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a IHF Institute for Hormone and Fertility Research, University of Hamburg, 22529 Hamburg, Germany
b Department of Reproductive Endocrinology, Deutsches Primatenzentrum, 37077 Göttingen, Germany
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Therefore, to complement the previous study on relaxin, we report here the structure and tissue-specific expression of the RLF from the marmoset at the mRNA and protein levels. Surprisingly, it was found that in this species the RLF gene is transcribed into two different mRNA forms. Besides the form also described for the cow [6], human [7, 8], pig [9], mouse [10], and sheep [11], an additional variant was characterized that contains an extra exonic sequence. Differential splicing leads to a transcript that, instead of the conventional heterotrimeric B-C-A domain structure, encodes a truncated protein comprising the B-domain followed by a short novel peptide region and then a stop codon. Analysis of genomic DNA showed that in the marmoset, although the splice donor and acceptor sites are similar to those in the other species for which genomic information is available, this intronic region in the marmoset is much larger and includes a novel short exon.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A cDNA library constructed in the lambda vector Uni-Zap-XR (Stratagene, La Jolla, CA) using RNA from adult marmoset testes [12] (generous gift of Dr. Lorraine Kerr, Edinburgh), with a complexity of 1.3 x 106, was screened by conventional hybridization of duplicate nylon membrane plaque lifts, using as probe an internal human RLF cDNA fragment [8]. Fifteen positive clones were obtained from a total of 50 000 clones screened, indicating an mRNA frequency of 0.03%. Two independent bacteriophage clones were purified, and the inserted recombinant DNA was transferred to pBS.SK plasmid vectors by in vivo phagemid excision. The complete cDNA inserts were then subjected to double-stranded DNA sequencing.
RNA Extraction and Hybridization Analysis
Total RNA was extracted as described previously [5] from various marmoset tissues as indicated in the figure legends. For Northern hybridization analysis, 10 µg total RNA per lane was electrophoresed on MOPS (3-[N-morpholino]propanesulfonic acid buffer)/formaldehyde gels [13] and transferred by capillary action overnight to nylon membranes (Nytran; Schleicher&Schüll, Dassel, Germany). After UV cross-linking, these were hybridized using as probes the full-length marmoset RLF cDNA insert, as well as a subcloned DNA fragment corresponding to the novel exon 1A (see later). Whereas the former probe was radiolabeled by random-primed DNA polymerase I (Klenow fragment) in the presence of [
-32P]dCTP (Amersham-Buchler, Braunschweig, Germany) according to the method of Feinberg and Vogelstein [14], the smaller probe was labeled using T7 DNA polymerase and a sequence-specific primer (28R; see Table 1). For this reaction, 100 ng template DNA and 100 ng oligonucleotide 28R in a total of 20 µl TE (10 mM Tris-HCl, pH 7.5, 1 mM EDTA) buffer were denatured at 95°C for 5 min and allowed to anneal by cooling slowly to room temperature. This was then supplemented with 10 µl 5-strength buffer A (200 mM Tris-HCl, pH 7.5, 50 mM MgCl2, 250 mM NaCl, 250 µg/ml BSA), 10 µl dATP, dGTP, dTTP mix (each 100 µM), 2 µl 5 mM dithiothreitol, 5 µl [-32P]dCTP (Amersham-Buchler), and 1 U T7 DNA polymerase (Pharmacia, Freiburg, Germany) and incubated for 5 min at 37°C. Both probes were purified over nick columns (Pharmacia) before use and had a specific activity of > 109 cpm/µg DNA.
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Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Assays to Detect RLF Transcripts and Genomic Fragments
Since the majority of marmoset tissues were not available in sufficient quantities to provide RNA for repeated Northern hybridizations, RLF-specific transcripts were detected using an RT-PCR assay. First-strand cDNA synthesis was performed using oligo(dT) as primer and the components of the SuperScript cDNA synthesis kit (Gibco-BRL, Eggenstein, Germany), with 2 µg total RNA as template per 100-µl reaction. Five microliters of this cDNA was then used to program a PCR reaction, using gene-specific oligonucleotides (1S, 2AS; see Table 1 and Fig. 4, PCR-1) in a conventional protocol with denaturation at 95°C for 5 min followed by 30 cycles of denaturation (95°C, 1 min), annealing (58°C, 1 min), elongation (72°C, 1 min) and with a final elongation step of 10 min at 72°C. The PCR products were resolved on 1.2% agarose gels, transferred to nylon membranes (Nytran; Schleicher&Schüll), and hybridized against a 182-base pair (bp) internal probe, created independently by PCR using internal oligonucleotide primers 6AS and 7S (see Table 1) and otherwise the same protocol as above; they were then radiolabeled by the random-priming procedure described above. As a control for the quality of the cDNA used, RT-PCR reactions were also performed for the presence of transcripts encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as previously described [15].
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As shown below, the RT-PCR reactions indicated the production of two differently sized RLF-specific PCR products. Both DNA fragments were excised from the electrophoresis gels, subcloned into pGEM-T Easy plasmids (Promega, Madison, WI), and sequenced. This resulted in the demonstration of a novel splice product of the RLF gene, including 103 nucleotides from what appeared to be a novel exonic region. In order to confirm this observation and the precise location of the novel sequence, further PCR reactions were performed using genomic DNA as template. Genomic DNA was prepared from a male marmoset liver or from placenta using the High Pure PCR template preparation kit (Boehringer, Mannheim, Germany) according to the manufacturer's protocol. As primers for these PCR reactions, oligonucleotides were used that were derived from both the predicted exons 1 and 2 (20S, 18R; see Table 1 and Fig. 4, PCR-3), as well as from exon 1 and the novel exon 1A (1S, 14R; see Table 1 and Fig. 4, PCR-2). PCR conditions were as above for the shorter PCR reaction (PCR-2), using 0.7 µg genomic DNA as template. For the longer PCR reaction (PCR-3), conditions were altered to denaturing at 95°C for 30 sec, annealing at 55°C for 45 sec, and elongation at 68°C for 12 min, for 30 cycles altogether. The resulting PCR products were subcloned into pGEM-T Easy plasmids (Promega) and subjected to partial double-stranded sequencing to confirm identity, as well as restriction analysis. Finally, to obtain an exon 1A-specific DNA probe for Northern and in situ hybridization, a new PCR reaction was carried out using 20 ng of the longer product of the novel exon 1A-containing splice product from the initial RT-PCR reaction as template and using oligonucleotides 27S and 28R (Table 1) as primers. PCR reactions were carried out for 30 cycles of denaturation at 95°C for 1 min, annealing at 45°C for 45 sec, and elongation at 72°C for 45 sec. The PCR product of 103 bp corresponded only to exon 1A and was unable to hybridize with exons 1 or 2. This 103-bp DNA fragment was subcloned into the plasmid pGEM-T Easy (Promega) for subsequent reactions.
All PCR-derived subclones were completely sequenced on both strands, except for the long genomic product of oligonucleotides 20S and 18R. This approximately 5-kilobase (kb) product was also subcloned into pGEM-T Easy (Promega); it was sequenced unidirectionally not only from the 5' and 3' ends, but also from internal oligonucleotide primers (31S, 29R; see Table 1 and Fig. 4) to provide sequence information across the exon IA and exon 2 splice junctions, respectively.
Southern Analysis of Genomic DNA
In order to confirm the organization and structure of the RLF genomic locus in the marmoset, first the longer genomic clone PCR-3 (see Fig. 4) was subjected to restriction analysis using the enzymes EcoRI, BamHI, HindIII, SacI, NcoI, and PstI. The cleavage pattern confirmed the positions of restriction sites predicted from the partial DNA sequence and indicated additional sites for EcoRI, PstI, and NcoI within the longer second intronic sequence. Secondly, 20 µg genomic DNA per reaction from the liver of a male monkey or from placenta was digested overnight with restriction enzymes as indicated, electrophoresed on a 1% agarose gel, transferred to nylon membrane (Nytran), and hybridized against a 543-bp RLF cDNA fragment as probe. The probe included the full open reading frame of exon 1 and most of exon 2, and was prepared by PCR using primers 20S and 23AS (Table 1) in the presence of digoxigenin-dUTP (Boehringer-Mannheim) by means of a conventional PCR protocol (denaturing at 95°C for 1 min, annealing at 55°C for 1 min, elongation at 72°C for 1 min, for 30 cycles). Alternatively, the genomic PCR product PCR-2 (see Fig. 4) was used, which included exons 1 and 1A as well as the intervening intron 1. This was also labeled by PCR with digoxigenin-dUTP as above, using the original PCR primers. Prehybridization, hybridization, and washing followed precisely the recommendations of the Boehringer-Mannheim protocol, with the final washing steps using only moderate stringency at 60°C. The labeled bands were visualized by chemiluminescence.
Immunological Detection of RLF Peptide
In order to detect RLF at the protein level, immunohistochemistry was performed on 10-µm 4% paraformaldehyde-fixed and paraffin-embedded sections of marmoset testis and ovary, as described previously for relaxin [5] or for human RLF [8]. The primary antibody used, M1, was raised against a conserved peptide epitope within the B-domain of the human RLF molecule and is absolutely conserved between human and marmoset. This antibody has been thoroughly characterized in the human testis [8] and has been shown to provide signal localization identical to that of other antibodies raised against recombinant RLF protein and to in situ transcript hybridization. Alternatively, another antibody, K1, was used that had been raised in rabbits against the same peptide antigen.
As an additional control for the M1 antibody, marmoset RLF protein was expressed in a baculovirus-infected insect cell expression system, and the resulting protein was used in a Western blot procedure. For the baculovirus expression, 100 ng of the full-length normal marmoset RLF cDNA clone was used as template for a PCR reaction with oligonucleotides 5.1 and ex.1 (Table 1) as primers (30 cycles of denaturing at 95°C for 1 min, annealing at 60°C for 1 min, elongation at 72°C for 1 min). This was initially subcloned into the pGEM-T Easy vector (Promega), sequenced, and then recloned into the baculovirus expression vector pVL 1392 (Invitrogen, Leek, The Netherlands). With use of standard procedures [16], this was transfected into Sf9 cells together with linearized AcMNPV viral DNA (Invitrogen), and the resulting recombinant virus was used to infect "high-five" cells (Invitrogen). Optimal expression of the recombinant marmoset RLF as a secreted protein was obtained upon incubating these cells in Grace's insect medium (Invitrogen) containing 10% fetal calf serum. As control for the Western blotting, baculovirus-infected cells were used that expressed recombinant marmoset relaxin [5] from a similar construct (unpublished results).
RT-PCR analysis indicated the presence of alternatively spliced transcripts that predicted a C-terminally extended B-peptide structure. The new reading frame thus created a novel C-terminal peptide epitope: -D-E-S-H-S-A-A-Q-D-G-G-Q. This peptide was chemically synthesized (Severn Biotech, Kidderminster, UK), coupled via an extra N-terminal cysteine to keyhole limpet hemocyanin, and used to immunize rats according to a conventional procedure [17]. The polyclonal antisera obtained from six rats were tested against the immunizing antigen, and the two with the best apparent titer were used for immunohistochemistry.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The cloned full-length structure of the marmoset RLF cDNA, obtained from the testis cDNA library, is shown in Figure 1, together with the amino acid sequence of the deduced open reading frame. This is shown in comparison with the RLF sequences from the human, pig, cow, and mouse. The marmoset RLF is 89% homologous at the amino acid level with the human sequence, and to a lesser extent for the other species. With the human, the homology is almost 100% in the signal peptide and B-domains, the latter including the RALVR motif (Fig. 1, boxed) considered to be involved in receptor interaction. Also, the A-domain is extremely homologous. As might be expected, most variation appears to occur in the C-domain, which is not part of the holo-hormone for relaxin and insulin.
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Northern hybridization analysis of several different marmoset tissues indicated a positively hybridizing band in RNA from testis samples only (Fig. 2), with an apparent size that is in agreement with the size predicted from the cloned cDNA sequence plus 100–200 A residues of poly(A) tail. In order to evaluate RLF expression in other tissues, an RT-PCR assay was constructed with primers designed to span the intron splice junction as predicted from the genomic sequences of other species. The RT-PCR analysis, however, indicated that, in addition to the predicted product at 295 bp, a second, longer transcript at 398 bp was expressed in many tissues (Fig. 3). The ratio of expression of this larger form appeared to parallel the shorter standard form in all tissues where expression was detected. As control for experimental artifact, the cloned testicular full-length RLF cDNA was used as template (lanes 17 and 31) and gave rise only to the expected 295-bp band. As anticipated, maximal transcript expression was in the testis, with signals also in the ovarian corpora lutea and in the epididymis. The uterus, liver, skeletal muscle, and prostate were negative. A very weak signal was evident on longer exposure for the spleen, for the shorter transcript only.
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These RT-PCR products were cloned and sequenced; this indicated that the longer form included a novel sequence of 103 bp interpolated at a position equivalent to the intron splice junction in other species (Figs. 4 and 5). The new transcribed sequence appears to derive from a short additional exon (exon 1A), located within the genomic region of what was previously considered intron 1. This novel exon continues the open reading frame from the B-domain into a short novel peptide sequence, characterized by frequent large polar amino acids, and then a stop codon (Fig. 5). The longer novel transcript therefore encodes a truncated RLF protein, comprising the B-domain and a short, highly charged C-terminal extension.
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However, the novel 103-bp sequence indicated no homology with any of the intron sequences derived from the porcine [18], human [18], mouse [19, 20], or bovine (unpublished results) genes, although the predicted exon 1-exon 2 splice junction for the marmoset RLF mRNA occurred at the identical position within the transcript. In order to confirm that the novel transcript was indeed a differentially spliced product of the marmoset genome, and not some sort of artifact, a PCR analysis of marmoset genomic DNA was undertaken using different combinations of oligonucleotide primers, as indicated in Figure 4, with marmoset genomic DNA from the liver of a male monkey or from placenta as template. No products of the expected size of approximately 1–2 kb, based on the genomic structure in other species, were obtained with any primer combination (several other primers were used besides those indicated in Table 1; all proved negative). Instead, after modification of PCR conditions to encourage longer PCR products, DNA fragments of approximately 5 kb in length were obtained using exon 1- and exon 2-specific primers. These were subcloned and partially sequenced as indicated (Fig. 4). Sequence information from independent PCR reactions confirmed the structure of the RLF gene locus in the marmoset to be as shown in Figure 4, with a long intronic region including the novel 103-bp exon 1A sited 346 bp from the 5' splice junction. This is followed by a second intron (intron 1A) of approximately 4 kb. All putative intron splice acceptor and donor sites conformed to the consensus rules for such sequences, and the full sequence information has been deposited in the international EMBL database (accession numbers AJ011961 and AJ011962).
Since PCR analysis of genomic DNA failed to indicate any products implying a shorter intron sequence, similar to that in other species, it seems likely that the genomic structure obtained (Fig. 4) represents a single-copy gene in the marmoset genome. This was confirmed by Southern hybridization of genomic DNA using a 550-bp cDNA fragment including exons 1 and 2 (Fig. 6, left panel), as well as a genomic fragment derived from exons 1 and 1A, and intron 1 (Fig. 6, right panel), as probes. Restriction digestion of the cloned approximately 5-kb PCR genomic fragment indicated that the long intron additionally included single cleavage sites for EcoRI, NcoI, and PstI, but not for BamHI (not shown), in addition to those restriction sites shown in Figure 4. This explains the presence of two hybridizing bands for genomic DNA cleaved with the former enzymes, but not with the latter, with use of the full-length cDNA probe (Fig. 6, left panel). Using the PCR-2 genomic fragment as probe, which includes only exons 1 and 1A and the intervening intron 1, only single hybridizing bands were obtained. The small 250-bp PstI fragment (Fig. 6, left panel) is that predicted from the cDNA sequence within exon 2. Smaller cleavage products were not retained on the gel. Also the single approximately 4.5-kb BamHI cleavage product (Fig. 6) was as anticipated from the analysis of the cloned DNA fragments.
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It is unlikely that the Northern gel system used can resolve the two products of differential splicing with use of the full-length cDNA as probe (Fig. 2). Since, however, hybridization of similar blots using the 103-bp probe, comprising only exon 1A, failed to reveal any hybridizing bands (not shown), this would suggest that the novel alternative transcript is expressed at a level below the detection limit for Northern hybridization.
Immunohistochemical Detection of RLF Sequences in the Ovary and Testis
The antibody M1 was raised against a peptide epitope of the B-domain that is identical in human and marmoset RLF sequences. This antibody has been thoroughly characterized with respect to the human testis [8] and has been shown to be very specific for RLF expression in Leydig cells. As a further test of specificity in the marmoset system, Western blots were performed using marmoset RLF and relaxin precursor proteins biotechnologically expressed in insect cells infected with recombinant baculovirus (Fig. 7). Only the secreted recombinant RLF protein reacted with the M1 antibody (Fig. 7, lanes 4–6). The recombinant marmoset relaxin made from similar expression constructs failed to cross-react with this antibody (Fig. 7, lane 3), though it did react with specific anti-relaxin antibodies (Fig. 7, lane 1). The immunoreactive RLF protein had an apparent molecular mass of approximately 14 kDa, consistent with the loss of a signal peptide but with no further endoproteolytic processing. Unfortunately, this assay system is not yet sensitive enough to detect RLF in testis extracts (not shown).
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In the testis, immunohistochemical staining was strongest within the Leydig cells (Fig. 8A), as observed for other species. No specific signals were evident in any other testicular compartment (cf. control, Fig. 8B). In the ovary, as well as in the luteal cells of the midphase corpus luteum (Fig. 8E; control, Fig. 8F), clear specific immunohistochemical staining was observed also in the theca layer of antral follicles (Fig. 8C; control, Fig. 8D) and in some stromal cells (Fig. 8G). The latter had the morphological characteristics of luteinized cells. Control sections of testis and ovary, where the primary antibody had been replaced by the preimmune serum from the same animals, were negative (Fig. 8, B, D, and F). Similar results were also obtained using the alternative antibody K1 (not shown).
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Since the M1 antibody is specific for an internal epitope of the B-peptide domain, it will also recognize the novel product of the alternatively spliced RLF gene transcript. Polyclonal antibodies were therefore also raised against the novel C-terminal peptide encoded by the new exon 1A. Although these were shown to react against the immunogen, they failed to show any specific cross-reaction in tissue sections (not shown). This result appears to confirm the negative results of the Northern hybridization indicating that, if translated, the novel splice product is expressed at only a very low level.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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As has previously been shown for other species, the major site of RLF biosynthesis in the marmoset is the Leydig cells of the testis, much as in the human [8]. This has also been confirmed at the immunohistochemical level. In the female reproductive system, RLF mRNA can be detected only with use of a sensitive RT-PCR assay. Thus the marmoset conforms to the pattern in those species, such as the mouse [23] and human [24], in which only low levels of RLF are expressed in the ovary, sufficient to support local paracrine effects only. This situation thus differs from that in ruminants, where in both the sheep [11] and cow [6], very high levels of RLF gene expression can be detected in the theca cells and corpus luteum of the cycle and pregnancy. Using specific antibodies, we can also confirm for the marmoset, that as in other species, RLF is expressed within the theca interna cells of the follicle as well as in the luteal cells of the corpus luteum. Interestingly, RLF epitopes can also be detected in apparently luteinized mesenchymal stromal cells of the cyclic ovary, similar to results also in the mouse [23]. This pattern of expression is also quite similar to what has been shown for the related hormone relaxin in the female marmoset [5]; yet Western blots, using recombinant marmoset relaxin, show that there is absolutely no cross-reactivity of the M1 antibody with relaxin epitopes. Taken together, these results indeed indicate a degree of temporal and spatial overlap between RLF and relaxin in the primate female reproductive system, such that a consideration of both peptides is necessary for a complete understanding of the local physiology. In the adult male marmoset, there appears to be a more clear-cut difference in the expression of the two peptides. Only relaxin mRNA can be detected in the prostate [5], whereas in the testis only RLF is present. There is unfortunately no information on relaxin expression in the male tract, so that a comparison in these tissues is not possible. Nor is there any reliable information on relaxin receptors or functions in male reproductive tissues, so that the physiological significance of these findings remains obscure.
The most interesting finding in the present study is that in the marmoset there has apparently been an alteration in the genome in the RLF gene locus, introducing several kilobases of DNA into what in other species is a relatively short intron. This has led to the evolution of a novel alternatively spliced transcript that includes an extra exon of 103 nucleotides into the open reading frame of the mRNA. The consequence of this insertion is to introduce a stop codon into the open reading frame closely following the end of the B-peptide domain. If translated, this transcript would give rise to a truncated protein comprising a C-terminally extended B-peptide. A very similar alternative splice event has been reported also for relaxin in the human placenta [25]. Here also the product is a C-terminally extended B-peptide. It is not known whether such a peptide is biologically meaningful; but it is conceivable, since the B-peptide of both relaxin and RLF includes a receptor-binding motif, that such a C-terminally extended B-peptide might be able to interact with the relaxin or RLF receptor, or possibly with some other hormone-binding moiety. This could thus represent an interesting additional means of modulating the physiology of these peptide hormones.
Since the only evidence for the transcription of the novel splice variant is at the RT-PCR level, it could be argued that this might represent a transcription product generated in the opposite direction from the anti-sense strand, as has been shown for the basic fibroblast growth factor gene [26]. This could explain why the immunohistochemical study using the variant-specific antibody was negative. However, the splice junctions are identical to those of the normal functional transcript. Since splicing is an asymmetrical process, involving non-palindromic donor and acceptor sequences, this proves that the variant transcript must be a product of the sense strand, presumably generated from the same promoter.
Both the mouse and human RLF genes have been shown to be unusual inasmuch as they are located within the 3' region of another quite different gene: that for the signal transduction protein JAK3 [19, 27]. The exon 2 of the RLF gene is incorporated into the 3' untranslated region of the JAK3 transcripts. The inclusion in this locus in the marmoset of a much larger intronic domain could therefore also have consequences for the splicing and expression of the JAK3 gene in this species. It has not been possible to assess the JAK3 gene locus in the present investigation, but subsequent studies should therefore also look for a possibly altered pattern of expression of JAK3 in the marmoset compared to other species.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Richard Ivell, IHF Institute for Hormone and Fertility Research, University of Hamburg, Grandweg 64, 22529 Hamburg, Germany. FAX: 49 40 56190864; ivell{at}rrz.uni-hamburg.de
Accepted: September 22, 1998.
Received: July 21, 1998.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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