINE-1 elements was not the primary event causing loss of exon 10 inclusion. Rather, it could be a process secondary to the intronization of exon 10 in the LHR type 2.
Mechanism of Exon 10 Intronization in the Marmoset LHR
This study shows that in the marmoset and in the other NWMs a polypyrimidine stretch, important for positioning splicing factors such as U2AF, is interrupted at position –19 by the purine A replacing a T found in all other species investigated. Surprisingly, however, this nucleotide contributes only marginally to exon inclusion (Fig. 5), and only when the marmoset-specific C at position –10 is replaced by T, a significant increase in exon 10 inclusion is obtained. Correspondingly, a decrease in inclusion efficiency of the human exon 10 can be observed by replacing the human T at position –10 by the marmoset-specific C. This is peculiar because a nucleotide substitution of T by C does not interrupt the polypyrimidinic nature of the stretch and because U2AF (or a related splicing factor) is thought to bind equally well to C and T (24). Therefore, the sterical properties of the region might be important for splicing as well. When both nucleotide substitutions at positions –19 and –10 are combined, inclusion of marmoset exon 10 is additionally enhanced to 91%, whereas the level of inclusion of the human exon 10 remains the same. From these experiments on the 3'-region of intron 9 we conclude that both nucleotides at position –19 and position –10 contribute significantly to exon 10 inclusion.
Another important nucleotide change is located within exon 10. The substitution of G by A at position +26 resulted in destruction of potential binding sequence with a SC35 signature (Refs. 25, 26, 27 and Fig. 2). However, our RNAi experiments indicated clearly that SC35 itself is not the regulatory splicing factor. Based on these experiments, we followed the hypothesis that the secondary RNA structure could be involved in exon 10 usage. Recent studies have underlined the important function of such structures for effective splicing. Changes within this structure seem to hinder the access of basic splicing factors to selected sequences, resulting in diminished intron processing and thereby promoting exon skipping (28). Such a scenario has been recently shown for the human TAU gene, the 3'-splice site of which is particularly sensitive to the presence of structured RNA (29).
The secondary RNA structure prediction using MFOLD corroborates the results of the in vitro mutagenesis experiments. For the human exon 10 the 3'-splice site is readily accessible for the splicing machinery, thereby resulting in constitutive exon 10 usage. For the marmoset exon 10, the experimental data and the structural prediction suggest that exon skipping is supported by the aberrant 3'-splice site structure conformation. Only when at least the two point mutations at positions –10 and +26 are introduced does the RNA assume a secondary structure highly reminiscent of the human exon 10, in which the 3'-splice site is forming a stem together with the SC35 sequence signature (supplemental Fig. 2g). The changes at position –10 and +26 of the human exon 10 significantly disrupt the secondary RNA structure, but it is only when all three nucleotide exchanges are operated that exon 10 splicing is maximally reduced (Fig. 5 and supplemental Fig. 1g). Thus, both intronic and exonic sequences have a substantial role in constitutive inclusion of exon 10. The partial exchange of these sequences immediately results in alternative splicing of exon 10, as revealed by experimental data in which only single point mutations have been studied (Fig. 5).
The fact that three nucleotide changes had to be combined for constitutive inclusion or exclusion of exon 10 indicates that splicing is an event tightly controlled by sequence constraints. The synergistic effect of a combination of nucleotides has been observed in the regulation of other genomic regions such as exon 9 of the CFTR gene and exon 7 of the SMN2 gene, where these sequence elements have been named "composite exonic regulatory element of splicing" (CERES) (18). We speculate that alternative splicing of other exons of the LHR and of the other glycoprotein hormone receptor genes might depend on similar mechanisms, residing in the gene sequence.
In conclusion, this study identifies the genomic checkpoints for exon 10 usage and shows, for the first time, the involvement of a CERES responsible for exon 10 inclusion in the LHR mRNA. Mutations in the CERES, which change the secondary RNA structure and the accessibility of the 3'-splice site, result in exon 10 alternative splicing or skipping in the LHR type 2. We generated a human LHR constitutively skipping exon 10, and a marmoset LHR constitutively including exon 10. Understanding factors and structures involved in splicing of gonadotropin receptors provides the possibility to manipulate splicing, e.g. by knocking in a mutated LHR type 2 in transgenic mice to explore the consequences of altered splicing patterns in vivo.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Long-Template PCR
Long-template PCR was performed on 300–500 ng genomic DNA using the Expand Long Template PCR System (Roche, Penzberg, Germany) using the following primers:
The amplicons obtained were cloned into the TOPO-XL vector (Invitrogen, Karlsruhe, Germany) and completely sequenced using the shotgun technique.
Minigene Construction
For analyzing exon splicing of the large genomic regions covering exons 9–11, we employed the minigene approach described by Stoss et al. (30). Consequently both DNA fragments were excised by the restriction enzymes XhoI and NotI and cloned into the pTarget (Promega, Mannheim, Germany) expression vector.
Preparation of Expression Vectors for Exon Trap Experiments
Analysis of the shorter genomic clones containing exon 10 and 450, 300, 150, and 45 bp upstream and downstream of it were performed using the exon trapping system (GibcoBRL, Germany). Clones were generated by amplification of DNA fragments from human or marmoset genomic DNA using specific primers containing the restriction site XhoI within the forward primer or BamHI in the reverse primer. The amplicons were restricted by XhoI and BamHI and cloned into the exon trap vector pSPL3.
Generation of Chimeric Intron/Exon Constructs
To generate chimeric constructs, consisting of 3'- and 5'-intronic marmoset sequences joined to the human exon 10 and vice versa, we used the splicing by overlap extension by the PCR technique, first described by Horton et al. (31). In the present study, splicing by overlap extension by PCR involves five different PCRs to synthesize one chimeric construct. The intronic upstream element, representing 45 bp of the 3'-end of marmoset intron 9 was amplified using a forward primer (containing a Xho1 restriction site) for the human LHR 5'-CCGCTCGAGGAAGTCCAAAAGCTCAAATGC-3' or the marmoset LHR 45-bp intronic region 5'-CCGCTCGAGGAAGACCAAAAGCTCAAATGC-3' and a chimeric reverse primer for the human exon 10
5'-CTTTACATGCAAATACTTACAGTGTTTTGTTACTCACTTTC-3' (consisting of marmoset intronic and human exonic sequences) or a chimeric marmoset LHR 5'-CTTGCATGCAAATACTTACAGTGTTTCATTATTTTG-3'.
In the same way the 5'-element was generated, using a chimeric forward primer for the human exon 10 5'-ATAACCTCTCCTTTTTCAGACAGAATTTTTCACATTCCA-3' or a chimeric 5'-TAACCTCTTCTCTTTCAGACAGAATTTTTCATTTTC-3' for the marmoset exon 10 and the reverse primer 45 bp 5'-CGCGGATCCGGTGCACACAGAACAAGATA-3' containing a BamHI restriction site.
The chimeric constructs obtained were gel purified, restricted by BamHI and XhoI, and cloned into the exon trap vector pSPL3.
Mutagenesis
Mutagenesis was performed using the oligonucleotide site-directed mutagenesis kit from Stratagene (Amsterdam, The Netherlands) according to the manufacturer’s protocol.
Transient Transfection and RT-PCR Using the Exon Trap Vector pSPL3
COS-7 cells or COV434 cells, a human granulosa cell line kindly provided by Dr. C. de Geyter [University Women’s Hospital, Basel, Switzerland (32)], were seeded on petri dishes and cultured until they reached 40–50% confluence. Plasmid DNA (12 µg per dish) were used for transfection by lipofectamine (Invitrogen, Amsterdam, The Netherlands) (12 µl/dish). After 6 h transfection was stopped by adding DMEM supplemented with 20% fetal calf serum. The cells were lysed 48 h later using Ultraspec (AMS, Germany) and RNA isolated according to the manufacturer’s protocol. Reverse transcription was performed using the vector-specific primers SA2 or ß-actin primers. Subsequent PCR was performed with the primer combination SD6/SA2 and ß-actin forward/reverse using the following program: 94 C for 50 sec, 60 C for 50 sec, 72 C for 90 sec for 35 cycles. The PCRs were subjected to 2% agarose gel electrophoresis and documented using the Multi-Image Light system (Biozym, Oldendorf, Germany).
Densitometric Analysis
Evaluation of splicing patterns using the exon trap vector were performed by densitometric analyis of the RT-PCR bands using the ChemiImager software (Alpha Innotech Corp., Biozym, Germany).
Generation of Chimeric Marmoset LHR Constructs
The marmoset LHR cDNA plasmid was kindly provided by Professor I. Huthaniemi (Imperial College, London, UK). Sequence and functional characteristics of the marmoset LHR cDNA have been previously reported (13). We generated marmoset LHR chimeric constructs in which either the putative marmoset exon 10 or the human exon 10 were inserted in the expected place. Constructs were produced by overlapping PCR according to Zhang et al. (13) and Müller et al. (17). The following primers were used for the introduction of marmoset putative exon 10:
The amplicons were gel purified and cloned into the pGEM-T easy vector (Promega, Heidelberg, Germany). By restriction site-directed ligation the fragments containing either human or marmoset exon 10 were cloned into marmoset wild-type LHR cDNA, which then was cloned in the pTarget expression vector (Promega).
RNAi Oligonuleotides
We have designed three different RNAis that, in principle, are capable of interfering with SC35 synthesis:
Transfection experiments by lipofection were performed with varying doses of RNAi (5–10 nM) or plasmid DNA (100 ng to 1 µg plasmid DNA). Western blot were performed according to routine protocols, and for the detection of SC35 a monoclonal anti-SC35 antibody (Sigma S4045) was used.
Functional Studies
Recombinant marmoset CG was produced and calibrated as described by Müller et al. (20). Highly purified human LH (Calbiochem, Germany) was calibrated against the World Health Organization (WHO) international standard for LH 80/522 as described by Müller et al. (17). COS7 cells were transiently transfected with constructs containing the wild-type marmoset LHR cDNA or the chimeric constructs into which the human or marmoset exon 10 had been inserted at the expected place as described above. Cells were stimulated in triplicate 24 h after transfection for 6 h at 37 C with recombinant marmoset CG or human LH at concentrations of 0, 4, and 16 IU/liter (in terms of WHO LH 80/522) in 250 µl PBG (PBS solution, 1 mM glucose, pH 7.4) in the presence of 25 mM isobutylmethylxanthine (Sigma-Aldrich, Germany). After incubation the wells were frozen at –20 C and thawed, and the triplicates were pooled and boiled at 100 C for 2 min. cAMP production of the COS7 cells was measured by ELISA (IHF, Hamburg, Germany) as described previously (20). The pooled samples of triplicate stimulations were measured in duplicate. Protein content of the samples was determined by Bio-Rad DC protein assay (Bio-Rad Laboratories, Inc., Hercules, CA) in duplicate. Results were statistically assessed by the Friedman test using GraphPad Prism software (GraphPad Software, Inc., San Diego, CA).
Sequence Analysis
Sequence alignments were performed using the ClustalW program (http://www.ebi.ac.uk/Tools/). Exon splicing binding sites were identified using the ESE finder software (http://rulai.cshl.edu/tools/ESE/). Scoring of the splice sites was performed using Alex’s Splice Site Score Calculator (www.genet.sickkids.on.ca/
ali/splicesitescore.html). Other searches were performed using the toolbox of the Alternative Splicing Database project (http://www.ebi.ac.uk/asd/; 33) or MFOLD (http://www.bioinfo.rpi.edu/applications/mfold/rna/form1.cgi).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The marmoset sequence has been deposited at the EMBL with the accession no.: AM181324.
Disclosure Statement: The authors have nothing to disclose.
First Published Online May 15, 2007
Abbreviations: CERE, Composite exonic regulatory element of splicing; CG, chorionic gonadotropin; ESE, exon splicing enhancer; LHR, LH receptor; LINE, long interspersed nucleotide element; NWM, New World monkey.
Received for publication November 30, 2006. Accepted for publication May 7, 2007.
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G) calculations by the MFOLD program on the RNA secondary structures obtained did not resulted in any significant difference.