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a Department of Biology, Tufts University, Medford, Massachusetts 02155
b Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, Michigan 48202
c The Jackson Laboratory, Bar Harbor, Maine 04609
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ABSTRACT |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although little is known about the mechanisms inactivating and activating "paternal" mRNAs in germ cells, a number of RNA-binding proteins (RBPs) likely to be involved in mRNA translational control have been identified in the mammalian testis. Testis/brain (TB)-RBP, the mouse homologue of the human single-stranded DNA-binding protein translin, binds to the 3' untranslated regions (UTRs) of many testicular mRNAs, serving as a repressor of translation in in vitro assays [5, 6]. Other proteins that bind to the 3' UTRs of testicular mRNAs include two proteins of 53 kDa and 55 kDa that bind to specific 22- and 20-nucleotide (nt) sequences in the 3' UTRs of protamine mRNAs [7], another protein with properties of a translational inhibitor of the protamines [8], and SPNR, a 71-kDa protein that localizes to cytoplasmic microtubules of the testis [9].
Multiple poly(A) binding proteins are also expressed in the testis [10], one of which, a 70-kDa protein, binds to both translated and stored mRNAs in male germ cells [11]. Another germ cell-specific RNA-binding protein, SOD-RBP, binds to the 5' UTR of a testis-specific SOD-1 mRNA and can represses SOD-1 mRNA translation in vitro [12]. Mammalian homologues of the Xenopus germ cell-specific Y-box binding proteins are also abundant in rodent testes [13, 14]. Thus, a growing number of testicular RNA-binding proteins have been identified and are likely to facilitate the repression and activation of mRNA translation in male germ cells.
The vertebrate Y box proteins represent a family of nucleic acid-binding proteins containing an ~100 amino acid domain termed the cold shock domain (CSD), which contains sequences that are over 40% identical to the small bacterial cold shock proteins [15, 16] and binds single-stranded nucleic acids by their ribonucleoprotein (RNP)-1 and RNP-2-like motifs [17, 18]. The bacterial cold shock proteins act as transcription factors [19], a property conserved in the Y box proteins that bind the specialized CCAAT box elements (the Y box CTGATTGGC/TC/TAA) to either promote or repress transcription of a variety of genes [20–23]. The bacterial cold shock proteins also act as RNA chaperones, destabilizing RNA secondary structure [24]. In addition to the CSD, all vertebrate Y box proteins contain in their C-terminal region four basic/aromatic (B/A) islands that bind RNA [25, 26]. In Xenopus oocytes, the Y box protein FRGY2 (mRNP3+4) is abundant and found in cytoplasmic translationally repressed maternal mRNPs [27]. Immunologically related proteins have been identified in both the nuclei and cytoplasm of mammalian testes [13, 22, 23, 28, 29].
Many Y-box proteins have been cloned, including mouse MSY1, a protein sharing 85% identity with Xenopus FRGY1 [14]. MSY1 is most highly expressed in the testis but is restricted to neither the testis nor the germ cell [14]. No homologue of Xenopus FRGY2 had previously been isolated, leading to speculation that a direct mammalian homologue of FRGY2 may not exist. Here, we report the isolation of cDNAs encoding a germ cell-specific mouse testicular homologue of Xenopus FRGY2, which we name MSY2. We demonstrate that MSY2, like its Xenopus counterpart, is expressed in both male and female germ cells and is maternally inherited in the early embryo. However, unlike its Xenopus counterpart, MSY2 undergoes bulk degradation in the two-cell-stage embryo that is coincident with the loss of maternal mRNAs. In the testis, Msy2 is alternatively spliced, giving rise to polypeptides that differ in the amino-terminal region upstream of the CSD. In addition to stimulating the transcription of testis-specific genes [30], the abundant cytoplasmic localization and temporal expression pattern of MSY2 suggests that it functions in the storage and translation of mRNAs in both male and female mammalian germ cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A lambda gt 11 CD-1 mouse testis cDNA expression library (Clontech, Palo Alto, CA) was screened using antibodies to Xenopus mRNP3+4 ([22, 27, 31]; mRNP4 is identical to FRGY2) at a 1:5000 dilution. Complementary DNA clones encoding MSY2 were detected with horseradish peroxidase-conjugated secondary antibodies with diaminobenzidine (DAB)-H2O2 as substrate. To obtain the complete open reading frame, a mouse testis lambda Zap II cDNA library (kindly provided by Dr. E.M. Eddy, NIEHS, Research Triangle Park, NC) and a mouse genomic DNA library (Stratagene, La Jolla, CA) were screened with the Msy2 cDNAs. The nucleotide sequences of the Msy2 cDNAs and genomic DNA were analyzed using the GCG-Unix program (Genetics Computer Group, Madison, WI) and the Basic Local Alignment Search Tool (BLAST) from the National Center for Biological Information.
The 5' RACE (rapid amplification of cDNA ends) system (Life Technologies Inc., Gaithersburg, MD) was employed according to the manufacturer's specifications. Oligonucleotide primers located either in the 3' UTR (nt 1222–1204 and nt 1157–1139 of Msy2) or the CSD (nt 521–503 and nt 319–297) were used for reverse transcription and amplification using ELONGase (Life Technologies Inc.), and the products were cloned into the pCR 2.1 TA vector (Invitrogen Corp., Carlsbad, CA). The longest clones were selected by colony polymerase chain reaction (PCR) and found to contain two alternative 5' ends. Clones containing each of these 5' ends were isolated from 5' RACE performed with both primer sets. In addition, multiple clones derived from unprocessed pre-mRNAs were isolated, thereby providing definitive assignment of the alternate splice site.
Southern Blot Analysis
Aliquots of high-molecular weight genomic DNA (10 µg), prepared from spleens of CD-1 mice, were digested with the restriction enzymes BamHI, EcoRI, HindIII, or SacI. After electrophoresis on 0.7% agarose gels, the digested DNA fragments were transferred to nylon membranes (NEN; Dupont, Boston, MA) and hybridized to a 32P-labeled mouse Msy2 cDNA (nt 253–1533).
Isolation of Meiotic and Postmeiotic Germ Cells
Germ cell suspensions were prepared from 10 adult CD-1 mice by enzymatic dissociation of 10 pairs of testes with collagenase (1 mg/ml) at 34°C for 12 min, followed by digestion with trypsin (0.5 mg/ml) for 10 min at 34°C. The cell suspensions were fractionated by unit gravity sedimentation on a BSA gradient as previously described [11]. The purity of the enriched populations of pachytene spermatocytes, round spermatids, and elongated spermatids, determined by Nomarski microscopy, was between 85% and 90%.
RNA Isolation and Northern Blotting
Total RNA was isolated from prepubertal and adult testes of CD-1 mice, from enriched populations of meiotic and postmeiotic germ cells, and from brain, spleen, liver, heart, and kidney as previously described [32]. Northern blot hybridization was performed as described by Gu et al. [11] with a 1533-nt Msy2 cDNA.
Preparation of Tissue Extracts and Western Blot Analysis
Protein extracts from the testes, liver, brain, and kidneys of CD-1 mice were prepared as previously described [33]. For immunoblots, aliquots of protein extracts were resolved in 12% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes, after preincubation in 20 mM Tris-buffered saline (pH 7.5) containing 1% BSA and 0.05% Tween-20 (TTBS), were incubated with polyclonal antibodies against Xenopus mRNP3+4 in TTBS at a dilution of 1:20 000. MSY2 was detected using an enhanced chemiluminescence (ECL) Western blotting detection system (Amersham, Chicago, IL) according to the manufacturer's instructions.
Immunocytochemistry
The ovaries of 22-day-old C57BL/6J mice were fixed in Bouin's fixative. In addition, the ovaries of fetal mice were fixed on Days 16, 17, and 18 postcoitum (pc). Sections were prepared for standard double-antibody immunocytochemistry with peroxidase-conjugated antibody as the second antibody. The primary antibody against Xenopus mRNP3+4 was diluted 1:20 000 before application.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Initial attempts to isolate a murine homologue of FRGY2 by nucleic acid screening with FRGY2 failed; thus antibodies against Xenopus mRNP3+4 (FRGY2a+b) were used to isolate cDNAs from a mouse testis lambda gt11 expression library. The initial screening yielded a 900-base pair cDNA that was used to rescreen a second mouse testis cDNA library and a mouse genomic library. Overlapping cDNA clones extended the 5' sequence of the initial clone. Analysis of the genomic clone and 5' RACE products allowed final determination of the complete open reading frame of MSY2. The final sequence was derived from multiple PCR products compared to the genomic sequence and is shown in Figure 1A.
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5' RACE yielded clones of MSY2 and an alternatively spliced form, MSY2a, that differs from MSY2 in its amino terminus upstream of the CSD (Fig. 1B). MSY2 is derived from an open reading frame of 1080 nt that encodes a polypeptide of 360 amino acids (Fig. 1A), whereas the alternatively spliced form encodes a polypeptide of 282 amino acids. The predicted molecular weights of these polypeptides are 38 and 31 kDa, respectively. Immunoblot analysis of mouse testis extracts using antibodies against Xenopus mRNP3+4 (FRGY2), or a peptide of B/A island II of mRNP3 (FRGY2b), revealed two forms of the mouse germ cell Y box protein at 52 and 48 kDa, corresponding to MSY2 and its alternatively spliced form MSY2a, respectively [13, 29]. All known Y box proteins demonstrate significantly retarded mobility in SDS-PAGE, and in the case of FRGY2 this results from a highly extended conformation [31].
The predicted MSY2 protein was rich in arginine (11.9%) and proline residues (16%), indicating that an unmodified protein would be highly basic with an isoelectric point of 11.56. Computer analysis of the predicted MSY2 protein revealed seven putative phosphorylation sites for casein kinase II and one site for protein kinase C (Fig. 1A). Several of these are conserved from FRGY2, which is highly phosphorylated [22]. MSY2 also contained two putative nuclear localization signals imbedded in the second B/A island (Fig. 1A). These may facilitate shuttling of the protein between the nucleus and cytoplasm.
Sequence analysis of MSY2 confirmed it to be a member of the Y box family of proteins that include Xenopus FRGY1 and FRGY2, mouse MSY1, several human somatic homologues [16], and the recently reported goldfish Y box proteins, GFYP1 and GFYP2 [34]. Like all other Y box proteins, MSY2 contained the highly conserved N-terminal CSD (from amino acids 89–166; Fig. 1) with motifs related to RNP-1 and RNP-2 that bind single-stranded nucleic acids [25]. In addition, overall MSY2 shared 61% amino acid identity and 56% nucleotide identity with FRGY2. Therefore, the overall similarity of MSY2 to FRGY2 is relatively low as compared to the 85% amino acid identity shared by MSY1 and FRGY1. However, alignment of MSY2 and FRGY2 revealed, in addition to the CSD, a high degree of conservation of the C-terminal B/A islands that have been proven to bind RNA [22, 25, 26]. The conservation of the B/A islands was further emphasized by alignment of MSY2, FRGY2, GFY2, MSY1, FRGY1, and GFY1, revealing the B/A islands (Fig. 2, underlined) to be uniquely conserved for the two different Y box proteins.
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MSY2 Was Encoded by a Single Copy Gene
To determine the size of the Msy2 gene family, mouse genomic DNA was digested with a single restriction enzyme (BamHI, EcoRI, HindIII, or SacI), and the DNA fragments were analyzed by Southern blotting (Fig. 3). Two or three DNA fragments with sizes ranging from 2 to 10 kilobases (kb) were detected. Mapping of the Msy2 genomic clone with BamHI yielded similar DNA fragments, suggesting that MSY2 is transcribed from a single copy gene (data not shown).
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Expression of Msy2 mRNA Was Developmentally Regulated during Spermatogenesis
To determine the temporal expression of Msy2 mRNA in mouse testes, Northern blot hybridizations were performed with RNA samples from the testes of prepubertal and adult mice, and from enriched germ cell populations. Msy2 mRNA was barely detected in testes from 6-day-old mice (Fig. 4A, lane 1). A low level of Msy2 mRNA is seen in testes from 12-day-old mice, the age at which meiosis begins (Fig. 4A, lane 2). In 17-day-old mice, a developmental stage in which the testis is enriched with meiotic pachytene spermatocytes, the expression of Msy2 mRNA increased significantly (Fig. 4A, lane 3). Maximal levels of Msy2 mRNA were detected in testes from 22-day-old mice and persisted throughout adulthood (Fig. 4A, lanes 4–6). A similar pattern of mRNA expression has been observed for Msy1 [14]. Analysis of the expression of Msy2 mRNAs in enriched populations of meiotic and postmeiotic germ cells showed that pachytene spermatocytes contained lower levels of Msy2 mRNA (Fig. 4B, lane 1) than did postmeiotic germ cells (Fig. 4B, lanes 2 and 3) or control adult testes (Fig. 4B, lane 4). The expression pattern of MSY2 mRNA in individual germ cell types (Fig. 4B) is consistent with that seen in extracts of total testes (Fig. 4A). The inability to detect Msy2 mRNA or protein [13, 29] (Fig. 5) in the somatic tissues suggests that the expression of MSY2 is testis-specific in the male germ cell development.
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In Male Mice, MSY2 Was Abundant in Testes
To determine the extent of MSY2 expression in adult mice, total RNA from somatic tissues and testis was analyzed by Northern blotting with a Msy2 cDNA. An abundant and broad Msy2 mRNA band of about 1.8 kb was detected in the testis (Fig. 5), but not in the brain, spleen, liver, heart, or kidney (Fig. 5). Similar results were observed when the hybridization was performed under reduced stringency or after overexposure of the gel. Although multiple Y box proteins were expressed in the tissues tested, the Msy2 cDNA did not cross-hybridize to other Y box mRNAs such as Msy1. The inability to detect Msy2 mRNA in the somatic tissues suggests that the expression of Msy2 mRNA is testis-specific.
Western blotting of extracts from brain, liver, and kidney were performed to further evaluate whether MSY2 protein could be detected in somatic tissues. Antibodies against Xenopus mRNP3+4 detected MSY2 in protein extracts of testes (Fig. 6, lanes 1–2), but not in extracts from brain (Fig. 6, lanes 3–4), kidney (Fig. 6, lanes 5–6), or liver (Fig. 6, lanes 7–8). The more slowly migrating Xenopus mRNP3+4 is also shown (Fig. 6, lane c). The Western blots were in agreement with the Northern blots and further indicated that MSY2 is a germ cell-specific protein.
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In Mouse Ovaries, MSY2 Was Exclusively Expressed in Oocytes
The localization of MSY2 in mouse ovaries and early embryos was determined by immunocytochemistry. In the 22-day-old mouse ovary, all oocytes, regardless of stage of development, were strongly positive for MSY2 immunostaining. Figure 7 shows positively staining oocytes in graafian follicles, preantral follicles, and primordial follicles. No cells other than oocytes were immunopositive. Diplotene oocytes in the ovaries of 18-day pc fetuses were immunopositive, but pachytene oocytes in 17-day pc fetuses were not (Fig. 8). Metaphase II oocytes were immunopositive just before ovulation as were single cell embryos (not shown), but MSY2 levels decreased greatly in 2-cell-stage embryos in the oviduct (Fig. 7).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Highly specific antibodies to FRGY2 identify mouse testis polypeptides of 52 and 48 kDa in SDS-PAGE corresponding to MSY2 and its alternatively spliced form MSY2a [13, 29]; however, their cDNA-predicted molecular masses are 38 and 31 kDa, respectively. FRGY2 exhibits a similar disparity in migration relative to its true molecular mass because of its highly extended conformation [31]. Subcellular fractionation has demonstrated MSY2a to be primarily restricted to the cytoplasm, while MSY2 is found in both nuclear and cytoplasmic compartments [23], suggesting that the alternate N-terminal end may modify MSY2 localization and possibly its function.
Comparison of cDNA-derived sequences of Y box proteins from mice, frogs, and goldfish demonstrates the relationship of the germ cell-restricted Y box proteins MSY2, FRGY2, and GFY2 and their divergence from the non-germ cell-restricted forms (Fig. 2). Among vertebrate Y box proteins, the CSD is more than 90% conserved from Xenopus FRGY2 to human YB-1 across its ~100 amino acids [22]. However, divergence of the germ cell-restricted Y box proteins is apparent in MSY2, FRY2, and GFY2 at two positions that are invariant in all other known vertebrate Y box proteins (Fig. 2, Q95 and F136 of MSY2 found as K56 and Y97 in MSY2). The coincident substitution of these otherwise invariant CSD amino acids supports a close relationship between MSY2 and FRGY2. Stronger support for MSY2 as the homologue of FRGY2 is derived from the conservation of the B/A islands (Fig. 2, underlined). The position and sequence of the four B/A islands is conserved between MSY2, FRGY2, and GFY2 and divergent from those in MSY1, FRGY1, and GFY1. In particular, B/A islands II and IV of FRGY2 are each > 90% identical in MSY2 and FRGY2. Since these FRGY2 B/A islands bind [26], it is highly probably that these motifs also participate in MSY2 RNA binding.
Sequence analysis of MSY2 protein predicts seven putative sites for phosphorylation by casein kinase II and one site for protein kinase C, raising the likelihood that the protein could be phosphorylated in vivo, as is FRGY2 [27]. MSY2 also contains several predicted nuclear localization signals. This is not surprising since the transcription factor MSY2 is located both in the nucleus and the cytoplasm [23], functioning as an mRNA-binding storage protein in the cytoplasm and in the nucleus.
MSY2 and FRGY2 also share a pattern of germ cell-restricted expression in contrast to the widespread expression of MSY1 and FRGY1 in both somatic and germ cells [14, 40]. Northern blot analysis using Msy2 cDNA detected a broad band of mRNAs of about 1.8 kb in the testis, but not in somatic RNAs (Fig. 5). Similarly, FRGY2 [40] was detected solely in male and female germ cells. Other vertebrate Y box genes such as FRGY1 and MSY1 are widely expressed in both germ cells and somatic tissues although not translated in all cases [30]. Detection of MSY2 exclusively in the germ cells of rodent testes and ovaries, but not in somatic tissues, and the absence of Msy2 mRNA in testes from 6-day-old mice, in which most of the cell types are somatic, suggests that MSY2 expression is germ cell-specific.
The expression of MSY2 is developmentally regulated during spermatogenesis. Kwon et al. [13] demonstrated that the mammalian protein immunologically related to FRGY2 (i.e., MSY2) is present in meiotic pachytene spermatocytes and accumulates markedly in postmeiotic spermatids, the cell types in which most of the "paternal" mRNA is stored for translation in later stages. Immunocytochemical studies by Oko et al. [29] demonstrated that in mouse and rat testes, MSY2 (previously called p48/52) is a predominant germ cell cytoplasm protein with maximal amounts in postmeiotic round spermatids, further establishing its role as an mRNA-binding storage protein. Our Northern blot hybridization confirms these observations, since Msy2 mRNA is detected in testes from 12-day-old mice and reaches a maximum in testes from 22-day-old mice (Fig. 4). Although Msy2 mRNA is detected in testes from 12-day-old mice, immunocytochemical and Western blot analyses first detect MSY2 protein in meiotic pachytene spermatocytes [29]. This may result from the limited sensitivity of the protein detection methods or may be indicative of translational regulation. Both possibilities are likely since MSY2 can be detected in the nuclei of germ cells by gel shift assays but not by immunocytochemical methods; and preliminary data indicate that most of the total Msy2 mRNA in mouse testes is nonpolysomal (unpublished results).
The abundance and temporal appearance of MSY2 in oocytes suggest a role in the storage of maternal mRNAs. MSY2 is developmentally regulated in mouse oocytes. MSY2 appears as oocytes enter the prolonged diplotene (dictyate) stage and persists until after fertilization in the single-stage embryo. MSY2 is not detected by our immunocytochemical methods in the two-cell embryo even though the antibody recognizes modified embryonic forms of FRGY2 [27]. This suggests that significant turnover of MSY2 occurs in the two-cell embryo. The timing of this event is coincident with the massive degradation of maternal mRNAs [41, 42], suggesting that degradation of MSY2 may trigger decay of maternal mRNAs. Western blot analyses indicate that testicular and oocyte MSY2 proteins have identical electrophoretic mobilities (data not shown). Any role MSY2 may play in transcription during oogenesis or after fertilization awaits analysis.
On the basis of the high levels of MSY2 in mouse male and female germ cells and its interactions with both DNA and RNA, we propose that MSY2 first functions in the earlier stages of germ cell development as a DNA-binding protein directing germ cell-specific gene transcription [23, 30], and it subsequently serves as a RNA-binding protein in the cytoplasm [13, 29], where it stores mRNAs and regulates their translation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Norman B. Hecht, Center for Research on Reproduction and Women's Health and Department of Obstetrics and Gynecology, University of Pennsylvania Medical Center, 752 Clinical Research Building/6142, Philadelphia, PA 19104–6142, FAX: 215 573 5408; or Mary T. Murray, Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, MI 48202.
3 Present address: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461
Accepted: July 10, 1998.
Received: May 28, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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J. E. Knight, E. N. Narus, S. L. Martin, A. Jacobson, B. M. Barnes, and B. B. Boyer mRNA Stability and Polysome Loss in Hibernating Arctic Ground Squirrels (Spermophilus parryii) Mol. Cell. Biol., September 1, 2000; 20(17): 6374 - 6379. [Abstract] [Full Text]
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M.-A. Mastrangelo and K. C. Kleene Developmental expression of Y-box protein 1 mRNA and alternatively spliced Y-box protein 3 mRNAs in spermatogenic cells in mice Mol. Hum. Reprod., September 1, 2000; 6(9): 779 - 788. [Abstract] [Full Text] [PDF]
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S. Soyal, A Amleh, and J Dean FIGalpha, a germ cell-specific transcription factor required for ovarian follicle formation Development, January 11, 2000; 127(21): 4645 - 4654. [Abstract] [PDF]
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 P. Ruzanov, V. Evdokimova, N. Korneeva, J. Hershey, and L. Ovchinnikov Interaction of the universal mRNA-binding protein, p50, with actin: a possible link between mRNA and microfilaments J. Cell Sci., January 10, 1999; 112(20): 3487 - 3496. [Abstract] [PDF]
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