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BOR - Papers in Press, published online ahead of print June 29, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.042788
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BIOLOGY OF REPRODUCTION 73, 840–847 (2005)
DOI: 10.1095/biolreprod.105.042788
© 2005 by the Society for the Study of Reproduction, Inc.

Meiotic Messenger RNA and Noncoding RNA Targets of the RNA-Binding Protein Translin (TSN) in Mouse Testis1

Yoon Shin Cho 3, Naoko Iguchi 3, Juxiang Yang 3, Mary Ann Handel 4, and Norman B. Hecht 2, 3

Center for Research on Reproduction and Women's Health,3 University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 The Jackson Laboratory,4 Bar Harbor, Maine 04609


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In postmeiotic male germ cells, TSN, formerly known as testis brain-RNA binding protein, is found in the cytoplasm and functions as a posttranscriptional regulator of a group of genes transcribed by the transcription factor CREM-tau. In contrast, in pachytene spermatocytes, TSN is found predominantly in nuclei. Tsn-null males show a reduced sperm count and high levels of apoptosis in meiotic cells, suggesting a critical function for TSN during meiosis. To identify meiotic target RNAs that associate in vivo with TSN, we reversibly cross-linked TSN to RNA in testis extracts from 17-day-old and adult mice and immunoprecipitated the complexes with an affinity-purified TSN antibody. Extracts from Tsn-null mice were used as controls. Cloning and sequencing the immunoprecipitated RNAs, we identified four new TSN target mRNAs, encoding diazepam-binding inhibitor-like 5, arylsulfatase A, a tetratricopeptide repeat structure-containing protein, and ring finger protein 139. In contrast to the population of postmeiotic translationally delayed mRNAs that bind TSN, these four mRNAs are initially expressed in pachytene spermatocytes. In addition, anti-TSN also precipitated a nonprotein-coding RNA (ncRNA), which is abundant in nuclei of pachytene spermatocytes and has a putative polyadenylation signal, but no open reading frame. A second similar ncRNA is adjacent to a GGA repeat, a motif frequently associated with recombination hot spots. RNA gel-shift assays confirm that the four new target mRNAs and the ncRNA specifically bind to TSN in testis extracts. These studies have, for the first time, identified both mRNAs and a ncRNA as TSN targets expressed during meiosis.

gametogenesis, gene regulation, meiosis, spermatogenesis, testis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Spermatogenesis is a process requiring massive gene expression that accompanies the cellular differentiation that generates haploid spermatozoa. During male germ cell development, the expression of many genes is regulated by intrinsically controlled genetic programs and by interactive processes with Sertoli cells, often mediated by extrinsic factors such as testosterone and FSH [1]. Posttranscriptional regulation of temporally expressed genes plays an especially important role in the differentiating gamete during the latter stages of spermatogenesis, because transcription terminates while the haploid round spermatid is transforming into the spermatozoon [2].

TSN (formerly known as testis brain-RNA binding protein), the mouse orthologue of the human protein Translin, is abundant in testis and brain. As a DNA binding protein, TSN binds breakpoint junctions of chromosomal translocations [3, 4], and as an RNA binding protein it mediates intracellular (from nucleus to cytoplasm) and intercellular (between spermatids) mRNA transport, which controls the time and, perhaps, the location of translation of specific mRNAs in postmeiotic germ cells [5–7]. Multimeric complexes of TSN, the TER ATPase, and a testis-enriched kinesin, KIF17b (also known as KIF17), transport mRNAs encoding proteins such as AKAP4 and the protamines from nuclei to cytoplasm, and through intercellular bridges as nontranslated RNP complexes [7]. Following RNA release from the RNA-protein complexes in later-stage spermatids, the mRNAs are translated, suggesting an in vivo involvement of TSN in the movement, stability, and translational suppression of specific mRNAs in male germ cells [6].

Although TSN shuttles between the nucleus and cytoplasm in association with an evolutionarily related TSN-binding protein, TRAX (also known as TSNAX; translin-associated factor X), it is primarily found in the nuclei of pachytene spermatocytes and in the cytoplasm of postmeiotic male germ cells [8]. Its nuclear functions in pachytene spermatocytes are unknown. Kasai and colleagues [9] have proposed that the human TSN orthologue plays a role in DNA recombination, repair, and metabolism, but deficiencies in somatic recombination or DNA repair are not readily detectable in mice that are lacking TSN [10]. However, the marked increase in apoptosis of spermatocytes in Tsn-null mice suggests a role for TSN during meiosis, perhaps functioning as a posttranscriptional regulator in spermatocytes [10].

Testis mRNAs encoding protamine 1 (Prm1), protamine 2 (Prm2), transition protein 1 (Tnp1), transition protein 2 (Tnp2), A-kinase anchoring protein 4 (Akap4), and glyceraldehyde 3-phosphate dehydrogenase-S (Gapds), and brain mRNAs encoding tau, ligatin, {alpha}-Ca2+-calmodulin-dependent protein kinase II, and myelin basic protein have been reported to be target mRNAs of TSN [11–14]. To date, all of the known testicular TSN target mRNAs are transcribed in postmeiotic germ cells by the transcription factor cAMP-responsive element modulator (CREM)-tau and are subcellularly transported in association with the kinesin KIF17b [7]. However, no target molecules of TSN from premeiotic, meiotic, or somatic cells of the testis have been identified.

Both the abundance of TSN in meiotic spermatocyte chromatin ([8] and unpublished observations) and the disruption of spermatogenesis at the meiotic phase in mice that are lacking TSN provide a compelling rationale to identify RNA-binding partners of TSN in pachytene spermatocytes. Therefore, we used cross-linking and immunoprecipitation of testis extracts with anti-TSN to identify new meiotic RNA targets of TSN. Four new TSN target mRNAs and a TSN-binding nuclear nonprotein-coding-RNA (ncRNA) expressed in pachytene spermatocytes were discovered by this strategy.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of Dissociated Germ Cells and In Vivo Cross-Linking

Single-cell suspensions of male germ cells were prepared from decapsulated testes from 17-day-old and adult wild-type or adult Tsn-null mice as described [7]. For the in vivo cross-linking [7, 15], cells were resuspended in 1x PBS containing 1% formaldehyde and incubated at room temperature for 10 min with slow mixing. To terminate the cross-linking reaction, 1 M glycine (pH 7) was added to a final concentration of 0.25 M and incubated at room temperature for 5 min. Cross-linked cells were washed twice with ice-cold PBS, harvested by centrifugation, and used immediately or stored at –80°C. Procedures involving animals were conducted under the approval of the Institutional Animal Care and Use Committee of the University of Pennsylvania in accordance with the Guide for Care and Use of Laboratory Animals.

Immunoprecipitation of TSN-RNP Complexes

Immunoprecipitation and cloning were performed following published procedures [7, 15, 16]. Cross-linked cells were resuspended in 6 ml of low-salt RIPA buffer (50 mM Tris-HCl pH 7.4, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.05% SDS, 1 mM EDTA, and 150 mM NaCl) containing RNAsin (300 U/ml) (Promega, Madison, WI), and protease inhibitor cocktail (10 µl/ml) (Sigma, St. Louis, MO). The cell suspension was disrupted by three 10-sec sonications (with a 2-mm probe at a setting of 1 with a Heat Systems/Ultrasonics Model W-220F Cell Disruptor [Heat Systems-Ultrasonics Inc., Farmingdale, NY]) and centrifuged at 13 000 x g at 4°C for 15 min. The supernatant fraction was incubated with 15 U of RNase T1 at 37°C for 10 min, and then precleared with 200 µl of protein A beads at 4°C for 1 h with slow rotation. Precleared supernatants were incubated with 20 µg of affinity-purified TSN antibody and 100 µl of protein A beads at 4°C for 4 h with slow mixing. Protein A beads were removed by centrifugation at 1000 x g at 4°C for 5 min and washed six times with 10 ml of RIPA buffer at alternating high-salt (1 M NaCl) and low-salt (150 mM) concentrations. RNA was purified with phenol and precipitated with isopropanol, with 0.5 µl of glycogen carrier. The immunoprecipitated RNAs were pelleted by centrifugation, resuspended in 10 µl of diethyl pyrocarbonate (DEPC) water, and further purified with Trizol.

Cloning of Immunoprecipitated RNAs

RNA linkers for RNA ligation, 5'-OH AGG GAG GAC GAU GCG G 3'-OH (5'-RNA linker) and 5'-P CGA GAU GGC GGC UUC CUG C 3'-puromycin (3'-RNA linker), were purchased from Dharmacon (Chicago, IL) [16]. RNA ligation reactions were performed according to the manufacturer's protocol (Ambion, Austin, TX). The purified RNA and the 5'-RNA linker were incubated at 16°C for 1 h in ligation mixture, the 3'-RNA linker was added, and the incubation continued at 16°C overnight. The RNA ligation mixture was treated with RQ1DNase (0.05 U/µl) (Promega) in the presence of RNAsin (1 U/µl) at 37°C for 20 min, precipitated, and resuspended in 10 µl of DEPC-treated water. Reverse transcription (RT) was performed to generate {alpha}-32P-dCTP labeled cDNAs from ligated RNAs using the reverse primer 5'-GCA GGA AGC CGC CAT CTC G-3' radiolabeled cDNAs (larger than 60 nucleotides [nt]) were separated on 8 M urea, 8% acrylamide gels, and purified by removing a prominent side reaction of 5'-3' RNA linkers only (about 35 nt). The cDNAs were recovered from sliced gels following incubation in 350 µl of 1 M sodium acetate pH 5.2, 1 mM EDTA at 37°C for 1 h with shaking. After precipitation and resuspension, the cDNAs were used for polymerase chain reaction (PCR) amplification with the forward primer 5'-AGG GAG GAC GAT GCG G-3' and reverse primer 5'-GCA GGA AGC CGC CAT CTC G-3', and cloned into the pCR4-TOPO vector (Invitrogen, Carlsbad, CA). The PCR products were sequenced and the sequences were analyzed with the National Center for Biotechnology Information's Basic Local Alignment Search Tool (BLAST; http://www.ncbi.nlm.nih.gov/) or Ensembl (http://www.ensembl.org/).

RT-PCR and Real-Time RT-PCR Assay

Nuclei and cytoplasm were isolated as previously described [17]. Reverse transcription with random decamer primers was performed with the RETROScript RT kit (Ambion) after treatment of 1 µg of purified RNA with RNase-free amplification-grade DNase I (0.1 U/µl) followed by heat-EDTA inactivation of the DNase I. The resulting cDNAs were used as templates for PCR with gene-specific primers and puReTaq Ready-To-Go PCR beads (Amersham Pharmacia, Piscataway, NJ) for 30 cycles. The PCR products were separated on 2% agarose gels and visualized by ethidium bromide staining.

Real-time RT-PCR was performed using the SYBR Green PCR Master Mix and the 7900 Thermal cycler at typical amplification parameters (50°C for 2 min, 95°C for 10 min, followed by 40 cycles at 95°C for 15 sec and 60°C for 1 min). Duplicate assays of two experiments were performed using cDNAs generated from total RNA prepared from highly enriched (>85% purity) populations of pachytene spermatocytes and round spermatids isolated by STAPUT sedimentation, and the fold differences were determined by comparing the delta Ct of each gene normalized with Gapdh. Three separate real-time RT-PCR replicates were performed for subcellular quantification of the ncRNAs. All primers were designed with Primer Express 1.5 software (Applied Biosystems, Foster City, CA), and checked by PCR to ensure that they generated single bands of the predicted size. Amplification efficiency for each primer pair was calculated and used for relative quantification. Primer sets used in the PCR and real-time RT-PCR are listed in Table 1 in Supplement 1 (available at http://www.biolreprod.org).


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  		TABLE 1. Real-time RT-PCR quantification of mRNAs in pachytene spermtocytes and round spermatids.a

RNA Gel-Shift Assays

Probe sequences of about 60 to 120 nt for each gene listed in Supplement 2 (available at http://www.biolreprod.org) were transcribed in vitro with {alpha}-32P-CTP using the Maxiscript kit (Ambion). The radiolabeled probes were purified by electrophoresis in denaturing PAGE. Forty-thousand cpm of each probe were incubated at 70°C for 10 min and cooled to room temperature before use. The RNA-binding assay was performed as previously described [12]. Briefly, the probes were incubated for 15 min with 30 µg of S100 fraction from testes of adult wild-type or adult TSN-deficient mice in RNA gel-shift solution (20 mM Hepes pH 7.5, 3 mM MgCl2, 40 mM KCl, 5% glycerol, and 2 mM dithiothreitol), followed by digestion with 1 U of RNase T1 for 10 min, and incubation with 0.25 mg/ ml of heparin for 10 min. The RNA-protein complexes were separated by 4% PAGE at 4°C. For supershift assays, 1 µg of anti-TSN rabbit immunoglobulin G (IgG) or normal rabbit IgG (Sigma) (dissolved in PBS) were added and incubated for 10 min. All experiments were performed at room temperature except where noted. The gels were exposed for PhosphorImaging, and signals were detected with the STORM model 860 (Molecular Images, Mountain View, CA).

Cloning of the Full-Length cDNA of Nct1

Because the Nct1 cDNA was isolated as a 178-base pair fragment, 5' and 3'-rapid amplification of cDNA ends (RACE) was used to determine the full-length RNA. The 5'- and 3'-RACE assays were performed with total RNA from adult mouse testes using a FirstChoice RLM-RACE Kit (Ambion) following the manufacturer's instructions. PCR was performed using the primers listed in Supplement 3 (available at http://www.biolreprod.org). The fragments obtained from the RLM-RACE assays were subcloned into a pCR4-TOPO vector (Invitrogen, Carlsbad, CA) and sequenced.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Messenger RNAs Encoding a Diazepam-Binding Inhibitor-Like 5 Protein, a Tetratricopeptide Repeat Protein, Arylsulfatase A, and a Patched Related Protein Translocated in Renal Cancer/Ring Finger Protein Are Targets of TSN

Immunohistochemistry with an affinity-purified antibody to recombinant mouse TSN revealed that TSN is found predominantly in nuclei in pachytene spermatocytes and in the cytoplasm in diplotene and metaphase spermatocytes, and in postmeiotic germ cells [8]. After subcellular fractionation and isolation of nuclei from testes of 17-day-old mice, most of the TSN appears soluble (data not shown), suggesting that TSN is not tightly bound in nuclei. To determine whether TSN interacts with specific RNAs in meiotic cells as it does in later-stage postmeiotic cells [7], we used reversible formaldehyde cross-linking and immunoprecipitation with affinity-purified anti-TSN to selectively precipitate TSN complexes from extracts of 17-day-old and adult mice. RT-PCR and sequencing were used to amplify and identify bound RNAs. To ensure that the cross-linking and immunoprecipitation procedures were specific, four known TSN target mRNAs and four mRNAs lacking the TSN binding sites were used as binding and nonbinding controls in a parallel experiment with adult testes. In addition, extracts prepared from adult Tsn-null mice were also cross-linked and immunoprecipitated. Confirming the specificity of the cross-linking and immunoprecipitation, the four TSN-binding mRNAs Prm1, Tnp1, Tnp2, and Gapds were detected in immunoprecipitated RNAs enriched from adult wild-type, but not from adult Tsn-null mouse testes (Fig. 1A), whereas four mRNAs lacking the TSN binding elements Clu (clusterin), Gapdh, Msy2, and Pgk2 were not precipitated from the cross-linked extracts of either the wild-type or TSN-deficient mice (Fig. 1B) [7].



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  		FIG. 1. TSN binds to four new target mRNAs. Following cross-linking and immunoprecipitation, RNAs were detected by RT-PCR or sequencing. M, markers; C, control RT-PCR using purified total testis RNA from adult wild-type mice; KO, immunoprecipitation with anti-TSN from testis extract of adult TSN-null mice; WT, immunoprecipitations with anti-TSN from testis extracts of adult wild-type mice

Four new mRNAs encoding diazepam-binding inhibitor-like 5 (Dbil5), a sequence (4921521J11Rik) encoding a tetratricopeptide repeat (TPR) structure-containing protein, and sequences encoding arylsulfatase A (Arsa), and a patched-related protein translocated in renal cancer (Rnf139) were immunoprecipitated in extracts from testes of 17-day-old and adult wild-type mice, but not from adult Tsn-null mice. The specific binding for the TSN was confirmed by RT-PCR analysis against adult wild-type and Tsn-null mice testes (Fig. 1C). Sequence analyses indicated the four new mRNAs all contain binding sites for TSN (listed in Supplement 4, available at http://www.biolreprod.org).

To determine whether the four newly identified mRNAs could bind TSN, RNA gel-shift assays were performed (Fig. 2). RNA-protein complexes with similar electrophoretic mobilities were observed with extracts from adult wild-type testes (Fig. 2, lane 3, arrowhead), but not with extracts from adult Tsn-null mice (Fig. 2, lane 2) for the four new immunoprecipitated mRNAs and transcript c, a subclone of the 3' untranslated region of Prm2 mRNA known to bind TSN [12]. No similar complexes were observed with a multimeric CA repeat sequence RNA probe that does not bind TSN. As a further test of the specificity of TSN binding to each of the putative target RNA probes, anti-TSN was used. When anti-TSN was added to the gel-shift mixtures, decreases of the RNA-protein complexes were detected for the probes recognizing transcript c, Dbil5, 4921521J11Rik, Arsa, and Rnf139, but not the CA repeat (Fig. 2, lane 5). No decreases in the RNA-protein complex were observed when normal rabbit IgG was used in place of the affinity-purified anti-TSN (Fig. 2, lane 4). Taken together, these results indicate specific interactions between TSN and the mRNAs encoding Dbil5, 4921521J11Rik, Arsa, and Rnf139.



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  		FIG. 2. RNA gel-shift assay for mRNAs identified by TSN cross-linking immunoprecipitation. Lane 1, radiolabeled probe alone; lane 2, radiolabeled probe and testis extract from adult Tsn-null mice; lane 3, radiolabeled probe and testis extract from adult wild-type mice; lane 4, radiolabeled probes and testis extract from adult wild-type mice incubated with normal rabbit IgG; lane 5, radiolabeled probe and testis extract from adult wild-type mice incubated with anti-TSN. Arrowheads indicate the RNA-TSN complex

The Four New mRNAs Are Initially Expressed in Pachytene Spermatocytes

To determine the temporal pattern of expression of the newly identified TSN target mRNAs, we performed real-time RT-PCR using cDNAs generated from total RNA prepared from highly enriched (>85% purity) populations of pachytene spermatocytes and round spermatids isolated by STAPUT sedimentation (Table 1). Using Gapdh for normalization of mRNA levels and Prm2 as a background mRNA marker for pachytene spermatocytes (the protamines are first expressed in postmeiotic male germ cells), Dbil5, 4921521J11Rik, Arsa, and Rnf139 transcripts are detected in both pachytene spermatocytes and round spermatids. This indicates that, in contrast to the previously identified CREM-tau regulated TSN target mRNAs [7], these four new TSN target mRNAs are expressed in pachytene spermatocytes.

A Nonprotein-Coding RNA (Nct1) Binds TSN

In addition to the four TSN-binding mRNAs, the cross-linking immunoprecipitation experiments with extracts of 17-day-old and adult mice also identified a 178-nt ncRNA. A BLAST search in the Ensembl mouse genomic database for homologous sequences revealed two similar genomic sequences (AL683814) mapping to mouse chromosome 2, band E1. One showed 100% identity to the isolated clone, and the second was partially homologous with the 3' region, showing greater than 99% identity with a total of 122 of 178 nt matching. We used RLM-RACE to determine that the 178-nt transcript was part of a 685-nt transcript (Fig. 3A). This sequence, which we designated Non-Coding in Testis 1 (Nct1), contains a putative polyadenylation signal at 596–601 nt (in bold), but no open reading frame. Two expressed sequence tag (EST) sequences (CF198309 and CF105565) from a preleptotene spermatocyte cDNA library prepared with cells isolated from 18-day-old mice showed a 100% match to the GenBank sequence. A database search indicated both of the ESTs were derived from a single genomic sequence, a finding we confirmed by RT-PCR with a primer set (listed in Supplement 5, available at http://www.biolreprod.org) and sequencing of the PCR product. The second sequence, Nct2, located 360 nt downstream of Nct1, is also a noncoding gene (Fig. 3B). Both Nct1 (AY940662) and Nct2 (AY940663) lack introns, and the Nct2 EST and genomic sequence have poly(A) stretches in their 3' ends but no polyadenylation signal (Fig. 3A). Sequence analysis of the DNA immediately upstream of the start of Nct2 transcription reveals a GGA triplet repeat, a motif frequently associated with gene regulatory regions, or recombination hot spot sites (or a combination of these) [18]. Gel-shift assays confirm the interaction of TSN and the Nct1 RNA (Fig. 3C).



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  		FIG. 3. Analysis of nonprotein-coding RNAs Nct1 and Nct2. A) Nucleotide sequences of Nct1 and Nct2, a second nonprotein-coding RNA located downstream of Nct1. Asterisks indicate nucleotide identity between Nct1 and Nct2. The original sequence determined from the immunoprecipitated RNA is boxed. A putative polyadenylation signal sequence in Nct1 is black-boxed. Arrows indicate the positions of the primers used for RLM-RACE. B) Schematic presentation of the genomic region of mouse chromosome 2 with the Nct1 and Nct2 sequences. The shaded regions indicate the homologous regions between Nct1 and Nct2. C) RNA gel-shift assay. Lane 1, radiolabeled probe alone; lane 2, radiolabeled probe and testis extract from adult wild-type mice: lane 3, radiolabeled probe and testis extract from adult wild-type mice incubated with normal rabbit IgG; lane 4, radiolabeled probe and testis extract from adult wild-type mice incubated with anti-TSN. Arrowheads indicate the RNA-TSN complex

Nct1 and Nct2 Are Enriched in Pachytene Spermatocyte Nuclei

To determine the expression pattern of Nct1 and Nct2, RT-PCR was performed with purified RNAs from a number of mouse tissues. Nct1 and Nct2 are detected in RNAs prepared from total testis, but not from brain, heart, intestine, kidney, liver, lung, spleen, ovary, or kitw/kitw-v mutant testes (which contain somatic cells and some spermatogonia but lack later-stage germ cells), suggesting an enrichment of these ncRNAs in male germ cells (Fig. 4A). This is supported by assays of RNA from highly purified populations of pachytene spermatocytes and round spermatids. Nct1 and Nct2 are detected in pachytene spermatocytes, but not in round spermatids or in germ cell-deficient testes from mice with the Kit gene mutation (Fig. 4B).



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  		FIG. 4. Expression pattern of Nct1 and Nct2 in tissues, cell types, and subcellular fractions. A) RT-PCR was used to assay Nct1 and Nct2 in total RNA from brain, heart, intestine, kidney, liver, lung, spleen, testis, kit mutant testis, and ovary. B) RT-PCR was used to assay RNA prepared from STAPUT-purified pachytene spermatocytes and round spermatids (cellular purity is confirmed by near background level of Prm2 mRNA in the pachytene spermatocyte RNA; see Table 1) and kit mutant testis. C) Real-time RT-PCR was used to quantitate Nct1 and Nct2 in purified cytoplasmic and nuclear fractions of adult mouse testes. To assess purity of fractionation, 28S rRNA, Prm2 mRNA, and Pgk2 mRNA were employed as controls for cytoplasm and a micro-RNA precursor (pre-mir34) for the nuclear fraction. Results represent the mean ± SD from three separate experiments

We assessed the presence of Nct1 and Nct2 in highly purified testicular nuclear and cytoplasmic preparations. As controls for purity of the subcellular fractions, we used the precursor of microRNA 34 (pre-miR34, Rfam accession number MI0000404) as a nuclear marker and 28s rRNA as a cytoplasmic marker. Using a subcellular fractionation procedure in which more than 80% of the miR34 precursor was in the nuclear fraction and >90% of the 28S rRNA was in the cytoplasm, more than half of Nct1 and an enrichment of Nct2 RNA (compared to control mRNAs) were in nuclei (Fig. 4C). We propose that both Nct1 and Nct2 transcripts are associated with TSN in pachytene spermatocyte nuclei, because TSN is in pachytene spermatocyte nuclei and binds Nct1, both Nct1 and Nct2 contain the same TSN binding sequence, and the ncRNAs are not detectable in round spermatids or in testes lacking germ cells (Figs. 3 and 4).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The starting point for this investigation was our observations that TSN is found predominantly in the nucleus in pachytene spermatocytes. We sought to identify nuclear RNA binding partners of TSN that might be distinct from those bound to cytoplasmic TSN in spermatids. We have identified four mRNAs and one ncRNA that bind TSN and are present in pachytene spermatocytes. These findings have implications for the roles of TSN in mRNA transport and regulation as well as in meiotic processes of the nucleus.

TSN may well have meiotic functions similar to its functions later in spermatogenesis; namely, RNA binding for transport, stability, and translational control. Consistent with this hypothesis, we identified four meiotic mRNA targets of TSN. These encode widely different proteins, all with sequences from well-characterized multifunctional proteins or motifs. Two of the encoded proteins, DBIL5 and ARSA, are known to be present in germ cells, whereas the other two sequences identify proteins not previously found in spermatogenic cells. All of the transcripts are expressed at higher levels in round spermatids than in pachytene spermatocytes, but the time of their translation is not known.

DBIL5, encoded on chromosome 1, shares homology with endozepine, a testis-specific isoform of the ubiquitous acyl-CoA binding protein, which is involved in transporting and donating acyl-CoA to acyl-CoA-consuming systems such as ß-lipid oxidation [19], in intracellular signaling mechanisms [20], and in promoting steroidogenesis [21]. In rat testis, a closely related gene, endozepine-like peptide, is abundant in germ cells and undergoes posttranscriptional regulation [19, 22].

ARSA, encoded by a gene on chromosome 15, is a lysosomal enzyme involved in the degradation of sulfated glycolipids. It is localized in the acrosomal region of round and elongating spermatids and on the sperm surface where it facilitates sperm-zona pellucida binding [23]. In mouse testes, the majority of Arsa mRNA is found in ribonucleoprotein particles, which are proposed to regulate its translation. Arsa mRNA levels in male germ cells increase in late pachytene and secondary spermatocytes, are maintained in round spermatids, and decrease in late-elongating spermatids [24].

The deduced amino acid sequence from the nucleotide sequence 4921521J11Rik predicts a protein with a TPR motif, encoded from a gene on chromosome 8. The TPR is a degenerate 34-amino acid sequence producing a series of antiparallel amphipatic {alpha} helices capable of mediating protein-protein interactions. TPR-containing proteins have biological functions as diverse as cell cycle regulation, transcriptional control, protein transport, and neurogenesis [25], and several are expressed in male germ cells. For example, the germ cell-specific form of a widely expressed TPR protein SPAG1 (also known as TPIS) of unknown function is expressed as a novel, longer transcript [26]. A rat kinesin light-chain KLC3 associates via its TPRs with the mitochondria of elongating spermatids [27]. A cofactor in the p300 coactivator complex, STRAP, which has a tandem series of six TPR motifs, facilitates p53 (also known as TRP53) activity in response to stress, by interfering with the MDM2-dependent down-regulation of p53 [28]. From this finding, we speculate that the increase in apoptosis in spermatocytes in Tsn-null mice [10] could be related to improper expression of this TPR motif protein. No significant homology exists between the TPR motif protein identified by 4921521J11Rik and other known TPR proteins. However, that many TPR-containing proteins are expressed in male germ cells, and orthologues with >90% conserved amino acid sequences have been isolated, suggest that this TPR motif-containing protein could play an important role during spermatogenesis.

The Rnf139 sequence on chromosome 15 predicts an mRNA that is part of a 668-amino acid multiple-membrane-spanning protein with a ring finger domain in its C-terminus [29]. Chromosomal translocation of this gene leads to hereditary renal cell carcinoma. RNF139 is similar to the patched family of proteins with a putative sterol-sensing domain and extracellular loop capable of interaction with the hedgehog protein. Two patched proteins, PTCH and PTCH2, have been identified in human and mouse. PTCH2 is expressed in the primary and secondary spermatocytes of testis and interacts with desert hedgehog (DHH), which is expressed specifically in Sertoli cells in the testis and is required for germ cell development [30, 31]. Whether RNF139 is involved in germ cell development through the interaction with proteins such as DHH, as occurs with PTCH2, is not known. Overexpression of Dtrc8, the Drosophila counterpart, inhibits growth, consistent with its presumed role as a tumor suppressor gene [32]. Because mouse Rnf139 is expressed ubiquitously (NCBI and Genomics Institute of the Novartis Research Foundation SymAtlas databases), disruption of the precise regulation of this gene may cause the growth retardation and the increased apoptosis in pachytene spermatocytes observed in Tsn-null mice [10].

If TSN functions to transport, stabilize, or suppress the expression of one or more of these mRNAs, misregulation of the temporal expression of one or more of these meiotic mRNAs could contribute to the increased apoptosis of pachytene spermatocytes in Tsn-null mice [10]. The four meiotic target mRNAs appear to be under different transcriptional, and perhaps posttranscriptional, regulation than the translationally regulated TSN target mRNAs transcribed by CREM-tau in postmeiotic cell types [7]. Although the Dbil5, Arsa, Rnf139, and 4921521J11Rik transcripts continue to be expressed in postmeiotic cell types (Table 1), they lack a CREM-tau binding element in their promoters. Moreover, their TSN-binding sites are in coding regions rather than in the untranslated regions as is observed in the CREM-tau target transcripts [7, 14]. Whether this indicates different posttranscriptional regulatory pathways for TSN in meiotic and postmeiotic cells remains to be established.

Given the known functions of TSN in mRNA dynamics during spermatogenesis, it was surprising that this screen for binding partners also identified an ncRNA, Nct1, which is closely linked on chromosome 2 to another ncRNA, Nct2. Although the functions of these unique RNAs in pachytene spermatocytes remain to be elucidated, this interesting finding prompts speculation about meiosis-specific functions of TSN and ncRNAs. The functions of much of the DNA in the mammalian genome are unknown, and indeed, it has been estimated that about 97%–98% of the transcriptional output of the human genome encodes ncRNA [33]. Although some ncRNAs originate from ultraconserved genomic sequences, their genomic sources and organization are still largely unknown [34]. Nonprotein-coding RNAs have been proposed to have diverse but functionally important roles in a wide range of biological events [35–37], including chromosome architecture, dosage compensation and X-chromosome inactivation, genomic imprinting, stress responses, regulation of protein activities in transcriptional control, neuronal function, developmental regulation, and RNA and protein localization. Of special relevance to this analysis of meiotic germ cells, Nishant et al. [38] reported that a recombination hot spot was located near the genomic site specifying an ncRNA transcript. Although the function of the RNA is unknown, the transcription of an ncRNA close to a recombination hot spot is consistent with the open chromatin domains that are often presumed to be essential for recombination. Additionally, the human TSN orthologue has been proposed to bind to chromosomal translocation breaks via binding to genomic hot spots [3]. Some proposed mechanisms for meiotic homologue recognition and pairing invoke requirements for transcription, RNA intermediates, or both [39, 40]. The nuclear location of both TSN and the Nct1 and Nct2 RNAs in pachytene spermatocytes could facilitate their interactions in recombination, chromosome pairing, or transcriptional regulation during meiosis.

The mouse genome has also been reported to have an abundance of ncRNAs, many of which are unspliced, single-exon RNA polymerase II-mediated transcripts carrying a polyadenylation signal [41]. In contrast, one-third of the noncoding cDNAs were reported to have a 15–20 poly(A) stretch derived from genomic DNA sequences [41]. Of the two testicular ncRNAs, Nct1 and Nct2, Nct1 contains a polyadenylation signal, whereas Nct2 lacks a polyadenylation signal but contains a poly(A) sequence in its 3' region, suggesting that Nct2 might have originated by reverse transcription of Nct1. Because we cloned the 3' region of Nct1 by RLM-RACE, this suggests the Nct1 RNA has a poly(A) tail. Nct2 show significant homology or complementarity to coding sequences in the mouse genome, suggesting they are unlikely to function as antisense RNAs. The discovery of Nct1 and Nct2 in the nuclei of pachytene spermatocytes and their absence in the somatic tissues we have examined (Fig. 4) suggests an involvement in processes unique to meiosis. Because GGA repeat structures are implicated in developing tertiary nucleic acid structure to facilitate recombination [18], the existence of a GGA repeat adjacent to the start of transcription of Nct2 (Fig. 3) raises the intriguing possibility that Nct1 and Nct2 are located within a recombination hot spot in the mouse genome, or that they mediate recombination, or both. Creation of mice lacking such noncoding sequences may further define their function(s).


   ACKNOWLEDGMENTS
 
We thank Dr. J. McCarrey and D.A. O'Brien for RNA from STAPUT-enriched pachytene spermatocytes and round spermatids.


   FOOTNOTES
 
1 Supported by National Institutes of Health grant HD28832. Y.S.C. and N.I. contributed equally to this study. Back

2 Correspondence: Norman B. Hecht, Center for Research on Reproduction and Women's Health, University of Pennsylvania School of Medicine, 1310 Biomedical Research Building II/III, 421 Curie Boulevard, Philadelphia, PA 19104-6080. FAX: 215 573 5408; nhecht{at}mail.med.upenn.edu Back

Received: 14 April 2005.

First decision: 9 May 2005.

Accepted: 24 June 2005.


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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