DAZL Binds to the Transcripts of Several Tssk Genes in Germ Cells

BMB reports

DAZL binds to the transcripts of several Tssk genes in germ cells

Mei Zeng, Wenqian Deng, Xinying Wang, Weimin Qiu ,Yanyan Liu, Huaqin Sun, Dachang Tao, Sizhong Zhang & Yongxin Ma* Department of Medical Genetics, West China Hospital; Division of Human Morbid Genomics, State Key Laboratory of Biotherapy, Sichuan University, Chengdu, 610041, P.R.China.

The Dazl gene encodes a germ-cell-specific RNA-binding pro- cluding a modest reduction in germ cell numbers before birth tein which is essential for spermatogenesis. It has been pro- and a failure to progress from Aaligned to A1 spermatogonia (4, posed that this protein (DAZL) binds to RNA in the cytoplasm 5). Although some cells progress beyond this point, the fur- of germ cells and controls spermatogenesis. Using the specific thest that cells have been seen to advance along the meiotic nucleic acids associated with proteins (SNAAP) technique, we pathway is to leptotene of meiotic prophaseⅠ (6). This in- identified 17 target mRNAs bound by mDAZL. Among these dicates that DAZL has several roles: in mitotic proliferation be- transcripts, we focused on TSSK2, which encodes a testis-spe- fore meiosis, during spermatogonial development and in the cific serine/threonine kinase. To date, five TSSK family mem- leptotene to zygotene transition. Heterozygous DAZL-deficient bers have been cloned, and all are exclusively expressed in mice have an increased percentage of malformed spermato- the testis. We demonstrated that in addition to the TSSK1 zoa, indicating a gene dosage effect and suggesting that DAZL 3’UTR , the 3’UTRs of TSSKs 2 and 4 were bound by human may also be involved in the control of major morphological and mouse DAZL, and that human DAZL (hDAZL) bound to changes during the post-meiotic phase of spermatogenesis the 3’UTR of human TSSK5 (hTSSK5). Our results suggest that when elongated spermatids and mature motile spermatozoa the Dazl gene may play different roles in human and mouse are formed (4). spermatogenesis by regulating different members of the down- It has been proposed that DAZL binds to RNA in the cyto- stream gene family. [BMB reports 2008; 41(4): 300-304] plasm of germ cells. Previous studies in which DAZL cofractio- nated with a polysome pool indicated that the DAZL-bound mRNA might be translationally active (7, 8). Consistent with INTRODUCTION this viewpoint, Collier reported that DAZL interacted with PABP and augmented the translation of target mRNAs in vivo During spermatogenesis immature male germ cells go through (9). On the other hand, an interaction of DAZL with Pum2, a a complex series of differentiation steps involving mitotic and potential translation repressor, has also been observed (10). meiotic cell divisions and morphogenesis. These differ- DAZL can also bind to the dynein motor complex and may entiation steps rely on regulating the time- and region-specific play a role in transport of specific mRNAs (11). These results expression of genetic information. Of all the genes, those in suggest that DAZL can influence both the active transport and the DAZ family are considered critical for germ cell develop- translational activity of mRNAs to which it binds. ment and to be master regulators of germline gene expression Although some mRNAs bound by mDAZL have been identi- in a wide range of animals, including humans. The DAZ fam- fied, others may not yet have been encountered. The current ily consists of two autosomal genes Boule and Dazl (DAZ-like) study was undertaken to explore the biological functions of and a Y-chromosomal Daz gene cluster. The presence of an DAZL during spermatogenesis, and to identify some novel tar- RNA-binding domain, indicative of RNA-protein interaction, is get mRNAs. Our results show that some of these targets play the main characteristic of all DAZ family members (1-3). important roles in spermatogenesis. In male mice, loss of DAZL results in multiple defects in- RESULTS

*Corresponding author. Tel: 86-28-85164009; Fax: 86-28-85164009; Identification of novel mRNA substrates bound by mDAZL E-mail: [email protected] or [email protected] protein We utilized the SNAAP technique and identified 17 mRNAs Received 3 December 2007, Accepted 24 December 2007 bound by mDAZL fused to GST (GST- mDAZL) (Table 1). We Keywords: Dazl gene, RNA-binding protein, Spermatogenesis, used GST as a control protein; we did not use any other Target mRNAs, TSSK RNA-binding protein as a control protein, because the targets

300 BMB reports http://bmbreports.org DAZL binds to the transcripts of several Tssk genes in germ cells Mei Zeng, et al.

of many RNA-binding proteins are known and some was bound by mDAZL (Fig. 1B). RNA-binding proteins may have conjunct targets. Sequencing showed all 17 transcripts contained the motif G/C (U) n in their Identification of the mDAZL binding site in the 3’UTR of 3’UTR region. Among these transcripts, Tpx-1 was also identi- mTSSK2 fied by Jiao et al (12). Other mRNAs are probably novel targets The specific sequence bound by mDAZL was identified using of mDAZL. It has been reported that some targets mRNAs are smaller segments of the mTSSK2 3’ UTR. Because previous translated into proteins that are involved in cell growth studies have shown that mDAZL binds to the sequence G/C regulation. Rap2c is a member of the Ras family that regulates (U) n (18-21), the 3’UTR of the mTSSK2 was divided into four a variety of cellular functions including cell growth, differ- small segments and the G/C (U) n sequence was further sub- entiation and apoptosis (13). Nbn is a G2 DNA damage divided please check (Fig. 1C). The mDAZL binding site in 3’ checkpoint (14). ERCC1 (excision repair cross com- UTR of mTSSK2 proved to be in the 100-160 bp 3’ UTR that plementation group 1) is a subunit of the nucleotide excision contains the G/C (U) n sequence (Fig. 1D). repair complex, which can perform DNA strand incision cor- rection of DNA damage (15). Some mRNAs are specifically ex- Mouse DAZL also binds to the 3’UTRs of mTSSK1 and 4 pressed in the germ cells of the testis and play important roles Five members in the TSSK family have been cloned, and the during spermatogenesis. TSSK2, TPX-1 and SPNR are reported expression of each is almost exclusively confined to the ma- to be expressed specifically in the testis. Both Dnmt3l and ture testis. All members contain the serine/threonine protein Sox17 are testes-specific splicing proteins in mammals. SPNR kinase domain and have very high homology at the amino encodes a cytoplasmic RNA-binding protein essential for sper- acid level. These kinases occupy different locations in mature matogenesis (16). testes (21-26), have different 3’UTR structures and may be regulated by different genes during spermatogenesis. We test- The 3’UTR of mTSSK2 is bound by mDAZL ed whether other members of this family could be bound by TSSK2 encodes a testis-specific serine/threonine kinase which mDAZL and found that the 3’UTRs of mTSSK1 and mTSSK4 plays a key role during the post-meiotic phase of spermato- mRNAs were bound, but not those of mTSSK3 and 5 (Fig. 2A). genesis (17). We observed that mDAZL bound to the 3’UTR of Competitive binding assays further demonstrated that the mTSSK2 mRNA, but GST did not (Fig. 1A), and we performed 3’UTRs of mTSSK1 and mTSSK4 were bound by mDAZL (Fig. competitive EMSA assays confirming that the 3’UTR of mTSSK2 2B).

Fig. 1. Binding experiments on the 3’UTRs of mTSSK2 RNAs and Fig. 2. Binding experiments on four members of the TSSK family GST or mDAZL protein (A, B) (A) About sixty femtomoles of di- and GST or mDAZL protein (A, B) (A) About sixty femtomoles of goxigenin-labeled of TSSK2 3’UTR was incubated with various digoxigenin-labeled mTSSK1, 3, 4 and 5 RNAs were incubated concentrations of mDAZL or GST protein: 0, 200, 300 and 400 with GST or mDAZL protein: 400 nM GST protein (lanes 2, 5, 8 nM mDAZL protein (lanes 1, 2, 3 and 4); 200, 300, 400 nM and 11); 400 nM mDAZL protein (lanes 3, 6, 9 and 12). (B) GST protein (lanes 5, 6 and 7). (B) Competitive RNA-binding ex- Competitive RNA-binding experiments on mTSSK1 and 4. About periments with unlabeled mTSSK2 RNA. Sixty femtomoles of di- sixty femtomoles of digoxigenin-labeled mTSSK1 and 4 RNAs goxigenin-labeled of mTSSK2 3’UTR was incubated with mDAZL were incubated with mDAZL at a final concentration of 400 nM, protein at a final concentration of 400 nM. Lanes 1, 2, 3, 4 con- and the following amounts of unlabeled competitor RNAs: 100 tained 0, 100, 1000, 5000 fmol of unlabeled competitor mTSSK2 fmol (lanes 2 and 6), 1000 fmol (lanes 3 and 7), 5000 fmol RNA, respectively. Identification of the mDAZL binding site in the (lanes 4 and 8). Identification of the mDAZL binding site in the 3’UTR of mTSSK2 (C, D) (C) The 3’UTR of mTSSK2 RNAs was 3’UTR of mTSSK1 (C, D) (C) The mTSSK1 3’UTR was divided in- divided into four smaller segments: T2-1, T2-2, T2-3 and T2-4. to four small segments: T1-1, T1-2, T1-3 and T1-4. (D) Sixty fem- (D) About sixty femtomoles of digoxigenin-labeled T2-1, T2-2, T2-3 tomoles of digoxigenin-labeled T1-1, T1-2, T1-3 and T1-4 RNAs and T2-4 RNAs were incubated with mDAZL protein at a final were incubated with mDAZL protein at a final concentration of concentration of 400 nM. 400 nM. http://bmbreports.org BMB reports 301 DAZL binds to the transcripts of several Tssk genes in germ cells Mei Zeng, et al.

Identification of the mDAZL binding site in the 3’UTR of DISCUSSION TSSK1 The 3’ UTR of TSSK4 is very small, and TSSK1 and 2 have the DAZL encodes an RNA-binding protein and is expressed in the highest homology in their kinase domains. To determine the cytoplasm of germ cells throughout gonadal development in mDAZL binding site of TSSK1, its 3’ UTR was divided into three the mouse (28). Previous studies suggest that mDAZL plays dif- segments (Fig. 2C) and the G/C（U）n sequence was cut into a ferent roles in different stages by binding to different mRNA small segment on its own. As in the case of TSSK2, the mDAZL targets. The fact that heterozygous DAZL-deficient mice have binding site of TSSK1 was located in the 110-160 bp 3’UTRs an increased fraction of malformed spermatozoa suggests that (Fig. 2D), which also contains the G/C（U）n sequence. mDazl play an important role in spermiogenesis (4). Although previous studies have identified some key targets of mDAZL, Human DAZL can bind to the 3’UTRs of hTSSK1, 2, 4 and 5 no targets are known that play an important role during this There is 87.25% identity between the amino acid sequences of stage. Among our identified target mRNAs, members of TSSK human and mouse DAZL and their RNA recognition motif are family play significant roles during spermiogenesis. Previous completely homologous. In mice, the DAZ gene family is rep- studies indicated that mRNA storage with subsequent transla- resented by the single-copy autosomal DAZL gene. In humans tional activation, or stability of the protein, are not involved in it consists of two autosomal genes BOULE and DAZL and a the regulation of TSSK 1 and 2 activity (17), but DAZL can Y-chromosomal DAZ gene. So DAZL proteins may have differ- bind to the dynein motor complex and may play a role in ent targets in mice and human. In humans, five members of transport of specific mRNAs (11). Our results show that DAZL the TSSK family have also been cloned; all five are expressed can bind to 3’UTR of TSSK1 and 2, and therefore may function exclusively in the testis (21-25) and are highly homologous to in the transport of TSSK1 and 2 mRNAs. mDAZL; however the structures of their 3’UTRs differ from Among the identified targets, we focused on the TSSK fam- that of mDAZL. We observed that human DAZL bound to the ily, which plays a crucial role in phosphorylation events dur- 3’UTRs of hTSSK1, 2, 4 and 5, but not to that of hTSSK3 (Fig. ing spermiogenesis. Although the mechanisms of post-meiotic 3A). Competitive binding assays further demonstrated that the chromatin condensation are not well known, phosphorylation 3’UTRs of hTSSK1, 2, 4 and 5 mRNAs were bound by hDAZL events appear to play a central role in chromatin remodeling (Fig. 3B). during mammalian spermiogenesis. Moreover, TSSK4 specifically phosphorylate histones H1, H2A, H2AX, and H3, and targeted disruption of the TSSK4 gene caused male sterility in mice, accompanied by reduced sperm numbers, impaired DNA condensation, and abnormal morphology and impaired motility of the spermatozoa. These results suggest that TSSK4 is required for proper post-meiotic chromatin remodeling (21). Since we demonstrated that the 3’UTR of TSSK4 is bound by DAZL, its expression may be regulated by the latter. We showed above that DAZL could bind to the 3’UTRs of several members of the TSSK family, and that they bound to three similar members of the TSSK family in mice and human. The amino acid sequences of human and mouse DAZL protein shares very high homology, and their RNA recognition motifs are almost identical; however the mouse DAZ gene family consists of the single-copy autosomal DAZL gene, whereas the human genome contains the DAZ gene family on the long arm of the Y chromosome (27) in addition to the autosomal DAZL Fig. 3. Binding experiments on the 3’UTRs of five members of gene. The cellular locations of human and mouse DAZL pro- the human TSSK family and GST or hDAZL protein. (A) About teins are also not completely the same; in mice the protein is sixty femtomoles of digoxigenin-labeled hTSSK1, 2, 3, 4 and 5 predominantly cytoplasmic throughout germ cell develop- RNAs were incubated with GST or hDAZL protein at a final con- ment, whereas, during human spermatogenesis, the pattern of centration of 400 nM. GST protein (lanes 2, 5, 8 11 and 14), hDAZL protein (lanes 3, 6, 9, 12 and 15). (B) Competitive DAZL of expression is dynamic, with the protein first localized RNA-binding experiments were performed on hTSSK1, 2, 4 and to the nuclei of gonocytes in male embryos, and then to the 5. About sixty femtomoles of digoxigenin-labeled hTSSK1 , 2, 4 cytoplasm of premeiotic germ cells in the adult testis (28). So and 5 RNAs were incubated with hDAZL protein at a final con- the Dazl gene may play different roles in human and mouse centration of 400 nM, and the following amounts of unlabeled competitor RNAs: 100 fmol (lanes 2, 6, 10 and 14), 1000 fmol spermatogenesis. Furthermore the structures of the 3’UTRs of (lanes 3, 7, 11 and 15), 5000 fmol (lanes 4, 8, 12 and 16). these members of the TSSK family differ in mice and humans.

302 BMB reports http://bmbreports.org DAZL binds to the transcripts of several Tssk genes in germ cells Mei Zeng, et al.

Statistical analysis of consensus sequences in the targets of thione beads and removing unbound proteins, the washed mouse and human DAZL protein have shown that the motif beads were incubated with 300 μg of testis total extract pre- G/C (U) n is over-represented within the 3’UTR regions of the cleared with 20 μl of glutathione Sepharose beads. After bind- targets of mDAZL protein by Reynolds N et al. Since our 17 ing at 4°C for 1 h, a wash was carried out with RBB/0.25% TX identified targets all have the motif G/C (U) n in the 3’UTRs of followed by a 10-min wash in RBB/0.25% TX containing 1 their mRNAs, we did analyze point mutations in the consensus mg/ml heparin. The beads were subsequently washed 4 times sequences. The fact that there is no G/C (U) n sequence in the in RBB/0.25% TX and bound RNA was then extracted by 3’UTR of mTSSK5 probably accounts in part for why mDAZL standard procedures. Copurifying RNAs were identified by dif- did not bind to its 3’UTR; however hDAZL also did not bind to ferential display according to Jiao et al. (12). the 3’UTR of hTSSK5. Members of the TSSK family have different locations; TSSK1, 2 and 4 are expressed in germ cells, but Electrophoretic mobility shift assay TSSK3 is mainly expressed in the androgen-producing Leydig The electromobility shift protocol was as described (29, 30). cells (20, 21, 23-25). The detailed location of TSSK5 is not yet RNA synthesis and labeling were performed with an SP6/T7 known, but it is reported to be only expressed in the testis. Transcription and DIG RNA Labeling Kit (Roche). The binding However, in all organisms studied thus far, the expression of buffer for mDAZL-TSSK interaction studies was buffer A (50 Dazl is specific to germ cells throughout gonadal development mM Tris-Hcl pH7.0, 150 mM NaCl, 67 μg/μl yeast tRNA, 0.25 (4), and this is probably in part the reason why mDAZL could mg/ml bovine serum albumin, 1.5 μg/μl heparim)+ 2% 2- not bind to TSSK3. All these results suggest that Dazl plays dif- mercaptoethanol. All RNAs used in the EMSAs were heat- ferent roles during spermatogenesis in humans and mice by treated at 90oC for 3 min, followed by rapid cooling on ice. regulating different members of the downstream gene family. Potential protein-RNA complexes were allowed to form at Moreover different members of the same gene family may be 37oC for 10 min. Following incubation, 2.5 μl of 6× loading subject to divergent regulatory mechanism. The fact that buffer (TaKaRa) was added, and the samples were immediately mDAZL binds to the mRNAs of these members implies that loaded on to a 1% agarose gel in 1 × TBE buffer. The gel was the latter may be regulated by mDAZL, perhaps by conferring subjected to electrophoresis on ice until separation was stability on their mRNAs, or by active transport or translational achieved. The RNA was then electroblotted to a positively regulation. Since all our experiments were performed in vitro, charged nylon membrane and subsequently crosslinked to the it will be necessary to test whether they are actually regulated membrane by short-wave ultraviolet radiation. RNA on the by Dazl in vivo. membrane was detected by washing the blot with a detergent solution, followed by a block solution and probing with an- MATERIALS AND METHODS ti-digoxigenin-AP conjugate to bind digoxigenin-labeled RNA. The blot was washed again, and substrate (CDP-Star) added to Preparation of total testis extract initiate the chemiluminescence reaction. The membrane was The method of preparation of total testis extracts has been de- wrapped with a plastic wrap and exposed to X-ray film. scribed by Jiao et al (12); testes were obtained from 4-week-old C57/B16 mouse that expressed DAZL normally throughout go- Acknowledgments nadal development. This work was supported by grants (No.90408025, No.30770812 and No.30500186) from the National Natural Science Founda- Generation of glutathione S-transferase fusion proteins tion of China, and one from the National High-Tech Research The pGEX-mDAZL plasmid encoding a glutathione S-trans- and Development Program of China (2007AA02Z127). ferase (GST)-mDAZL fusion protein was constructed by placing the mDAZL coding region into the pGEX-5X-3 vector. 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