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Molecular cloning and characterization of a novel Y-box gene from planarian Dugesia japonica
Biochemical and Biophysical Research Communications xxx (2018) 1e6
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Molecular cloning and characterization of a novel Y-box gene from planarian Dugesia japonica Yahong Han 1, Zhenbiao Xu 1, Yanan Liu, Da Wei, Jing Zhang, Le Xue, Xiangmin Zhang, Jie Qin, Hongkuan Deng, Linxia Song* College of Life Science, Shandong University of Technology, 266 Xincun West Road, Zibo, Shandong, 255049, China
a r t i c l e i n f o
a b s r a c t
Article history: Received 9 September 2018 Accepted 21 September 2018 Available online xxx
Y-box binding protein (YB protein) is an ancient conserved multifunctional DNA/RNA-binding protein. A novel YB protein DjY2 gene from planarian Dugesia japonica was cloned by RACE method and characterized. This cDNA contains 689 bp with a putative open reading frame of 197 amino acids. It has a predicted molecular mass of 22.14 kDa and an isoelectric point of 9.67. Whole-mount in situ hybridization and relative quantitative real-time PCR were used to study the spatial and temporal expression pattern of DjY2 in the process of planarian regeneration. Results showed that DjY2 was expressed in many parts of the body in intact planarian, but the expression level was low in head and pharynx. The transcripts of DjY2 was significantly increased both at the head parts and the tail parts after amputation, especially at the site of cutting. The spatial expression gradually recovered to the state of intact planarian with the time of regeneration. Our results indicated that DjY2 might participate in the process of regeneration in planarian. © 2018 Elsevier Inc. All rights reserved.
Keywords: Y-box binding protein DjY2 Planarian Regeneration
1. Introduction The Y-box binding proteins (YB proteins) are a family of gene regulators that contain a conserved nucleic acid-binding domain, the cold shock domain (CSD) [1]. As a multifunctional DNA/RNAbinding protein, YB protein involves in many cellular activities, including regulation of transcription and translation, DNA replication and repair，chromatin remodeling and pre-mRNA splicing [2]. Human YB-1 was the first cloned YB protein that binds to the Ybox motif in the promoter of major histocompatibility complex (MHC) class II genes [3]. Studies showed that YB-1 has a variety of cell functions. It participates in oncogenic transformation of cells, tumor metastasis, multidrug resistence, inflammatory processes, viral infections and embronic development [4e7]. So far many genes of YB proteins have been identified in different eukaryotes, such as Xenopus laevis, Drosophila melanogaster, mouse and the other animals [8e12]. YB protein consists of three domains: a variable N-terminal domain, a CSD and a C-terminal tail domain.
* Corresponding author. E-mail address: [email protected] (L. Song). 1 Co-first authors.
The N-terminal domain has high content of alanine and proline (A/ P domain), which is thought to be a trans-activation domain. The CSD consists of an antiparallel five-stranded b-barrel, which recognizes both DNA and RNA [1]. This domain is highly conserved from bacteria to eukaryotes. The C-terminal tail domain consists of alternate regions of predominantly basic or acidic amino acids, each of which are about 30 amino acids in length, called a B/A repeat [13]. This domain is thought to be involved in protein-protein interactions. It also contains several sites for phosphorylation by casein kinase II [13]. Planarian is a kind of platyhelminthes with strong regeneration abilities. This abilities are due to the pluripotent adult stem cells called neoblasts, which are almost all over the body except pharynx and the area in front of eyespots [14]. When planarian is injured, neoblasts migrate to the wound area and begin to divide, forming a tissue without pigment, which is called a blastema [15]. Then the cells in blastema differentiate into cells in damaged tissue for repairing. Generally it takes seven days for the injured planarian to grow into a complete individual after cutting. This feature makes planarian a good model for studying the molecular mechanisms of stem cell differentiation, development, proliferation and regulation. In planarian, two YB genes have been isolated, DjY1 and DeY1 [16,17]. In regenerating tissues the expression of DjY1 is specifically up-regulated. Its transcripts are localized first in the blastema, and
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later in the auricles, the differentiating sensory organs [16]. While the transcripts of DeY1 are not found in the blastema during regeneration, they are found in the testes of sexual planarians. Its transcripts are localized in spermatogonia, spermatocytes and spermatids, but not in spermatozoa [17]. In this paper, we isolated and characterized a novel cDNA encoding YB protein from D. japonica using rapid amplification of cDNA ends (RACE) technology，we named it DjY2. Using Wholemount in situ hybridization and relative quantitative real-time PCR, we found the transcripts of DjY2 are predominantly expressed in most parts of the body except head and pharynx in intact worm. But after amputation, its transcripts level is significantly upregulated and its expression is concentrated in the blastemas, suggesting DjY2 may be involved in the process of planarian regeneration. 2. Materials and methods 2.1. Planarian An asexual strain Dugesia ZB-1, which was from Zibo, Shandong Province, China, cultured in commercially mineral water, 20e24 C in biochemical incubator. The worms were fed with beef liver two times a week and starved for a week before experiments. 2.2. Primer design NCBI EST database was searched using human YB-1 gene (Genbank No. J03827) as the query sequence. A fragment with higher homology (Genbank No. BP185287) in Dugesia japonica was found. According to this sequence, primers DjY2-1 and DjY2-2 were designed using Primer Premier 5.0 software. 2.3. Extraction of total RNA and cloning of DjY2 gene Total RNA was extracted using Trizol reagent (Invitrogen) and reverse transcription (reverse transcriptase was purchased from TaKaRa) was carried out with oligo(dT)15 as primer. The middle fragment F1 was obtained through PCR using DjY2-1 and DjY2-2 primers and cloned into pMD-T18 vector. Sanger sequencing technolgy was employed to sequence the PCR fragments. RACE experiments were performed to obtain the 30 end fragment F3 and 50 end fragment F5 using RACE kit (TaKaRa). Combined the
sequences of F5, F1 and F3 to get the full length sequence information of DjY2 gene. Then the full length DjY2 gene was obtained by PCR amplification using DjY2-A1 and DjY2-A2 as primers. DNA and protein sequences were analyzed using blastn and blastp at NCBI Web Server (http://www.ncbi.nlm.nih.gov/blast). The open reading frame (ORF) was predicted at ExPASy Server (http://web. expasy.org/translate). Multiple sequence alignment was carried out using GeneDoc software.
2.4. Whole-mount in situ hybridization Planarians about 4e7 mm in length were selected for in situ hybridization experiment. PCR primers (ISH-1 and ISH-2) were designed according to the ORF sequence of DjY2 cDNA. T7 promoter sequence was added to the end of the reverse primer. A DIG-labeled antisense probe was synthesized with purified PCR products as template using RNA polymerase (Roche) by in vitro transcription reaction. Planarians were cut into three parts: head, trunk and tail, then fixed with 4% PFA and bleached in 6% H2O2 in methanol under light overnight. In situ hybridization was performed at 56 C in hybridization buffer (50% formamide, 5 SSC, 1 mg/ml yeast RNA, 1% Tween-20, 100 mg/ml heparin, 5 mM DTT, 1 Denhardt's, 10% Dextran Sulfate) for at least 12 h. Then incubated with blocking solution for 1 h and with anti-DIG-AP conjugated antibody solution (1:2000 in blocking solution) for 2 h at room temperature. At last a freshly prepared NBT/BCIP solution was used for colorimetrical detection.
2.5. Quantitative real-time PCR (qPCR) Total RNAs were isolated from the intact planarians and the regenerating planarians of 1e7 days after amputation. Planarians were cut into two parts: head and tail, then total RNAs were extracted and cDNAs were synthesized using 2 mg RNAs as template. qPCR was performed using ABI 7500 fluorescence quantitative PCR instrument with SYBR green transcription system from TAKARA company. DjActin gene was selected as the endogenous standardization. qPCR datasets was carried out using 2^-△△Ct method and subjected to a Student's t-test (P < 0.05 was considered significant difference, P < 0.01 was considered extremely significant difference). The results are presented as means ± SD. Primers used in this experiments are listed in Table 1.
Table 1 Primer sequences used in this study. Primer name PCR for F1 fragment DjY2-1 DjY2-2 RACE DjY2-5GSP1 DjY2-5GSP2 DjY2-3GSP1 DjY2-3GSP2 Full length gene DjY2-A1 DjY2-A2 Real-time PCR qPCR-DjY2-1 qPCR-DjY2-2 qPCR-DjActin-1 qPCR-DjActin-2 In situ hybridization probe ISH-1 ISH-2
Sequence 50 -GTTAAGTATGTCTTCAGATGG-30 50 -GATATCCCCCACCACTACTTC-30 50 -ACCAGGTTGGCATCTACTTATTG-30 50 -ATTATACCATTTAACTTTTCCGG-30 50 -CCTAATGGTGATGCGGTGCT-30 50 -GGCCGTGGACGTAGTTACTT-30 50 -CAGATCTTTTTGAAATTTTGAA-30 50 -TCCATATAAAAGAACATAAATATA-30 50 -TGCTCCTAACAATGATTTCCAA-30 50 -TCCCCCACCACTACTTCCAC-30 50 -ACCAGCAGATTCCATACCCA-30 50 -TTATGTTACGTTGCCCTCGA-30 50 -ATGTCTTCTGATGGTGAAGAA-30 50 -GATCACTAATACGACTCACTATAGGGTTATACACCTCTGGTTTGATA-30
Please cite this article in press as: Y. Han, et al., Molecular cloning and characterization of a novel Y-box gene from planarian Dugesia japonica, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.09.136
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Fig. 1. Nucleotide sequence and the deduced amino acid sequence of the D. japonica DjY2 cDNA. The putative translation start codon is in bold letters and underlined, the stop codon is indicated by an asterisk and underlined. There are two stop codons (TGA and TAG) in italics with shadow in front of the start codon ATG. The nucleotide sequence reported here has been submitted to the Genbank Database with accession number KY799178.
3. Results 3.1. Cloning and sequence analysis of DjY2 gene Full length DjY2 gene was cloned by RACE experiments. Its cDNA is 689 bp in length with a putative ORF of 594 bp, which encodes a peptide of 197 amino acids (Fig. 1). The predicted molecular mass is 22.14 kDa and an isoelectric point is 9.67. There are two stop codons in front of the start codon ATG (Fig. 1). Bioinformatics analysis revealed that there is a CSD at the region of 22e90 amino acids. This domain is 61% identity with that of human YB-1. The multiple alignment of DjY2 with YB proteins from other species is shown in Fig. 2. The RNP-1 core residues GYGF and the RNP-2 like motif VFV are also found in CSD of DjY2. The GYGF sequence is conserved among all the eukaryotic proteins, while the VFV sequence is a little different between these proteins. Sequence analysis showed that DjY2 contains five putative casein kinase II phosphorylation sites and seven protein kinase C phosphorylation sites, suggesting that DjY2 could be highly phosphorylated in vivo. 3.2. Temporal and spatial expression pattern of DjY2 in intact and regenerating planarians In order to identify the temporal and spatial expression pattern of DjY2, whole-mount in situ hybridization was used to examine
the intact planarians and the regenerating planarians of 1, 3, 5, 7 days after amputation. Worms were cut into three parts: head, trunk and tail (Fig. 3). In intact planarians, DjY2 was expressed in many parts of the body, but the expression level was low in head and pharynx. DjY2 transcripts gradually migrated to the blastema of wound after amputation. At 1 day after amputation, the expression level of DjY2 in the blastema was obviously higher than that in other parts. At 3 days after amputation, the expression signal was mainly concentrated in the region of the blastema while the signal was very weak in other parts. At 5 days after amputation, the expression gradually spread to other parts of the body. The expression pattern of DjY2 gradually recovered to its intact state when the cutting worm became a complete individual at 7 days after amputation. 3.3. Expression of DjY2 is upregulated during regeneration In order to study the expression level of DjY2 in the process of regeneration, planarians were cut into two parts: head and tail. They were respectively collected at 1e7 days after amputation. The intact worms were as control. Total RNAs were isolated from the amputated worms and RT-qPCRs were performed. As shown in Fig. 4, the transcription level of DjY2 in the process of regeneration was higher than that in the intact worms. The highest level was observed at 1 day after amputation, then a slight decline appeared
Please cite this article in press as: Y. Han, et al., Molecular cloning and characterization of a novel Y-box gene from planarian Dugesia japonica, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.09.136
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Fig. 2. Multiple alignment of DjY2 sequences and the other YB protein members. Black backgrounds indicate identical residues and grey backgrounds indicate conserved residues. The cold-shock DNA-binding domain are indicated from amino acid (;) to amino acid (*). The sequences of RNP-1(GYGF) and RNP-2 (VFV) motif are underlined. The accession numbers of the aligned proteins are as follows: YB-1 (J03827 from Homo sapiens), rBYB1 (AB046928 from Rattus norvegicus), MSY-1 (M62867 from Mus musculus), FRGY1 (M38382 from Xenopus laevis), YPS (U49120 Drosophila melanogaster), SmYB (U39883 from Schistosoma mansoni), DeY1 (AJ439094 from Dugesia etrusca), DjY1(X99748 from Dugesia japonica), DjY2(KY799178 from Dugesia japonica).
Fig. 3. Expression patterns of DjY2 in intact and regenerating planarians at different days of regeneration. A. An intact planarian. Positive signals were detected in many parts of the body, but were very low in head and pharynx. B-E, F-I, J-M represented the expression of DjY2 in regenerating planarians of head, trunl and tail at 1, 3, 5 and 7 days after amputation. Scale bars represnet 200 mm.
Please cite this article in press as: Y. Han, et al., Molecular cloning and characterization of a novel Y-box gene from planarian Dugesia japonica, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.09.136
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Fig. 4. Expression analysis of DjY2 mRNA by qPCR at different time points. The time points are 0, 1, 2, 3, 4, 5, 6, 7 days after amputation from left to right. “0” represents the intact planarians. The expression level was normalized with DjActin as the endogenous standardization. Each experiment was repeated three times. Standard deviation is represented by error bars. Asterisk means significant difference. One means P < 0.05, two means P < 0.01. A. The expression level of the head parts after amputation. B. The expression level of the tail parts after amputation.
in the next few days. The expression patterns of head parts were almost the same as that of tail parts. 4. Discussion YB protein is the most evolutionary conserved DNA/RNAbinding protein with a conserved CSD. Many YB proteins were found in eukaryotes. Human YB-1 is the most studied YB protein with a wide variety of cellular functions. But research on YB proteins in invertebrates is not as much as that in vertebrates. Planarian is a kind of invertebrate with bilateral symmetry and three germ layers. Due to its remarkable ability of regeneration, a lot of research work has been carried out using this model animal. In this paper, we cloned a full-length cDNA of YB protein named DjY2 from D. japonica. Similar to other YB proteins in eukaryotes, DjY2 has three domains with the conserved CSD, indicated it belongs to the family members of YB protein. The identity of CSD is 62% between DjY2 and DjY1, 64% between DjY2 and DeY1, but the C-terminal structure of DjY2 has low similarity with DjY1 and DeY1. The C-terminal tail domain is thought to be involved in proteinprotein interactions and stabilization of protein-nucleic acid interaction [13]. These structural alterations may be due to the different interactions with proteins and nucleic acids, which are necessary for their pleiotropic functions of YB proteins. YB proteins fall into three subfamilies according to their different cell localization, functions and synthesis period. There are more than one YB proteins exist in human, mouse and X. laevis [2]. In mouse, five YB proteins have four different amino termini, three very similar but non-identical CSD, and four different carboxyl termini [18]. All the five YB mRNAs are expressed higher in testis than in somatic tissues. Msy2 mRNA is expressed only in male and female germ cell [19]. Msy1 mRNA is expressed at high levels in kidney and heart, and Msy3S mRNA is expressed at high levels in skeletal muscle [18]. In X. laevis, FRG Y1 mRNA is expressed in oocytes, embryos, and the adult tissues, whereas FRG Y2 mRNA is only expressed in testis and immature oocytes [20]. In planarian, DjY1 is expressed at the blastema. Its transcripts appear rapidly at the regenerative tissue and increase in number as the blastema grows. As regeneration nears completion, there is a general decrease in transcript level except in the differentiating structures, specifically in the auricles [16]. Our results showed that DjY2 is not only expressed in the regenerating tissue, but also in the intact one. Unlike DjY1, the transcripts level of DjY2 is very low in the head
area, and is not detected in the auricles, no matter in the regenertating stages or the non-regenerating stages. Although expressed in the same regeneration stage, the different expression pattern between DjY1 and DjY2 suggests that they may perform different roles in the regeneration process in planarian. In this paper, our experiments results showed the transcripts of DjY2 significantly increased at the site of cutting, reached to the highest level at 1 day after amputation in both head parts and tail parts, indicated DjY2 might involve in the process of regeneration like DjY1. YB protein is an ancient conserved multifunctional protein, maybe more than one YB protein members exist in D. japonica like other species. Up to now, many genes and signaling pathways related with regeneration were found in planarian [21e24]. So what's the relationship between YB proteins and the genes participate in the process of planarian regeneration? What are the functions of these YB proteins and where do they play roles in the cell? More research work needs to be carried out to reveal the specific function and molecular mechanism of planarian YB proteins in the future. Declaration of interest We have no conflicts of interest with other people or organizations. Acknowledgements This study was supported by the National Natural Science Foundation of China (31550005,31350004) and the Natural Science Foundation of Shandong Province, China (ZR2011HM065). References [1] A.P. Wolffe, Structural and functional properties of the evolutionarily ancient Y-box family of nucleic acid binding proteins, Bioessays 16 (1994) 245e251. [2] I.A. Eliseeva, E.R. Kim, S.G. Guryanov, L.P. Ovchinnikov, D.N. Lyabin, Y-boxbinding protein 1 (YB-1) and its functions, Biochemistry (Mosc) 76 (2011) 1402e1433. [3] D.K. Didier, J. Schiffenbauer, S.L. Woulfe, M. Zacheis, B.D. Schwartz, Characterization of the cDNA encoding a protein binding to the major histocompatibility complex class II Y box, Proc. Natl. Acad. Sci. U.S.A. 85 (1998) 7322e7326. [4] V. Evdokimova, C. Tognon, T. Ng, P. Ruzanov, N. Melnyk, D. Fink, A. Sorokin, L.P. Ovchinnikov, E. Davicioni, T.J. Triche, P.H. Sorensen, Translational activation of snail1 and other developmentally regulated transcription factors by YB-1 promotes an epithelial-mesenchymal transition, Canc. Cell 15 (2009)
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402e415. [5] B. Schittek, K. Psenner, B. Sauer, F. Meier, T. Iftner, C. Garbe, The increased expression of Y box-binding protein 1 in melanoma stimulates proliferation and tumor invasion, antagonizes apoptosis and enhances chemoresistance, Int. J. Canc. 120 (2007) 2110e2118. [6] T. Ohga, T. Uchiumi, Y. Makino, K. Koike, M. Wada, M. Kuwano, K. Kohno, Direct involvement of the Y-box binding protein YB-1 in genotoxic stressinduced activation of the human multidrug resistance 1 gene, J. Biol. Chem. 273 (1998) 5997e6000. [7] Z.H. Lu, J.T. Books, T.J. Ley, YB-1 is important for late-stage embryonic development, optimal cellular stress responses, and the prevention of premature senescence, Mol. Cell Biol. 25 (2005) 4625e4637. [8] M.T. Murray, D.L. Schiller, W.W. Franke, Sequence analysis of cytoplasmic mRNA-binding proteins of Xenopus oocytes identifies a family of RNA-binding proteins, Proc. Natl. Acad. Sci. U.S.A. 89 (1992) 11e15. [9] H.A. Thieringer, K. Singh, H. Trivedi, M. Inouye, Identification and developmental characterization of a novel Y-box protein from Drosophila melanogaster, Nucleic Acids Res. 25 (1997) 4764e4770. [10] S.R. Tafuri, M. Familari, A.P. Wolffe, A mouse Y box protein, MSY1, is associated with paternal mRNA in spermatocytes, J. Biol. Chem. 268 (1993) 12213e12220. [11] C.E. Grant, R.G. Deeley, Cloning and characterization of chicken YB-1: regulation of expression in the liver, Mol. Cell Biol. 13 (1993) 4186e4196. [12] V.M. Evdokimova, C.L. Wei, A.S. Sitikov, P.N. Simonenko, O.A. Lazarev, K.S. Vasilenko, V.A. Ustinov, J.W. Hershey, L.P. Ovchinnikov, The major protein of messenger ribonucleoprotein particles in somatic cells is a member of the Y-box binding transcription factor family, J. Biol. Chem. 270 (1995) 3186e3192. [13] K. Kohno, H. Izumi, T. Uchiumi, M. Ashizuka, M. Kuwano, The pleiotropic functions of the Y-box-binding protein, YB-1, Bioessays 25 (2003) 691e698. nchez Alvarado, Bromodeoxyuridine specifically labels [14] P.A. Newmark, A. Sa the regenerative stem cells of planarians, Dev. Biol. 220 (2000) 142e153.
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Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Molecular cloning and characterization of a novel Y-box gene from planarian Dugesia japonica Yahong Han 1, Zhenbiao Xu 1, Yanan Liu, Da Wei, Jing Zhang, Le Xue, Xiangmin Zhang, Jie Qin, Hongkuan Deng, Linxia Song* College of Life Science, Shandong University of Technology, 266 Xincun West Road, Zibo, Shandong, 255049, China
a r t i c l e i n f o
a b s r a c t
Article history: Received 9 September 2018 Accepted 21 September 2018 Available online xxx
Y-box binding protein (YB protein) is an ancient conserved multifunctional DNA/RNA-binding protein. A novel YB protein DjY2 gene from planarian Dugesia japonica was cloned by RACE method and characterized. This cDNA contains 689 bp with a putative open reading frame of 197 amino acids. It has a predicted molecular mass of 22.14 kDa and an isoelectric point of 9.67. Whole-mount in situ hybridization and relative quantitative real-time PCR were used to study the spatial and temporal expression pattern of DjY2 in the process of planarian regeneration. Results showed that DjY2 was expressed in many parts of the body in intact planarian, but the expression level was low in head and pharynx. The transcripts of DjY2 was significantly increased both at the head parts and the tail parts after amputation, especially at the site of cutting. The spatial expression gradually recovered to the state of intact planarian with the time of regeneration. Our results indicated that DjY2 might participate in the process of regeneration in planarian. © 2018 Elsevier Inc. All rights reserved.
Keywords: Y-box binding protein DjY2 Planarian Regeneration
1. Introduction The Y-box binding proteins (YB proteins) are a family of gene regulators that contain a conserved nucleic acid-binding domain, the cold shock domain (CSD) [1]. As a multifunctional DNA/RNAbinding protein, YB protein involves in many cellular activities, including regulation of transcription and translation, DNA replication and repair，chromatin remodeling and pre-mRNA splicing [2]. Human YB-1 was the first cloned YB protein that binds to the Ybox motif in the promoter of major histocompatibility complex (MHC) class II genes [3]. Studies showed that YB-1 has a variety of cell functions. It participates in oncogenic transformation of cells, tumor metastasis, multidrug resistence, inflammatory processes, viral infections and embronic development [4e7]. So far many genes of YB proteins have been identified in different eukaryotes, such as Xenopus laevis, Drosophila melanogaster, mouse and the other animals [8e12]. YB protein consists of three domains: a variable N-terminal domain, a CSD and a C-terminal tail domain.
* Corresponding author. E-mail address: [email protected] (L. Song). 1 Co-first authors.
The N-terminal domain has high content of alanine and proline (A/ P domain), which is thought to be a trans-activation domain. The CSD consists of an antiparallel five-stranded b-barrel, which recognizes both DNA and RNA [1]. This domain is highly conserved from bacteria to eukaryotes. The C-terminal tail domain consists of alternate regions of predominantly basic or acidic amino acids, each of which are about 30 amino acids in length, called a B/A repeat [13]. This domain is thought to be involved in protein-protein interactions. It also contains several sites for phosphorylation by casein kinase II [13]. Planarian is a kind of platyhelminthes with strong regeneration abilities. This abilities are due to the pluripotent adult stem cells called neoblasts, which are almost all over the body except pharynx and the area in front of eyespots [14]. When planarian is injured, neoblasts migrate to the wound area and begin to divide, forming a tissue without pigment, which is called a blastema [15]. Then the cells in blastema differentiate into cells in damaged tissue for repairing. Generally it takes seven days for the injured planarian to grow into a complete individual after cutting. This feature makes planarian a good model for studying the molecular mechanisms of stem cell differentiation, development, proliferation and regulation. In planarian, two YB genes have been isolated, DjY1 and DeY1 [16,17]. In regenerating tissues the expression of DjY1 is specifically up-regulated. Its transcripts are localized first in the blastema, and
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Please cite this article in press as: Y. Han, et al., Molecular cloning and characterization of a novel Y-box gene from planarian Dugesia japonica, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.09.136
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later in the auricles, the differentiating sensory organs [16]. While the transcripts of DeY1 are not found in the blastema during regeneration, they are found in the testes of sexual planarians. Its transcripts are localized in spermatogonia, spermatocytes and spermatids, but not in spermatozoa [17]. In this paper, we isolated and characterized a novel cDNA encoding YB protein from D. japonica using rapid amplification of cDNA ends (RACE) technology，we named it DjY2. Using Wholemount in situ hybridization and relative quantitative real-time PCR, we found the transcripts of DjY2 are predominantly expressed in most parts of the body except head and pharynx in intact worm. But after amputation, its transcripts level is significantly upregulated and its expression is concentrated in the blastemas, suggesting DjY2 may be involved in the process of planarian regeneration. 2. Materials and methods 2.1. Planarian An asexual strain Dugesia ZB-1, which was from Zibo, Shandong Province, China, cultured in commercially mineral water, 20e24 C in biochemical incubator. The worms were fed with beef liver two times a week and starved for a week before experiments. 2.2. Primer design NCBI EST database was searched using human YB-1 gene (Genbank No. J03827) as the query sequence. A fragment with higher homology (Genbank No. BP185287) in Dugesia japonica was found. According to this sequence, primers DjY2-1 and DjY2-2 were designed using Primer Premier 5.0 software. 2.3. Extraction of total RNA and cloning of DjY2 gene Total RNA was extracted using Trizol reagent (Invitrogen) and reverse transcription (reverse transcriptase was purchased from TaKaRa) was carried out with oligo(dT)15 as primer. The middle fragment F1 was obtained through PCR using DjY2-1 and DjY2-2 primers and cloned into pMD-T18 vector. Sanger sequencing technolgy was employed to sequence the PCR fragments. RACE experiments were performed to obtain the 30 end fragment F3 and 50 end fragment F5 using RACE kit (TaKaRa). Combined the
sequences of F5, F1 and F3 to get the full length sequence information of DjY2 gene. Then the full length DjY2 gene was obtained by PCR amplification using DjY2-A1 and DjY2-A2 as primers. DNA and protein sequences were analyzed using blastn and blastp at NCBI Web Server (http://www.ncbi.nlm.nih.gov/blast). The open reading frame (ORF) was predicted at ExPASy Server (http://web. expasy.org/translate). Multiple sequence alignment was carried out using GeneDoc software.
2.4. Whole-mount in situ hybridization Planarians about 4e7 mm in length were selected for in situ hybridization experiment. PCR primers (ISH-1 and ISH-2) were designed according to the ORF sequence of DjY2 cDNA. T7 promoter sequence was added to the end of the reverse primer. A DIG-labeled antisense probe was synthesized with purified PCR products as template using RNA polymerase (Roche) by in vitro transcription reaction. Planarians were cut into three parts: head, trunk and tail, then fixed with 4% PFA and bleached in 6% H2O2 in methanol under light overnight. In situ hybridization was performed at 56 C in hybridization buffer (50% formamide, 5 SSC, 1 mg/ml yeast RNA, 1% Tween-20, 100 mg/ml heparin, 5 mM DTT, 1 Denhardt's, 10% Dextran Sulfate) for at least 12 h. Then incubated with blocking solution for 1 h and with anti-DIG-AP conjugated antibody solution (1:2000 in blocking solution) for 2 h at room temperature. At last a freshly prepared NBT/BCIP solution was used for colorimetrical detection.
2.5. Quantitative real-time PCR (qPCR) Total RNAs were isolated from the intact planarians and the regenerating planarians of 1e7 days after amputation. Planarians were cut into two parts: head and tail, then total RNAs were extracted and cDNAs were synthesized using 2 mg RNAs as template. qPCR was performed using ABI 7500 fluorescence quantitative PCR instrument with SYBR green transcription system from TAKARA company. DjActin gene was selected as the endogenous standardization. qPCR datasets was carried out using 2^-△△Ct method and subjected to a Student's t-test (P < 0.05 was considered significant difference, P < 0.01 was considered extremely significant difference). The results are presented as means ± SD. Primers used in this experiments are listed in Table 1.
Table 1 Primer sequences used in this study. Primer name PCR for F1 fragment DjY2-1 DjY2-2 RACE DjY2-5GSP1 DjY2-5GSP2 DjY2-3GSP1 DjY2-3GSP2 Full length gene DjY2-A1 DjY2-A2 Real-time PCR qPCR-DjY2-1 qPCR-DjY2-2 qPCR-DjActin-1 qPCR-DjActin-2 In situ hybridization probe ISH-1 ISH-2
Sequence 50 -GTTAAGTATGTCTTCAGATGG-30 50 -GATATCCCCCACCACTACTTC-30 50 -ACCAGGTTGGCATCTACTTATTG-30 50 -ATTATACCATTTAACTTTTCCGG-30 50 -CCTAATGGTGATGCGGTGCT-30 50 -GGCCGTGGACGTAGTTACTT-30 50 -CAGATCTTTTTGAAATTTTGAA-30 50 -TCCATATAAAAGAACATAAATATA-30 50 -TGCTCCTAACAATGATTTCCAA-30 50 -TCCCCCACCACTACTTCCAC-30 50 -ACCAGCAGATTCCATACCCA-30 50 -TTATGTTACGTTGCCCTCGA-30 50 -ATGTCTTCTGATGGTGAAGAA-30 50 -GATCACTAATACGACTCACTATAGGGTTATACACCTCTGGTTTGATA-30
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Fig. 1. Nucleotide sequence and the deduced amino acid sequence of the D. japonica DjY2 cDNA. The putative translation start codon is in bold letters and underlined, the stop codon is indicated by an asterisk and underlined. There are two stop codons (TGA and TAG) in italics with shadow in front of the start codon ATG. The nucleotide sequence reported here has been submitted to the Genbank Database with accession number KY799178.
3. Results 3.1. Cloning and sequence analysis of DjY2 gene Full length DjY2 gene was cloned by RACE experiments. Its cDNA is 689 bp in length with a putative ORF of 594 bp, which encodes a peptide of 197 amino acids (Fig. 1). The predicted molecular mass is 22.14 kDa and an isoelectric point is 9.67. There are two stop codons in front of the start codon ATG (Fig. 1). Bioinformatics analysis revealed that there is a CSD at the region of 22e90 amino acids. This domain is 61% identity with that of human YB-1. The multiple alignment of DjY2 with YB proteins from other species is shown in Fig. 2. The RNP-1 core residues GYGF and the RNP-2 like motif VFV are also found in CSD of DjY2. The GYGF sequence is conserved among all the eukaryotic proteins, while the VFV sequence is a little different between these proteins. Sequence analysis showed that DjY2 contains five putative casein kinase II phosphorylation sites and seven protein kinase C phosphorylation sites, suggesting that DjY2 could be highly phosphorylated in vivo. 3.2. Temporal and spatial expression pattern of DjY2 in intact and regenerating planarians In order to identify the temporal and spatial expression pattern of DjY2, whole-mount in situ hybridization was used to examine
the intact planarians and the regenerating planarians of 1, 3, 5, 7 days after amputation. Worms were cut into three parts: head, trunk and tail (Fig. 3). In intact planarians, DjY2 was expressed in many parts of the body, but the expression level was low in head and pharynx. DjY2 transcripts gradually migrated to the blastema of wound after amputation. At 1 day after amputation, the expression level of DjY2 in the blastema was obviously higher than that in other parts. At 3 days after amputation, the expression signal was mainly concentrated in the region of the blastema while the signal was very weak in other parts. At 5 days after amputation, the expression gradually spread to other parts of the body. The expression pattern of DjY2 gradually recovered to its intact state when the cutting worm became a complete individual at 7 days after amputation. 3.3. Expression of DjY2 is upregulated during regeneration In order to study the expression level of DjY2 in the process of regeneration, planarians were cut into two parts: head and tail. They were respectively collected at 1e7 days after amputation. The intact worms were as control. Total RNAs were isolated from the amputated worms and RT-qPCRs were performed. As shown in Fig. 4, the transcription level of DjY2 in the process of regeneration was higher than that in the intact worms. The highest level was observed at 1 day after amputation, then a slight decline appeared
Please cite this article in press as: Y. Han, et al., Molecular cloning and characterization of a novel Y-box gene from planarian Dugesia japonica, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.09.136
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Fig. 2. Multiple alignment of DjY2 sequences and the other YB protein members. Black backgrounds indicate identical residues and grey backgrounds indicate conserved residues. The cold-shock DNA-binding domain are indicated from amino acid (;) to amino acid (*). The sequences of RNP-1(GYGF) and RNP-2 (VFV) motif are underlined. The accession numbers of the aligned proteins are as follows: YB-1 (J03827 from Homo sapiens), rBYB1 (AB046928 from Rattus norvegicus), MSY-1 (M62867 from Mus musculus), FRGY1 (M38382 from Xenopus laevis), YPS (U49120 Drosophila melanogaster), SmYB (U39883 from Schistosoma mansoni), DeY1 (AJ439094 from Dugesia etrusca), DjY1(X99748 from Dugesia japonica), DjY2(KY799178 from Dugesia japonica).
Fig. 3. Expression patterns of DjY2 in intact and regenerating planarians at different days of regeneration. A. An intact planarian. Positive signals were detected in many parts of the body, but were very low in head and pharynx. B-E, F-I, J-M represented the expression of DjY2 in regenerating planarians of head, trunl and tail at 1, 3, 5 and 7 days after amputation. Scale bars represnet 200 mm.
Please cite this article in press as: Y. Han, et al., Molecular cloning and characterization of a novel Y-box gene from planarian Dugesia japonica, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.09.136
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Fig. 4. Expression analysis of DjY2 mRNA by qPCR at different time points. The time points are 0, 1, 2, 3, 4, 5, 6, 7 days after amputation from left to right. “0” represents the intact planarians. The expression level was normalized with DjActin as the endogenous standardization. Each experiment was repeated three times. Standard deviation is represented by error bars. Asterisk means significant difference. One means P < 0.05, two means P < 0.01. A. The expression level of the head parts after amputation. B. The expression level of the tail parts after amputation.
in the next few days. The expression patterns of head parts were almost the same as that of tail parts. 4. Discussion YB protein is the most evolutionary conserved DNA/RNAbinding protein with a conserved CSD. Many YB proteins were found in eukaryotes. Human YB-1 is the most studied YB protein with a wide variety of cellular functions. But research on YB proteins in invertebrates is not as much as that in vertebrates. Planarian is a kind of invertebrate with bilateral symmetry and three germ layers. Due to its remarkable ability of regeneration, a lot of research work has been carried out using this model animal. In this paper, we cloned a full-length cDNA of YB protein named DjY2 from D. japonica. Similar to other YB proteins in eukaryotes, DjY2 has three domains with the conserved CSD, indicated it belongs to the family members of YB protein. The identity of CSD is 62% between DjY2 and DjY1, 64% between DjY2 and DeY1, but the C-terminal structure of DjY2 has low similarity with DjY1 and DeY1. The C-terminal tail domain is thought to be involved in proteinprotein interactions and stabilization of protein-nucleic acid interaction [13]. These structural alterations may be due to the different interactions with proteins and nucleic acids, which are necessary for their pleiotropic functions of YB proteins. YB proteins fall into three subfamilies according to their different cell localization, functions and synthesis period. There are more than one YB proteins exist in human, mouse and X. laevis [2]. In mouse, five YB proteins have four different amino termini, three very similar but non-identical CSD, and four different carboxyl termini [18]. All the five YB mRNAs are expressed higher in testis than in somatic tissues. Msy2 mRNA is expressed only in male and female germ cell [19]. Msy1 mRNA is expressed at high levels in kidney and heart, and Msy3S mRNA is expressed at high levels in skeletal muscle [18]. In X. laevis, FRG Y1 mRNA is expressed in oocytes, embryos, and the adult tissues, whereas FRG Y2 mRNA is only expressed in testis and immature oocytes [20]. In planarian, DjY1 is expressed at the blastema. Its transcripts appear rapidly at the regenerative tissue and increase in number as the blastema grows. As regeneration nears completion, there is a general decrease in transcript level except in the differentiating structures, specifically in the auricles [16]. Our results showed that DjY2 is not only expressed in the regenerating tissue, but also in the intact one. Unlike DjY1, the transcripts level of DjY2 is very low in the head
area, and is not detected in the auricles, no matter in the regenertating stages or the non-regenerating stages. Although expressed in the same regeneration stage, the different expression pattern between DjY1 and DjY2 suggests that they may perform different roles in the regeneration process in planarian. In this paper, our experiments results showed the transcripts of DjY2 significantly increased at the site of cutting, reached to the highest level at 1 day after amputation in both head parts and tail parts, indicated DjY2 might involve in the process of regeneration like DjY1. YB protein is an ancient conserved multifunctional protein, maybe more than one YB protein members exist in D. japonica like other species. Up to now, many genes and signaling pathways related with regeneration were found in planarian [21e24]. So what's the relationship between YB proteins and the genes participate in the process of planarian regeneration? What are the functions of these YB proteins and where do they play roles in the cell? More research work needs to be carried out to reveal the specific function and molecular mechanism of planarian YB proteins in the future. Declaration of interest We have no conflicts of interest with other people or organizations. Acknowledgements This study was supported by the National Natural Science Foundation of China (31550005,31350004) and the Natural Science Foundation of Shandong Province, China (ZR2011HM065). References [1] A.P. Wolffe, Structural and functional properties of the evolutionarily ancient Y-box family of nucleic acid binding proteins, Bioessays 16 (1994) 245e251. [2] I.A. Eliseeva, E.R. Kim, S.G. Guryanov, L.P. Ovchinnikov, D.N. Lyabin, Y-boxbinding protein 1 (YB-1) and its functions, Biochemistry (Mosc) 76 (2011) 1402e1433. [3] D.K. Didier, J. Schiffenbauer, S.L. Woulfe, M. Zacheis, B.D. Schwartz, Characterization of the cDNA encoding a protein binding to the major histocompatibility complex class II Y box, Proc. Natl. Acad. Sci. U.S.A. 85 (1998) 7322e7326. [4] V. Evdokimova, C. Tognon, T. Ng, P. Ruzanov, N. Melnyk, D. Fink, A. Sorokin, L.P. Ovchinnikov, E. Davicioni, T.J. Triche, P.H. Sorensen, Translational activation of snail1 and other developmentally regulated transcription factors by YB-1 promotes an epithelial-mesenchymal transition, Canc. Cell 15 (2009)
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