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Testis |
Department of Environment and Natural Sciences, Graduate School of Environment and Information Sciences,3
and Department of Chemistry and Bioengineering, Graduate School of Engineering,4 Yokohama National University, Yokohama 240-8501, Japan
Laboratory of Cytogenetics, Division of Bioscience, Graduate School of Environmental Earth Science,5
and Chromosome Research Unit, Faculty of Science,6 Hokkaido University, Sapporo 060-0810, Japan
Department of Genetic Information,7 School of Medicine, Tokai University, Isehara 259-1153, Japan
Department of Obstetrics and Gynecology, Center for Research on Reproduction and Women's Health,8 School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6142
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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spermatid, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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During murine spermatogenesis, the developing haploid genome becomes transcriptionally dormant [3, 4]. Many mRNAs that are required for nuclear shaping and the processes of spermiogenesis during the haploid stage are stored in the cytoplasm of spermatids, until they are recruited for translation days later. This tight translational regulation of specific mRNAs permits changes in protein levels to occur in the absence of new transcription.
One of the mechanisms regulating translational activation or repression of eukaryotic mRNAs involves dynamic changes in the length of their poly(A) tails in the cytoplasm [5]. This phenomenon, known as cytoplasmic polyadenylation mediated by cytoplasmic polyadenylation element binding (CPEB) protein, has been well studied during Xenopus oocyte maturation, in which poly(A) addition is associated with translational stimulation [6–9]. In mammalian oogenesis and spermatogenesis, translational regulation is modulated by a change in adenylation. During oogenesis, the poly(A) tail of tissue-type plasminogen activator mRNA is deadenylated in the cytoplasm, resulting in translational silencing [10]. In contrast, poly(A) shortening is associated with the translational activation of protamine mRNA during late spermatogenesis [11]. The specific poly(A) elongation and shortening in the cytoplasm are mediated by interactions of specific cis elements, the cytoplasmic polyadenylation element (CPE), and trans factors, RNA-binding proteins (CPEB). In addition to CPEB, the human and mouse have two additional members of this family of proteins, named KIAA0940 and KIAA1673. Although their precise functions are unknown, they may act as translational regulators, dividing their functions both spatially and temporally. Considering the complexity of posttranscriptional gene regulation during spermatogenesis, many additional germ cell-specific RNA-binding proteins are likely to have important functions in male germ cells.
A large number of RNA-binding proteins share an evolutionarily conserved, 80- to 90-amino acid domain known as an RNA-recognition motif (RRM). This domain includes two ribonucleoprotein identifier sequences, RNP-1 and RNP-2 [12, 13]. In the present study, we have applied a degenerate polymerase chain reaction (PCR) strategy to clone another RRM-containing protein, a CPEB-related protein (CPEB2), from mouse. We report the characterization of CPEB2 below.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Total RNA was extracted from male mouse (BALB/cJ) brains. After purification by Oligo d(T25) dynabeads (Dynal, Oslo, Norway), poly(A) mRNA (2 µg) was reverse transcribed with AMV reverse transcriptase (Life Science, St. Petersburg, FL) with d(T)15 primer according to the manufacturer's protocol. The PCR reaction mixture (10 µl) contained 3 ng of single-stranded cDNA, 1x PCR buffer (Applied Biosystems, Foster City, CA), 1.5 mM MgCl2, 250 µM dNTP, 100 pmol of each primer, and 0.25 unit of AmpliTaq DNA polymerase (Applied Biosystems). To clone RRM fragments, PCR was performed on the mouse brain cDNAs by using primers based on an alignment of the eight- and six-amino acid stretches of RNP-1 and RNP-2, respectively (the primer sequences are shown in Table 1). The PCR conditions were 94°C for 30 sec; 30 cycles of 94°C for 20 sec, 55°C for 20 sec, and 72°C for 30 sec; and then 72°C for 7 min. The PCR products were cloned into the PCR II TOPO vector with the TOPO TA cloning kit (Invitrogen, Carlsbad, CA). Nucleotide sequences were determined by the dideoxynucleotide chain-termination method using an automated sequencing system (model ABI 373A; Applied Biosystems). Sequence data were compiled, and DNA and protein sequences were aligned using the BLAST 2 (http://www.ncbi.nlm.nih.gov/gorf/b12.html) and FASTA (http://www.ebi.ac.uk/fasta33/) programs.
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Cloning of Full-Length cDNAs by Phage Library Screening
A mouse seminiferous tubule cDNA UniZAP XR phage library (Stratagene, La Jolla, CA) was screened by using a 32P-labeled CPEB2 RRM-region probe (145 base pairs [bp]) according to standard protocols [14]. Positive plaques were isolated, and plasmids (pBS SK) containing CPEB2 cDNA fragments of at least 1500 nucleotides were excised by helper phage. Sequence analyses were carried out as described above.
Reverse transcription (RT)-PCR was employed to verify the presence of 5' upstream sequences obtained from an EST database search. Ten nanograms of DNase-treated mRNA from testis were reverse transcribed with CPEB2B primer using RT-PCR beads (Amersham Pharmacia Biotech, Piscataway, NJ) in 50 µl. One microliter of the product was PCR amplified using CPEB2A and CPEB2B primers with pfu Turbo DNA polymerase (Stratagene). The cycling reaction was performed in a PCR thermal cycler (Applied Biosystems) at 94°C for 4 min; 30 cycles of 95°C for 1 min, 58°C for 1 min, and 72°C for 1 min; and then 72°C for 5 min.
Northern Blot Analysis and RT-PCR
Total RNA (15 µg) from each tissue sample was separated on a 1.5% agarose-formaldehyde gel and transferred to a Hybond-N+ membrane (Amersham) by upward capillary transfer. The membrane was air-dried, ultraviolet (UV) cross-linked, and prehybridized at 60°C for 4 h in 0.25 M Na2HPO4, 1 mM EDTA, 7% SDS, and 100 µg/ml of sonicated salmon sperm DNA. The membrane was hybridized at 60°C overnight in the same solution, and the 1515-bp cDNA (shown in capital letters in Fig. 1a) in pBS SKII was labeled with [32P]dCTP by random priming. Following hybridization, the membrane was washed to a final stringency of 0.2x SSC (1x SSC: 0.15 M sodium chloride and 0.015 M sodium citrate) at 60°C for 20 min and then exposed and analyzed by FLA2000 BioImage analyzer (Fuji Photo Film, Tokyo, Japan).
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Total RNAs from various mouse tissues previously digested with DNase (10 ng) were reverse transcribed and PCR amplified using the CPEB2-M1P2 and CPEB2-M2P1 primers (Table 1) by Ready-To-Go RT-PCR beads (Amersham). Conditions of RT and PCR were 42°C for 30 min; 95°C for 5 min; 35 cycles of 95°C for 30 sec, 58°C for 30 sec, and 72°C for 30 sec; and then 72°C for 10 min.
mRNA In Situ Hybridization
Sense and antisense riboprobes of 1683-bp mCPEB2 cDNA corresponding to nucleotides 259–1942 were labeled with digoxigenin as outlined in the DIG System protocol (Roche Diagnostics, Basel, Switzerland). Male mice were anesthetized, and testes were removed and fixed with 4% paraformaldehyde (PFA)/PBS. After dehydration, the testes were embedded into paraffin and sectioned (thickness, 8–10 µm). The deparaffinized sections were treated with proteinase K solution (5 µg/ml) for 10 min at 37°C and postfixed with 4% PFA/PBS. After washing with PBS followed by treatment with 0.1 M triethanolamine/0.25% acetic anhydride for 10 min at room temperature, the sections were subjected to in situ hybridization. The mRNA in situ hybridization histochemistry was carried out on tissue sections as outlined in the DIG System protocol with a hybridization temperature of 58°C in a buffer containing 50% formamide.
Chromosome Mapping of CPEB2 Gene by Fluorescence In Situ Hybridization
The direct R-banding fluorescence in situ hybridization (FISH) method was used for determination of mouse chromosomal localization of the CPEB2 gene. Preparation of R-banded chromosomes and FISH were performed as described by Matsuda et al [15] and by Matsuda and Chapman [16]. The same 1683-bp cDNA fragment of CPEB2 used for mRNA in situ hybridization was labeled by nick translation with biotin-labeled 16-dUTP (Roche). The hybridized biotinylated probes were reacted with goat anti-biotin antibody (Vector Laboratories, Burlingame, CA) and then stained with fluoresceinated donkey anti-goat immunoglobulin G (Nordic Immunological Laboratories, Tilburg, Netherlands) at a 1:500 dilution for 1 h at 37°C. The hybridization signals were visualized with Nikon filter sets B-2A and UV-2A (Nikon Corporation, Tokyo, Japan). Kodak Ektachrome ASA 100 films (Eastman Kodak, Rochester, NY) were used for microphotography.
Northwestern Blot Assay for RNA Binding
The Northwestern blot assay for RNA binding was performed as described by Siomi et al. [17] with some modifications. Partially purified CPEB2 RRM-Zn region recombinant proteins expressed in bacteria were separated by 12% SDS-PAGE and transferred electrophoretically to Immobilon polyvinylidene fluoride membranes (Millipore, Billerica, MA). The proteins on the membranes were renatured for 30 min at room temperature in PBS plus 0.5% Tween 20 and 100 µM ZnSO4 and washed twice with PBS. The membranes were then blocked in binding buffer (10 mM Tris-HCl [pH 7.4], 300–500 mM NaCl, 1 mM EDTA, and 1% BSA) overnight at 4°C. The membranes were hybridized with 106 cpm of four [32P]-labeled RNA homopolymers [poly(A), poly(G), poly(C), and poly(U)] and tRNA (20 µg) in the binding buffer for 90 min at room temperature. After five washes (5 min each) with the binding buffer (500 ml), the membranes were dried and subjected to autoradiography.
Transfection of GFP-CPEB2 Expression Plasmid
The full-length coding sequence of CPEB2 cDNA was subcloned into the pEGFP C1 vector, which produces a fusion protein with the green fluorescent protein at the N-terminus of CPEB2. HeLa cells were grown in Dulbecco modified Eagle medium supplemented with 10% fetal calf serum. Cells were transiently transfected with the plasmid construct using Polyfect transfection reagent (Qiagen, Hilden, Germany) according to the manufacturer's protocol. Cells were grown on slide coverglasses (Matsunami Glass, Tokyo, Japan) and fixed at 24-h posttransfection using 4% PFA/PBS, washed two times in PBS, and mounted. Thirty minutes before fixation, 0.6 µg/ml of Hoechst 33258 was added to the cultures for nuclear staining.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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One of the cloned CPEB2 amplification products (no. 51-36) of 145 bp in length contained an open reading frame (ORF) whose deduced amino acid sequence revealed the presence of RNP-2 and RNP-1 sequences at the N- and C-termini, respectively, with several identifier sequences between the RNP-1 and RNP-2 motifs [12, 13]. The sequence of no. 51-36 differed from those of similar genes in human and mouse databases. To further establish if no. 51-36 is a novel RRM protein, it was used as a hybridization probe to isolate longer cDNA clones from a mouse seminiferous tubule cDNA library. After screening 4 x 106 phage, we obtained two positive phage clones. Nucleotide sequence analysis of the two cDNA inserts, containing sequences identical to that of no. 51-36, yielded 1771- and 1700-bp sequences, respectively. Both had essentially the same sequence except for the lack of 71 bp at the 5' region in one cDNA and encoded a single ORF of 501 amino acids (Fig. 1a). The clone had translatable sequence until the proximal upstream sequence. This suggested that the clone might be incomplete at the N-termini.
To address the putative translation initiation site, we conducted a BLAST homology search against the mouse database and found a mouse EST clone (GenBank accession no. BB638853) and a protein coding mRNA predicted from its genome sequence (XM132075). Both extended for 170 bp toward the 5' end. Combining this information, we obtained a 1942-bp continuous ORF encoding 522 amino acid residues (Fig. 1a) with two putative RRMs and C4- and C2H2-type Zn-finger motifs. The RT-PCR for the entire coding sequence (1566 bp) gave a single band with the expected length (Fig. 1b), confirming the existence of the mRNA in the testis. The initiation ATG codon was located at nucleotides 113–115, which reside in a Kozak consensus sequence [18]. A predicted 0.3-kilobase (Kb) 3'-noncoding sequence included a poly(A) tail and a polyadenylation signal (AATAAA). From our protein database survey against deduced amino acid sequences, the protein showed significant homology to Xenopus, mouse, and human CPEBs, proteins involved in translational regulation of specific mRNAs, and with KIAA0940 and KIAA1673. Therefore, we named the protein CPEB2.
When we compared the amino acid sequence homologies of CPEB family proteins, CPEB2, KIAA0940, and KIAA1673 were much closer to each other than to CPEB. Particularly, amino acid sequences in the RRM-Zn finger region, which might be required for specific RNA binding, were almost identical. Next, we examined genetic distance as calculated by the Kimura corrected distance algorithm among CPEB family proteins from various organisms (Fig. 1d). From the tree, CPEB family proteins can be divided into two subgroups. One includes CPEB2-KIAA0940-KIAA1673, Drosophila CG5735, and Caenorhabditis elegans Cpb-1 and Cpb-2. A second group includes CPEB proteins from Spisula, Aplysia, zebrafish, Drosophila, Xenopus, mouse, and human proteins.
Chromosome Mapping of the Mouse CPEB2 Gene
The chromosomal location of the CPEB2 gene was determined by R-banding FISH using a 1683-bp cDNA fragment as a probe (Fig. 2). The CPEB2 gene localized to mouse chromosome 5B distal [19]. From the human genome database, human CPEB2 gene was found on chromosome 4q32-33 (GenBank accession no. NT_022941.2).
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Expression of mCPEB2 mRNA
To determine the expression profile of CPEB2 mRNA, we carried out Northern and RT-PCR analyses. Northern hybridization for samples from various mouse tissues showed that the CPEB2 probe gives rise to a prominent, approximately 2.5-kb transcript in testis exclusively (Fig. 3a). Because we originally cloned CPEB2 from a brain cDNA library, we next examined the expression of CPEB2 by RT-PCR, which demonstrated that all tissues examined (cerebrum, cerebellum, salivary gland, thymus, liver, spleen, kidney, intestine, testis, and ovary) expressed CPEB2 mRNA (Fig. 3b). These data suggested that although the expression of CPEB2 is below the detection level by Northern blot analysis in all tissues but testis, it is ubiquitously expressed in mouse tissues.
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Next, we determined the cell type(s) expressing the cpeb2 mRNA in testis by mRNA in situ hybridization. The 1683-bp cDNA fragment was used for generating antisense and sense riboprobes. The sense probe did not give any hybridization signals at any stages of spermatogenesis (Fig. 4, b and d). Lower magnification (100x) revealed that the distribution of hybridization signals obtained by antisense probe displayed a stage-specific pattern (Fig. 4a). Higher magnification (200x) revealed that spermatogonia and spermatocytes did not express detectable levels of CPEB2 mRNA and that CPEB2 mRNA was confined primarily to round spermatids of steps 1–7 (Fig. 4c), showing the haploid expression. These cells were identified by their location within the seminiferous tubule, their nuclear morphology, and the germ cell composition of the tubule [20].
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CPEB2 Binds to poly(U)
Despite much effort to express recombinant CPEB2 protein in Escherichia coli, we were unable to produce a soluble form of the protein. Therefore, to examine the RNA-binding ability of CPEB2, we used denaturing conditions and Northwestern blot analysis (Fig. 5). CPEB2 binds to poly(U), but not to poly(A), poly(G), or poly(C), in the presence of 300 mM NaCl. The binding of CPEB2 to poly(U) requires renaturation with 100 µM ZnSO4. The binding after incubation with ZnSO4 suggests that CPEB2 might coordinate Zn2+ into its Zn-finger motif in its C-terminus. The binding is reduced as the salt concentration is increased. We conclude that CPEB2 binds to poly(U) preferentially and that the two RRMs and Zn finger are sufficient for binding.
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CPEB2 Protein Localizes in Cytoplasm
To determine the subcellular localization of CPEB2, transfections were performed in HeLa cells using a construct producing a fusion protein of CPEB2 and green fluorescent protein (GFP). Cell and nuclear shapes were identified through nuclear staining by Hoechst 33258 and phase-contrast microscopy. Fluorescent microscopy revealed that the CPEB2-GFP fusion protein was exclusively localized in the cytoplasm of the HeLa cells (Fig. 6). Control transfections with GFP alone did not give any localization.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The deduced amino acid sequence of CPEB2 indicates that it is a novel member of the CPEB protein family. Originally, CPEB was described as a protein that binds the CPE sequences in the 3' untranslated region (UTR) of c-mos mRNA serving as a key regulator of Xenopus oocyte maturation and is involved in translational regulation [7]. The CPEB contains two RRMs and a Zn-finger motif as an RNA-binding domain. Both of the RRM domains and the Zn finger are required to bind CPE [21]. Similarly, CPEB2 may form a Zn-finger structure, because ZnSO4 is facilitated in the renaturation of recombinant CPEB2 protein in the Northwestern blotting analysis with poly(U) (Fig. 5). In addition, recombinant CPEB2 protein binds to Zn-chelate columns (data not shown), as described by Meetei and Rao [22].
Most of the sequence of the human genome is available in the database. Searching the nucleotide sequence of CPEB2 against this database, we found that the human and mouse genomes contain four CPEB-related protein genes, which are on human chromosome 4 (hCPEB2), chromosome 5 (hKIAA1673), chromosome 10 (hKIAA0940), and chromosome 15 (hCPEB). The mouse CPEB2 is on chromosome 5. By comparing amino acid sequences among this family of proteins, we found that the amino acid sequences of N-termini were less conserved than those of the C-termini, which contain the RNA-binding domains (Fig. 1c). In particular, the RNA-binding domains of CPEB2, KIAA0940, and KIAA1673 are highly homologous, suggesting that they might share a similar recognition sequence. In contrast, the N-termini, where the sequences are more divergent, may provide specific functions.
The CPEB-related proteins are expressed in many metazoans as well as vertebrates. To determine the sequence relationship of CPEB2 to other members of CPEB family, we employed the Kimura corrected distance algorithm to construct an unrooted phylogenetic tree based on the RRM and Zn finger (Fig. 1d). Amino acid comparisons demonstrated that the family can be subdivided into two groups. One is CPEB and its orthologues that primarily participate in oogenesis. Another contains CPEB2, KIAA0940, KIAA1673, Cpb-1, and Cpb-2. Both Cpb-1 and Fog-1 are members of CPEB-related proteins, which control key steps during spermatogenesis in C. elegans [23] and are not essential for oogenesis. The high expression of CPEB2 in postmeiotic cells of testis suggests a primary role of CPEB2 during late stages of spermatogenesis.
Possible Function of CPEB2
In addition to the similarity of the primary sequences, CPEB2 shares many similar features with CPEB. In transfections, CPEB2 is localized to the cytoplasm and binds to poly(U) RNA oligomers, as seen for CPE [21], suggesting that CPEB2, like CPEB, may have similar translational regulation functions.
The translational regulation of specific mRNAs is critical in oogenesis and early embryogenesis. Although a number of different mechanisms are probably responsible for the translational control of different mRNAs, one of the best-studied mechanisms is cytoplasmic polyadenylation [8, 9]. Most mRNA is polyadenylated in the nucleus, and a poly(A) tail of approximately 200 nucleotides assures efficient translation in the cytoplasm. In addition, deadenylation of the poly(A) tail of some of mRNAs occurs in the cytoplasm by an uncharacterized mechanism, which likely is different from that regulating the degradation of mRNAs [10]. The mRNAs with short poly(A) tails of approximately 20 nucleotides become translationally quiescent in oocytes. Responding to stimulation, the poly(A) tails are extended again up to approximately 200 nucleotides, and translation is activated. Cytoplasmic polyadenylation requires two cis-acting elements in the 3' UTR of specific mRNAs: a ubiquitous hexanucleotide AAUAAA, which is also necessary for nuclear polyadenylation; and a CPE with a general sequence of UUUUUAAU or UUUUUAU. The protein CPEB functions as a CPE-binding protein in the cytoplasm [6, 7].
In spermatogenesis, the length of poly(A) in mRNAs also correlates with translational activity. Kashiwabara et al. [24] has shown poly(A) elongation of acrosin, sc32, sc38, and actin mRNAs in round spermatids. These mRNAs contain CPE or CPE-like sequences in their 3' UTRs, suggesting that CPE-dependent translational regulation occurs during spermatogenesis as well as oogenesis. Recently, Tay and Richter [25] reported that CPEB-deficient mice lack mature oocytes and, interestingly, that most spermatocytes arrest at the pachytene stage of meiosis. In male germ cells, CPEB mRNA is expressed in pachytene spermatocytes and round spermatids, and CPEB regulates translation of two synaptonemal complex protein mRNAs (SCP1 and SCP3) during meiosis. This finding argues that translational regulation by cytoplasmic polyadenylation is essential during spermatogenesis. Because CPEB2 is abundantly expressed in testis and its expression is restricted to haploid spermatids, we propose that CPEB2 regulates the translation of stored mRNAs during spermiogenesis.
The C. elegans genome contains three CPEB-related protein genes, Cpb-1, Cpb-2, and Fog-1 [23, 26]. Both Cpb-1 and Fog-1 have key functions at different steps of spermatogenesis. Fog-1 has two isoforms, L (long form) and S (short form). Fog-1(L) specifies the sperm cell fate, whereas Fog-1(S) seems to be inactive. Cpb-1 is essential for the progression of spermatogenesis. These CPEB proteins may regulate different mRNAs at different stages of spermatogenesis. Similarly, the expression of CPEB and CPEB2 is regulated both temporally and spatially. The CPEB is known to function during meiosis, and it controls translation of mRNAs encoding synaptonemal complex proteins. Because CPEB2 mRNA is expressed at a later stage of spermatogenesis in postmeiotic male germ cells, we propose that CPEB2 regulates translation of mRNAs necessary for spermiogenesis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Yasuyuki Kurihara, Department of Environment and Natural Sciences, Graduate School of Environment and Information Sciences, Yokohama National University, Tokiwa-dai, Hodogaya, Yokohama 240-8501, Japan. FAX: 81 45 339 4263; kurihara{at}mac.bio.bsk.ynu.ac.jp
Received: 22 January 2003.
First decision: 24 February 2003.
Accepted: 4 March 2003.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1.56 kb). c) Amino acid homologies between CPEB family members. Because no data are available regarding mouse KIAA0940 and KIAA1673, we used the human data. All sequence data were aligned by the CLASTALX program, and homology percentages were calculated. We compared the homology in the entire coding region and the RNA-binding regions (two RRMs and Zn-finger region). d) Unrooted tree calculated by using the Kimura-corrected distance algorithm with the CLUSTALX program (ftp://ftp-igbmc.u-strasbg.fr/pub/ClustalX/) and drawn using the neighbor-joining method with the Phylip Drawtree application
