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Biology of Reproduction 66, 475-485 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Identification of Target Messenger RNA Substrates for the Murine Deleted in Azoospermia-Like RNA-Binding Protein1

Xinfu Jiaoa, Panayiota Trifillis3,a, and Megerditch Kiledjian2,a

a Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, New Jersey 08854-8082


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The murine autosomal deleted in azoospermia-like protein (mDAZL) is a germ cell-restricted RNA-binding protein essential for sperm production. Homozygous disruption of the mDAZL gene results in the absence of germ cells beyond the spermatogonial stage. Progress into the function of DAZL in spermatogenesis has been hampered without identification of the cognate mRNA substrates that it binds to and regulates. Using the isolation of specific nucleic acids associated with proteins (SNAAP) technique recently developed in our lab, we identified mRNAs from testis that were specifically bound by mDAZL. One mRNA encoded the Tpx-1 protein, a testicular cell adhesion protein essential for the progression of spermatogenesis. A 26-nucleotide region necessary and sufficient to bind mDAZL was found within additional mRNAs isolated by the screen. These included mRNA encoding Pam, a protein associated with myc; GRSF1, an mRNA-binding protein involved in translation activation, and TRF2, a TATA box-binding protein-like protein involved in transcriptional regulation. Each mRNA containing the mDAZL binding site was specifically bound by mDAZL. A similar sequence is also present in the Cdc25A mRNA, a threonine/tyrosine phosphatase involved in cell cycle progression. The mDAZL and Cdc25A homologues are functionally linked in Drosophila and are necessary for spermatogenesis. Our demonstration that Tpx-1 and Cdc25A mRNAs are bound by mDAZL suggests that mDAZL regulates a subset of mRNAs necessary for germ cell development and cell cycle progression. Understanding how mDAZL regulates the target mRNAs will provide new insights into spermatogenesis, strategies for therapeutic intervention in azoospermic patients, and novel approaches for male contraception.

developmental biology, gene regulation, spermatogenesis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Approximately 2% of males worldwide are affected by infertility resulting from the absence of sperm production (azoospermia). Cytological analysis of patients with azoospermia revealed a deletion on the Y chromosome in a significant proportion of these patients, implying a genetic component to the phenotype in some individuals [1]. This region was proposed to contain an azoospermia factor (AZF). Subsequent analysis of this region revealed 3 distinct genes as AZF candidates [2], 1 of which was termed deleted in azoospermia (DAZ) [3]. Approximately 10% of azoospermic males contain a deletion on the Y chromosome in a region encoding DAZ. The DAZ gene is Y chromosome linked and is present only in Old World monkeys and hominoids. In addition, these species and all other mammals contain an ancestral autosomal DAZ-like gene, DAZL, which is approximately 80% identical to DAZ [4–7]. More recently, a predecessor to the DAZL gene was also identified [8], indicating that there are multiple members in the DAZ family of proteins. All members of the DAZ protein family are expressed exclusively in germ cells [3, 8–10]. The significance of DAZL in spermatogenesis was demonstrated by a homozygous disruption of the murine DAZL (mDAZL) gene in mice, which resulted in the absence of germ cells in both sexes [10]. Gametogenesis progressed only up to the mitotic spermatogonial stage of development, suggesting a requirement of mDAZL for the development and survival of germ cells [10]. Similarly, the Drosophila homologue of mDAZL, termed Boule, is essential for spermatogenesis; primary spermatocytes fail to progress through the G2/M transition in mutant flies [11]. Expression of the human DAZ transgene in mDAZL knockout mice confers partial rescue of spermatogenesis, demonstrating an overlap in the function of both proteins [12].

Germ cells undergo spermatogenesis in association with the somatic Sertoli cells. Sertoli cells serve as support units for the developing germ cells and provide nutrition and fate-determining signals [13]. Much of this influence is thought to be mediated through direct Sertoli-germ cell contact, which is required for spermatogenesis [14]. Several molecules, including ß1,4-galactosyltransferase [15], N-cadherin [16], and testis-specific protein 1 (Tpx-1) [17], have been identified as candidate adhesion proteins. Antibodies to all 3 proteins inhibit Sertoli-germ cell binding to various degrees in vitro. Chromosomal breakpoints within the Tpx-1 gene have also been identified within infertile men [18], further implicating a functional role for this protein in spermatogenesis. Tpx-1 is a 243-amino acid testis-specific protein expressed exclusively in germ cells and is a member of the cysteine-rich secretory protein family [19]. The secreted protein subsequently associates with the spermatogenic cell surface [20]. Tpx-1 gene expression appears to begin at the pachytene stage and persists throughout spermatogenesis to the elongating spermatid stage [20–22]. Expression of the Tpx-1 protein is delayed relative to the mRNA and is more intense in later stages of spermatogenesis, suggesting its expression is translationally regulated [20].

A critical level of gene expression is governed by posttranscriptional regulation. Messenger RNA processing occurs in association with RNA-binding proteins within ribonucleoprotein complexes [23]. The processing events include capping at the 5' end, removal of intervening sequences by splicing, polyadenylation at the 3' end, transport of the mRNA out of the nucleus, and ultimately translation into protein [24–27]. Inherent throughout this process is the turnover of the mRNA. All mRNAs have a distinct half-life that is dictated by both cis elements and trans factors [28]. The DAZ family of proteins contains a ribonucleoprotein (RNP)-motif type of RNA-binding domain (RBD) at their amino terminus, and they bind RNA [29–31]. Their localization to the cytoplasm and association with polysomes suggests a functional role in mRNA stability or translational regulation [10, 29]. This suggestion is further supported by the functional link between Boule and Twine in Drosophila. Twine is a Cdc25 phosphatase necessary for the G2/M transition in spermatocytes whose translation is dependent on the presence of the Drosophila DAZL homologue, Boule [32].

To begin addressing the components involved in spermatogenic defects arising in the absence of the DAZ family of proteins, we set out to identify the substrate target mRNAs bound by mDAZL. We recently described the isolation of specific nucleic acids associated with proteins (SNAAP) approach, which employed copurification of unknown mRNAs bound by an RNA-binding protein and subsequent identification of the mRNA by reverse transcription (RT) and polymerase chain reaction (PCR) using differential display technology [33]. We now report the identification of a testis-specific substrate mRNA bound by mDAZL and the delineation of the mDAZL-binding sequence. The presence of this sequence in other mouse mRNAs enabled identification of a subset of mRNAs that are bound by and potentially regulated by the DAZ family of proteins, including Cdc25A phosphatase. Identification of DAZL-bound substrate mRNAs broadens and illustrates the potential spectrum of processes in gametogenesis regulated by this family of proteins and provides valuable insight into the function of this RNA-binding protein family in the pathogenesis of azoospermia.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of Testis Total Extract

Testes were extracted from 6-wk-old Balb/C mice, washed twice in PBS and placed into lysis buffer (20 mM Hepes, pH 7.6, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol [DTT], 2 mg/ml leupeptin, 0.5% aprotinin). The tissue was diced with a razor blade followed by sonication. Insoluble matter was removed with a 5-min spin at 15 000 x g at 4°C, and supernatant was collected, supplemented with 5% glycerol, and frozen in aliquots at -70°C.

Generation of Glutathione S-Transferase Fusion Proteins

The pGEX-mDAZL plasmid encoding the glutathione S-transferase (GST)-mDAZL fusion protein was constructed by placing the mDAZL coding region into the pGEX-6P-1 vector (Pharmacia, Piscataway, NJ). The coding region was reverse transcribed and amplified from mouse testis RNA with primers that placed BamHI and EcoRI restriction sites on the 5' and 3' ends, respectively, and was inserted into the same sites within the vector. The GST-{alpha}CP1 and GST-RBD expression plasmids have previously been described [33, 34]. Fusion proteins were expressed in Escherichia coli BL21 cells, and extract was prepared and treated with micrococcal nuclease to degrade bacterial RNA as previously described [33].

SNAAP Screen

The SNAAP screen was carried out as described by Trifillis et al. [33]. Micrococcal nuclease-treated GST-fusion protein (50 µg) was bound to 40 µl of glutathione beads at 4°C for 15 min in a 1-ml total volume containing RNA-binding buffer (RBB; 10 mM Tris-HCl, pH 7.5, 1.5 mM MgCl2, 250 mM KCl, 0.5 mM DTT, 2 mg/ml leupeptin, and 0.5% [v/v] aprotinin) with 0.5% Triton X-100 (RBB/0.5% TX). Unbound protein was removed with four 1-ml washes with RBB/0.5% TX and two 1-ml washes with RBB. The washed beads containing the fusion protein were resuspended in 350 µl of RBB and incubated with 300 µg of testis total extract precleared with 20 µl of glutathione Sepharose beads. Binding was carried out at 4°C for 1 h followed by a rinse with RBB/0.25% TX and a 10-min wash in RBB/0.25% TX containing 1 mg/ml heparin. The beads were subsequently rinsed 4 times in RBB/0.25% TX and bound RNA was isolated by boiling for 3 min in 200 µl Tris-EDTA/1% SDS as previously described [33]. Copurifying RNAs were identified by the differential display technique (GenHunter, Nashville, TN) as previously described [33] using the 3 anchored 3' primers H-T11A, H-T11C, and H-T11G and 12 distinct 5' primers. The 5' primers used were H-AP1 through H-AP8 (GenHunter), DAP28 (GTTTTCGCAG), DAP29 (GATCCAGTAC), DAP30 (GATCACGTAC), and DAP31 (GATCTGACAC). The bands representing RNAs specifically bound by mDAZL were cloned into the pGEM-T vector (Promega, Madison, WI) and sequenced.

Electrophoretic Mobility Shift Assays

Electrophoretic mobility shift assays were carried out with approximately 0.5 ng of in vitro transcribed [32P]UTP uniformly labeled RNA (~10 000 cpm) per reaction. Binding reactions were carried out in RBB with the indicated amount of GST-fusion protein in a 20-µl total volume. Following a 20-min binding reaction at room temperature, 1.5 units of RNase T1 (Roche, Indianapolis, IN) was added to the reaction and incubated for 10 min at room temperature. Addition of RNase T1 degrades regions of the RNA not complexed with protein and enables a clearer identification of RNA-protein complexes. Heparin is subsequently added to a final concentration of 2 mg/ml for 10 min at room temperature to minimized nonspecific RNA-protein interactions. Competitor RNA or oligonucleotide was added at the beginning of the reaction where indicated. The complexes were resolved on a 5% polyacrylamide gel (60:1 acrylamide:bis) in 0.5x Tris-borate-EDTA buffer at 8 V/cm.

RT-PCR and Riboprobe Generation

Mouse Tpx-1 full-length cDNA and the 3' untranslated region (UTR) were amplified from mouse testis RNA RT product using the Advantage cDNA Polymerase Mix (Clontech, Palo Alto, CA) and cloned into the pGEM-T vector (Promega) to generate pGEM-Tpx cDNA and pGEM-Tpx 3' UTR, respectively. The Tpx-1 full-length cDNA was amplified with primers 5'-CCTCCGTGAGCAACGATAACC-3' and 5'-GGTAGCTTGAGTTCTTTATTGAAAG-3'. The Tpx-1 3‘ UTR was amplified with 5'-TAACATGCCCAGTGTGCAGC-3' and 5'-ATTGAAAGAAGTGATTATCTGTG-3'. RNA transcripts corresponding to the 5' half of the Tpx-1 cDNA were generated by T7 RNA polymerase from the pGEM-Tpx cDNA plasmid linearized with ApaLI (fragment A). Fragments within the 3' UTR were generated by SP6 RNA polymerase from the pGEM-Tpx 3' UTR plasmid linearized with NcoI (fragment B), AvaII (fragment C), and AseI (fragment D). Fragment E was transcribed by T7 RNA polymerase from a PCR-generated template (5'-TAATACGACTCACTATAGGGTAATATCTTTCAGAAC-3' and 5'-CAATTCCAAAGTTGTTATAC-3'). Uniformly 32P-labeled riboprobes were generated and gel purified on an 8% denaturing polyacrylamide gel as previously described [35].

Gene-specific PCR amplifications in Figure 1B were carried out for Tpx-1 with 5'-TAACATGCCCAGTGTGCAGC-3' and 5'-ATTGAAAGAAGTGATTATCTGG-3' and for Dmc1 with 5'-TAGGTGGGGAATTGGTACAG-3' and 5'-ATGCTGGATGAGAAGTCTGG-3'. PCR amplifications in Figure 5 were carried out with the following primer sets: for Pam (protein associated with myc), 5'-ACACGCAGATCCTTTGTCTA-3' and 5'-GTTTTATGACATGTACATTC C-3'; for clone D2, 5'-AATCGCCCAAATGGTGACAC-3' and 5'-AGCACCTCCTCGTTAAATAC-3'; for GRSF1 (G-rich specificity factor 1), 5'-TACAGCTTTATCCACGATC-3' and 5'-AGAAACTTGGCCTCCCTAAG-3'; and for Trf2 (TATA box-binding protein related factor 2; also referred to as TLP or TRP), 5'-ACTCAAACTTGGTTGGTTAG-3' and 5'-AACTTTTATTTGTGCACATTCAA-3'. Amplification of the Cdc25A RT product was carried out with 5'-GAAGTTCCGCACCAAGAGC-3' and 5'-GTTAAGGTCATCCACGAGG-3'. The Cdc25C RT product was amplified with 5'-ATGAAGCATCTGGGCAGTCC-3' and 5'-GAGAACGGCACATTCGAGGG-3'.



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  		FIG. 1. Isolation of Tpx-1 mRNA as a substrate for mDAZL. A) Mouse testis mRNA bound by GST-mDAZL (lane 2), GST-{alpha}CP1 (lane 3), or GST-RBD (lane 4) were isolated, reverse transcribed with H-T11G, amplified by PCR with the same 3' primer and a combination of four 5' primers (H-AP5 to H-AP8), and resolved by denaturing 5% PAGE. Bands that were reproducibly bound by mDAZL and not the control proteins in 3 independent experiments are denoted by an arrow. The indicated bands were extracted, cloned, and sequenced. Band A corresponds to the 277-nucleotide 3' termini of the Tpx-1 mRNA, band B is a 231-nucleotide fragment of the Tpx-1 mRNA 3' terminus, band C is a 169-nucleotide segment of a novel gene, and band D is a 104-nucleotide fragment of Cappß1. DNA size markers are shown on the left in nucleotides. B) Mouse testis mRNA bound to GST-mDAZL, GST-{alpha}CP1, and GST-RBD were reverse transcribed with an oligo(dT) primer and amplified by PCR using primers specific for mouse Tpx-1 (lanes 2–5) or mouse Dmc1 as a negative control (lanes 7 and 8). RT-PCR amplification products using mouse testis total RNA are shown in lanes 5 and 8. Migration of the Tpx-1 and Dmc1 products are indicated by the arrows, and the DNA size markers are shown on the left in base pairs



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  		FIG. 5. Alignment of the Tpx-1, Pam, Trf2, GRSF1, Cappß1, Pa7/C8, H47, and clone D2 DBS sequences; a consensus sequence is shown at the bottom. A preference for each position was designated when a particular nucleotide was represented 4 or more times or when 2 nucleotides each were represented at least 3 times. N, any nucleotide

Riboprobes used in Figure 5B were transcribed with T7 RNA polymerase from PCR-generated templates using primers that introduce a T7 promoter at the 5' end. The primer sets are as follows: Pam, 5'-TAATACGACTCACTATAGGGGTAATAAGTATCACACTGTATA-3' and 5'-CTTAACCTAGAAGTTTAAATGA-3'; clone D2, 5'-TAATACGACTCACTATAGGGGTGGCGTGACAAGGACAG-3' and 5'-CTAAATATTTCCATAAGCCTC-3'; GRSF1, 5'-TAATACGACTCACTATAGGGATCACACCAAAGCAACAG A-3' and 5'-CTATCCCTAAGTAATCAAATAC-3'; and Trf2, 5'-TAATACGACTCACTATAGGGTAGTCTTTTACATTTTTGTATGC-3' and 5'-GATCTTTTCCAAACTTTTATTTG-3'.

Oligonucleotides used in the binding studies were end labeled with {gamma}-32P-dATP using T4 polynucleotide kinase (New England Biolabs, Beverly, MA), and unincorporated nucleotides were removed with a G-25 spin column (Pharmacia).

Binding of in vitro transcribed RNA or oligonucleotides to the GST fusion protein were carried out as described above for the SNAAP screen except the nucleic acid was used directly without testis extract.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Tpx-1 mRNA Is Specifically Bound by mDAZL

The mDAZL protein is necessary for spermatogenesis and is presumed to regulate a subset of mRNAs essential for this process. We utilized the SNAAP technique [33] to identify mRNAs from mouse testis extract that were specifically bound by an mDAZL protein fused to the GST domain (GST-mDAZL). The immobilized GST-mDAZL fusion protein was incubated with testis total extract, and mRNP complexes bound by GST-mDAZL were extracted. Testis total extract, which contains both protein and RNA, was used rather than just purified mRNA to mimic the natural repertoire of RNA-binding proteins and mRNA. Use of total extract provides the natural complement of competitor proteins, increases the specificity of the protein-cognate mRNA binding, and enables the identification of true substrate mRNA [33]. RNA bound by GST-mDAZL and RNA bound to an unrelated control protein were each individually isolated and subjected to RT-PCR using differential display analysis [36] (see Materials and Methods). Messenger RNAs specifically bound by GST-mDAZL were determined by a direct comparison of the amplified band profiles derived from GST-mDAZL-bound mRNA with those obtained from mRNA bound to unrelated control RNA-binding proteins.

The PCR profile derived using the H-T11G 3' primer and a set of four 5' primers (H-AP5 to H-AP8) is shown in Figure 1A. A comparison of the bands obtained from RNA bound to the individual proteins permits a determination of which bands correspond to RNA specifically bound by mDAZL (lane 2) but not 2 other RNA-binding proteins, {alpha}CP1 (lane 3) and the hetergeneous nuclear RNP U RBD (lane 4). As expected, several of the bands were equally associated with 2 or all 3 RNA-binding proteins, indicating that they were nonspecifically bound and were not further pursued. Four bands appeared to be bound specifically by mDAZL (arrows). Isolation and sequencing of these bands revealed that the top 2 bands (A and B) both corresponded to distinct fragments within the Tpx-1 3' UTR. Band C corresponded to a novel sequence, and band D corresponded to an mRNA encoding the actin capping protein ß1 subunit, Cappß1 [37]. Because a combination of 5' primers was used, the distinct Tpx-1 bands, A and B, resulted from amplification by 2 different 5' primers (H-AP8 and H-AP6, respectively). Tpx-1 was also identified a third time with another set of primers (data not shown). Tpx-1 is an abundant mRNA; however, despite its abundance significantly lower levels were reproducibly associated with the control proteins (bands A and B in lanes 3 and 4). In total, 22 bands were identified with this assay system as substrates that were reproducibly bound by mDAZL but not by the control protein. Seven of the isolated mRNAs corresponded to previously identified genes, and 15 did not correspond to known genes in the GenBank database. In addition to Tpx-1 and Cappß1, the remaining known mRNAs were Pam [38]; GRSF1 [39]; Trf2 [40–43]; minor histocompatability antigen precursor H47 [44]; and proteosome {alpha}7/C8 subunit (P{alpha}7/C8; GenBank accession no. AF055983).

Tpx-1 is a testis-specific protein [19] involved in testicular cell adhesion, and at least 1 of its functions is to mediate the binding of spermatogenic cells to Sertoli cells during spermatogenic differentiation in vitro [20]. The testis specificity, its repeated reproducible detection as substrate of mDAZL by SNAAP, and its function made Tpx-1 an interesting candidate gene to analyze. The specific binding of mDAZL to the Tpx-1 mRNA was further confirmed (Fig. 1B). Testis extract was incubated with GST-mDAZL, and bound mRNA was eluted, reverse transcribed, and amplified by PCR using Tpx-1 cDNA primers. Tpx-1 was specifically bound by mDAZL (lane 2) but not by the 2 control RNA-binding proteins (lanes 3 and 4). To ensure that mDAZL is not a promiscuous RNA-binding protein that associates with all mRNA, the PCR was also carried out with primers to the abundant germ cell-restricted Dmc1 mRNA [45]. Despite the presence of this mRNA in testis total extract (lane 8), it was not bound by mDAZL (lane 7). We concluded that mDAZL can specifically bind the Tpx-1 mRNA.

Identification of the mDAZL Binding Site

The approach employed above enables identification of mRNAs that are specifically bound by mDAZL but does not provide information with regard to the binding site. The Tpx-1 mRNA is 1481 bases long. The mDAZL binding site was determined using a similar GST-mDAZL copurification strategy as described above except RNAs consisting of distinct segments of the Tpx-1 mRNA were generated in vitro and used. RNAs corresponding to the 5' UTR and most of the coding region or the 3' UTR of the Tpx-1 cDNA were transcribed in the presence of 32P-UTP. The labeled RNAs were subsequently incubated with GST-mDAZL or the control GST-RBD, and copurifying RNAs were isolated and resolved on a polyacrylamide gel. As shown in Figure 2A, the mDAZL-binding site is not contained within the 5' portion of the mRNA; insignificant binding was detected to this RNA (lane 2). In contrast, efficient binding of mDAZL to the 3' UTR of Tpx-1 was detected (lane 5). The binding specificity is further underscored by the failure of the GST-RBD control protein to bind this RNA (lane 6). These data demonstrate that the binding site for mDAZL is contained within the Tpx-1 3' UTR. This binding is direct and additional proteins are not required for mDAZL to bind the Tpx-1 mRNA since recombinant protein in the absence of testis extract was used.



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  		FIG. 2. Murine DAZL binds the Tpx-1 mRNA 3' UTR. A) GST pull-down assays were carried out with 4 µg of the fusion protein and 32P-labeled Tpx-1 RNA segments. RNA bound to GST-mDAZL (mDAZL) or GST-RBD (RBD) were eluted and resolved by denaturing PAGE. A fraction of each RNA was included in the input lanes. The identity of each fragment is indicated (C). B) GST pull-down assays were carried out as described above using the full length Tpx-1 3' UTR (lanes 1–3), the 3' UTR lacking the 61-nucleotide fragment E (lanes 4–6), or a control RNA derived from the firefly luciferase coding region (lanes 7–9). C) The RNAs used in A and B are schematically shown with the 5' UTR, coding, and 3' UTR regions indicated. The RNA fragments are labeled with the corresponding letter and size in nucleotides. The binding results are summarized on the right: +, binding; -, lack of binding

The specific sequence bound by mDAZL was further identified using smaller segments of the Tpx-1 3' UTR. A 200-nucleotide region (fragment C) was specifically bound by mDAZL (Fig. 2A, lanes 8 and 9), but fragment D, which is shorter by 61 nucleotides on the 3' end, was not (Fig. 2A, lane 11), indicating that this 61-nucleotide region is necessary for binding. Binding analysis carried out with the 61-nucleotide fragment E showed that this region alone could be bound by mDAZL (Fig. 2A, lane 14). Therefore, fragment E is necessary and sufficient for mDAZL binding. Fragment E also contains the primary mDAZL binding site within the Tpx-1 mRNA; a 3' UTR containing a deletion of this 61-nucleotide region was not bound by mDAZL (Fig. 2B, fragment F, lane 5) nor was an unrelated RNA (Fig. 2B, lanes 8 and 9).

Specific binding of mDAZL to the 61-nucleotide fragment E was also detected using electrophoretic mobility shift analysis and is not restricted to the protein copurification assay. In vitro transcribed fragment E was incubated with an increasing amount of mDAZL protein followed by treatment with RNase T1. The RNase step removes regions of the RNA that are not bound by protein and retains a minimal RNA-protein ribonucleoprotein complex. As shown in Figure 3A, 2 complexes were detected with purified recombinant GST-mDAZL protein (lanes 2–4) that are lacking with the 2 control recombinant RNA-binding proteins (lanes 5–10). The lower band was nonspecific and unrelated to the GST-mDAZL protein; it was also present with the control proteins. Although the exact nature of the 2 upper bands has not been addressed, they most likely came from a monomer and homodimer of GST-mDAZL binding to the RNA, because mDAZL has been shown to homodimerize [30]. The binding of GST-mDAZL is specific to the 61-nucleotide fragment E; it is competed by self RNA (Fig. 3B, lanes 2–5) but not by an unrelated RNA (Fig. 3B, lanes 6–8). These data demonstrate that with at least 2 distinct binding assay systems, mDAZL can specifically bind the 61-nucleotide fragment E contained within the Tpx-1 3' UTR.



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  		FIG. 3. Specificity of mDAZL binding to the 61-nucleotide fragment E of the Tpx-1 3' UTR. A) An electrophoretic mobility shift assay was carried out using 32P-UTP uniformly labeled fragment E RNA with an increasing amount of protein ranging from 1 to 4 µg of GST-mDAZL (lanes 2–4), GST-RBD (lanes 5–7), or GST-{alpha}CP1 (lanes 8–10). Following RNase treatment, the RNA-protein complex was resolved on a 5% polyacrylamide gel and visualized by autoradiography. The mDAZL complexes are indicated on the left. The input RNA is shown in lane 1. B) An electrophoretic mobility shift assay was carried out as described above with 2 µg of GST-mDAZL protein bound to 32P-UTP uniformly labeled fragment E in the absence (lane 2) or presence (lanes 3–8) of competitor RNA. Lane 1 contains the input RNA only, lanes 3–5 were competed with unlabeled self RNA, and lanes 6–8 contained unlabeled RNA generated from the polylinker region of pcDNA3 (control). Lanes 3 and 6 contain 1.25 pmol, lanes 4 and 7 contain 2.5 pmol, and lanes 5 and 8 contain 3.6 pmol of competitor RNA

The binding site within fragment E was further narrowed using oligonucleotides. In many cases, RNA-binding proteins can bind single-stranded DNA in addition to RNA [23, 33, 46]; therefore, delineation of the mDAZL binding site within fragment E was carried out using deoxyoligonucleotides spanning both halves of this region. Each oligonucleotide was 32P-labeled at the 5' end and tested for its ability to be bound by GST-mDAZL. Testing the 5' and 3' fragments of this region individually demonstrated that the 3' half (fragment E2) was specifically bound by mDAZL whereas the 5' half (fragment E1) was not (Fig. 4A; compare lanes 2 and 5). Therefore, the mDAZL-binding region is contained within the 31-nucleotide 3' segment.



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  		FIG. 4. Identification of the DBS. A) GST pull-down assays were carried out as described for Figure 2 except 32P-labeled deoxyoligonucleotides were used as binding substrates. The sequence of fragment E (Fig. 2) is shown as are the 2 oligonucleotides. A summary of the binding results is shown on the right. The 31-nucleotide 3' fragment region (fragment E2) contains the binding region. B) An electrophoretic mobility shift assay was carried out with uniformly labeled fragment E as described for Figure 3 with 2 µg of GST-mDAZL protein and competed with various unlabeled deoxyoligonucleotides containing substitution mutations within fragment E2. Lanes 3, 5, 7, 9, 11, and 13 contain 4.5 pmol, and lanes 4, 6, 8, 10, 12, and 14 contain 9 pmol of competitor RNA. The E2 fragment and the various substitution mutations within this fragment are schematically shown at the bottom. The boxed area includes the substitution mutations, and the dashes represent nucleotides that correspond to the wild-type sequence

Further fine tuning of the essential sequences for mDAZL binding within the 31-nucleotide region was carried out by a competition assay. The 61-nucleotide fragment E RNA was radiolabeled and used in an electrophoretic mobility shift assay in the presence of a series of competitor oligonucleotides that each contained 7 nucleotide substitutions. As expected, the 31-nucleotide fragment E2 oligonucleotide competed for mDAZL binding (Fig. 4B, lanes 3 and 4). The mutant oligonucleotide containing substitutions at the 5' end (E2m1; Fig. 4B, lanes 5 and 6) was still able to compete for GST-mDAZL binding, implying that this region is not critical for binding, because the mutant oligonucleotide is still competent to bind the protein and sequester it from the labeled Tpx-1 RNA. However, mutations in the remaining regions of the sequence seem to have an adverse effect on binding. Mutant E2m2 (Fig. 4B, lanes 7 and 8), E2m4 (Fig. 4B, lanes 11 and 12), and E2m5 (Fig. 4B, lanes 13 and 14) were less effective in their ability to compete for GST-mDAZL binding, and mutant E2m3 had an intermediate competition phenotype. These mutant oligonucleotides have lost their ability to be bound by GST-mDAZL, suggesting that the 26-nucleotide region spanning nucleotides 1120–1145 of the Tpx-1 RNA are critical for GST-mDAZL binding. This region is referred to hereinafter as the mDAZL-binding site (DBS).

Presence of the DBS in Other mRNAs

Sequence searches of the clones identified by the SNAAP screen were carried out using the ClusterW pairwise alignment [47] to determine whether additional mRNAs also contained the 26-nucleotide DBS sequence. All of the remaining 6 previously identified mRNAs also contained a DBS-like sequence in the 3' UTR, as did 1 of the unknown genes (clone D2, GenBank accession no. AK014447)) present as multiple expressed sequence tags (ESTs) in the database. Pam is a 510-kDa protein of unknown function [38], GRSF1 is an RNA-binding protein that functions in translation initiation [39], Trf2 is a TATA box-binding protein (TBP)-like protein highly expressed in testis [40–43], and Cappß1 is a component of a heterodimeric actin capping protein [37]. The H47 mRNA encodes a minor histocompatability antigen precursor possibly involved in graft rejection [44], and P{alpha}7/C8 is an mRNA encoding a putative murine proteosome subunit listed in GenBank. An alignment of the DBS sequences contained within the isolated substrate mRNAs is shown in Figure 5. Conserved sequences were used to derive a consensus that reveals the binding site to be particularly rich in uracil and adenine. The uracil-rich nature of the binding site is consistent with results of a previous report demonstrating preferential binding of mDAZL to poly(U) ribohomopolymers [29] or a uracil-rich sequence [48].

We next tested whether other DBS-containing mRNAs, in addition to Tpx-1, can also be bound by mDAZL. Four of the remaining 7 DBS-containing mRNAs where chosen for further study. The specific association of the DBS-containing mRNA with mDAZL protein is shown in Figure 6A. Messenger RNAs from all 4 genes tested (Pam, D2, GRSF1, and Trf2) could be specifically copurified with mDAZL from testis extract (lanes 2, 6, 10, and 14) but not by control proteins as detected by RT-PCR analysis with gene-specific primers. Binding was not detected to an unrelated mRNA, Dmc1 (lane 18). To determine whether binding was direct or mediated through additional proteins, RNAs containing the respective DBS regions were generated and tested. 32P-labeled in vitro-transcribed RNA was incubated with GST-mDAZL in the absence of testis extract, and bound RNA was isolated and resolved by denaturing PAGE. As seen in Figure 6B, mDAZL can bind each RNA directly and does not require additional proteins (lanes, 2, 5, 8, 11, and 14) but does not bind a control RNA (lane 17). The RNAs are not promiscuously bound by mDAZL because they are not bound by an unrelated RNA-binding protein (lanes 3, 6, 9, 12, and 15). The binding is mediated through the DBS because oligonucleotides consisting of the respective DBS sequences were sufficient to bind mDAZL (data not shown). Furthermore, the 3' UTR of the remaining 3 DBS-containing genes were also able to compete for mDAZL binding to the Tpx-1 RNA in mobility shift assays (data not shown). These data support the conclusion that mDAZL can specifically bind mRNAs containing a DBS and affirms the predictive potential of the DBS in identifying mDAZL substrates.



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  		FIG. 6. mDAZL-binding to RNA containing a DBS. A) Primer pairs specific for the indicated genes were used to amplify reverse-transcribed mRNA that was copurified with GST-mDAZL (mDAZL) or the 2 controls, GST-{alpha}CP1 ({alpha}CP1) and GST-RBD (RBD). Amplification of mRNA isolated from total extract is shown (Total; lanes 5, 9, 13, 17, and 21). The mDAZL protein can bind to the DBS-containing mRNA but not the Dmc1 control mRNA. DNA size markers are shown in lane 1, and the corresponding sizes are denoted on the left. B) The DBS-containing regions of Txp-1 (61 nucleotides), Pam (97 nucleotides), D2 (100 nucleotides), GRSF1 (100 nucleotides), and Trf2 (100 nucleotides) or the pcDNA3 polylinker (150 nucleotides; control) were transcribed in vitro in the presence of 32P-UTP. The RNA was incubated with the indicated fusion proteins, and specifically bound RNAs were eluted and resolved by denaturing PAGE as described for Figure 2

Murine DAZL Can Specifically Bind Cdc25A Phosphatase

The Drosophila homologue of mDAZL, Boule, is functionally linked to the meiotic cell division cycle protein Cdc25 phosphatase (Twine) and appears to regulate the expression of Twine mRNA translation. In mammals, there are 3 Cdc25 genes, Cdc25A, Cdc25B, and Cdc25C [49–51]. In the testis, Cdc25A is expressed in mitotic and meiotic germ cells, Cdc25B is predominantly in somatic cells, and Cdc25C is expressed in the late stages of germ cell development [51–54]. To determine whether any of the murine Cdc25 phosphatase mRNAs are potential ligands for mDAZL, we searched for a DBS sequence in these mRNAs. A DBS-like sequence is present in the Cdc25A 3' UTR but not in the remaining 2 genes. We next determined whether mDAZL could bind the Cdc25A mRNA. Total testis extract was incubated with GST-mDAZL, and copurifying RNAs were isolated, reverse transcribed, and amplified by PCR with gene-specific primers. Results from the 2 germ cell Cdc25 forms are presented in Figure 7. Despite the presence of both Cdc25A and Cdc25C mRNAs in the testis extract (lanes 5 and 9, respectively), the Cdc25A mRNA is efficiently bound by mDAZL but the Cdc25C mRNA is not (compare lanes 2 and 6). The specificity of mDAZL binding to the Cdc25A mRNA is further demonstrated by the lack of detectable binding to this mRNA by the nonspecific RNA-binding protein in lane 4. These data suggest a functional role for mDAZL in the progression of the mitotic spermatogonia into the meiotic spermatocyte in mammals, as is the case in Drosophila. A low, but reproducible, level of binding to Cdc25A was also detected by the {alpha}CP1 protein (lane 3), which is an avid poly(C)-binding protein [55–58]. Sequence search of the Cdc25A mRNA revealed 3 poly(C)-rich regions in the mRNA that could be potential {alpha}CP1 binding sites positioned at nucleotides 467–492, 1875–1891, and 2777–2800. The functional significance of {alpha}CP1 binding to Cdc25A is currently unclear.



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  		FIG. 7. Cdc25A is specifically bound by mDAZL. Messenger RNA copurifying with GST-mDAZL was tested for the presence of Cdc25A and Cdc25C mRNAs with gene-specific primers as described for Figure 5A. Cdc25A but not Cdc25C was bound by mDAZL. The DNA markers and corresponding sizes are shown on the left


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The presence of mDAZL throughout spermatogenesis suggests that it regulates multiple target mRNAs, either simultaneously or at different stages of sperm development, that are essential for proper spermatogenesis. In the present study, we obtained evidence that the mDAZL protein can specifically bind to at least 9 distinct mRNAs: Tpx-1, Pam, GRSF1, Trf2, Cappß1, P{alpha}7/C8, H47, clone D2, and Cdc25A. We also isolated a consensus binding site for mDAZL that will enable identification of additional potential mDAZL target mRNAs as sequences become available in the databases.

The following observations suggest that one of the functions of mDAZL is to regulate the expression of the Tpx-1 mRNA. First, mDAZL can directly bind to the 3' UTR of the Tpx-1 mRNA. Second, the temporal expression pattern of both proteins is consistent with Tpx-1 being a downstream target of mDAZL. The mDAZL protein is present in the early stages of the mitotic spermatogonia and is most abundant during the pachytene stage of meiosis and thereafter [10, 59, 60]. Tpx-1 mRNA expression appears to begin at the pachytene stage and persists throughout spermatogenesis to the elongating spermatid stage [20–22]. Sperm cell development in mDAZL knockout mice ceases within the mitotic spermatogonia stage and is unable to progress into meiosis [10]. Third, the potential functional significance for Tpx-1 in spermatogenesis was recently suggested by the identification of 3 infertile men that carry a chromosomal breakpoint within the Tpx-1 gene [18]. Therefore, Tpx-1 could be critical for spermatogenesis, and the demonstration that mDAZL specifically binds this mRNA implies that expression of Tpx-1 could be regulated by mDAZL perhaps by conferring stability onto the mRNA or translational regulation.

Mice containing a homozygous disruption of the mDAZL gene have sparse tubule populations just prior to birth and an absence of germ cells at 9 wk of age [10]. These data indicate that the initial action of mDAZL is in the mitotic spermatogonial stage because defects are observed prior to birth. In mice, the meiotic stage of spermatogenesis does not initiate until about 8 days of age [61]. Thus, it is unlikely that Tpx-1 is the initial target mRNA for progression into meiosis. Therefore, what is the significance of regulating Tpx-1 mRNA expression by mDAZL in the pathogenesis of azoospermia? Tpx-1 appears to be important in the attachment of spermatogenic cells with Sertoli cells [17], and this attachment is obligatory for spermatogenesis [13, 14, 62]. Aberrant expression of Tpx-1 could result in the loss of, or improper, interactions between these 2 cell types. The demonstration that mDAZLnull/mDAZL+ heterozygous mice contain 4-fold higher amounts of abnormal sperm cells, which accounts for 61% of the total sperm produced in these animals [10], is consistent with a defect in Sertoli-germ cell interactions. Sertoli cells are essential for the fate-determining signals that regulate the incidence of apoptosis and the eventual phagocytosis of degenerating spermatogenic cells [13, 63]. The influence of the Sertoli cells may be diminished in the heterozygous animals, which could account for the high incidence of abnormal sperm that are not purged. Of the 2 proteins in addition to Tpx-1 that are postulated to be essential for spermatogenic and Sertoli cell attachment, the N-cadherin mRNA also contains a DBS-like sequence, suggesting that it too could be regulated by mDAZL.

There is strong evidence that Cdc25A is also a substrate for mDAZL regulation. The Drosophila Cdc25 homologue Twine has been functionally linked to the Drosophila mDAZL homologue Boule. Boule was recently shown to be required for proper translational regulation of Twine mRNA [32]; however, it is not known whether it regulates Twine mRNA directly or indirectly. Our demonstration that mDAZL can bind the Cdc25A mRNA suggests that mDAZL also regulates this mRNA in mice and that the regulation is direct. The Cdc25 proteins are threonine/tyrosine phosphatases, which function on cyclin-dependent kinases and influence the cell cycle [64]. Cdc25A is expressed in spermatogonia and is at its highest level during the early meiotic stages of spermatogenesis in the primary and secondary spermatocytes but is not present in the later stages, suggesting that Cdc25A is important for the transition into meiosis [51]. This function is similar to that postulated for Twine in Drosophila germ cell development. We noticed a DBS-like sequence within the 3' UTR of Twine (nucleotides 1610–1641), suggesting that Boule directly binds the Twine mRNA and regulates its expression.

The Cdc25A and Cdc25C proteins are both approximately 55% similar to Twine at the protein level. However, the similarity of the expression profile between Cdc25A and Twine and the presence of a DBS in the 3' UTR of both these mRNAs suggest that Cdc25A is the functional homologue of Twine in mammals. This hypothesis is further supported by the recent demonstration that mice containing a knockout of the Cdc25C gene are fertile [65], precluding Cdc25A as a critical mRNA regulated by mDAZL in fertility. Our demonstration that mDAZL binds the 3' UTR of the Cdc25A mRNA indicates that this gene is an essential mDAZL target in vertebrates and regulates the progression of spermatogonia into spermatocytes.

{alpha}CP1, which has an affinity for Cdc25A, is also involved in both mRNA stability [35] and translation [66–70]. Whether it has a functional role in the regulation of Cdc25A needs to be determined. The binding of {alpha}CP1 to the Cdc25A mRNA could also explain why this mRNA was not identified as an mDAZL substrate in the initial SNAAP screen. Bands that were common to both the target mDAZL protein and the control {alpha}CP protein were not evaluated further and would have been scored as false negative. An exhaustive screen for mDAZL substrates has not yet been carried out.

In humans, the Y-chromosome-encoded DAZ protein is expressed in the early spermatogonial stage, whereas the autosomal DAZL protein is expressed in later stages, suggesting that each protein contributes to distinct aspects of this process [71]. The presence in mice of only an autosomal copy suggests that mDAZL is involved in multiple functions throughout gametogenesis. Perhaps 1 function is to regulate the progression into meiosis by the control of Cdc25A and the subsequent differentiation by the regulation of Tpx-1 expression.

Although Tpx-1 is the only testis-specific mDAZL substrate mRNA that we have thus far identified, the remaining isolated mRNAs could also be essential in spermatogenesis. Pam was originally identified as a protein associated with c-myc [38]. Expression of a c-myc transgene in the mouse testes results in the arrest of spermatogenic cell differentiation, leading to sterility [72]. Therefore, a protein that interacts with c-myc could have significant effects on the function of c-myc in spermatogenesis. GRSF1 is a cytoplasmic RNA-binding protein originally identified by its binding preference to guanosine-rich sequences [73]. It was subsequently shown to upregulate the translation of influenza virus and is most likely involved in general translational control [39]. Misregulation of GRSF1 could adversely affect the translation of various genes essential for spermatogenesis. Trf2 is a TBP-like protein that can associate with the basal transcription factor TFIIA and inhibit transcription of promoters containing a TATA-box and is thought to activate transcription from promoters containing nonconsensus TATA boxes [40–43]. Despite its apparent ubiquitous low level of expression in all cells tested, Trf2 mRNA is overexpressed in the testes [41–43], suggesting that it is an important component of gene expression in this organ. While this manuscript was under review, Zhang et al. [74] reported that mice containing a disrupted Trf2 gene are sterile because of a spermiogenesis defect. This observation is consistent with our hypothesis that mDAZL binds to and potentially regulates the expression of the Trf2 mRNA.

Of the 22 clones that were identified in the SNAAP screen, all 8 with a known complete mRNA sequence contain a discernable DBS. The remaining 14 isolated clones are novel and have no corresponding ESTs or incomplete ESTs that do not appear to span the entire 3' UTR. Therefore, it is unclear whether they contain a DBS. It is also conceivable that some of the mRNAs do not have a DBS and copurified with mDAZL only because of their association with an mDAZL interacting protein. In such a scenario, mDAZL does not directly bind these mRNAs but is associated with them through a larger multiprotein RNP complex. A protein bound directly or indirectly to an mRNA can still exert an influence on the regulation of that mRNA. However, these clones have not been further characterized to independently confirm that they are true mDAZL substrates, and they could represent false-positive results.

Identification of mDAZL substrate mRNAs will now enable a detailed mechanistic assessment of the consequence of mDAZL binding to each target mRNA. The DBS is contained in the 3' UTR of each DBS-containing mRNA isolated thus far. The 3' UTR location of the DBS and the cytoplasmic polysome association of mDAZL [29] indicate that it regulates expression of its substrate mRNAs by regulating translation or stability of the mRNA. Both possible mechanisms are currently under investigation for the individual substrate mRNAs. The results of these investigations will provide valuable insights into the role of mDAZL and the related Y-chromosome homologue DAZ in azoospermia and may lead to novel avenues for therapeutic intervention.


   ACKNOWLEDGMENTS
 
We thank C. Tovar and B. Babiarz for providing mouse testes and D. Denhardt for providing the DAP28–DAP31 primers. We are grateful to N.D. Rodgers and Z. Wang for critical reading of the manuscript.


   FOOTNOTES
 
First decision: 20 June 2001.

1 Supported by the Johnson and Johnson Discovery Award and NIH grant HD39744 to M.K. Back

2 Correspondence: Megerditch Kiledjian, Department of Cell Biology and Neuroscience, Rutgers University, 604 Allison Rd., Piscataway, NJ 08854-8082. FAX: 732 445 0104; kiledjia{at}biology.rutgers.edu Back

3 Current address: PTC Therapeutics, South Plainfield, NJ 07080. Back

Accepted: September 27, 2001.

Received: May 18, 2001.


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