- Email: [email protected]
Protein arginine methyltransferase 1 mediates regeneration in Dugesia japonica
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Protein arginine methyltransferase 1 mediates regeneration in Dugesia japonica Yichao Zhang a, b, 1, Weiwei Wu a, b, 1, Lili Gao a, b, Meishan Chen a, b, Xi Liu a, b, Mujie Huang a, b, Ao Li a, b, Kang Zheng a, b, Dongwu Liu b, Hongkuan Deng a, b, Bosheng Zhao a, Baohua Liu b, c, **, Qiuxiang Pang a, b, * a b c
Laboratory of Developmental and Evolutionary Biology, School of Life Sciences, Shandong University of Technology, Zibo, Shandong, 255049, China Anti-aging & Regenerative Medicine Research Institution, School of Life Sciences, Shandong University of Technology, Zibo, Shandong, 255049, China Shenzhen University of Health Science Center, Shenzhen, Guangdong, 518060, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 December 2019 Accepted 14 January 2020 Available online xxx
As a typical organism of platyhelminth, Dugesia japonica attracts more and more attention for its strong regenerative ability. Protein arginine methyltransferase (PRMT) family is composed of a class of enzymes with methyltransferase activities, which play fundamental roles in vivo in many important physiological processes. PRMT1 is a predominant type I PRMT, which has been reported to be expressed in Schmidtea mediterranea. Nevertheless, the existence and the specific biological functions of PRMT1 in Dugesia japonica need further investigation. In this study, we acquired the full-length sequence of DjPRMT1 and confirmed it was a conserved protein. Thereafter, whole-mount in situ hybridization results showed DjPRMT1 was mainly expressed in neoblasts of adult worms, and obvious aggregation of DjPRMT1 was observed at the wound site in early stages of regeneration. Silencing of the DjPRMT1 gene retarded the movement of planarians with decreased DjPIWI-A expression, and DjPRMT1 knockdown also affected planarian regeneration with slightly attenuated proliferation around the blastema of posterior-facing wounds regeneration. In summary, these preliminary results demonstrated DjPRMT1 was involved in the regeneration of planarian. © 2020 Elsevier Inc. All rights reserved.
Keywords: Planarian (Dugesia japonica) DjPRMT1 Regeneration Proliferation
1. Introduction As early as 40 years ago, the phenomenon of arginine methylation in proteins had been noted [1]. But not until the 1990s, when the first arginine methyltransferase gene was isolated by yeast twohybrid experiments to verify B-cell translocation gene 1 (BTG1) and TPA in NIH3T3 cells (TIS21) interactions, did the genetic research begin to flourish [2]. Researchers predict that 0.6%e1.6% of the genes encode methyltransferases in the genome of Homo sapiens, Mus musculus, Caenorhabditis elegans, Arabidopsis thaliana,
* Corresponding author. Anti-aging & Regenerative Medicine Research Institution, School of Life Sciences, Shandong University of Technology, Zibo, Shandong, 255049, China. ** Co-corresponding author. Anti-aging & Regenerative Medicine Research Institution, School of Life Sciences, Shandong University of Technology, Zibo, Shandong, 255049, China. E-mail addresses: [email protected] (B. Liu), [email protected] (Q. Pang). 1 Co-first author: Yichao Zhang, Weiwei Wu.
Saccharomyces cerevisiae, and Escherichia coli [3]. A total of 12 PRMTs have been identified, including ten in mammals and two in yeast [4]. PRMTs can be divided into four groups (types I, II, III, and IV) according to the type of arginine methylation of the substrate molecule [5]. PRMT1 is a predominant type I PRMT in mammals [6], which functions as a S-adenosylmethionine dependent methyltransferase. PRMT1 has four conserved domains (motif I, post I, II, and III) with a conserved ‘double E’ loop and a THW (Thr-His-Trp) loop [2]. The core sequence motif I, post I, II, and III are VLD/EVGXGXG, V/IXG/AXD/E, F/ I/VDI/L/K, and LR/KXXG, respectively [7]. These conservative sequences form the main structure of the SAM-binding pocket [2]. Alternative mRNA splicing of the PRMT gene results in several isoforms of PRMT1 that differ in their amino terminus regions, and its total activity accounts for 85% of the total activity of protein arginine methyltransferase in cultured rat fibroblast cells and mouse liver [8,9]. Studies have shown that PRMT1 is mainly located in the nucleus, and a certain amount of substrates are located in the cytoplasm and other organelles [6]. In mammals, PRMT1 exhibits a
https://doi.org/10.1016/j.bbrc.2020.01.087 0006-291X/© 2020 Elsevier Inc. All rights reserved.
Please cite this article as: Y. Zhang et al., Protein arginine methyltransferase 1 mediates regeneration in Dugesia japonica, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.087
2
Y. Zhang et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
very wide substrate specificity, its substrates include many RNA binding and transporting proteins, transcription factors, nuclear matrix proteins, and cytokines [6]. By regulating the methylation level of the substrate molecules, PRMT1 plays an important role in many intracellular biological pathways, such as cell differentiation, proliferation [10], migration and apoptosis [11]. PRMT1 also functions in DNA damage responses (DDR). Decreased PRMT1 activities in cells lead to spontaneous DNA damage, missing checkpoints [12], increased sensitivity to ionizing radiation [13], chromosomal instability [12], and production of aneuploids and polyploids [14]. The conditioned knockout of PRMT1 in mouse embryonic fibroblasts (MEFs) leads to the death of embryos, which failed to survive beyond 7.5 days post coitum [14]. Numerous studies confirmed that PRMT1 is closely related to many diseases, including cardiovascular diseases [15], cancer [16], and viral infection [17]. Planarians have the ability to regenerate their whole body from any small amputated fragment of the body. In this paper, we cloned the full-length of PRMT1 homolog from planarians (Dugesia japonica) and named it DjPRMT1 through the informatics analysis. Using in situ hybridization, we found that the mRNA of DjPRMT1 was mainly expressed in neoblasts, and obvious aggregation of DjPRMT1 was observed at the wound site in the early stages of regeneration. By comparing the subcellular localization of DjPRMT1 to that of mammals, we speculated that DjPRMT1 might perform similar functions with the PRMT1 in mammals. The RNAi of DjPRMT1 resulted in regeneration failure and reduced mobility of planarians. DjPRMT1 (RNAi) did not affect neoblasts differentiation in small wounds with eyespot repair, but affected cell proliferation at the blastema. Collectively, these results indicated that DjPRMT1 was involved in the regeneration of planarian. 2. Materials and methods 2.1. Animals and culture conditions Freshwater planarians (D. japonica) were collected from a
fountain in Quanhetou, Boshan, China. The planarians were kept in autoclaved tap water at 20 C and fed with liver of buffalo one time every week. Before the experiments, they were starved for at least one week.
2.2. Sequence analysis DjPRMT1 was analyzed for coding probability using the DNATools program. The detection of conserved domains of DjPRMT1 was performed with NCBI CD-search (http://www.ncbi.nlm.nih. gov/Structure/cdd/wrpsb.cgi). Multiple protein sequences were aligned by the clustalW method using the MegAlign program in the DNASTAR software. The phylogenetic tree was constructed using neighbor joining method with 1000 bootstrap replicates by MEGA7 software.
2.3. Cell culture, westernblot, and immunofluorescence The Sf9 insect cell line (a kind gift from K. Yang, Sun Yat-sen University) was cultured at 27 C in TNM-FH medium supplemented with 10% fetal bovine serum (Gibco), 100 mg/mL penicillin, and 30 mg/mL streptomycin. Sf9 cells (5 105) were transfected with 2 mg plasmid of the transient expression vector pIB/V5-FLAGDjPRMT1 [18] and incubated for 48 h. The cells were fixed with 4% paraformaldehyde and permeabilized with 0.25% Triton X-100, followed by incubation with anti-FLAG (1:200, Abmart) as the primary antibodies and anti-mouse antibody (1:200, Thermo) as the secondary antibodies. Finally, the samples were incubated with 5 mM DRAQ5 nuclear stain (1:1000, Abcam), and a laser scanning confocal microscope (Leica TCS SP2, Germany) was used to detect the immunofluorescence. H3P antibody (LOT: 2900699) was purchased from EMD millipore corp.
Fig. 1. Multiple sequences alignment of deduced DjPRMT1 and other PRMT1s from different species by the MegAlign software in DNASTAR. GenBank accession numbers of PRMT1 homologues used in this part were listed as following: C.elegans (NP_507909.1), D.rerio (AAH44522.1), H.sapiens (AAH19268.2), H.vulgaris (CDG67776.1), M.musculus (AAH51953.1), P.abelii (NP_001126260.1), X.tropicalis (AAH74614.1), and S.mediterranea (AEX33823.1). Conserved amino acids were labeled with shade. PRMT1 harbors a conserved S-adenosylmethionine binding domain which is characterized by four consensus domains, motif I, post I, II and III. There are two key loops, including the ‘double E’ loop and the THW loop. The red box marks DjPRMT1. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: Y. Zhang et al., Protein arginine methyltransferase 1 mediates regeneration in Dugesia japonica, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.087
Y. Zhang et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
3
developed using NBT and BCIP (1:50, Roche) for a few hours until signals were visible. Finally, the planarians were washed with ethanol of 100% and 50% respectively to eliminate nonspecific background staining [19]. All samples were observed with a Nikon SMZ 1500 stereomicroscope.
2.5. RNA interference (RNAi) The template and the double-stranded RNA (dsRNA) of DjPRMT1 were synthesized as previously described [20]. Then the worms were soaked in water with DjPRMT1 dsRNA (10 ng/mL) and GFP dsRNA (10 ng/mL) every two days for three times. Two days after the last treatment, we performed endogenous transcript detection and amputation. For morphometric analysis, the planarians were fixed following 9 days of regeneration and photographed every other day. Photos were taken by a Nikon SMZ 1500 stereomicroscope (Nikon, Japan).
2.6. Statistical analysis Data were presented as means ± SD with at least three independent experiments (n 3) by GraphPad PRISM (version 7.0). The paired experimental and control groups were compared using Student’s t-test or one-way analysis of variance. Significance was accepted at p < 0.05. 3. Results 3.1. Homology analysis
Fig. 2. The spatial and temporal expression patterns of DjPRMT1 in intact adult and regenerating planarians. (A) The expression of DjPRMT1 and DjPIWI-A expression in the adult planarians. White bars represent 1 mm. (B) The expression of DjPRMT1 mRNAs in regenerating planarians at different time points (0.5, 1, 3, 5, 7, 9, 12 days) of regeneration. White bars represent 500 mm.
2.4. In situ hybridization (ISH) Planarians with appropriate sizes were selected and washed in the dishes on ice. Planarians were treated with 5% N-acetyl-Lcysteine for 5 min until they were fully stretched and then fixed for 20 min at room temperature in 4% paraformaldehyde. Subsequently, the planarians were washed with phosphate-buffered saline containing 0.1% Triton X-100 (PBST) and hatched in Reduction buffer (50 mM DTT, 1% NP-40, 0.5% SDS in PBS) for 15 min at 37 C. The planarians were dehydrated with 100% and 50% methanol before the bleaching (1:5, 30% H2O2: methanol) at room temperature under light irradiation. The planarians were permeabilized with proteinase K (10 mg/ml) for 10 min and then fixed again by 4% paraformaldehyde for 20 min at room temperature. Afterwards, acetylation was performed by triethanolamine before prehybridization (50% formamide, 5 Х SSC, 1 mg/mL yeast RNA, 1% Tween-20, 100 mg/mL heparin, 5 mM DTT, 1 Denhardt’s in PBST) at 53 C for 2e4 h and hybridization (10% dextran sulfate in prehybridization solution) at 53 C for 28e36 h. The planarians were washed several times in the washing buffer I (pre-hybridization solution), II (Pre-hybridization solution: 2 SSC ¼ 1:1), III (2 SSC), and Ⅳ (0.2 SSC) successively and incubated with an anti-DIG antibody (1:1000, Roche) overnight. The color was
We obtained and confirmed the full cDNA sequence of DjPRMT1 by using RACE and phylogenetic analysis. To further understand the structural characteristics of DjPRMT1, we aligned DjPRMT1 with PRMT1 sequences from a number of animals (Fig. 1). DjPRMT1 showed a high similarity with the PRMTs of many other species, including C.elegans (59.2%), D.rerio (69.8%), H.sapiens (68.6%), H.vulgaris (63.6%), M.musculus (68.6%), P.abelii (68.6%), R.norvegicus (68.9%), X.tropicalis (69.2%), S.mediterranea (96.0%). DjPRMT1 harbors a conserved S-adenosylmethionine binding domain characterized by numerous consensus domains, including motif I, post I, II, and III. There are two conservative loop structures, the ‘double E’ loop located between motif II and III, and the THW loop located in the close proximity of the c-terminus. These results indicated that DjPRMT1 was conservative during evolution.
3.2. Localization of DjPRMT1 mRNA in intact adult and regenerating planarians To investigate the function of DjPRMT1, whole-mount in situ hybridization was performed firstly to confirm the spatial and temporal expression patterns of the DjPRMT1 mRNAs in intact adult planarians. The mRNA of DjPRMT1 was detected in the whole body, which is similar to the expression position of DjPIWI-A, a marker gene of planarian neoblasts (Fig. 2A). Based on these observations, we detected the spatial and temporal expression pattern of the DjPRMT1 mRNAs in regenerating adult planarians. As shown in Fig. 2B, in the planarians cut into three pieces (head, trunk, and tail), DjPRMT1 was aggregated in the wound area (red arrow) during the first three days of the regeneration process. Not until the fifth day of regeneration did DjPRMT1 return to the state of full-body distribution. The distribution pattern suggested that DjPRMT1 might be related to the regeneration process of the planarian.
Please cite this article as: Y. Zhang et al., Protein arginine methyltransferase 1 mediates regeneration in Dugesia japonica, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.087
4
Y. Zhang et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
Fig. 3. Regeneration of DjPRMT1 RNAi planarian. (A) Experimental time points of DjPRMT1 interference, amputation, and observation. (B) DjPRMT1 interference efficiency. Data were analyzed by Student’s t-test (*p < 0.05) (C) Phenotype of DjPRMT1 RNAi in intact planarians. Left panel shows a dorsal view of a control (GFP RNAi) planarian. Right panel shows a dorsal view of a DjPRMT1 RNAi planarian. (D-F) Planarians were amputated into three fragments: head, trunk, and tail. As compared with control (GFP RNAi) planarians, DjPRMT1 RNAi showed severe regeneration defects. White bars represent 1 mm. (G-I) These graphs represent the area in the red box in Figure D, E, and F. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
3.3. DjPRMT1 gene knockdown leads to pleiotropic defects in regeneration To verify whether DjPRMT1 is contributed to regeneration, we evaluated the effects of DjPRMT1 silencing on intact animals and planarian fragments which were amputated 2 days post the last application of RNAi. The procedure was performed according to the method in Fig. 3A. After the silencing of DjPRMT1, we detected that 70% of DjPRMT1 was successfully disturbed (Fig. 3B), while no distinction was observed between intact control and DjPRMT1knockdown planarians (Fig. 3C). However, the regeneration process was severely affected in some of the DjPRMT1-knockdown planarians, especially the tail regeneration process. Posterior-facing wounds defects were observed in more than 35% of planarians fragments. Less than 10% of the planarians had head defects at anterior-facing wounds. The severity of the regenerating defect can
be observed by magnifying the defect in the red box (Fig. 3DeI). In comparison with the control group, the crawling ability of DjPRMT1-silenced planarians was reduced during the whole regeneration process, as they twisted and curled up in-situ (Video 1). Our results confirmed that DjPRMT1 participated in planarian regeneration. Supplementary video related to this article can be found at https://doi.org/10.1016/j.bbrc.2020.01.087. 3.4. Localization of DjPRMT1 protein in sf9 Mammalian PRMT1 functions in regulating the methylation of various proteins in the nucleus and cytoplasm. We transfected the SF9 cells with PIB/V5-FLAG-DjPRMT1 and confirmed that DjPRMT1 was indeed expressed (Fig. 4A). We observed that the recombinant DjPRMT1 protein predominantly localized in the nucleus with a
Please cite this article as: Y. Zhang et al., Protein arginine methyltransferase 1 mediates regeneration in Dugesia japonica, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.087
Y. Zhang et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
5
Fig. 4. DjPRMT1 affects neoblasts proliferation rather than differentiation. (A) Localization of DjPRMT1 in sf9 cells. Merged image (pink) shows that DjPRMT1 (red) was colocalized with nucleus (blue). White bars represent 4 mm. Lysates of Sf9 cells isolated from pIB/V5-FLAG transfected Sf9 cells or pIB/V5-FLAG-DjPRMT1 transfected Sf9 cells were detected with anti-FLAG and anti-b-actin antibodies. b-actin was used as a loading control. (B) The transcription levels of DjPRMT1 and DjPIWI-A in planarians treated with X-ray irradiation at different time points (0, 1, 3, 5 days). The transcription patterns were measured by qPCR. (C) DjPIWI-A and DjPRMT1 expression in X-ray irradiation treated planarians by ISH. Planarians with a GFP probe were used as the negative control. The positive signal was revealed with blue taint. White bars represent 500 mm. (D) The expression of DjPIWI-A was reduced after silencing of DjPRMT1. Data were analyzed by Student’s t-test (***p < 0.001). Error bars indicate standard deviations (SD) from three independent experiments. (E) The proliferation capacity of neoblasts at blastema (delineated by a discontinuous red line) were compared after silencing of GFP and DjPRMT1. White bars represent 0.5 mm. (F) Quantification of green pixel intensity in the blastema of control and DjPRMT1 RNAi planarians. Data were analyzed by Student’s t-test (*p < 0.05, ns ¼ not significant). (G) The eye resection of DjPRMT1 (RNAi) planarians recovers on the seventh day (n ¼ 10). Black bars represent 2 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
small amount in the cytoplasm (Fig. 4A), which is consistent with the expression of mammalian PRMT1. Therefore, we speculated that DjPRMT1 might function in a similar way to those of mammals.
3.5. DjPRMT1 affects neoblasts proliferation rather than differentiation We speculate that DjPRMT1 may be mainly located in neoblasts based on the mRNA expression patterns. Neoblasts, the only proliferating cells in the animal, are particularly sensitive to irradiation. Thus, we detected DjPRMT1 expression using X-ray irradiated adult planarians. Both the qPCR and ISH results showed that DjPIWI-A was gradually disappeared in intact adult planarians treated by X-ray irradiation, while DjPRMT1 could be detected in
the nerves after one day post X-ray irradiation (Fig. 4C, red arrow), though the expression of DjPRMT1 was gradually decreased (Fig. 4B and C). These results are consistent with those in previous reports [21]. These results confirmed DjPRMT1 was indeed expressed in neoblasts, and DjPIWI-A and DjPRMT1 were partially co-expressed. However, it should be noted that DjPIWI-A expression was slightly decreased in planarians subjected to DjPRMT1 RNAi (Fig. 4D), suggesting that DjPRMT1 may influence the survival of undifferentiated neoblasts or directly affect DjPIWI-A gene expression. Immunohistochemical analysis was performed with a H3p antibody to compare the cell division of neoblasts in worms subjected to GFP RNAi and DjPRMT1 RNAi. The positive signal was obviously weakened in the posterior-facing wounds of the planarian subjected to DjPRMT1 RNAi, but anterior-facing wounds
Please cite this article as: Y. Zhang et al., Protein arginine methyltransferase 1 mediates regeneration in Dugesia japonica, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.087
6
Y. Zhang et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
were not significant change (Fig. 4E and F). Moreover, the eyes were successfully reformed in the planarian subjected to DjPRMT1 RNAi which performed eye resection (Fig. 4G). This procedure, which causes small wounds to the planarians, does not cause the neoblasts to proliferate continuously but requires them to maintain the ability to differentiate [22]. The above results indicated that DjPRMT1 was expressed in the neoblasts and might be involved in tissue regeneration and renewal through influence cell proliferation, especially the posterior-facing wounds.
signaling pathways in planarian regeneration.
4. Discussion
We thank everyone of our laboratory for their technical assistance and helpful suggestions. This study was supported by the National Natural Science Foundation of China (31172074, 31572263, 31701015), the Natural Science Foundation of Shandong Province, China (ZR2017MC066), and the Key Science and Technology Project in Institutions of Higher Education of Shandong Province, China (J17KZ003).
Planarians are triploblastic, acoelomate, unsegmented, dorsoventrally flattened soft-bodied, and nonparasitic organisms, without circulatory, respiratory, or skeletal structures [23]. Because of the large population of adult stem cells, planarians have a remarkable ability to regenerate [24]. Therefore, with the application of various technologies and the development of genomic reagents, the molecular mechanisms by which pluripotent somatic stem cells (neoblasts) replenish, repair, and regenerate planarian tissues and organs have been explored [25]. The regeneration process of the planarian requires a series of cell proliferation and differentiation, and a large number of regeneration-related proteins need to be expressed and properly modified after translation. Previous studies have shown that depletion of PRMT1 inhibits cell proliferation [26], cell cycle progression delay, checkpoint defects, cell growth arrest, and cell death in mammals [11,27,28], while the DjPRMT1-silenced planarians do not regenerate completely and properly after amputating, partially due to the incorrect cell division at the wound site. Our results showed that the recombinant protein DjPRMT1 was predominantly located in the nucleus and a small amount was found in the cytoplasm. This expression pattern is consistent with the localization of mammalian PRMT1 [6], providing the first hint that DjPRMT1 may have the same functions in regulating proliferation and differentiation by targeting similar substrates in the planarian. Although we found that DjPRMT1(RNAi) did not affect the differentiation process of precursor stem cells in small wounds with eyespot repair, we speculate that DjPRMT1 may regulate the differentiation from neoblast to multiple precursor stem cells. Planarian displays robust regenerative properties driven by neoblasts, a stem cell population capable of developing into a complete individual [29]. Previous investigations have shown that PRMT1 affects the proliferation and differentiation of mouse muscle stem cells [30,31], liver cells [32], and intestinal epithelial cells [33]. Muscle-specific PRMT1 deficiency leads to muscle atrophy and muscle loss [34]. We observed reduced mobility and in situ contorted body phenotypes in the planarian subjected to DjPRMT1 RNAi. After muscle injury in mammals, deficiency of PRMT1 can cause the myogenic differentiation unterminated, leading to failure of muscle regeneration [30]. Thus, DjPRMT1 may have similar roles. PRMT1 deficiency lead to the decline in athletic ability during regeneration of planarians, may also be caused by muscle atrophy, loss of muscle, or failure to regenerate muscle [35]. In summary, we cloned the DjPRMT1 gene of Dugesia japonica and showed it was a highly evolutionarily conserved protein. DjPRMT1 was predominantly concentrated in neoblasts and showed obvious aggregation at blastema in the early stages of wound regeneration. By interfering with DjPRMT1, the wound area of the planarians cannot be completely repaired especially in the posterior regeneration. We also find that DjPRMT1-silenced planarians showed reduced mobility. Our results provide valuable information for further investigations of the potential molecular mechanisms of DjPRMT1 in the regenerative process of planarians. Future studies will focus on the precise regulation of DjPRMT1
Declaration of competing interest We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted. Acknowledgements
References [1] W.K. Paik, S. Kim, Enzymatic methylation of protein fractions from calf thymus nuclei, Biochem. Biophys. Res. Commun. 29 (1967) 14e20. [2] W.J. Lin, J.D. Gary, M.C. Yang, et al., The mammalian immediate-early TIS21 protein and the leukemia-associated BTG1 protein interact with a proteinarginine N-methyltransferase, J. Biol. Chem. 271 (1996) 15034e15044. [3] J.E. Katz, M. Dlakic, S. Clarke, Automated identification of putative methyltransferases from genomic open reading frames, Mol. Cell. Proteom. : MCP 2 (2003) 525e540, https://doi.org/10.1074/mcp.M300037-MCP200. [4] C. Peng, C.C. Wong, The story of protein arginine methylation: characterization, regulation, and function, Expert Rev. Proteomics 14 (2017) 157e170, https://doi.org/10.1080/14789450.2017.1275573. [5] M.T. Bedford, S.G. Clarke, Protein arginine methylation in mammals: who, what, and why, Mol. Cell 33 (2009) 1e13, https://doi.org/10.1016/ j.molcel.2008.12.013. [6] J. Tang, A. Frankel, R.J. Cook, et al., PRMT1 is the predominant type I protein arginine methyltransferase in mammalian cells, J. Biol. Chem. 275 (2000) 7723e7730. [7] X. Cheng, R.E. Collins, X. Zhang, Structural and sequence motifs of protein (histone) methylation enzymes, Annu. Rev. Biophys. Biomol. Struct. 34 (2005) 267e294, https://doi.org/10.1146/annurev.biophys.34.040204.144452. [8] M.R. Pawlak, C.A. Scherer, J. Chen, et al., Arginine N-methyltransferase 1 is required for early postimplantation mouse development, but cells deficient in the enzyme are viable, Mol. Cell. Biol. 20 (2000) 4859e4869. [9] S. Wang, X. Tan, B. Yang, et al., The role of protein arginine-methyltransferase 1 in gliomagenesis, BMB Rep. 45 (2012) 470e475, https://doi.org/10.5483/ BMBRep.2012.45.8.022. [10] W.K. Hua, Y.I. Chang, C.L. Yao, et al., Protein arginine methyltransferase 1 interacts with and activates p38alpha to facilitate erythroid differentiation, PLoS One 8 (2013), e56715, https://doi.org/10.1371/journal.pone.0056715. [11] Y. Hirata, K. Katagiri, K. Nagaoka, et al., TRIM48 promotes ASK1 activation and cell death through ubiquitination-dependent degradation of the ASK1negative regulator PRMT1, Cell Rep. 21 (2017) 2447e2457, https://doi.org/ 10.1016/j.celrep.2017.11.007. [12] F.M. Boisvert, U. Dery, J.Y. Masson, et al., Arginine methylation of MRE11 by PRMT1 is required for DNA damage checkpoint control, Genes Dev. 19 (2005) 671e676, https://doi.org/10.1101/gad.1279805. [13] Q. Yuan, R. Tian, H. Zhao, et al., Multiple Arginine Residues Are Methylated in Drosophila Mre11 and Required for Survival Following Ionizing Radiation, vol. 8, G3, Bethesda, Md, 2018, pp. 2099e2106, https://doi.org/10.1534/ g3.118.200298. [14] Z. Yu, T. Chen, J. Hebert, et al., A mouse PRMT1 null allele defines an essential role for arginine methylation in genome maintenance and cell proliferation, Mol. Cell. Biol. 29 (2009) 2982e2996, https://doi.org/10.1128/mcb.00042-09. [15] M.C. Stuhlinger, P.S. Tsao, J.H. Her, et al., Homocysteine impairs the nitric oxide synthase pathway: role of asymmetric dimethylarginine, Circulation 104 (2001) 2569e2575. [16] L. Liu, X. Zhao, L. Zhao, et al., Arginine methylation of SREBP1a via PRMT5 promotes de novo lipogenesis and tumor growth, Cancer Res. 76 (2016) 1260e1272, https://doi.org/10.1158/0008-5472.can-15-1766. [17] M.S. Eram, Y. Shen, M. Szewczyk, et al., A potent, selective, and cell-active inhibitor of human type I protein arginine methyltransferases, ACS Chem. Biol. 11 (2016) 772e781, https://doi.org/10.1021/acschembio.5b00839. [18] A. Li, H. Zhao, Q. Lai, et al., Posttranslational modifications of baculovirus protamine-like protein P6.9 and the significance of its hyperphosphorylation for viral very late gene hyperexpression, J. Virol. 89 (2015) 7646e7659, https://doi.org/10.1128/jvi.00333-15. [19] Y. Han, A. Li, L. Gao, et al., Identification and characterization of a phospholipid scramblase encoded by planarian Dugesia japonica, Gene 602 (2017) 43e49, https://doi.org/10.1016/j.gene.2016.11.029.
Please cite this article as: Y. Zhang et al., Protein arginine methyltransferase 1 mediates regeneration in Dugesia japonica, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.087
Y. Zhang et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx [20] L. Gao, Y. Han, H. Deng, et al., The role of a novel C-type lectin-like protein from planarian in innate immunity and regeneration, Dev. Comp. Immunol. 67 (2017) 413e426, https://doi.org/10.1016/j.dci.2016.08.010. [21] N. Shibata, T. Hayashi, R. Fukumura, et al., Comprehensive gene expression analyses in pluripotent stem cells of a planarian, Dugesia japonica, Int. J. Dev. Biol. 56 (2012) 93e102, https://doi.org/10.1387/ijdb.113434ns. [22] S.A. LoCascio, S.W. Lapan, P.W. Reddien, Eye absence does not regulate planarian stem cells during eye regeneration, Dev. Cell 40 (2017) 381e391, https://doi.org/10.1016/j.devcel.2017.02.002, e383. [23] E. Salo, J.F. Abril, T. Adell, et al., Planarian regeneration: achievements and future directions after 20 years of research, Int. J. Dev. Biol. 53 (2009) 1317e1327, https://doi.org/10.1387/ijdb.072414es. [24] P.W. Reddien, A. Sanchez Alvarado, Fundamentals of planarian regeneration, Annu. Rev. Cell Dev. Biol. 20 (2004) 725e757, https://doi.org/10.1146/ annurev.cellbio.20.010403.095114. [25] D.J. Forsthoefel, P.A. Newmark, Emerging patterns in planarian regeneration, Curr. Opin. Genet. Dev. 19 (2009) 412e420, https://doi.org/10.1016/ j.gde.2009.05.003. [26] K.A. Akter, M.A. Mansour, T. Hyodo, et al., FAM98A is a novel substrate of PRMT1 required for tumor cell migration, invasion, and colony formation, Tumour Biol.: the journal of the International Society for Oncodevelopmental Biology and Medicine 37 (2016) 4531e4539, https://doi.org/10.1007/s13277015-4310-5. [27] N. Cheung, T.K. Fung, B.B. Zeisig, et al., Targeting aberrant epigenetic networks mediated by PRMT1 and KDM4C in acute myeloid leukemia, Cancer Cell 29 (2016) 32e48, https://doi.org/10.1016/j.ccell.2015.12.007.
7
[28] S. Infantino, B. Benz, T. Waldmann, et al., Arginine methylation of the B cell antigen receptor promotes differentiation, J. Exp. Med. 207 (2010) 711e719, https://doi.org/10.1084/jem.20091303. [29] A. Sanchez Alvarado, Stem cells and the planarian Schmidtea mediterranea, Comptes Rendus Biol. 330 (2007) 498e503, https://doi.org/10.1016/ j.crvi.2007.05.005. [30] R.S. Blanc, G. Vogel, X. Li, et al., Arginine methylation by PRMT1 regulates muscle stem cell fate, Mol. Cell. Biol. 37 (2017), https://doi.org/10.1128/ mcb.00457-16. [31] N.Y. Shen, S.Y. Ng, S.L. Toepp, et al., Protein arginine methyltransferase expression and activity during myogenesis, Biosci. Rep. 38 (2018), https:// doi.org/10.1042/bsr20171533. [32] Y. Lim, Y.H. Kwon, N.H. Won, et al., Multimerization of expressed proteinarginine methyltransferases during the growth and differentiation of rat liver, Biochim. Biophys. Acta 1723 (2005) 240e247, https://doi.org/10.1016/ j.bbagen.2005.02.015. [33] H. Matsuda, Y.B. Shi, An essential and evolutionarily conserved role of protein arginine methyltransferase 1 for adult intestinal stem cells during postembryonic development, Stem Cells (Dayton, Ohio) 28 (2010) 2073e2083, https://doi.org/10.1002/stem.529. [34] S. Choi, H.J. Jeong, H. Kim, et al., Skeletal muscle-specific Prmt1 deletion causes muscle atrophy via deregulation of the PRMT6-FOXO3 axis, Autophagy (2019) 1e13, https://doi.org/10.1080/15548627.2019.1569931. [35] M.L. Scimone, L.E. Cote, P.W. Reddien, Orthogonal muscle fibres have different instructive roles in planarian regeneration, Nature 551 (2017) 623e628, https://doi.org/10.1038/nature24660.
Please cite this article as: Y. Zhang et al., Protein arginine methyltransferase 1 mediates regeneration in Dugesia japonica, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.087
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Protein arginine methyltransferase 1 mediates regeneration in Dugesia japonica Yichao Zhang a, b, 1, Weiwei Wu a, b, 1, Lili Gao a, b, Meishan Chen a, b, Xi Liu a, b, Mujie Huang a, b, Ao Li a, b, Kang Zheng a, b, Dongwu Liu b, Hongkuan Deng a, b, Bosheng Zhao a, Baohua Liu b, c, **, Qiuxiang Pang a, b, * a b c
Laboratory of Developmental and Evolutionary Biology, School of Life Sciences, Shandong University of Technology, Zibo, Shandong, 255049, China Anti-aging & Regenerative Medicine Research Institution, School of Life Sciences, Shandong University of Technology, Zibo, Shandong, 255049, China Shenzhen University of Health Science Center, Shenzhen, Guangdong, 518060, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 December 2019 Accepted 14 January 2020 Available online xxx
As a typical organism of platyhelminth, Dugesia japonica attracts more and more attention for its strong regenerative ability. Protein arginine methyltransferase (PRMT) family is composed of a class of enzymes with methyltransferase activities, which play fundamental roles in vivo in many important physiological processes. PRMT1 is a predominant type I PRMT, which has been reported to be expressed in Schmidtea mediterranea. Nevertheless, the existence and the specific biological functions of PRMT1 in Dugesia japonica need further investigation. In this study, we acquired the full-length sequence of DjPRMT1 and confirmed it was a conserved protein. Thereafter, whole-mount in situ hybridization results showed DjPRMT1 was mainly expressed in neoblasts of adult worms, and obvious aggregation of DjPRMT1 was observed at the wound site in early stages of regeneration. Silencing of the DjPRMT1 gene retarded the movement of planarians with decreased DjPIWI-A expression, and DjPRMT1 knockdown also affected planarian regeneration with slightly attenuated proliferation around the blastema of posterior-facing wounds regeneration. In summary, these preliminary results demonstrated DjPRMT1 was involved in the regeneration of planarian. © 2020 Elsevier Inc. All rights reserved.
Keywords: Planarian (Dugesia japonica) DjPRMT1 Regeneration Proliferation
1. Introduction As early as 40 years ago, the phenomenon of arginine methylation in proteins had been noted [1]. But not until the 1990s, when the first arginine methyltransferase gene was isolated by yeast twohybrid experiments to verify B-cell translocation gene 1 (BTG1) and TPA in NIH3T3 cells (TIS21) interactions, did the genetic research begin to flourish [2]. Researchers predict that 0.6%e1.6% of the genes encode methyltransferases in the genome of Homo sapiens, Mus musculus, Caenorhabditis elegans, Arabidopsis thaliana,
* Corresponding author. Anti-aging & Regenerative Medicine Research Institution, School of Life Sciences, Shandong University of Technology, Zibo, Shandong, 255049, China. ** Co-corresponding author. Anti-aging & Regenerative Medicine Research Institution, School of Life Sciences, Shandong University of Technology, Zibo, Shandong, 255049, China. E-mail addresses: [email protected] (B. Liu), [email protected] (Q. Pang). 1 Co-first author: Yichao Zhang, Weiwei Wu.
Saccharomyces cerevisiae, and Escherichia coli [3]. A total of 12 PRMTs have been identified, including ten in mammals and two in yeast [4]. PRMTs can be divided into four groups (types I, II, III, and IV) according to the type of arginine methylation of the substrate molecule [5]. PRMT1 is a predominant type I PRMT in mammals [6], which functions as a S-adenosylmethionine dependent methyltransferase. PRMT1 has four conserved domains (motif I, post I, II, and III) with a conserved ‘double E’ loop and a THW (Thr-His-Trp) loop [2]. The core sequence motif I, post I, II, and III are VLD/EVGXGXG, V/IXG/AXD/E, F/ I/VDI/L/K, and LR/KXXG, respectively [7]. These conservative sequences form the main structure of the SAM-binding pocket [2]. Alternative mRNA splicing of the PRMT gene results in several isoforms of PRMT1 that differ in their amino terminus regions, and its total activity accounts for 85% of the total activity of protein arginine methyltransferase in cultured rat fibroblast cells and mouse liver [8,9]. Studies have shown that PRMT1 is mainly located in the nucleus, and a certain amount of substrates are located in the cytoplasm and other organelles [6]. In mammals, PRMT1 exhibits a
https://doi.org/10.1016/j.bbrc.2020.01.087 0006-291X/© 2020 Elsevier Inc. All rights reserved.
Please cite this article as: Y. Zhang et al., Protein arginine methyltransferase 1 mediates regeneration in Dugesia japonica, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.087
2
Y. Zhang et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
very wide substrate specificity, its substrates include many RNA binding and transporting proteins, transcription factors, nuclear matrix proteins, and cytokines [6]. By regulating the methylation level of the substrate molecules, PRMT1 plays an important role in many intracellular biological pathways, such as cell differentiation, proliferation [10], migration and apoptosis [11]. PRMT1 also functions in DNA damage responses (DDR). Decreased PRMT1 activities in cells lead to spontaneous DNA damage, missing checkpoints [12], increased sensitivity to ionizing radiation [13], chromosomal instability [12], and production of aneuploids and polyploids [14]. The conditioned knockout of PRMT1 in mouse embryonic fibroblasts (MEFs) leads to the death of embryos, which failed to survive beyond 7.5 days post coitum [14]. Numerous studies confirmed that PRMT1 is closely related to many diseases, including cardiovascular diseases [15], cancer [16], and viral infection [17]. Planarians have the ability to regenerate their whole body from any small amputated fragment of the body. In this paper, we cloned the full-length of PRMT1 homolog from planarians (Dugesia japonica) and named it DjPRMT1 through the informatics analysis. Using in situ hybridization, we found that the mRNA of DjPRMT1 was mainly expressed in neoblasts, and obvious aggregation of DjPRMT1 was observed at the wound site in the early stages of regeneration. By comparing the subcellular localization of DjPRMT1 to that of mammals, we speculated that DjPRMT1 might perform similar functions with the PRMT1 in mammals. The RNAi of DjPRMT1 resulted in regeneration failure and reduced mobility of planarians. DjPRMT1 (RNAi) did not affect neoblasts differentiation in small wounds with eyespot repair, but affected cell proliferation at the blastema. Collectively, these results indicated that DjPRMT1 was involved in the regeneration of planarian. 2. Materials and methods 2.1. Animals and culture conditions Freshwater planarians (D. japonica) were collected from a
fountain in Quanhetou, Boshan, China. The planarians were kept in autoclaved tap water at 20 C and fed with liver of buffalo one time every week. Before the experiments, they were starved for at least one week.
2.2. Sequence analysis DjPRMT1 was analyzed for coding probability using the DNATools program. The detection of conserved domains of DjPRMT1 was performed with NCBI CD-search (http://www.ncbi.nlm.nih. gov/Structure/cdd/wrpsb.cgi). Multiple protein sequences were aligned by the clustalW method using the MegAlign program in the DNASTAR software. The phylogenetic tree was constructed using neighbor joining method with 1000 bootstrap replicates by MEGA7 software.
2.3. Cell culture, westernblot, and immunofluorescence The Sf9 insect cell line (a kind gift from K. Yang, Sun Yat-sen University) was cultured at 27 C in TNM-FH medium supplemented with 10% fetal bovine serum (Gibco), 100 mg/mL penicillin, and 30 mg/mL streptomycin. Sf9 cells (5 105) were transfected with 2 mg plasmid of the transient expression vector pIB/V5-FLAGDjPRMT1 [18] and incubated for 48 h. The cells were fixed with 4% paraformaldehyde and permeabilized with 0.25% Triton X-100, followed by incubation with anti-FLAG (1:200, Abmart) as the primary antibodies and anti-mouse antibody (1:200, Thermo) as the secondary antibodies. Finally, the samples were incubated with 5 mM DRAQ5 nuclear stain (1:1000, Abcam), and a laser scanning confocal microscope (Leica TCS SP2, Germany) was used to detect the immunofluorescence. H3P antibody (LOT: 2900699) was purchased from EMD millipore corp.
Fig. 1. Multiple sequences alignment of deduced DjPRMT1 and other PRMT1s from different species by the MegAlign software in DNASTAR. GenBank accession numbers of PRMT1 homologues used in this part were listed as following: C.elegans (NP_507909.1), D.rerio (AAH44522.1), H.sapiens (AAH19268.2), H.vulgaris (CDG67776.1), M.musculus (AAH51953.1), P.abelii (NP_001126260.1), X.tropicalis (AAH74614.1), and S.mediterranea (AEX33823.1). Conserved amino acids were labeled with shade. PRMT1 harbors a conserved S-adenosylmethionine binding domain which is characterized by four consensus domains, motif I, post I, II and III. There are two key loops, including the ‘double E’ loop and the THW loop. The red box marks DjPRMT1. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: Y. Zhang et al., Protein arginine methyltransferase 1 mediates regeneration in Dugesia japonica, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.087
Y. Zhang et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
3
developed using NBT and BCIP (1:50, Roche) for a few hours until signals were visible. Finally, the planarians were washed with ethanol of 100% and 50% respectively to eliminate nonspecific background staining [19]. All samples were observed with a Nikon SMZ 1500 stereomicroscope.
2.5. RNA interference (RNAi) The template and the double-stranded RNA (dsRNA) of DjPRMT1 were synthesized as previously described [20]. Then the worms were soaked in water with DjPRMT1 dsRNA (10 ng/mL) and GFP dsRNA (10 ng/mL) every two days for three times. Two days after the last treatment, we performed endogenous transcript detection and amputation. For morphometric analysis, the planarians were fixed following 9 days of regeneration and photographed every other day. Photos were taken by a Nikon SMZ 1500 stereomicroscope (Nikon, Japan).
2.6. Statistical analysis Data were presented as means ± SD with at least three independent experiments (n 3) by GraphPad PRISM (version 7.0). The paired experimental and control groups were compared using Student’s t-test or one-way analysis of variance. Significance was accepted at p < 0.05. 3. Results 3.1. Homology analysis
Fig. 2. The spatial and temporal expression patterns of DjPRMT1 in intact adult and regenerating planarians. (A) The expression of DjPRMT1 and DjPIWI-A expression in the adult planarians. White bars represent 1 mm. (B) The expression of DjPRMT1 mRNAs in regenerating planarians at different time points (0.5, 1, 3, 5, 7, 9, 12 days) of regeneration. White bars represent 500 mm.
2.4. In situ hybridization (ISH) Planarians with appropriate sizes were selected and washed in the dishes on ice. Planarians were treated with 5% N-acetyl-Lcysteine for 5 min until they were fully stretched and then fixed for 20 min at room temperature in 4% paraformaldehyde. Subsequently, the planarians were washed with phosphate-buffered saline containing 0.1% Triton X-100 (PBST) and hatched in Reduction buffer (50 mM DTT, 1% NP-40, 0.5% SDS in PBS) for 15 min at 37 C. The planarians were dehydrated with 100% and 50% methanol before the bleaching (1:5, 30% H2O2: methanol) at room temperature under light irradiation. The planarians were permeabilized with proteinase K (10 mg/ml) for 10 min and then fixed again by 4% paraformaldehyde for 20 min at room temperature. Afterwards, acetylation was performed by triethanolamine before prehybridization (50% formamide, 5 Х SSC, 1 mg/mL yeast RNA, 1% Tween-20, 100 mg/mL heparin, 5 mM DTT, 1 Denhardt’s in PBST) at 53 C for 2e4 h and hybridization (10% dextran sulfate in prehybridization solution) at 53 C for 28e36 h. The planarians were washed several times in the washing buffer I (pre-hybridization solution), II (Pre-hybridization solution: 2 SSC ¼ 1:1), III (2 SSC), and Ⅳ (0.2 SSC) successively and incubated with an anti-DIG antibody (1:1000, Roche) overnight. The color was
We obtained and confirmed the full cDNA sequence of DjPRMT1 by using RACE and phylogenetic analysis. To further understand the structural characteristics of DjPRMT1, we aligned DjPRMT1 with PRMT1 sequences from a number of animals (Fig. 1). DjPRMT1 showed a high similarity with the PRMTs of many other species, including C.elegans (59.2%), D.rerio (69.8%), H.sapiens (68.6%), H.vulgaris (63.6%), M.musculus (68.6%), P.abelii (68.6%), R.norvegicus (68.9%), X.tropicalis (69.2%), S.mediterranea (96.0%). DjPRMT1 harbors a conserved S-adenosylmethionine binding domain characterized by numerous consensus domains, including motif I, post I, II, and III. There are two conservative loop structures, the ‘double E’ loop located between motif II and III, and the THW loop located in the close proximity of the c-terminus. These results indicated that DjPRMT1 was conservative during evolution.
3.2. Localization of DjPRMT1 mRNA in intact adult and regenerating planarians To investigate the function of DjPRMT1, whole-mount in situ hybridization was performed firstly to confirm the spatial and temporal expression patterns of the DjPRMT1 mRNAs in intact adult planarians. The mRNA of DjPRMT1 was detected in the whole body, which is similar to the expression position of DjPIWI-A, a marker gene of planarian neoblasts (Fig. 2A). Based on these observations, we detected the spatial and temporal expression pattern of the DjPRMT1 mRNAs in regenerating adult planarians. As shown in Fig. 2B, in the planarians cut into three pieces (head, trunk, and tail), DjPRMT1 was aggregated in the wound area (red arrow) during the first three days of the regeneration process. Not until the fifth day of regeneration did DjPRMT1 return to the state of full-body distribution. The distribution pattern suggested that DjPRMT1 might be related to the regeneration process of the planarian.
Please cite this article as: Y. Zhang et al., Protein arginine methyltransferase 1 mediates regeneration in Dugesia japonica, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.087
4
Y. Zhang et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
Fig. 3. Regeneration of DjPRMT1 RNAi planarian. (A) Experimental time points of DjPRMT1 interference, amputation, and observation. (B) DjPRMT1 interference efficiency. Data were analyzed by Student’s t-test (*p < 0.05) (C) Phenotype of DjPRMT1 RNAi in intact planarians. Left panel shows a dorsal view of a control (GFP RNAi) planarian. Right panel shows a dorsal view of a DjPRMT1 RNAi planarian. (D-F) Planarians were amputated into three fragments: head, trunk, and tail. As compared with control (GFP RNAi) planarians, DjPRMT1 RNAi showed severe regeneration defects. White bars represent 1 mm. (G-I) These graphs represent the area in the red box in Figure D, E, and F. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
3.3. DjPRMT1 gene knockdown leads to pleiotropic defects in regeneration To verify whether DjPRMT1 is contributed to regeneration, we evaluated the effects of DjPRMT1 silencing on intact animals and planarian fragments which were amputated 2 days post the last application of RNAi. The procedure was performed according to the method in Fig. 3A. After the silencing of DjPRMT1, we detected that 70% of DjPRMT1 was successfully disturbed (Fig. 3B), while no distinction was observed between intact control and DjPRMT1knockdown planarians (Fig. 3C). However, the regeneration process was severely affected in some of the DjPRMT1-knockdown planarians, especially the tail regeneration process. Posterior-facing wounds defects were observed in more than 35% of planarians fragments. Less than 10% of the planarians had head defects at anterior-facing wounds. The severity of the regenerating defect can
be observed by magnifying the defect in the red box (Fig. 3DeI). In comparison with the control group, the crawling ability of DjPRMT1-silenced planarians was reduced during the whole regeneration process, as they twisted and curled up in-situ (Video 1). Our results confirmed that DjPRMT1 participated in planarian regeneration. Supplementary video related to this article can be found at https://doi.org/10.1016/j.bbrc.2020.01.087. 3.4. Localization of DjPRMT1 protein in sf9 Mammalian PRMT1 functions in regulating the methylation of various proteins in the nucleus and cytoplasm. We transfected the SF9 cells with PIB/V5-FLAG-DjPRMT1 and confirmed that DjPRMT1 was indeed expressed (Fig. 4A). We observed that the recombinant DjPRMT1 protein predominantly localized in the nucleus with a
Please cite this article as: Y. Zhang et al., Protein arginine methyltransferase 1 mediates regeneration in Dugesia japonica, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.087
Y. Zhang et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
5
Fig. 4. DjPRMT1 affects neoblasts proliferation rather than differentiation. (A) Localization of DjPRMT1 in sf9 cells. Merged image (pink) shows that DjPRMT1 (red) was colocalized with nucleus (blue). White bars represent 4 mm. Lysates of Sf9 cells isolated from pIB/V5-FLAG transfected Sf9 cells or pIB/V5-FLAG-DjPRMT1 transfected Sf9 cells were detected with anti-FLAG and anti-b-actin antibodies. b-actin was used as a loading control. (B) The transcription levels of DjPRMT1 and DjPIWI-A in planarians treated with X-ray irradiation at different time points (0, 1, 3, 5 days). The transcription patterns were measured by qPCR. (C) DjPIWI-A and DjPRMT1 expression in X-ray irradiation treated planarians by ISH. Planarians with a GFP probe were used as the negative control. The positive signal was revealed with blue taint. White bars represent 500 mm. (D) The expression of DjPIWI-A was reduced after silencing of DjPRMT1. Data were analyzed by Student’s t-test (***p < 0.001). Error bars indicate standard deviations (SD) from three independent experiments. (E) The proliferation capacity of neoblasts at blastema (delineated by a discontinuous red line) were compared after silencing of GFP and DjPRMT1. White bars represent 0.5 mm. (F) Quantification of green pixel intensity in the blastema of control and DjPRMT1 RNAi planarians. Data were analyzed by Student’s t-test (*p < 0.05, ns ¼ not significant). (G) The eye resection of DjPRMT1 (RNAi) planarians recovers on the seventh day (n ¼ 10). Black bars represent 2 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
small amount in the cytoplasm (Fig. 4A), which is consistent with the expression of mammalian PRMT1. Therefore, we speculated that DjPRMT1 might function in a similar way to those of mammals.
3.5. DjPRMT1 affects neoblasts proliferation rather than differentiation We speculate that DjPRMT1 may be mainly located in neoblasts based on the mRNA expression patterns. Neoblasts, the only proliferating cells in the animal, are particularly sensitive to irradiation. Thus, we detected DjPRMT1 expression using X-ray irradiated adult planarians. Both the qPCR and ISH results showed that DjPIWI-A was gradually disappeared in intact adult planarians treated by X-ray irradiation, while DjPRMT1 could be detected in
the nerves after one day post X-ray irradiation (Fig. 4C, red arrow), though the expression of DjPRMT1 was gradually decreased (Fig. 4B and C). These results are consistent with those in previous reports [21]. These results confirmed DjPRMT1 was indeed expressed in neoblasts, and DjPIWI-A and DjPRMT1 were partially co-expressed. However, it should be noted that DjPIWI-A expression was slightly decreased in planarians subjected to DjPRMT1 RNAi (Fig. 4D), suggesting that DjPRMT1 may influence the survival of undifferentiated neoblasts or directly affect DjPIWI-A gene expression. Immunohistochemical analysis was performed with a H3p antibody to compare the cell division of neoblasts in worms subjected to GFP RNAi and DjPRMT1 RNAi. The positive signal was obviously weakened in the posterior-facing wounds of the planarian subjected to DjPRMT1 RNAi, but anterior-facing wounds
Please cite this article as: Y. Zhang et al., Protein arginine methyltransferase 1 mediates regeneration in Dugesia japonica, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.087
6
Y. Zhang et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
were not significant change (Fig. 4E and F). Moreover, the eyes were successfully reformed in the planarian subjected to DjPRMT1 RNAi which performed eye resection (Fig. 4G). This procedure, which causes small wounds to the planarians, does not cause the neoblasts to proliferate continuously but requires them to maintain the ability to differentiate [22]. The above results indicated that DjPRMT1 was expressed in the neoblasts and might be involved in tissue regeneration and renewal through influence cell proliferation, especially the posterior-facing wounds.
signaling pathways in planarian regeneration.
4. Discussion
We thank everyone of our laboratory for their technical assistance and helpful suggestions. This study was supported by the National Natural Science Foundation of China (31172074, 31572263, 31701015), the Natural Science Foundation of Shandong Province, China (ZR2017MC066), and the Key Science and Technology Project in Institutions of Higher Education of Shandong Province, China (J17KZ003).
Planarians are triploblastic, acoelomate, unsegmented, dorsoventrally flattened soft-bodied, and nonparasitic organisms, without circulatory, respiratory, or skeletal structures [23]. Because of the large population of adult stem cells, planarians have a remarkable ability to regenerate [24]. Therefore, with the application of various technologies and the development of genomic reagents, the molecular mechanisms by which pluripotent somatic stem cells (neoblasts) replenish, repair, and regenerate planarian tissues and organs have been explored [25]. The regeneration process of the planarian requires a series of cell proliferation and differentiation, and a large number of regeneration-related proteins need to be expressed and properly modified after translation. Previous studies have shown that depletion of PRMT1 inhibits cell proliferation [26], cell cycle progression delay, checkpoint defects, cell growth arrest, and cell death in mammals [11,27,28], while the DjPRMT1-silenced planarians do not regenerate completely and properly after amputating, partially due to the incorrect cell division at the wound site. Our results showed that the recombinant protein DjPRMT1 was predominantly located in the nucleus and a small amount was found in the cytoplasm. This expression pattern is consistent with the localization of mammalian PRMT1 [6], providing the first hint that DjPRMT1 may have the same functions in regulating proliferation and differentiation by targeting similar substrates in the planarian. Although we found that DjPRMT1(RNAi) did not affect the differentiation process of precursor stem cells in small wounds with eyespot repair, we speculate that DjPRMT1 may regulate the differentiation from neoblast to multiple precursor stem cells. Planarian displays robust regenerative properties driven by neoblasts, a stem cell population capable of developing into a complete individual [29]. Previous investigations have shown that PRMT1 affects the proliferation and differentiation of mouse muscle stem cells [30,31], liver cells [32], and intestinal epithelial cells [33]. Muscle-specific PRMT1 deficiency leads to muscle atrophy and muscle loss [34]. We observed reduced mobility and in situ contorted body phenotypes in the planarian subjected to DjPRMT1 RNAi. After muscle injury in mammals, deficiency of PRMT1 can cause the myogenic differentiation unterminated, leading to failure of muscle regeneration [30]. Thus, DjPRMT1 may have similar roles. PRMT1 deficiency lead to the decline in athletic ability during regeneration of planarians, may also be caused by muscle atrophy, loss of muscle, or failure to regenerate muscle [35]. In summary, we cloned the DjPRMT1 gene of Dugesia japonica and showed it was a highly evolutionarily conserved protein. DjPRMT1 was predominantly concentrated in neoblasts and showed obvious aggregation at blastema in the early stages of wound regeneration. By interfering with DjPRMT1, the wound area of the planarians cannot be completely repaired especially in the posterior regeneration. We also find that DjPRMT1-silenced planarians showed reduced mobility. Our results provide valuable information for further investigations of the potential molecular mechanisms of DjPRMT1 in the regenerative process of planarians. Future studies will focus on the precise regulation of DjPRMT1
Declaration of competing interest We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted. Acknowledgements
References [1] W.K. Paik, S. Kim, Enzymatic methylation of protein fractions from calf thymus nuclei, Biochem. Biophys. Res. Commun. 29 (1967) 14e20. [2] W.J. Lin, J.D. Gary, M.C. Yang, et al., The mammalian immediate-early TIS21 protein and the leukemia-associated BTG1 protein interact with a proteinarginine N-methyltransferase, J. Biol. Chem. 271 (1996) 15034e15044. [3] J.E. Katz, M. Dlakic, S. Clarke, Automated identification of putative methyltransferases from genomic open reading frames, Mol. Cell. Proteom. : MCP 2 (2003) 525e540, https://doi.org/10.1074/mcp.M300037-MCP200. [4] C. Peng, C.C. Wong, The story of protein arginine methylation: characterization, regulation, and function, Expert Rev. Proteomics 14 (2017) 157e170, https://doi.org/10.1080/14789450.2017.1275573. [5] M.T. Bedford, S.G. Clarke, Protein arginine methylation in mammals: who, what, and why, Mol. Cell 33 (2009) 1e13, https://doi.org/10.1016/ j.molcel.2008.12.013. [6] J. Tang, A. Frankel, R.J. Cook, et al., PRMT1 is the predominant type I protein arginine methyltransferase in mammalian cells, J. Biol. Chem. 275 (2000) 7723e7730. [7] X. Cheng, R.E. Collins, X. Zhang, Structural and sequence motifs of protein (histone) methylation enzymes, Annu. Rev. Biophys. Biomol. Struct. 34 (2005) 267e294, https://doi.org/10.1146/annurev.biophys.34.040204.144452. [8] M.R. Pawlak, C.A. Scherer, J. Chen, et al., Arginine N-methyltransferase 1 is required for early postimplantation mouse development, but cells deficient in the enzyme are viable, Mol. Cell. Biol. 20 (2000) 4859e4869. [9] S. Wang, X. Tan, B. Yang, et al., The role of protein arginine-methyltransferase 1 in gliomagenesis, BMB Rep. 45 (2012) 470e475, https://doi.org/10.5483/ BMBRep.2012.45.8.022. [10] W.K. Hua, Y.I. Chang, C.L. Yao, et al., Protein arginine methyltransferase 1 interacts with and activates p38alpha to facilitate erythroid differentiation, PLoS One 8 (2013), e56715, https://doi.org/10.1371/journal.pone.0056715. [11] Y. Hirata, K. Katagiri, K. Nagaoka, et al., TRIM48 promotes ASK1 activation and cell death through ubiquitination-dependent degradation of the ASK1negative regulator PRMT1, Cell Rep. 21 (2017) 2447e2457, https://doi.org/ 10.1016/j.celrep.2017.11.007. [12] F.M. Boisvert, U. Dery, J.Y. Masson, et al., Arginine methylation of MRE11 by PRMT1 is required for DNA damage checkpoint control, Genes Dev. 19 (2005) 671e676, https://doi.org/10.1101/gad.1279805. [13] Q. Yuan, R. Tian, H. Zhao, et al., Multiple Arginine Residues Are Methylated in Drosophila Mre11 and Required for Survival Following Ionizing Radiation, vol. 8, G3, Bethesda, Md, 2018, pp. 2099e2106, https://doi.org/10.1534/ g3.118.200298. [14] Z. Yu, T. Chen, J. Hebert, et al., A mouse PRMT1 null allele defines an essential role for arginine methylation in genome maintenance and cell proliferation, Mol. Cell. Biol. 29 (2009) 2982e2996, https://doi.org/10.1128/mcb.00042-09. [15] M.C. Stuhlinger, P.S. Tsao, J.H. Her, et al., Homocysteine impairs the nitric oxide synthase pathway: role of asymmetric dimethylarginine, Circulation 104 (2001) 2569e2575. [16] L. Liu, X. Zhao, L. Zhao, et al., Arginine methylation of SREBP1a via PRMT5 promotes de novo lipogenesis and tumor growth, Cancer Res. 76 (2016) 1260e1272, https://doi.org/10.1158/0008-5472.can-15-1766. [17] M.S. Eram, Y. Shen, M. Szewczyk, et al., A potent, selective, and cell-active inhibitor of human type I protein arginine methyltransferases, ACS Chem. Biol. 11 (2016) 772e781, https://doi.org/10.1021/acschembio.5b00839. [18] A. Li, H. Zhao, Q. Lai, et al., Posttranslational modifications of baculovirus protamine-like protein P6.9 and the significance of its hyperphosphorylation for viral very late gene hyperexpression, J. Virol. 89 (2015) 7646e7659, https://doi.org/10.1128/jvi.00333-15. [19] Y. Han, A. Li, L. Gao, et al., Identification and characterization of a phospholipid scramblase encoded by planarian Dugesia japonica, Gene 602 (2017) 43e49, https://doi.org/10.1016/j.gene.2016.11.029.
Please cite this article as: Y. Zhang et al., Protein arginine methyltransferase 1 mediates regeneration in Dugesia japonica, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.087
Y. Zhang et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx [20] L. Gao, Y. Han, H. Deng, et al., The role of a novel C-type lectin-like protein from planarian in innate immunity and regeneration, Dev. Comp. Immunol. 67 (2017) 413e426, https://doi.org/10.1016/j.dci.2016.08.010. [21] N. Shibata, T. Hayashi, R. Fukumura, et al., Comprehensive gene expression analyses in pluripotent stem cells of a planarian, Dugesia japonica, Int. J. Dev. Biol. 56 (2012) 93e102, https://doi.org/10.1387/ijdb.113434ns. [22] S.A. LoCascio, S.W. Lapan, P.W. Reddien, Eye absence does not regulate planarian stem cells during eye regeneration, Dev. Cell 40 (2017) 381e391, https://doi.org/10.1016/j.devcel.2017.02.002, e383. [23] E. Salo, J.F. Abril, T. Adell, et al., Planarian regeneration: achievements and future directions after 20 years of research, Int. J. Dev. Biol. 53 (2009) 1317e1327, https://doi.org/10.1387/ijdb.072414es. [24] P.W. Reddien, A. Sanchez Alvarado, Fundamentals of planarian regeneration, Annu. Rev. Cell Dev. Biol. 20 (2004) 725e757, https://doi.org/10.1146/ annurev.cellbio.20.010403.095114. [25] D.J. Forsthoefel, P.A. Newmark, Emerging patterns in planarian regeneration, Curr. Opin. Genet. Dev. 19 (2009) 412e420, https://doi.org/10.1016/ j.gde.2009.05.003. [26] K.A. Akter, M.A. Mansour, T. Hyodo, et al., FAM98A is a novel substrate of PRMT1 required for tumor cell migration, invasion, and colony formation, Tumour Biol.: the journal of the International Society for Oncodevelopmental Biology and Medicine 37 (2016) 4531e4539, https://doi.org/10.1007/s13277015-4310-5. [27] N. Cheung, T.K. Fung, B.B. Zeisig, et al., Targeting aberrant epigenetic networks mediated by PRMT1 and KDM4C in acute myeloid leukemia, Cancer Cell 29 (2016) 32e48, https://doi.org/10.1016/j.ccell.2015.12.007.
7
[28] S. Infantino, B. Benz, T. Waldmann, et al., Arginine methylation of the B cell antigen receptor promotes differentiation, J. Exp. Med. 207 (2010) 711e719, https://doi.org/10.1084/jem.20091303. [29] A. Sanchez Alvarado, Stem cells and the planarian Schmidtea mediterranea, Comptes Rendus Biol. 330 (2007) 498e503, https://doi.org/10.1016/ j.crvi.2007.05.005. [30] R.S. Blanc, G. Vogel, X. Li, et al., Arginine methylation by PRMT1 regulates muscle stem cell fate, Mol. Cell. Biol. 37 (2017), https://doi.org/10.1128/ mcb.00457-16. [31] N.Y. Shen, S.Y. Ng, S.L. Toepp, et al., Protein arginine methyltransferase expression and activity during myogenesis, Biosci. Rep. 38 (2018), https:// doi.org/10.1042/bsr20171533. [32] Y. Lim, Y.H. Kwon, N.H. Won, et al., Multimerization of expressed proteinarginine methyltransferases during the growth and differentiation of rat liver, Biochim. Biophys. Acta 1723 (2005) 240e247, https://doi.org/10.1016/ j.bbagen.2005.02.015. [33] H. Matsuda, Y.B. Shi, An essential and evolutionarily conserved role of protein arginine methyltransferase 1 for adult intestinal stem cells during postembryonic development, Stem Cells (Dayton, Ohio) 28 (2010) 2073e2083, https://doi.org/10.1002/stem.529. [34] S. Choi, H.J. Jeong, H. Kim, et al., Skeletal muscle-specific Prmt1 deletion causes muscle atrophy via deregulation of the PRMT6-FOXO3 axis, Autophagy (2019) 1e13, https://doi.org/10.1080/15548627.2019.1569931. [35] M.L. Scimone, L.E. Cote, P.W. Reddien, Orthogonal muscle fibres have different instructive roles in planarian regeneration, Nature 551 (2017) 623e628, https://doi.org/10.1038/nature24660.
Please cite this article as: Y. Zhang et al., Protein arginine methyltransferase 1 mediates regeneration in Dugesia japonica, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.087