Isolation, Expression, and Chromosomal Localization of the Human Mitochondrial Capsule Selenoprotein Gene (MCSP)

GENOMICS

32, 184–190 (1996) 0104

ARTICLE NO.

Isolation, Expression, and Chromosomal Localization of the Human Mitochondrial Capsule Selenoprotein Gene (MCSP) HANNE AHO,* MICHAEL SCHWEMMER,* DIRK TESSMANN,* DEREK MURPHY,* GENEVIEVE MATTEI,† WOLFGANG ENGEL,* AND IBRAHIM M. ADHAM*,1 *Institut fu¨r Humangenetik der Universita¨t, Gosslerstrasse 12D, 37073 Go¨ttingen, Germany; and †INSERM U. 242, CHU Timone, F-13385 Marseille, Cedex 5, France Received August 15, 1995; accepted November 17, 1995

The mitochondrial capsule selenoprotein (MCS) (HGMW-approved symbol MCSP) is one of three proteins that are important for the maintenance and stabilization of the crescent structure of the sperm mitochondria. We describe here the isolation of a cDNA, the exon–intron organization, the expression, and the chromosomal localization of the human MCS gene. Nucleotide sequence analysis of the human and mouse MCS cDNAs reveals that the 5*- and 3*-untranslated sequences are more conserved (71%) than the coding sequences (59%). The open reading frame encodes a 116-amino-acid protein and lacks the UGA codons, which have been reported to encode the selenocysteines in the N-terminal of the deduced mouse protein. The deduced human protein shows a low degree of amino acid sequence identity to the mouse protein (39%). The most striking homology lies in the dicysteine motifs. Northern and Southern zooblot analyses reveal that the MCS gene in human, baboon, and bovine is more conserved than its counterparts in mouse and rat. The single intron in the human MCS gene is approximately 6 kb and interrupts the 5*-untranslated region at a position equivalent to that in the mouse and rat genes. Northern blot and in situ hybridization experiments demonstrate that the expression of the human MCS gene is restricted to haploid spermatids. The human gene was assigned to q21 of chromosome 1. q 1996 Academic Press, Inc.

INTRODUCTION

The mitochondria in the sperm differ from those in somatic cells in their morphology and arrangement (Hrudka, 1977; De Martino et al., 1979; Otani et al., 1988). They are flattened, elongated, and arranged cirThe nucleotide sequence data reported in this paper have been deposited with the GenBank/EMBL Data Libraries under Accession No. X89960 for the human MSCP cDNA and X89961 for the human MCSP gene. 1 To whom correspondence should be addressed. Telephone: 0049551/397590. Fax: 0049-551/399303.

0888-7543/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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cumferentially into a tight helical coil around the taildense fibers of the mature sperm. Maintenance and stabilization of this crescent-shaped structure is thought to be due to proteins that are found in the capsule associated with the outer membranes of the sperm mitochondria. Pallini et al. (1979) reported that the bull sperm mitochondria capsule consists essentially of three polypeptide chains with molecular weights of 31, 29, and 20 kDa. The 20-kDa protein is rich in cysteine (17.9%) and proline (26.5%) and contains selenium. Selenium was also shown to play a role in the organization of the mitochondria around the sperm tail-dense fibers in dietary experiments. Rats on a selenium-deficient diet show reduced sperm motility and disorganization of the sperm mitochondria (Wu et al., 1979; Calvin et al., 1981; Wallace et al., 1983). Furthermore, studies using radioactively labeled selenium show that selenium is incorporated into a 17-kDa protein, localized in sperm mitochondria (Calvin et al., 1981, 1987). This protein was shown to be identical to the previously described 20-kDa cysteine-rich protein of the bull sperm mitochondria and was designated mitochondria capsule protein or mitochondria capsule selenoprotein (MCS) (for review see Kleene, 1994). Kleene et al. (1990) isolated and sequenced a truncated cDNA clone for the mouse MCS.2 The nucleotide sequence analysis of the mouse MCS cDNA revealed that the reading frame encodes a 143-amino-acid protein and lacks an in-frame UGA codon, which codes for selenocysteine in several selenoproteins. They have further characterized several cDNA clones and the gene of the mouse MCS and reported that the mouse MCS encodes a 197amino-acid protein with a molecular weight of 21 kDa and that its reading frame contains three UGA codons in the 5* end (Karimpour et al., 1992). The mouse MCS gene is present as a single copy per haploid genome and is composed of two exons. Northern blot analyses using RNA extracted from purified spermatogenic cells 2 The HGMW-approved symbol for the gene described in this paper is MCSP.

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and from testes of staged prepubertal mice suggested that the MCS gene is first expressed in late meiotic cells and that the level of the mRNA increases in early haploid cells (Kleene et al., 1990), while in situ hybridization shows that the mouse mRNA is first detectable in early haploid cells and persists until the end of the haploid phase (Shih and Kleene, 1992). We have cloned the human MCS cDNA and characterized the exon–intron structure of the gene. The human gene is expressed in postmeiotic stages of spermatogenic cells. In situ hybridization to metaphase chromosomes reveals that the gene maps to the q21 region of chromosome 1. MATERIALS AND METHODS Isolation of human MCS cDNA and genomic clones. A total of 6 1 105 recombinants from a human testis cDNA library (Clontech) were screened by the plaque hybridization method (Benton and Davis, 1977) with a radiolabeled rat MCS cDNA fragment (unpublished result). Hybridization was carried out at 587C overnight in the following solution: 61 SSC/51 Denhardt’s solution/0.1% SDS and 100 mg/ml denatured salmon sperm DNA. Washing was performed at 587C with 21 SSC/0.1% SDS for 2 h. One positive clone (HS10) was plaque-purified, and its cDNA fragment was subcloned into the EcoRI site of the pGEM 3Zf (/) vector (Promega). A genomic cosmid library h2a (Poustka et al., 1984) was screened with a 32P-labeled human MCS cDNA fragment using the method described by Troen (1987). DNA from cosmid clones that hybridized to the cDNA probes were digested with various restriction endonucleases and electrophoresed on agarose gels. Appropriate genomic fragments containing exonic sequences were subcloned into pGEM plasmid. Amplification of the 3* end of the MCS cDNA. Total RNA (200 ng) from human testis was reverse transcribed with Superscript reverse

FIG. 1. The nucleotide sequence and the deduced amino acid sequence of the human MCS cDNA. Numbering of nucleotide and amino acid sequences is given to the right, the dicysteine motifs are double underlined, and the exon–intron boundary in the human MCS is indicated by an arrow. The stop codon is marked with asterisks, and the polyadenylation signals are underlined.

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FIG. 2. Comparison of deduced amino acid sequences of human and mouse MCS (Kleene et al., 1990). The sequences were aligned by the DNA STAR computer program to optimize amino acid sequence identity. Matching amino acid residues are printed between the two sequences. Gaps indicated as dashes are introduced to maximize homology of pairing amino acids. transcriptase (BRL-GIBCO) at 427C for 30 min using primer (dT)17 . The cDNA was used as template for amplification with primer (dT)17 and a cDNA-specific primer located at positions 562 to 582 of the cDNA (Fig. 1). An amplified fragment was purified from preparative acrylamide gel and directly sequenced (Innis et al., 1988) using a cDNA-specific primer. Long-range PCR amplification. One microgram of human genomic DNA prepared from peripheral blood lymphocytes (Miller et al., 1988) was used as template to amplify the intron and exon–intron boundaries of the human MCS gene. The PCR was performed with primers, located at positions 41 to 61 and 517 to 537 of the cDNA sequence (Fig. 1), and the GeneAmp XL PCR kit (Perkin–Elmer Cetus Instruments) according to the manufacturer’s recommendations. Thermal cycling was carried out first for 16 cycles, with denaturation at 947C for 1 min and annealing/extension at 607C for 12 min, followed by 21 cycles, with denaturation at 947C for 1 min and annealing/extension at 607C for 15 s. The amplified DNA fragment was purified from a 0.6% agarose gel, subcloned into pGEM-T vector (Promega), and sequenced with vector primers. DNA sequence analysis. Both strands of DNA were sequenced with fluorescently labeled primers on an ABI 373 sequencer (Applied Biosystems) and/or manually with Sequenase Version 2.0 (USB). The sequence data were analyzed with the DNA STAR computer program. Primer extension. Primer extension was performed as described previously (Domenjoud et al., 1990). Total RNA was isolated from human testis using the acid guanidinium thiocyanate–phenol–chloroform extraction method (Chomczynski and Sacchi, 1987). A synthetic oligonucleotide complementary to positions 55 to 75 (Fig. 1) was 5*-endlabeled with [t-32P]-ATP and T4 polynucleotide kinase. Fifty micrograms of total RNA was mixed with 5 ng of the labeled oligonucleotide in 60 ml hybridization buffer (0.3 M NaCl, 10 mM Tris, pH 7.5, 1 mM EDTA). After denaturation, the mixture was allowed to cool slowly to 427C, followed by ethanol precipitation. Primer extension was carried out at 427C for 30 min using Superscript reverse transcriptase (BRLGIBCO). The extended product was extracted with phenol–chloroform and ethanol precipitation and analyzed on electrophoresis sequencing gel consisting of 6% polyacrylamide/6 M urea. Northern analysis. Total RNA from different human tissues (testis, muscle, brain, kidney, and liver) and from testes of different species (human, baboon, rat, mouse, and bull) was size-fractionated

FIG. 3. Nucleotide sequence of the determined exon–intron boundaries of the human MCS gene. Nucleotide sequences of exons are shown in uppercase letters, and intronic sequences are in lowercase letters.

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FIG. 4. Determination of the transcription start site of the human MCS gene. A synthetic oligonucleotide was end-labeled, hybridized to testicular RNA, and extended with reverse transcriptase (lane 5). The reaction was electrophoresed and sized against sequencing reactions (lanes 1–4). The size of the primer extension product is indicated. by electrophoresis on 1% agarose gels containing formaldehyde (Lehrach et al., 1977) and transferred to nylon membranes (Amersham). The Northern blots containing RNA from human tissues and from the testes of the various species were hybridized with 32P-labeled human MCS cDNA at 65 and 607C for 16 h in hybridization solution and washed to final stringency at 657C for 15 min in 0.21 SSC/0.1% SDS and at 607C for 1 h in 11 SSC/0.1% SDS, respectively. Southern analysis. Human genomic DNA was digested with various restriction endonucleases, fractionated in 1% agarose gel, and transferred onto nylon filters. The blot was hybridized with radiolabeled human MCS cDNA and washed to final stringency at 657C for 15 min in 0.21 SSC/1.0% SDS. In situ hybridization. Samples from human testes were fixed in 4% paraformaldehyde at 47C for 16 h and embedded in paraffin. Four-micrometer sections were cut, dewaxed in xylene, and then processed essentially as described by Dressler and Gruss (1989). [35S]UTP-labeled riboprobes were synthesized using T7 or SP6 RNA polymerase (Promega) from linearized pGEM-plasmid templates containing the human MCS cDNA. Hybridization was carried out for 16 h at 557C and washed to final stringency at 607C in 21 SSC/ 50% formamide. Tissue sections were exposed to X-ray film (Amersham) for 20 h and to NTB2 emulsion (Kodak) for 2 to 5 days. Chromosomal localization. For chromosomal mapping of the human MCS gene, in situ hybridization with the human MCS cDNA was performed using metaphase spreads from cultured lymphocytes as previously described (Mattei et al., 1985). The specific activity of the probe was 1.5 1 108 dpm/mg, its final concentration was 100 ng/ ml, and the slides were exposed for 15 days at 47C. R-banding was performed by the fluorochrome–photolysis–Giemsa method.

of 0.75 kb was identified, subcloned into the pGEM 3Zf(/) vector, and sequenced. The nucleotide sequence showed that the cDNA did not terminate with a poly(A) tract, nor did it contain a polyadenylation signal. To determine the missing 3* sequences, a rapid amplification of cDNA ends by the polymerase chain reaction was performed. The PCR product was directly sequenced. The complete nucleotide sequence of the human MCS is 747 bp long and contains three ATGs at positions 5, 57, and 126. The third ATG occurred inframe with the termination codon TGA at position 474, predicting a translation product of 116 amino acids. In position 03 of the third ATG is a purine, which agrees with the consensus sequence for the translation initiation site of most eukaryotic genes (Kozak, 1989). The 5*-untranslated sequence is 125 nucleotides long, and the 3*-untranslated sequence, which possesses two polyadenylation signals AATAAA, is 274 bp long. The nucleotide sequences of the human and mouse MCS cDNAs exhibit a sequence identity of 69%. The identity between the two sequences is higher in the 5*- and 3*untranslated regions (71%) than in the coding sequences (59%). The deduced amino acid sequence of the human MCS can be conveniently divided into two domains. The amino domain, consisting of amino acids 1 to 74, is rich in cysteine (26%). This domain contains nine repeats, consisting of 7 or 8 amino acids, with the consensus sequence K.X.N.Q.C.C.X.P (X is any amino acid). Eighteen of the 20 cysteine residues occur as dicysteine motifs in these repeats. Comparison of the human with the mouse MCS showed only a moderate degree of amino acid sequence identity (39%). The most striking homology is found in the dicysteine motifs (Fig. 2). The human protein is shorter than the mouse protein, which is due to a deletion of several repeats in the N-terminus of the human protein. Characterization of the Human MCS Gene We isolated a cosmid clone MS1 by screening a human cosmid library with the 32P-labeled cDNA. South-

RESULTS

Isolation and Characterization of Human MCS cDNA The human testis cDNA library was screened at low stringency using a 0.85-kb rat MCS cDNA probe (unpublished result). A single clone HS10 with an insert

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FIG. 5. RNA blot hybridization analysis of human MCS mRNA. (A) Total RNA (15 mg/lane) from testis (1), muscle (2), brain (3), kidney (4), and liver (5) was hybridized with 32P-labeled human MCS cDNA. (B) The blot was subsequently washed and rehybridized with human elongation factor-2 cDNA (Hanes et al., 1992) to ensure that all samples contained intact RNA.

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FIG. 6. Localization of the MCS transcript in the human testis. Paraffin sections were hybridized in situ with 35S-labeled human MCS sense RNA (A) or antisense RNA (B and C) transcript. (B) Dark-field photomicrograph of tubules showing specific hybridization to the central tubular region. (C) Bright-field photomicrograph of the tubule section demonstrates the concentration of silver grains in postmeiotic stages of the spermatogenic cell.

ern blots and nucleotide sequencing of the exonic fragments revealed that the cosmid clone MS1 contains a part of intron 1, exon 2, and 3*-flanking sequences. To determine the nucleotide sequence of the exon–intron boundaries, we performed long-range PCR amplification with human genomic DNA. An amplified fragment of 6.5 kb was obtained and subcloned, and exon–intron boundaries were sequenced (Fig. 3). The predicted 5* splice donor and 3* splice acceptor sites are in good agreement with the consensus sequence for these sites (Shapiro and Senapathy, 1987). A comparison of available intronic sequences of the mouse and human MCS genes revealed that 60 nucleotides located 3* to exon 1 and 40 nucleotides 5* to exon 2 exhibit 73 and 67% similarity, respectively. The nucleotide sequence of the exons, exon–intron boundaries, and 3*-flanking region of the human MCS are reported in the EMBL Data Library and have been assigned the Accession No. X89961. To determine the length of exon 1 and thereby the length of the 5*-untranslated region, primer extension was performed on testicular RNA with a 21-mer oligonucleotide complementary to nucleotide sequences at positions 55 to 75 (Fig. 1), and a single major band of 119 nucleotides was obtained (Fig. 4). On the basis of the nucleotide sequence and primer extension analyses, we conclude that the 5*-untranslated region is 169 bp and that the human MCS gene consists of two exons and one intron, like the mouse MCS gene. Exon 1 is 149 bp and contains only the 5*-untranslated sequence. Exon 2 is 642 bp and includes the coding sequence and the 3*-untranslated region. The single intron in the human MCS gene is approximately 6 kb and interrupts the 5*-untranslated region at a position equivalent to that in the mouse gene.

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Expression and Conservation of the MCS Gene Total RNA was isolated from human testis, muscle, brain, kidney, and liver and subjected to Northern blot analysis (Fig. 5). A 1-kb transcript for the MCS gene was found only in the testis. To identify the cellular localization of the MCS transcript, in situ hybridization was carried out on testis sections with an antisense MCS RNA probe. As shown in Fig. 6, a strong accumulation of silver grains was restricted to the cell layers close to the lumen. This area corresponds to the histological localization of the postmeiotic cells (round and elongated spermatids). No signal above background was present among the diploid spermatogenic cells and the Sertoli cells. These results indicate that the human MCS mRNA is expressed mainly in the postmeiotic stages. To assess whether the MCS gene is conserved in mammalian species and to demonstrate whether the gene is expressed in testes of different mammalian species, the human MCS cDNA was used as a probe in low-stringency Northern blot hybridization experiments. Strong hybridization signals were obtained with testicular RNA from baboon and bull after 16 h (Fig. 7), and much weaker signals were obtained after prolonged exposure (14 days) with RNA from rat and mouse (data not shown). At the genomic DNA level, no hybridization signal could be obtained with the DNA of these latter species (data not shown). Copy Number of the MCS Gene in the Human Genome A Southern blot of human genomic DNA digested with different restriction endonucleases was probed with the

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FIG. 6—Continued

human cDNA. As shown in Fig. 8, one band was detected in EcoRI and HindIII, while one strong and one weak band were detected in PstI, PvuII, and BamHI digests. Restriction maps of a 6.5-kb PCR-amplified fragment containing the intronic sequence of the human MCS gene reveal that the intronic sequence contains one, two, and three restriction sites for PvuII, PstI, and BamHI, respectively. These results demonstrate that the haploid human genome contains a single copy of the MCS gene. Chromosomal Localization of the Human MCS Gene For chromosomal assignment of the MCS gene, in situ hybridization was performed on human metaphase chromosomes with the MCS cDNA. In the 100 metaphases examined, 192 silver grains were scored, and

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40 of these (20.8%) were located on chromosome 1. The distribution of grains on this chromosome was not random; 67.5% mapped to the q21 band of the long arm of chromosome 1 (Fig. 9). DISCUSSION

This report describes the molecular cloning of the human homolog of the mouse MCS gene by screening a human testes cDNA library with a rat MCS cDNA fragment under low-stringency conditions. The nucleotide sequence analysis of the human and mouse cDNAs reveals that the 5*- and 3*-untranslated sequences are more conserved (71%) than the coding sequences (59%). The first 28 bp and the last 75 bp of the human cDNA sequence show remarkable conservation with the corre-

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FIG. 7. Expression and conservation of the MCS gene in different mammalian species. (A) Northern blot containing RNA from testes of human (1), baboon (2), rat (3), and bovine (4) that was hybridized with 32P-labeled human MCS cDNA. (B) The blot was subsequently washed and rehybridized with the human elongation factor-2 cDNA.

sponding sequences of the mouse cDNA (100 and 88%, respectively). The extremely high conservation of these sequences in the 5*- and 3*-untranslated regions between human and mouse may be an indication of the importance of these sequences in the control of translation initiation and regulation of mRNA stability. Moreover, there is an ATTTA element in the conserved sequence of the 3*-untranslated region that has been recognized as a motif controlling mRNA degradation of some genes (Cleveland and Yen, 1989). The amino acid sequence of the mouse MCS was first deduced from a truncated cDNA sequence (Kleene et al., 1990). This sequence consists of 143 amino acids and shares 39% sequence identity with the corresponding human MCS. The homology is mainly concentrated in the dicysteine motifs. Further full-length mouse cDNA clones have been sequenced that have been reported to encode a 197-amino-acid protein and to contain three UGA codons in the 5* end, which presumably encode selenocysteines in the mouse MCS (Karimpour et al., 1992). Two UGAs are present at the equivalent

FIG. 8. Southern blot analysis of the human MCS gene. The human genomic DNA was digested with PvuII (1), PstI (2), HindIII (3), EcoRI (4), and BamHI (5) and probed with the human MCS cDNA. The length of the DNA fragments are shown to the left.

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FIG. 9. Chromosomal localization of the human MCS gene. Distribution of grains on chromosome 1 after hybridization with the MCS cDNA. Region 1q21 has the largest number of grains.

position in the human MCS cDNA but are located in a reading frame different than that which encodes the human MCS protein. Similar results have been found by analysis of the rat MCS cDNA (unpublished results). Northern blot and in situ hybridization results reveal that the human MCS gene is expressed in haploid spermatid cells. The mouse MCS mRNA is expressed in early spermatids and remains translationally inactive until the end of the spermatid stage (Shih and Kleene, 1992). Further experiments have demonstrated that the mouse MCS mRNA is translationally repressed with a long poly(A) tail in round spermatids and then becomes translationally active through the shortening of the poly(A) tail in elongating spermatids (Kleene, 1989; Kleene et al., 1990). Southern blot hybridization indicates that the haploid human genome contains a unique MCS gene and, as shown by in situ hybridization, maps to region q21 of chromosome 1. This region contains the genes for H1, H2, and H4 histones, family 2; glucosidase beta (GBAP); apolipoprotein A-II (AOA2); nemaline myopathy 1 disease (NEM1); pyruvate kinase, liver (PKL); and the interleukin 6 receptor (IL6R) (Bruns and Dracopoli, 1993; Dracopoli et al., 1994). The rat MCS gene was assigned to rat chromosome 2 (unpublished result). Since the genes for IL6R and PKL were mapped to rat chromosome 2 and mouse chromosome 3 (Yamada et al., 1994), it can be assumed that the mouse MCS gene maps to chromosome 3. ACKNOWLEDGMENT We thank Angelika Winkler for secretarial help. This work was supported by the Bundesministerium for Forschung and Technologie (01KY9104).

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