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RNF151, a testis-specific RING finger protein, interacts with dysbindin
ABB Archives of Biochemistry and Biophysics 465 (2007) 157–163 www.elsevier.com/locate/yabbi
RNF151, a testis-specific RING finger protein, interacts with dysbindin Hong Nian, Cuihong Fan, Shangying Liao, Yuqiang Shi, Keying Zhang, Yixun Liu, Chunsheng Han * State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 10080, China Received 27 April 2007, and in revised form 12 May 2007 Available online 4 June 2007
Abstract RING finger proteins play important roles in spermatogenesis. Here, we report that a novel RING finger protein RNF151, with a C3HC4-type RING finger domain, a putative nuclear localization signal (NLS), and a TRAF-type zinc finger domain, was exclusively expressed in the mouse testis and developmentally regulated during spermatogenesis. While RNF151 mRNA was present in round spermatids, its protein was expressed in elongating spermatids of the stage VIII–IX seminiferous tubules. The NLS together with the RING domain were necessary and sufficient for the nuclear localization of RNF151-EGFP in transfected cells. Yeast two-hybrid screening identified the physical interaction of mouse RNF151 and dysbindin, which was confirmed by the co-immunoprecipitation of the proteins and by their co-localization in intact cells. As dysbindin has lately been shown to be involved in membrane biogenesis and fusion, a key process for acrosome formation, we propose that RNF151 may play a role in acrosome formation. 2007 Elsevier Inc. All rights reserved. Keywords: RING finger protein; RNF151; Spermatogenesis; Testis; Spermatids; Dysbindin
Mammalian spermatogenesis is a multi-step process in which highly specialized haploid sperms are produced from diploid spermatogonial stem cells (SSCs)1 in the testis [1,2]. As a first step, SSCs undergo mitotic division and differentiation to produce intermediate types of spermatogonia and primary spermatocytes. Subsequently, spermatocytes divide meiotically giving rise to round spermatids. The round spermatids then initiate a complex differentiation process named spermiogenesis in which elongated spermatids and mature sperms are generated and released into the lumen of the seminiferous tubules. During the whole process, the expressions of and the interactions between diverse gene families such as transcription factors, cell cycle regulators, enzymes, and so on, are required and regulated in an ordered manner [2]. Some of these genes or their alternative transcripts are specifically expressed in the testis, and the number of testis-specific genes was estimated *
Corresponding author. Fax: +86 10 64807105. E-mail address: [email protected] (C. Han). 1 Abbreviations used: NLS, nuclear localization signal; SSCs, spermatogonial stem cells; ZFPs, zinc finger proteins; RNFs, RING finger proteins. 0003-9861/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2007.05.013
to be close to 4% of the whole mouse genome [3]. Identification of these genes and their roles is important in understanding the mechanism of spermatogenesis. The zinc finger proteins (ZFPs) constitute one of the largest gene families in mammalian genomes. According to a recent report, 1573 sequences containing 46 different conserved zinc finger domains are listed in the InterPro database [4]. One sub-family of the ZFPs are the RING finger proteins (RNFs) characteristic of a cysteine-rich domain CX2CX(9–39)CX(1-3)HX(2-3)CX2CX(4-48)CX2C that is cable of chelating two zinc ions using a cross-brace structure. RNFs are involved in biological processes such as signal transduction, transcriptional regulation, ubiquitination, and apoptosis [5,6]. Up to now, more than 30 RNFs have been reported to be involved in spermatogenesis in human, mice, and rats. For examples, RNF17, a germ cell-specific gene, is a component of a novel germ cell nuage and is required for the differentiation of male germ cells [7]. MEX, a testis-specific E3 ubiquitin ligase, promotes death receptor-induced apoptosis [8]. Other RNFs expressed in the testis include SNURF [9], ZNF313 [10], ZNF230 [11], OIP1 [12], Haprin [13], to name a few. Except for sharing
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the common RING domains and the testis expression specificity, these RNFs are diverse in terms of their sequences, structures, and functions. Here, we report the expression, localization of a novel testis-specific RING finger protein, RNF151 and its interaction with protein dysbindin. We hypothesize that RNF151 might participate in acrosome formation of spermatids. Materials and methods Animals Adult CD-1 mice were obtained from the Experimental Animal Center, Chinese Academy of Sciences. The animals were treated in accordance with the NIH Guide for the Care and Use of Laboratory Animals. All protocols are approved by the Committee of Animal Care and Use of the Institute of Zoology, Chinese Academy of Sciences.
RNA isolation and RT-PCR Total RNA was isolated from various mouse tissues and mouse testes at different stages of development. RT was performed by using 5 lg total RNA, oligo(dT), and SuperScript III RNase H Reverse Transcriptase (Invitrogen). Detailed procedure followed the manufacturer’s protocol. The primers used to detect RNF151 mRNA expression were 5 0 -CAGCATATGAGTGGTGGGTACGATCT-3 0 (sense) 5 0 -CTACTCGAGACTTTGGCCCTGCGTAC-3 0 (antisense). For PCR, reaction mixture was first heated at 94 C for 2 min. 25 cycles were then carried out with the following parameters: denaturing at 94 C for 20 s, annealing at 58 C for 30 s, extension at 72 C for 30 s. Reaction was finished with a final extension at 72 C for 10 min.
In situ hybridization In situ hybridizations were carried out as previously described [14]. Briefly, a 483 bp fragment corresponding to the protein coding sequence of RNF151 was subcloned into the pGEM-T vector (Promega). Digoxigenin (DIG)-labeled antisense and sense RNA probes were prepared using the DIG RNA-Labeling Kit (Roche). Hybridizations to testis sections were carried out at 55 C for 24 h, and color signals were developed using alkaline phosphatase-conjugated anti-DIG antibody with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate as substrates.
Preparation of GST–RNF151 fusion protein and its rabbit polyclonal antiserum
stained with alkaline phosphate red substrate (Vector Laboratories) and were counterstained with hematoxylin. Pre-immune serum was used as a negative control.
Fluorescent protein-tagging studies RNF151 ORF and different truncated forms (Fig. 3) were subcloned into the pEGFP–N1 vector (Clontech) to produce GFP fusion proteins. The dysbindin ORF was subcloned into pDsRed-N1 to produce RFP fusion proteins. GC-2 cells were plated (3 · 105 cells/well) in six-well plates containing sterilized glass cover slips and transfected the following day using Lipofectamine 2000 by following manufacturer’s manuals (Invitrogen). Twenty-four hours after transfection, cells were fixed with 4% paraformaldehyde for 15 min and then permeabilized with 0.2% Triton X100 for 10 min. After washing with PBS, Hoechst 33258 (Sigma) was added in a final concentration of 1 lg/ml to stain the nucleus for 10 min, mounted on microscope slides. Protein subcellular localizations were examined under a laser confocal microscope (Zeiss).
Yeast two-hybrid screening Yeast two-hybrid screening was performed by using the Matchmaker library construction and screening kit (Clontech, Cat. No. K1615-1). The bait plasmid was constructed by subcloning full-length RNF151 cDNA into pGBKT7. To construct the testis cDNA library, mRNA (1 lg) isolated from 5 adult mice was used to synthesize the first-strand cDNA using an oligo(dT) primer. Double-strand cDNA was synthesized with SMARTIII and CDSIII anchors. The AD fusion library construction and two-hybrid screen were carried out in one step by co-transforming the yeast strain AH109 with ds cDNA, pGADT7-Rec and pGBKT7. Colonies were picked from SD/-Ade/-His/-Leu/-Trp/X-a-Gal selection plates after 5 days. The inserts of selected positive clones were sequenced and identified by searching the NCBI BLAST database.
Co-immunoprecipitation of in vitro translated proteins The in vitro transcription/translation reactions were performed using the TNT Quick Coupled Transcripton/Translation Systems (Promega). The reactions were carried out according to manufacturer’s protocols. For co-immunoprecipitation analysis, 10 ll in vitro translated (35S-methionine-labeled) RNF151 was incubated with 10 ll radio-labeled dysbindin protein and then immunoprecipitated with the appropriate antibody (antiMyc or anti-HA antibody). The co-precipitated labeled proteins were detected by autoradiography following SDS–PAGE.
Co-immunoprecipitation of proteins from cell lysate
The RNF151 cDNA fragments encoding the N-terminal 68 amino acids (16–83) was subcloned into pGEX-4T-1 (Pharmacia Biosciences). The GST–RNF151 fusion protein was expressed in Escherichia coli BL21 and purified using a GSTrap FF column (Amersham Pharmacia) according to the manufacturer’s instructions. 500 lg of purified protein was emulsified in complete Freund’s adjuvant and injected into healthy rabbits, and 3 boosting injections with 500 lg protein emulsified in incomplete Freund’s adjuvant ensued with 3-week intervals in between.
293-T cells were transfected with pFLAG-CMV-4-RNF151 and/or pEGFP–N1-dysbindin. 24 h later, cells were harvested and lysed in lysis buffer (1% Nonidet P-40, 50 mM Tris–HCl, pH 7.4, 10% glycerol, 150 mM NaCl, 1 mM EDTA, pH 8.0) with a protease inhibitor cocktail (Sigma). Subsequently, an aliquot of the cell lysate was incubated with anti-FLAG M2 agarose resin (Sigma) for 4 h at 4 C with agitation. The resin was then washed five times with PBS. The pulled-down protein complexes were then examined by Western blotting analysis using anti-GFP antibody (Santa Cruz). The expression of the proteins in the transfected cells were confirmed by Western blotting analysis using the anti-FLAG antibody (Sigma) and the anti-GFP antibody.
Immunohistochemistry
Results
Immunohistochemistry was performed as previously described [14] . Briefly, 5 lm paraffin sections of mouse testis were incubated with the rabbit antiserum (diluted 1:300 in 10% horse serum–PBS, RT, 30 min), the biotin-labeled secondary antibody (RT, 30 min) and the alkaline phosphatase-conjugated streptavidin (RT, 30 min) sequentially. Sections were
Structural features of RNF151 In search for testis-specific genes, one gene named RNF151 in the NCBI Unigene database (NM_026205)
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was identified as its ESTs are only distributed in the testis. An alignment of mouse RNF151 with orthologous protein sequences in Rattus norvegicus, Canis familiaris, Bos Taurus, Macaca mulatta, Homo sapiens, Pan troglodytes indicates that the RNF151 proteins are well-conserved among these species with an average identity of 48% over their full lengths (Fig. 1). Interestingly, all the RNF151 proteins have a C3HC4-type RING domain and one TRAF-type zinc finger domain. Analysis of mouse RNF151 amino acid sequence by pSORTII reveals one putative bipartite nuclear localization signal (NLS, 58RKEVTRRKMVEVNKLRK74) right after the RING domain. This putative NLS is well-conserved among all the RNF151 orthologs. Spatial and temporal expression of RNF151 mRNA RT-PCR analysis indicated that RNF151 mRNA expression was indeed restricted to the testes of the mouse (Fig. 2a). To examine whether its mRNA expression is developmentally regulated, total RNA samples were prepared from mouse testes of 0–70 days post partum (dpp). As shown in Fig. 2b, RNF151 mRNA expression was only detected in the testis of 21 dpp and thereafter, and the expression level seemed to keep increasing until 35 dpp. As the first wave of spermatogenesis after birth takes 35 days in the mouse and round spermatids start to appear 21 dpp, these results suggested that RNF151 mRNA might be expressed in round spermatids. Cell type distribution of RNF151 mRNA and protein in mouse testis In situ hybridization was employed to investigate cell type distribution of RNF151 mRNA. As shown in Fig. 2c and d, RNF151mRNA was present in round sper-
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matids of the stages VII–VIII seminiferous tubules. To study the protein expression of mouse RNF151, we developed polyclonal antiserum against the N-terminal 68 amino acids of the RNF151 produced from E. coli (Fig. 3a), and used it to perform immunohistochemical staining of the testis tissue sections of the adult mice. The specificity of the antiserum was confirmed by the Western blotting result as a single band of the predicted size (26 kDa) was detected in the mouse total testicular lysate (Fig. 3b). Immunohistochemical data indicated that RNF151 protein was present in the cytoplasm of elongating spermatids of stages VIII–IX seminiferous tubules (Fig. 3c and d), but not in tubules of other stages.
Subcellular localization of EGFP-RNF151 protein in transfected cells The identification of a putative NLS in RNF151 proteins suggests that they might function as nuclear proteins under certain conditions. To test this, we made a series of plasmid constructs from which EGFP fusion proteins of mouse full-length (ORF) RNF151 and its truncated forms could be expressed in mammalian cells. The cells to be transfected are the GC-2 cell line, immortalized preleptotene spermatocytes with SV40 large T antigen [15]. As shown by Fig. 4b, the full-length RNF151 fusion protein displayed a speckled nuclear distribution. When the Nterminal part containing the RING domain and NLS was removed, the protein was now localized in the cytoplasm (Fig. 4c). Adding back NLS to the truncated protein recovered its nuclear localization partially (Fig. 4d), suggesting that the RING domain also plays a role in transporting the protein into the nucleus. RING domain plus NLS only are sufficient for the nuclear localization of the fusion protein although a uniform but not speckled pattern was observed (Fig. 4e). When the TRAF-type zinc finger
Fig. 1. Amino acid sequence alignment RNF151 homologues. Sequence alignment of mouse (NP_080481), rat (XP_220225), dog (XP_853720), cattle (NP_001070506), rhesus monkey (XP_001082447), human (NP_777563), and chimpanzee (XP_001161952) was performed by using the EBI clustalw web tool. The C3HC4-type RING domain and one TRAF-type zinc finger domain are underlined with black lines, while the putative NLS is underlined with a blue line.
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Fig. 2. mRNA expression of mouse RNF151 gene. mRNA of mouse RNF151 was only detected in the testis (a) of 21 dpp and thereafter (b) by RT-PCR . The integrity of the extracted RNAs was indicated by mRNA expression of G3PDH. In situ hybridization showed that RNF151 mRNA was restricted to round spermatids (c, 100·; d, 400·).
Fig. 3. Immunohistochemical localization of RNF151 in mouse testis. GST fusion protein of the N-terminus amino acid (16–83) of RNF151 was purified from E. coli (a) and was used to generate a rabbit polyclonal antiserum that detected a protein of 26 kDa in the testis cell lysate (b). Anti-RNF151 antiserum was used as primary antibody to detect the expression of the RNF151. The presence of RNF151 was revealed by red staining. RNF151 was stained in the cytoplasm of elongating spermatids of stage VIII–IX seminiferous tubules (c, 200·; d, 600·).
domain was added to the RING plus NLS fragment, the speckled nuclear localization of the full-length protein was totally recovered (Fig. 4f). This implied that the TRAF-type zinc finger domain may play a role in the formation of the specked pattern indicative of protein selfaggregation. As expected, RING domain alone distributed uniformly in the transfected cells (Fig. 4g) while the C-terminal segment after the TRAF-type zinc fingers was only
localized in the cytoplasm (Fig. 4h). These data indicated that the RING domain plus NLS are necessary and sufficient for the nuclear localization of RNF151. RNF151 interacts with dysbindin Yeast two-hybrid system was employed to screen out potential binding partners of mouse RNF151. Of the 30
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Fig. 4. Subcellular distribution of RNF151 wild-type and six different truncated proteins in GC-2 cells. Green color is from the EGFP or EGFP-fusion proteins while the blue one is from the Hoechst 33258-stained cell nuclei. NLS, the putative nuclear localization signal; RING, the RING domain; TRAF, the TRAF-type zinc fingers domain. Magnification, 400·.
positive clones acquired, 4 clones encode full-length cDNAs for a protein named dysbindin, and 5 for RNF151 itself. The interaction between RNF151 and dysbindin was first confirmed by the growth of the yeast cells co-transfected with these two proteins on the SD/-Ade/His/-Leu/-Trp/X-a-Gal selection medium. The interaction was confirmed next by in vitro binding assays (Fig. 5a). In these assays, radioactive c-Myc-tagged RNF151 and HA-tagged dysbindin proteins were first synthesized separately by in vitro transcription and translation of the respective genes in the rabbit reticulocyte lysates in which 35 S-methionine was included. When the proteins were mixed, both proteins could be pulled down by either the c-Myc antibody or the HA antibody as a result of their physical interaction. The interaction between RNF151 and dysbindin was further confirmed by co-immunoprecipitation of FLAG-RNF151 and GFP–dysbindin that were expressed in 293-T cells (Fig. 5b). As another evidence for the interaction between these two proteins, GFP– RNF151 and RFP–dysbindin co-localized in the nuclei of transfected GC-2 cells (Fig. 5c). Taken together, these observations have indicated that dysbindin can be a bona fide binding partner of RNF151. Discussion Spermatogenesis, one of the most complex biological processes, involves active cell mitotic and meiotic divisions, morphological changes, and apoptosis. These activities are driven by the expressions of different gene families and
their protein interactions [16,17]. Gene expressions are especially active after the accomplishment of meiosis when round spermatids are ready to start an astonishing metamorphic journey to become the highly specialized sperms [18]. The testis is well-known for its possession of quite a number of tissue specific genes [3]. These genes are termed ‘‘Chauvinist genes’’ by Eddy [19], and are either transcribed specifically or with their transcripts post-transcriptionally processed in a testis-specific manner. Identification of the full complement of these genes will lay the solid ground for our further understanding and better manipulation of spermatogenesis. The RNFs are a subfamily of the zinc finger protein family. RNFs are very disparate in terms of their sequences and functions except for sharing the common RING domains [5]. RNFs participate in multiple processes such as gene expression regulation, enzymatic reactions, membrane traffic, protein complex formation, to name a few. To our knowledge, more than 30 RNFs have been reported to be involved in spermatogenesis in the mouse, rat, and human. In the present study, we have added to this growing list mouse RNF151, whose transcript is exclusively present in the round spermatids. We have also identified another testis-specific membrane bound RNF (data not shown), and try to make this testicular RNF list complete. In addition to the RING domains, RNFs usually possess other domains that together with the RING mediate protein–protein interactions. Except for the C3HC4-type RING domain, RNF151 also have a TRAF-type zinc finger domain. The mouse RNF151 shares low but
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Fig. 5. Interaction between RNF151 and dysbindin. (a) Co-immunoprecipitation of RNF151 and dysbindin produced by in vitro translation. Lane 1: RNF151 + anti-Myc antibody; lane 2: dysbindin + anti-HA antibody; lanes 3 and 4: RNF151 + dysbindin + anti-HA or Myc antibody. (b) Co-immunoprecipitation of RNF151 and dysbinding from transfected 293-T cells. 293-T cells were transfected with FLAG-RNF151 and EGFP–dysbindin. RNF151 proteins were immunoprecipitated with anti-FLAG antibody, and Western blot analyses were performed with anti-GFP antibody. (c) Co-localization of RNF151 and dysbindin proteins. GFP–RNF151 and RFP–dysbindin proteins were indicated by green and red colors, and their co-localization was demonstrated by yellow color in the overlaid image. Magnification, 400·. WB, Western blotting; IP, immunoprecipitation.
significant homologies with 3 TNF receptor associated factors (TRAF), TRAF3, 5, and 6. All TRAFs except for TRAF2 have a RING domain with a unique spacing pattern between the zinc chelating residuals (CX2CX(11,12)CX1HX2CX2CX(9,11)CX2C), and all of them except for TRAF1 possess 5–7 zinc fingers [20]. RNF151 also has the TRAF-type RING domain and zinc fingers. These features indicate that RNF151 proteins are distally related to the TRAF family more than to any other protein families. TRAFs are usually cytoplasmic proteins. The immunohistochemical staining of mouse RNF151 on testis sections indicated that it was predominantly cytoplas-
mic. However, the presence of a well-conserved NLS in RNF151 proteins suggests that they could relocate into the nucleus under certain conditions. Consistent with this hypothesis, the full-length mouse RNF151 with EGFP fused to its C-terminus was localized to the nuclei as speckles indicative of protein aggregates. Our data indicated that RING plus NLS are necessary and sufficient for RNF151 being localized in the nucleus, and that the TRAF-type zinc finger domain may mediate self-aggregation of the protein. Therefore, we speculate that, under normal condition, RNF151 stays in the cytoplasm either due to direct tethering by its interacting protein(s), and/or due to the unavailability of NLS to nuclear transporting proteins as a result of its interactions with other proteins. In the transfected cells, the protein translocates into the nucleus because the tethering protein is not present and/or the fused protein renders NLS accessible by nuclear transporting proteins. It is important to investigate whether and how such translocation occurs in vivo. Search for interacting target by using the yeast twohybrid system provide valuable information about the function of a novel gene. Thirty independent colonies resulted from interactions between mouse RNF151 and potential targets were acquired, out of which 5 contained full-length coding sequence of mouse RNF151 itself and 4 contained full-length sequences for dysbindin. It is not surprising that RNF151 interacts with itself in yeast cells for the following two reasons: First, RNFs are well known to form aggregates in cells. For example, TRAFs, to which RNF151 proteins bear significant sequence and structural similarities, form homotrimers [15]; Second, the speckled nuclear localization pattern of mouse RNF151 in transfected cells is indicative of self-aggregation. The interaction between RNF151 and dysbindin interacting was confirmed by the co-immunoprecipitation of the in vitro translated proteins or proteins expressed in transfected cells as well as by the proteins’ co-localization in transfected cells. However, we understand that interaction between RNF151 and dysbindin is not without any doubt because the yeast two-hybrid screening and assays used to confirm their interaction are all in vitro experiments. It would be more convincing to show that these two proteins co-immunoprecipitate from testicular extracts and co-localize in testicular tissue sections, both of which would be carried out when the antibody against dysbindin is available. It would also be interesting to examine whether the interaction between RNF151 and dysbindin is mediated by the RING domain of RNF151 and whether other testicular RNFs also bind to dysbindin so that the developmental importance of the two proteins’ interaction could be clarified. Dysbindin was first identified, by using the yeast twohybrid system, as a binding partner of dystrobrevin, a component of the dystrophin-associated protein complex (DPC) in both muscle and nonmuscle cells [21]. It has attracted wide interest because it is a susceptibility gene for Schizophrenia [22,23]. Dysbindin was lately reported
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to be a synaptic and microtubular protein that binds brain snapin, a SNARE-binding protein that mediated intracellular membrane fusion in both neuronal and non-neuronal cells [24,25]. Dysbindin is also a component of a multisubunit protein complex named BLOC-1 (for Biogenesis of lysosome-related organelles complex-1), which is required for the normal biogenesis of specialized organelles of the endosomal–lysosomal system, such as melanosomes and platelet dense granule [26]. Mutations in genes of dysbindin and the other components of BLOC-1 lead to the so-called Hermansky–Pudlak syndrome in human characterized by oculocutaneous albinism, prolonged bleeding, and pulmonary fibrosis due to abnormal vesicle trafficking to lysosomes and related organelles, such as melanosomes and platelet dense granules [27]. One key process of the spermiogenesis is the acrosome biogenesis started at the round spermatid stage. Acrosome formation involves membrane trafficking from Golgi apparatus and probably lysosomes [28,29]. It has been shown recently that the assembly of spermatid acrosome also depends on microtubule organization [30]. Based on these clues, it is reasonable to propose that RNF151 might regulate acrosome formation through interacting with multiple proteins participating in membrane biogenesis and microtubule organization. The best proof of this hypothesis is to see spermatogenesis stalked in the pre-acrosomal stage in knockout mice, a work currently undergoing in our laboratory. Acknowledgments Financial support was provided by the National Natural Science Foundation of China (Grant No. 30428030 and No. 90508008), the ‘‘Bai Ren Ji Hua’’ Fund of the Chinese Academy of Sciences. References [1] R.A.E.L.D. Russell, A.P. Sinha Hikim, E.D. Clegg (Eds.), Histological and Histopathological Evaluation of the Testis, Clearwater, Florida, 1990. [2] R.M. Sharpe (Ed.), Regulation of Spermatogenesis, New York, 1993. [3] N. Schultz, F.K. Hamra, D.L. Garbers, Proceedings of the National Academy of Sciences of the United States of America 100 (2003) 12201–12206. [4] T. Ravasi, T. Huber, M. Zavolan, A. Forrest, T. Gaasterland, S. Grimmond, D.A. Hume, Genome Research 13 (2003) 1430–1442. [5] K.L. Borden, Journal of Molecular Biology 295 (2000) 1103–1112. [6] P.S. Freemont, Current Biology 10 (2000) R84–R87.
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RNF151, a testis-specific RING finger protein, interacts with dysbindin Hong Nian, Cuihong Fan, Shangying Liao, Yuqiang Shi, Keying Zhang, Yixun Liu, Chunsheng Han * State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 10080, China Received 27 April 2007, and in revised form 12 May 2007 Available online 4 June 2007
Abstract RING finger proteins play important roles in spermatogenesis. Here, we report that a novel RING finger protein RNF151, with a C3HC4-type RING finger domain, a putative nuclear localization signal (NLS), and a TRAF-type zinc finger domain, was exclusively expressed in the mouse testis and developmentally regulated during spermatogenesis. While RNF151 mRNA was present in round spermatids, its protein was expressed in elongating spermatids of the stage VIII–IX seminiferous tubules. The NLS together with the RING domain were necessary and sufficient for the nuclear localization of RNF151-EGFP in transfected cells. Yeast two-hybrid screening identified the physical interaction of mouse RNF151 and dysbindin, which was confirmed by the co-immunoprecipitation of the proteins and by their co-localization in intact cells. As dysbindin has lately been shown to be involved in membrane biogenesis and fusion, a key process for acrosome formation, we propose that RNF151 may play a role in acrosome formation. 2007 Elsevier Inc. All rights reserved. Keywords: RING finger protein; RNF151; Spermatogenesis; Testis; Spermatids; Dysbindin
Mammalian spermatogenesis is a multi-step process in which highly specialized haploid sperms are produced from diploid spermatogonial stem cells (SSCs)1 in the testis [1,2]. As a first step, SSCs undergo mitotic division and differentiation to produce intermediate types of spermatogonia and primary spermatocytes. Subsequently, spermatocytes divide meiotically giving rise to round spermatids. The round spermatids then initiate a complex differentiation process named spermiogenesis in which elongated spermatids and mature sperms are generated and released into the lumen of the seminiferous tubules. During the whole process, the expressions of and the interactions between diverse gene families such as transcription factors, cell cycle regulators, enzymes, and so on, are required and regulated in an ordered manner [2]. Some of these genes or their alternative transcripts are specifically expressed in the testis, and the number of testis-specific genes was estimated *
Corresponding author. Fax: +86 10 64807105. E-mail address: [email protected] (C. Han). 1 Abbreviations used: NLS, nuclear localization signal; SSCs, spermatogonial stem cells; ZFPs, zinc finger proteins; RNFs, RING finger proteins. 0003-9861/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2007.05.013
to be close to 4% of the whole mouse genome [3]. Identification of these genes and their roles is important in understanding the mechanism of spermatogenesis. The zinc finger proteins (ZFPs) constitute one of the largest gene families in mammalian genomes. According to a recent report, 1573 sequences containing 46 different conserved zinc finger domains are listed in the InterPro database [4]. One sub-family of the ZFPs are the RING finger proteins (RNFs) characteristic of a cysteine-rich domain CX2CX(9–39)CX(1-3)HX(2-3)CX2CX(4-48)CX2C that is cable of chelating two zinc ions using a cross-brace structure. RNFs are involved in biological processes such as signal transduction, transcriptional regulation, ubiquitination, and apoptosis [5,6]. Up to now, more than 30 RNFs have been reported to be involved in spermatogenesis in human, mice, and rats. For examples, RNF17, a germ cell-specific gene, is a component of a novel germ cell nuage and is required for the differentiation of male germ cells [7]. MEX, a testis-specific E3 ubiquitin ligase, promotes death receptor-induced apoptosis [8]. Other RNFs expressed in the testis include SNURF [9], ZNF313 [10], ZNF230 [11], OIP1 [12], Haprin [13], to name a few. Except for sharing
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the common RING domains and the testis expression specificity, these RNFs are diverse in terms of their sequences, structures, and functions. Here, we report the expression, localization of a novel testis-specific RING finger protein, RNF151 and its interaction with protein dysbindin. We hypothesize that RNF151 might participate in acrosome formation of spermatids. Materials and methods Animals Adult CD-1 mice were obtained from the Experimental Animal Center, Chinese Academy of Sciences. The animals were treated in accordance with the NIH Guide for the Care and Use of Laboratory Animals. All protocols are approved by the Committee of Animal Care and Use of the Institute of Zoology, Chinese Academy of Sciences.
RNA isolation and RT-PCR Total RNA was isolated from various mouse tissues and mouse testes at different stages of development. RT was performed by using 5 lg total RNA, oligo(dT), and SuperScript III RNase H Reverse Transcriptase (Invitrogen). Detailed procedure followed the manufacturer’s protocol. The primers used to detect RNF151 mRNA expression were 5 0 -CAGCATATGAGTGGTGGGTACGATCT-3 0 (sense) 5 0 -CTACTCGAGACTTTGGCCCTGCGTAC-3 0 (antisense). For PCR, reaction mixture was first heated at 94 C for 2 min. 25 cycles were then carried out with the following parameters: denaturing at 94 C for 20 s, annealing at 58 C for 30 s, extension at 72 C for 30 s. Reaction was finished with a final extension at 72 C for 10 min.
In situ hybridization In situ hybridizations were carried out as previously described [14]. Briefly, a 483 bp fragment corresponding to the protein coding sequence of RNF151 was subcloned into the pGEM-T vector (Promega). Digoxigenin (DIG)-labeled antisense and sense RNA probes were prepared using the DIG RNA-Labeling Kit (Roche). Hybridizations to testis sections were carried out at 55 C for 24 h, and color signals were developed using alkaline phosphatase-conjugated anti-DIG antibody with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate as substrates.
Preparation of GST–RNF151 fusion protein and its rabbit polyclonal antiserum
stained with alkaline phosphate red substrate (Vector Laboratories) and were counterstained with hematoxylin. Pre-immune serum was used as a negative control.
Fluorescent protein-tagging studies RNF151 ORF and different truncated forms (Fig. 3) were subcloned into the pEGFP–N1 vector (Clontech) to produce GFP fusion proteins. The dysbindin ORF was subcloned into pDsRed-N1 to produce RFP fusion proteins. GC-2 cells were plated (3 · 105 cells/well) in six-well plates containing sterilized glass cover slips and transfected the following day using Lipofectamine 2000 by following manufacturer’s manuals (Invitrogen). Twenty-four hours after transfection, cells were fixed with 4% paraformaldehyde for 15 min and then permeabilized with 0.2% Triton X100 for 10 min. After washing with PBS, Hoechst 33258 (Sigma) was added in a final concentration of 1 lg/ml to stain the nucleus for 10 min, mounted on microscope slides. Protein subcellular localizations were examined under a laser confocal microscope (Zeiss).
Yeast two-hybrid screening Yeast two-hybrid screening was performed by using the Matchmaker library construction and screening kit (Clontech, Cat. No. K1615-1). The bait plasmid was constructed by subcloning full-length RNF151 cDNA into pGBKT7. To construct the testis cDNA library, mRNA (1 lg) isolated from 5 adult mice was used to synthesize the first-strand cDNA using an oligo(dT) primer. Double-strand cDNA was synthesized with SMARTIII and CDSIII anchors. The AD fusion library construction and two-hybrid screen were carried out in one step by co-transforming the yeast strain AH109 with ds cDNA, pGADT7-Rec and pGBKT7. Colonies were picked from SD/-Ade/-His/-Leu/-Trp/X-a-Gal selection plates after 5 days. The inserts of selected positive clones were sequenced and identified by searching the NCBI BLAST database.
Co-immunoprecipitation of in vitro translated proteins The in vitro transcription/translation reactions were performed using the TNT Quick Coupled Transcripton/Translation Systems (Promega). The reactions were carried out according to manufacturer’s protocols. For co-immunoprecipitation analysis, 10 ll in vitro translated (35S-methionine-labeled) RNF151 was incubated with 10 ll radio-labeled dysbindin protein and then immunoprecipitated with the appropriate antibody (antiMyc or anti-HA antibody). The co-precipitated labeled proteins were detected by autoradiography following SDS–PAGE.
Co-immunoprecipitation of proteins from cell lysate
The RNF151 cDNA fragments encoding the N-terminal 68 amino acids (16–83) was subcloned into pGEX-4T-1 (Pharmacia Biosciences). The GST–RNF151 fusion protein was expressed in Escherichia coli BL21 and purified using a GSTrap FF column (Amersham Pharmacia) according to the manufacturer’s instructions. 500 lg of purified protein was emulsified in complete Freund’s adjuvant and injected into healthy rabbits, and 3 boosting injections with 500 lg protein emulsified in incomplete Freund’s adjuvant ensued with 3-week intervals in between.
293-T cells were transfected with pFLAG-CMV-4-RNF151 and/or pEGFP–N1-dysbindin. 24 h later, cells were harvested and lysed in lysis buffer (1% Nonidet P-40, 50 mM Tris–HCl, pH 7.4, 10% glycerol, 150 mM NaCl, 1 mM EDTA, pH 8.0) with a protease inhibitor cocktail (Sigma). Subsequently, an aliquot of the cell lysate was incubated with anti-FLAG M2 agarose resin (Sigma) for 4 h at 4 C with agitation. The resin was then washed five times with PBS. The pulled-down protein complexes were then examined by Western blotting analysis using anti-GFP antibody (Santa Cruz). The expression of the proteins in the transfected cells were confirmed by Western blotting analysis using the anti-FLAG antibody (Sigma) and the anti-GFP antibody.
Immunohistochemistry
Results
Immunohistochemistry was performed as previously described [14] . Briefly, 5 lm paraffin sections of mouse testis were incubated with the rabbit antiserum (diluted 1:300 in 10% horse serum–PBS, RT, 30 min), the biotin-labeled secondary antibody (RT, 30 min) and the alkaline phosphatase-conjugated streptavidin (RT, 30 min) sequentially. Sections were
Structural features of RNF151 In search for testis-specific genes, one gene named RNF151 in the NCBI Unigene database (NM_026205)
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was identified as its ESTs are only distributed in the testis. An alignment of mouse RNF151 with orthologous protein sequences in Rattus norvegicus, Canis familiaris, Bos Taurus, Macaca mulatta, Homo sapiens, Pan troglodytes indicates that the RNF151 proteins are well-conserved among these species with an average identity of 48% over their full lengths (Fig. 1). Interestingly, all the RNF151 proteins have a C3HC4-type RING domain and one TRAF-type zinc finger domain. Analysis of mouse RNF151 amino acid sequence by pSORTII reveals one putative bipartite nuclear localization signal (NLS, 58RKEVTRRKMVEVNKLRK74) right after the RING domain. This putative NLS is well-conserved among all the RNF151 orthologs. Spatial and temporal expression of RNF151 mRNA RT-PCR analysis indicated that RNF151 mRNA expression was indeed restricted to the testes of the mouse (Fig. 2a). To examine whether its mRNA expression is developmentally regulated, total RNA samples were prepared from mouse testes of 0–70 days post partum (dpp). As shown in Fig. 2b, RNF151 mRNA expression was only detected in the testis of 21 dpp and thereafter, and the expression level seemed to keep increasing until 35 dpp. As the first wave of spermatogenesis after birth takes 35 days in the mouse and round spermatids start to appear 21 dpp, these results suggested that RNF151 mRNA might be expressed in round spermatids. Cell type distribution of RNF151 mRNA and protein in mouse testis In situ hybridization was employed to investigate cell type distribution of RNF151 mRNA. As shown in Fig. 2c and d, RNF151mRNA was present in round sper-
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matids of the stages VII–VIII seminiferous tubules. To study the protein expression of mouse RNF151, we developed polyclonal antiserum against the N-terminal 68 amino acids of the RNF151 produced from E. coli (Fig. 3a), and used it to perform immunohistochemical staining of the testis tissue sections of the adult mice. The specificity of the antiserum was confirmed by the Western blotting result as a single band of the predicted size (26 kDa) was detected in the mouse total testicular lysate (Fig. 3b). Immunohistochemical data indicated that RNF151 protein was present in the cytoplasm of elongating spermatids of stages VIII–IX seminiferous tubules (Fig. 3c and d), but not in tubules of other stages.
Subcellular localization of EGFP-RNF151 protein in transfected cells The identification of a putative NLS in RNF151 proteins suggests that they might function as nuclear proteins under certain conditions. To test this, we made a series of plasmid constructs from which EGFP fusion proteins of mouse full-length (ORF) RNF151 and its truncated forms could be expressed in mammalian cells. The cells to be transfected are the GC-2 cell line, immortalized preleptotene spermatocytes with SV40 large T antigen [15]. As shown by Fig. 4b, the full-length RNF151 fusion protein displayed a speckled nuclear distribution. When the Nterminal part containing the RING domain and NLS was removed, the protein was now localized in the cytoplasm (Fig. 4c). Adding back NLS to the truncated protein recovered its nuclear localization partially (Fig. 4d), suggesting that the RING domain also plays a role in transporting the protein into the nucleus. RING domain plus NLS only are sufficient for the nuclear localization of the fusion protein although a uniform but not speckled pattern was observed (Fig. 4e). When the TRAF-type zinc finger
Fig. 1. Amino acid sequence alignment RNF151 homologues. Sequence alignment of mouse (NP_080481), rat (XP_220225), dog (XP_853720), cattle (NP_001070506), rhesus monkey (XP_001082447), human (NP_777563), and chimpanzee (XP_001161952) was performed by using the EBI clustalw web tool. The C3HC4-type RING domain and one TRAF-type zinc finger domain are underlined with black lines, while the putative NLS is underlined with a blue line.
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Fig. 2. mRNA expression of mouse RNF151 gene. mRNA of mouse RNF151 was only detected in the testis (a) of 21 dpp and thereafter (b) by RT-PCR . The integrity of the extracted RNAs was indicated by mRNA expression of G3PDH. In situ hybridization showed that RNF151 mRNA was restricted to round spermatids (c, 100·; d, 400·).
Fig. 3. Immunohistochemical localization of RNF151 in mouse testis. GST fusion protein of the N-terminus amino acid (16–83) of RNF151 was purified from E. coli (a) and was used to generate a rabbit polyclonal antiserum that detected a protein of 26 kDa in the testis cell lysate (b). Anti-RNF151 antiserum was used as primary antibody to detect the expression of the RNF151. The presence of RNF151 was revealed by red staining. RNF151 was stained in the cytoplasm of elongating spermatids of stage VIII–IX seminiferous tubules (c, 200·; d, 600·).
domain was added to the RING plus NLS fragment, the speckled nuclear localization of the full-length protein was totally recovered (Fig. 4f). This implied that the TRAF-type zinc finger domain may play a role in the formation of the specked pattern indicative of protein selfaggregation. As expected, RING domain alone distributed uniformly in the transfected cells (Fig. 4g) while the C-terminal segment after the TRAF-type zinc fingers was only
localized in the cytoplasm (Fig. 4h). These data indicated that the RING domain plus NLS are necessary and sufficient for the nuclear localization of RNF151. RNF151 interacts with dysbindin Yeast two-hybrid system was employed to screen out potential binding partners of mouse RNF151. Of the 30
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Fig. 4. Subcellular distribution of RNF151 wild-type and six different truncated proteins in GC-2 cells. Green color is from the EGFP or EGFP-fusion proteins while the blue one is from the Hoechst 33258-stained cell nuclei. NLS, the putative nuclear localization signal; RING, the RING domain; TRAF, the TRAF-type zinc fingers domain. Magnification, 400·.
positive clones acquired, 4 clones encode full-length cDNAs for a protein named dysbindin, and 5 for RNF151 itself. The interaction between RNF151 and dysbindin was first confirmed by the growth of the yeast cells co-transfected with these two proteins on the SD/-Ade/His/-Leu/-Trp/X-a-Gal selection medium. The interaction was confirmed next by in vitro binding assays (Fig. 5a). In these assays, radioactive c-Myc-tagged RNF151 and HA-tagged dysbindin proteins were first synthesized separately by in vitro transcription and translation of the respective genes in the rabbit reticulocyte lysates in which 35 S-methionine was included. When the proteins were mixed, both proteins could be pulled down by either the c-Myc antibody or the HA antibody as a result of their physical interaction. The interaction between RNF151 and dysbindin was further confirmed by co-immunoprecipitation of FLAG-RNF151 and GFP–dysbindin that were expressed in 293-T cells (Fig. 5b). As another evidence for the interaction between these two proteins, GFP– RNF151 and RFP–dysbindin co-localized in the nuclei of transfected GC-2 cells (Fig. 5c). Taken together, these observations have indicated that dysbindin can be a bona fide binding partner of RNF151. Discussion Spermatogenesis, one of the most complex biological processes, involves active cell mitotic and meiotic divisions, morphological changes, and apoptosis. These activities are driven by the expressions of different gene families and
their protein interactions [16,17]. Gene expressions are especially active after the accomplishment of meiosis when round spermatids are ready to start an astonishing metamorphic journey to become the highly specialized sperms [18]. The testis is well-known for its possession of quite a number of tissue specific genes [3]. These genes are termed ‘‘Chauvinist genes’’ by Eddy [19], and are either transcribed specifically or with their transcripts post-transcriptionally processed in a testis-specific manner. Identification of the full complement of these genes will lay the solid ground for our further understanding and better manipulation of spermatogenesis. The RNFs are a subfamily of the zinc finger protein family. RNFs are very disparate in terms of their sequences and functions except for sharing the common RING domains [5]. RNFs participate in multiple processes such as gene expression regulation, enzymatic reactions, membrane traffic, protein complex formation, to name a few. To our knowledge, more than 30 RNFs have been reported to be involved in spermatogenesis in the mouse, rat, and human. In the present study, we have added to this growing list mouse RNF151, whose transcript is exclusively present in the round spermatids. We have also identified another testis-specific membrane bound RNF (data not shown), and try to make this testicular RNF list complete. In addition to the RING domains, RNFs usually possess other domains that together with the RING mediate protein–protein interactions. Except for the C3HC4-type RING domain, RNF151 also have a TRAF-type zinc finger domain. The mouse RNF151 shares low but
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Fig. 5. Interaction between RNF151 and dysbindin. (a) Co-immunoprecipitation of RNF151 and dysbindin produced by in vitro translation. Lane 1: RNF151 + anti-Myc antibody; lane 2: dysbindin + anti-HA antibody; lanes 3 and 4: RNF151 + dysbindin + anti-HA or Myc antibody. (b) Co-immunoprecipitation of RNF151 and dysbinding from transfected 293-T cells. 293-T cells were transfected with FLAG-RNF151 and EGFP–dysbindin. RNF151 proteins were immunoprecipitated with anti-FLAG antibody, and Western blot analyses were performed with anti-GFP antibody. (c) Co-localization of RNF151 and dysbindin proteins. GFP–RNF151 and RFP–dysbindin proteins were indicated by green and red colors, and their co-localization was demonstrated by yellow color in the overlaid image. Magnification, 400·. WB, Western blotting; IP, immunoprecipitation.
significant homologies with 3 TNF receptor associated factors (TRAF), TRAF3, 5, and 6. All TRAFs except for TRAF2 have a RING domain with a unique spacing pattern between the zinc chelating residuals (CX2CX(11,12)CX1HX2CX2CX(9,11)CX2C), and all of them except for TRAF1 possess 5–7 zinc fingers [20]. RNF151 also has the TRAF-type RING domain and zinc fingers. These features indicate that RNF151 proteins are distally related to the TRAF family more than to any other protein families. TRAFs are usually cytoplasmic proteins. The immunohistochemical staining of mouse RNF151 on testis sections indicated that it was predominantly cytoplas-
mic. However, the presence of a well-conserved NLS in RNF151 proteins suggests that they could relocate into the nucleus under certain conditions. Consistent with this hypothesis, the full-length mouse RNF151 with EGFP fused to its C-terminus was localized to the nuclei as speckles indicative of protein aggregates. Our data indicated that RING plus NLS are necessary and sufficient for RNF151 being localized in the nucleus, and that the TRAF-type zinc finger domain may mediate self-aggregation of the protein. Therefore, we speculate that, under normal condition, RNF151 stays in the cytoplasm either due to direct tethering by its interacting protein(s), and/or due to the unavailability of NLS to nuclear transporting proteins as a result of its interactions with other proteins. In the transfected cells, the protein translocates into the nucleus because the tethering protein is not present and/or the fused protein renders NLS accessible by nuclear transporting proteins. It is important to investigate whether and how such translocation occurs in vivo. Search for interacting target by using the yeast twohybrid system provide valuable information about the function of a novel gene. Thirty independent colonies resulted from interactions between mouse RNF151 and potential targets were acquired, out of which 5 contained full-length coding sequence of mouse RNF151 itself and 4 contained full-length sequences for dysbindin. It is not surprising that RNF151 interacts with itself in yeast cells for the following two reasons: First, RNFs are well known to form aggregates in cells. For example, TRAFs, to which RNF151 proteins bear significant sequence and structural similarities, form homotrimers [15]; Second, the speckled nuclear localization pattern of mouse RNF151 in transfected cells is indicative of self-aggregation. The interaction between RNF151 and dysbindin interacting was confirmed by the co-immunoprecipitation of the in vitro translated proteins or proteins expressed in transfected cells as well as by the proteins’ co-localization in transfected cells. However, we understand that interaction between RNF151 and dysbindin is not without any doubt because the yeast two-hybrid screening and assays used to confirm their interaction are all in vitro experiments. It would be more convincing to show that these two proteins co-immunoprecipitate from testicular extracts and co-localize in testicular tissue sections, both of which would be carried out when the antibody against dysbindin is available. It would also be interesting to examine whether the interaction between RNF151 and dysbindin is mediated by the RING domain of RNF151 and whether other testicular RNFs also bind to dysbindin so that the developmental importance of the two proteins’ interaction could be clarified. Dysbindin was first identified, by using the yeast twohybrid system, as a binding partner of dystrobrevin, a component of the dystrophin-associated protein complex (DPC) in both muscle and nonmuscle cells [21]. It has attracted wide interest because it is a susceptibility gene for Schizophrenia [22,23]. Dysbindin was lately reported
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to be a synaptic and microtubular protein that binds brain snapin, a SNARE-binding protein that mediated intracellular membrane fusion in both neuronal and non-neuronal cells [24,25]. Dysbindin is also a component of a multisubunit protein complex named BLOC-1 (for Biogenesis of lysosome-related organelles complex-1), which is required for the normal biogenesis of specialized organelles of the endosomal–lysosomal system, such as melanosomes and platelet dense granule [26]. Mutations in genes of dysbindin and the other components of BLOC-1 lead to the so-called Hermansky–Pudlak syndrome in human characterized by oculocutaneous albinism, prolonged bleeding, and pulmonary fibrosis due to abnormal vesicle trafficking to lysosomes and related organelles, such as melanosomes and platelet dense granules [27]. One key process of the spermiogenesis is the acrosome biogenesis started at the round spermatid stage. Acrosome formation involves membrane trafficking from Golgi apparatus and probably lysosomes [28,29]. It has been shown recently that the assembly of spermatid acrosome also depends on microtubule organization [30]. Based on these clues, it is reasonable to propose that RNF151 might regulate acrosome formation through interacting with multiple proteins participating in membrane biogenesis and microtubule organization. The best proof of this hypothesis is to see spermatogenesis stalked in the pre-acrosomal stage in knockout mice, a work currently undergoing in our laboratory. Acknowledgments Financial support was provided by the National Natural Science Foundation of China (Grant No. 30428030 and No. 90508008), the ‘‘Bai Ren Ji Hua’’ Fund of the Chinese Academy of Sciences. References [1] R.A.E.L.D. Russell, A.P. Sinha Hikim, E.D. Clegg (Eds.), Histological and Histopathological Evaluation of the Testis, Clearwater, Florida, 1990. [2] R.M. Sharpe (Ed.), Regulation of Spermatogenesis, New York, 1993. [3] N. Schultz, F.K. Hamra, D.L. Garbers, Proceedings of the National Academy of Sciences of the United States of America 100 (2003) 12201–12206. [4] T. Ravasi, T. Huber, M. Zavolan, A. Forrest, T. Gaasterland, S. Grimmond, D.A. Hume, Genome Research 13 (2003) 1430–1442. [5] K.L. Borden, Journal of Molecular Biology 295 (2000) 1103–1112. [6] P.S. Freemont, Current Biology 10 (2000) R84–R87.
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