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- Original Article -

Expression of a novel alternative transcript of the novel retinal pigment epithelial cell gene NORPEG in human testes

Wa Yuan, Ying Zheng, Ran Huo, Li Lu, Xiao-Yan Huang, Lan-Lan Yin, Jian-Min Li, Zuo-Min Zhou, Jia-Hao Sha

Key Laboratory of Reproductive Medicine, Department of Histology and Embroyology, Nanjing Medical University, Nanjing 210029, China

Abstract

Aim: To identify a novel alternative transcript of the novel retinal pigment epithelial cell gene (NORPEG) expressed in the human testis. Methods: A human testis cDNA microarray was established and hybridized with cDNA probes from human fetal testes, adult testes and human spermatozoa. Differentially expressed clones were sequenced and analyzed. One of these clones was a short transcript of NORPEG which we proceeded to analyze by RT-PCR. Results: The novel short alternative transcript of NORPEG was isolated and named sNORPEG. It was 3486 bp in length and contained a 2952-bp open reading frame, encoding a 110.4-kDa protein of 983 amino acids. Amino acid sequence analysis showed that the sNORPEG protein contains six ankyrin repeats and two coiled-coil domains. It shares a high homology with the NORPEG and ankycorbin proteins in both its sequence and motifs. Blasting the human genome database localized sNORPEG to human chromosome 5p13.2-13.3. Expression profiles showed that sNORPEG was expressed in human fetal testes, adult testes and spermatozoa. Moreover, sNORPEG was found to be ubiquitously expressed in human tissues. Conclusion: sNORPEG is expressed in different developmental stages of the testis and encodes a protein that may have roles in human testis development and spermatogenesis. (Asian J Androl 2005 Sep; 7: 277-288)

Keywords: alternative transcript; NORPEG; testis development; spermatogenesis

Correspondence to: Dr Jia-Hao Sha, Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing 210029, China.

Tel/Fax: +86-25-8686-2908
E-mail: shajh@njmu.edu.cn
Received 2004-08-11 Accepted 2005-02-12
DOI: 10.1111/j.1745-7262.2005.00040.x

1 Introduction

Actin cytoskeleton structures are essential for a wide variety of cell functions, including cell shape change, cell motility, cell adhesion, cell polarity and cytokinesis. Many actin-associated proteins with functions related to actin dynamics have been isolated and characterized. For example, the recently identified ankycorbin is an actin cytoskeleton-associated protein that may be involved in actin cytoskeleton maintenance and/or reorganization [1]. During testis development and spermatogenesis, actin and actin associated proteins play a crucial role in many important activities, including the Sertoli-germ cell adherens junction dynamics [2], spermiogenesis [3] and acrosomal transformation [4]. The investigation of proteins related to actin dynamics in testes may provide information about testicular function and physiology.

In this study we identified a short alternative transcript of the novel retinal pigment epithelial cell gene (NORPEG) in the human testis and named it sNORPEG. The discovery was based on the analysis of cDNA probe hybridizations with a human cDNA microarray constructed in this laboratory. We found that sNORPEG encodes a protein which is highly homologous with both the NORPEG protein and the recently identified actin-associated protein ankycorbin [1]. Both the NORPEG and ankycorbin proteins are thought to have functions related to the cellular cytoskeleton. This paper describes the novel alternative transcript sNORPEG in human testes and discusses its possible roles in testis development and spermatogenesis.

2 Materials and methods

2.1 cDNA microarray construction and hybridization

A human testis cDNA microarray was constructed in order to study gene expression in testis development. A total of 9216 positive phage clones were selected randomly from the Human Testis Insert l phage cDNA library (HL5503U; Clontech, Palo Alto, CA, USA) and amplified by PCR. The PCR products were spotted onto a membrane to make the human testis cDNA microarray. This microarray was hybridized with ³³P-labeled cDNA probes prepared from the mRNA of human fetal testes, adult testes and human spermatozoa. The microarray was scanned by an FLA-3000A plate/fluorescent image analyzer (Fuji Photo Film, Tokyo, Japan). The radioactive signal intensity of each spot was linearly scanned and read using the Array Gauge software (Fuji Photo Film, Tokyo, Japan). After subtraction of the background from an area where no PCR product was spotted, clones with intensities over 10 were considered positive. The hybridization intensities of the corresponding dots from adult and fetal samples were compared. If the intensity comparison between the samples yielded a difference ³3-fold, then the clones were considered differentially expressed. The cDNA clones showing differential expression patterns between fetal and adult testes were selected and analyzed. Protocols for human testis cDNA microarray construction, adult testis, fetal testis and spermatozoa cDNA probe preparation, hybridization, and signal analysis have been described elsewhere in detail [5-7].

2.2 Sequence identification and analysis

The cDNA clones that were found to be differentially expressed in adult testes and fetal testes were purified with mini-preps (QIAprep Spin Miniprep Kit, Qiagen, Hilden, Germany) and then sequenced by an ABI377 automatic sequencer (Perkin-Elmer, Norwalk, CT, USA). The sequence of the forward sequencing primer was 5'- CCATTGTGTTGGTACCCGGGAATTCG-3' and the sequence of the reverse sequencing primer was 5'-ATAAG-CTTGCTCGAGTCTAGAGTCGAC-3'. The results were analyzed with BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) and SMART (http://smart.embl-heidelberg.de/) database programs with the goal of identifying homologous genes and proteins. The nucleotide and the deduced amino acid sequences were also analyzed using Gene Runner software (http://www.generunner.com). Meanwhile, highly homologous proteins were compared by ClustalW (http://www.ebi.ac.uk/clustalw/). The novel NORPEG transcript sNORPEG was isolated and identified. The promoters of sNORPEG and NORPEG were analyzed by PROSCAN version 1.7 (http://bimas.dcrt.nih.gov/molbio/proscan/).

2.3 Expression profile of sNORPEG in different developmental stages of testis and spermatozoa

The expression profile of sNORPEG in a human adult testis (aged 43 years), fetal testis (gestational age ~6 months) and spermatozoa was determined using RT-PCR. Human adult and fetal testes total RNA was isolated using Trizol Reagent (Gibco BRL, Grand Island, New York, USA). Ejaculate spermatozoa from a normal male (WHO, 1999 criteria) were allowed to liquefy for 1 h at room temperature, washed twice in phosphate-buffered saline (pH 7.4) and the total RNA were extracted from the sediment.

Reverse transcription reactions from the extracted RNA samples were performed in 15 µL of reaction mixture. First 2 µL (about 5 µg) total RNA, 1 µL random hexamer primer (0.2 µg/mL, Sangon, Shanghai, China) and 6 µL diethyl pyrocarbonate (DEPC) treated water were mixed and incubated at 70 ºC for 5 min; then 3 µL AMV 5 × buffer, 0.75 µL dNTP (20 mmol/L), 0.25 µL Rnasin (40 U/µL), 1 µL AMV reverse transcriptase (Promega, Madison, USA) and 1 µL DEPC water were added and incubated at 42 ºC for 1.5 h, and then held at 90 ºC for 5 min. PCR was performed with sNORPEG specific primers. The specific primers were designed to overpass two introns to prevent contamination of genomic DNA. Primers were as follows: upstream 5'-TGCTGGC-TGTATGTTATGC-3', and downstream 5'-GGTAGT-ATCTTGGGCTGTC-3'. The amplified fragment of sNORPEG was 279 bp in size. The upstream primer was located in the exclusive exon of the sNORPEG transcript but the downstream primer was homologous with that of NORPEG. Primers were synthesized at BioAsia Company (Shanghai, China). Human b-actin was used as a positive control. The following human b-actin primers were used: upstream 5'-CGGTTGGCCTTGGGGTTCAGGGGG-3', and downstream 5'-ATCGTGGG-GGCGCCCCAGGCACCA-3'. The PCR thermal cycling conditions program consisted of an initial denaturation at 94 °C for 5 min, followed by 35 30-s cycles of denaturation at 94 °C, annealing at 54 °C for 30 s, extension at 72 °C for 1 min and an additional extension at 72 °C for 7 min. The PCR products were analyzed by 1.5 % (w/v) agarose gel electrophoresis.

2.4 Expression profile of sNORPEG in different tissues

The expression profile of sNORPEG in different tissues was assessed using RT-PCR. Multiple tissue cDNA panels were obtained from the commercial Human Multiple Tissue cDNA (MTC) Panel I and II kit (Cat# K1420-1 and K1421-2, Clontech), which included 16 human tissues (testis, skeletal muscle, liver, pancreas, brain, lung, kidney, heart, placenta, spleen, thymus, prostate, ovary, small intestine, colon and peripheral blood leukocytes). Primers and PCR conditions were the same as that described above. G3PDH was used as a positive control. Its upstream primer was 5'-TGAAGGTCGGAGTCA-ACGGATTTGGT-3', and downstream primer was 5'- CATGTGGGCCATGAGGTCCACCAC-3'. The desired fragment was 983 bp. PCR conditions were performed according to the manufacturer's instructions as follows: denaturation at 95 °C for 30 s, subsequent annealing and extension at 68 °C for 3 min. The first cycle had a denaturation period of 1 min. The last cycle had an extension period of 3 min at 68 °C. Thirty-six cycles of PCR were performed. The plasmid containing sNORPEG from the human testis large insert cDNA library (HL5503U, Clontech) was used as a positive control of PCR amplification. PCR products were analyzed by 2 % (w/v) agarose gel electrophoresis.

2.5 TA clone DNA sequencing

RT-PCR analysis revealed an unexpected fragment in the lung, testis and sperm. The PCR reaction products were gel-purified (QIAquick Gel Extraction Kit, Qiagen) and the purified PCR products were cloned into a pinpoint xa-1 T-vector (Cat# V2610, Promega). Positive clones after transformation were selected and sequenced (sequenced by BioAsia) with a PinPoint Vector Sequencing Primer (sequence 5'-CGTGACGCGGTGCAGGGCG-3', Promega). The publicly available blast program (http://www.ncbi.nlm.nih.gov/BLAST/) was utilized to compare the sequence with expressed sequence tag database (dbEST) and the human genome.

3 Results

3.1 cDNA microarray hybridization

One of the differentially expressed genes was isolated. The hybridization intensities of this gene in fetal and adult testes were 9.88 and 30.90, respectively. The intensity of this gene's expression in the adult testis was about 3-fold stronger than that in fetal testes (Figure 1). The hybridization intensity of the newly identified gene in spermatozoa was 15.54, indicating that it was expressed in germ cells. Subsequent analyses as described below indicated that this novel differentially expressed gene represented a short alternative transcript of NORPEG and therefore it was named sNORPEG.

3.2 Sequence identification and analysis of sNORPEG

The full cDNA length of sNORPEG was 3486 bp and had a 2952-bp open reading frame from 493 bp to 3444 bp, encoding a 110.4-kDa protein of 983 amino acids. The methionine at 493-495 bp was almost certainly the site of initiation because there was an up-stream stop code at 427-429 bp (Figure 2).

Blast analysis showed that sNORPEG (GenBank accession number AY317139) was highly homologous with NORPEG (GenBank accession number AF155135) and with the AB037755, BC028681 and AY354204 gene transcripts, all of which are classified as derived from the RAI14 gene (Retinoic Acid Induced gene) in GenBank and belong to the UniGene Cluster Hs. 368605. Blast search in the human genome database showed that the RAI14 gene consists of 23 exons and 22 introns and is localized to human chromosome 5p13.2-13.3. Splicing comparison of sNORPEG with its homologous genes indicated that sNORPEG had 20 exons. Exon 7 was its unique exon at the 5' terminus. The last exon of sNORPEG (103 bp) at the 3' terminus was shorter than that of NORPEG (1927 bp). Sequence analysis (http://l25.itba.mi.cnr.it/~webgene/wwwHC-polya.html) indicated that while NORPEG had a typical poly A signal at its 3' terminal (AATAAA, nt 4880-4885), sNORPEG had an atypical poly A signal (ACTAAA, nt 3465-3470) (Figure 2). Promoter scan software predicted that sNORPEG and NORPEG had different putative promoter regions. One of the predicted promoters (P1) was located at position -250 bp to -1 bp upstream of the 5' terminal of NORPEG. The transcripts of NORPEG and AB037755 could be driven by the P1 promoter. Another predicted promoter (P2) was located between -982 bp and -732 bp upstream of the 5' end of sNORPEG. The expression of sNORPEG, BC028681 and AY354204 could be initiated at the P2 promoter (Figure 3A). The above analysis indicated that sNORPEG (3486 bp) was a short alternative transcript of NORPEG (4925 bp).

Blast protein analysis showed that the sNORPEG protein was highly homologous with the NORPEG (98 % identity) and ankycorbin (84 % identity) proteins. The ankycorbin protein was encoded by a mouse NORPEG-homologous gene. The sNORPEG, NORPEG and ankycorbin proteins were found to contain 983, 980 and 979 amino acid residues, respectively. As shown in Figure 4, the sequence from the 13th amino acid residue to the end of the sNORPEG protein matched the sequence from the 16th amino acid residue to the end of the NORPEG protein.

Analysis of the amino acid sequence using SMART software (http://smart.embl-heidelberg.de/) revealed that the sNORPEG and NORPEG proteins had identical domains. They both contain six ankyrin repeats in the N-terminal region and two coiled-coil domains in the C-terminal region (Figure 5). Ankycorbin similarly has six ankyrin repeats and a long coiled-coil domain [1].

3.3 Expression profiles of sNORPEG

Expression profiles in different developmental stages of the testis and spermatozoa showed that sNORPEG is expressed in fetal testes, adult testes and spermatozoa (Figure 6). Multi-tissue PCR data indicated that sNORPEG is widely expressed in human tissues (Figure 7). In addition, unexpected 340 bp band was detected and sequenced (Figure 3C). Blast searches revealed that it was a novel EST of NORPEG (Figure 3B, EST id number: 25994518; GenBank accession number: CK433905).

4 Discussion

In the present study, a testis cDNA microarray was used to identify genes related to testis development and spermatogenesis. A novel short alternative transcript of NORPEG was cloned and identified with this method and given the name sNORPEG. Bioinformatics analysis and experimental results suggest that sNORPEG may play a role in testis development and spermatogenesis.

Sequence analysis showed that the sNORPEG protein contains six ankyrin repeats and two coiled-coil domains. The ankyrin repeat is one of the most common protein sequence motifs. It comprises approximately 33 amino acids and occurs in at least four consecutive copies [8-10]. Ankyrin repeats have been found in proteins as diverse as Cdk inhibitors, signal transduction and transcriptional regulators, cytoskeletal organizers, developmental regulators, and toxins [9]. It is generally assumed that the ankyrin repeats play an important role in protein-protein interactions [11]. The C-terminal domain contains the coiled-coil domain which is a highly versatile protein folding motif related to protein-protein interaction [12, 13]. The coiled-coil domain exists in some actin-binding proteins, such as tara [14], tropomyosin [15] and KRAP [16]. Thus it is likely that these two conserved domains are involved in mediating protein-protein interactions of the sNORPEG protein with its partner proteins.

The sNORPEG protein shows 84 % identity with ankycorbin and 98 % identity with NORPEG. These three homologous proteins have in common six ankyrin repeats and coiled-coil domains, suggesting that the sNORPEG protein may have a function similar to that of the NORPEG and ankycorbin proteins. Prior evidence suggests that the NORPEG protein is associated with the cytoskeleton [17]. Likewise, ankycorbin is highly concentrated at cortical actin cytoskeleton structures in terminal web and cell-cell adhesion sites and stress fibers. Ankycorbin appears to be an actin cytoskeleton-associated protein and may be involved in actin cytoskeleton maintenance and/or reorganization [1]. The homology of the sNORPEG protein to the NORPEG and ankycorbin proteins is consistent with our hypothesis that its function may be related to actin cytoskeleton dynamics and that it may play a role in the actin-related events that occur during testis development and spermatogenesis.

Actin filaments are concentrated in specific regions of spermatogenic cells and Sertoli cells. In spermatogenic cells they occur in intercellular bridges and in the subacrosomal space. In Sertoli cells they are abundant in the ectoplasmic specializations and in regions adjacent to the tubulobulbar processes of spermatogenic cells [18]. In the testis there exists an important cell-cell actin-based adherens junction, the dynamics of which are important in permitting the timely movement of germ cells across the epithelium [19]. Actin and actin-associated proteins are involved in regulating the Sertoli-germ cell actin-based adherens junction assembly and disassembly. It is possible that the sNORPEG protein may play an important role in this process. sNORPEG was not only expressed in human fetal testes, adult testes and spermatozoa, but also ubiquitously expressed in other human tissues. These findings suggest that the sNORPEG protein may also be involved in actin cytoskeleton dynamics in these tissues.

Alternative transcripts can be generated during gene expression by using promoters and transcription factors that activate transcription at different start sites upstream or downstream of the usual site, by incorporation of alternate exons, by germ cell-specific splicing events, and by using alternate initiation sites for polyadenylation [20]. Compared with NORPEG, sNORPEG has a different putative promoter and a shorter 3'-untranslated region. The sNORPEG transcript may be generated by an alternative promoter usage and an alternate polyadenylation signal. Our study revealed an additional unknown alternatively spliced variant of NORPEG that was only expressed in the lung, adult testis and spermatozoa. The restricted expression of this unknown splice variant suggests that its function would be specific to the lung and testis. The diversity of these alternative transcripts gives us new insight into the complex genetic regulation of NORPEG.

In summary, a novel mRNA transcript, sNORPEG, was identified which has several important conserved domains. The homologous motif properties of the protein encoded by sNORPEG and its expression in human fetal testes, adult testes and spermatozoa suggest that it may participate in actin cytoskeleton dynamics during testis development and spermatogenesis. Further study will be required to elucidate the functional role and regulatory mechanisms of the sNORPEG protein in testicular development and spermatogenesis.

Acknowledgment

This work  was supported by grants from China National 973 Program (No. G1999055901), National Natural Science Foundation of China (No. 30425006), the Foundation of Science and Technology of Jiangsu Province, China (No. BG2003028) and the Foundation of National Ministry of Science and Technology, China (No. 2004CCA06800).

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