NSun2 promotes cell migration through methylating autotaxin mRNA
2020; Elsevier BV; Volume: 295; Issue: 52 Linguagem: Inglês
10.1074/jbc.ra119.012009
ISSN1083-351X
AutoresXin Xu, Yihua Zhang, Junjie Zhang, Xiaotian Zhang,
Tópico(s)Galectins and Cancer Biology
ResumoNSun2 is an RNA methyltransferase introducing 5-methylcytosine into tRNAs, mRNAs, and noncoding RNAs, thereby influencing the levels or function of these RNAs. Autotaxin (ATX) is a secreted glycoprotein and is recognized as a key factor in converting lysophosphatidylcholine into lysophosphatidic acid (LPA). The ATX-LPA axis exerts multiple biological effects in cell survival, migration, proliferation, and differentiation. Here, we show that NSun2 is involved in the regulation of cell migration through methylating ATX mRNA. In the human glioma cell line U87, knockdown of NSun2 decreased ATX protein levels, whereas overexpression of NSun2 elevated ATX protein levels. However, neither overexpression nor knockdown of NSun2 altered ATX mRNA levels. Further studies revealed that NSun2 methylated the 3′-UTR of ATX mRNA at cytosine 2756 in vitro and in vivo. Methylation by NSun2 enhanced ATX mRNA translation. In addition, NSun2-mediated 5-methylcytosine methylation promoted the export of ATX mRNA from nucleus to cytoplasm in an ALYREF-dependent manner. Knockdown of NSun2 suppressed the migration of U87 cells, which was rescued by the addition of LPA. In summary, we identify NSun2-mediated methylation of ATX mRNA as a novel mechanism in the regulation of ATX. NSun2 is an RNA methyltransferase introducing 5-methylcytosine into tRNAs, mRNAs, and noncoding RNAs, thereby influencing the levels or function of these RNAs. Autotaxin (ATX) is a secreted glycoprotein and is recognized as a key factor in converting lysophosphatidylcholine into lysophosphatidic acid (LPA). The ATX-LPA axis exerts multiple biological effects in cell survival, migration, proliferation, and differentiation. Here, we show that NSun2 is involved in the regulation of cell migration through methylating ATX mRNA. In the human glioma cell line U87, knockdown of NSun2 decreased ATX protein levels, whereas overexpression of NSun2 elevated ATX protein levels. However, neither overexpression nor knockdown of NSun2 altered ATX mRNA levels. Further studies revealed that NSun2 methylated the 3′-UTR of ATX mRNA at cytosine 2756 in vitro and in vivo. Methylation by NSun2 enhanced ATX mRNA translation. In addition, NSun2-mediated 5-methylcytosine methylation promoted the export of ATX mRNA from nucleus to cytoplasm in an ALYREF-dependent manner. Knockdown of NSun2 suppressed the migration of U87 cells, which was rescued by the addition of LPA. In summary, we identify NSun2-mediated methylation of ATX mRNA as a novel mechanism in the regulation of ATX. mRNA methylation is an important modification type in physiological and pathological processes. NSun2, also called myc-induced SUN domain-containing protein, belongs to the NOP2/Sun domain family. As a major 5-methylcytosine (m5C) methyltransferase, NSun2 was initially considered to be a typical tRNA methyltransferase. Recently, increasing evidences suggest that NSun2-mediated m5C methylation extensively occur in mRNAs and noncoding RNAs (1Yuan S. Tang H. Xing J. Fan X. Cai X. Li Q. Han P. Luo Y. Zhang Z. Jiang B. Dou Y. Gorospe M. Wang W. Methylation by NSun2 represses the levels and function of microRNA 125b.Mol. Cell. Biol. 2014; 34 (25047833): 3630-364110.1128/MCB.00243-14Crossref PubMed Scopus (50) Google Scholar, 2Zhang X. Liu Z. Yi J. Tang H. Xing J. Yu M. Tong T. Shang Y. Gorospe M. Wang W. The tRNA methyltransferase NSun2 stabilizes p16INK(4) mRNA by methylating the 3′-untranslated region of p16.Nat. Commun. 2012; 3 (22395603): 71210.1038/ncomms1692Crossref PubMed Scopus (79) Google Scholar, 3Hussain S. Sajini A.A. Blanco S. Dietmann S. Lombard P. Sugimoto Y. Paramor M. Gleeson J.G. Odom D.T. Ule J. Frye M. 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LPA is a potent bioactive lipid that is capable of activating different signal pathways through interacting with the LPA receptors on cell surface. LPA can regulate a broad range of cell functions, such as cell survival, proliferation, and migration. The ATX-LPA axis plays an important role in cancer development, especially in cancer cell migration (15Liu S. Umezu-Goto M. Murph M. Lu Y. Liu W. Zhang F. Yu S. Stephens L.C. Cui X. Murrow G. Coombes K. Muller W. Hung M.C. Perou C.M. Lee A.V. et al.Expression of autotaxin and lysophosphatidic acid receptors increases mammary tumorigenesis, invasion, and metastases.Cancer Cell. 2009; 15 (19477432): 539-55010.1016/j.ccr.2009.03.027Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar, 16Nam S.W. Clair T. Campo C.K. Lee H.Y. Liotta L.A. Stracke M.L. 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Maluccio M.A. Autotaxin expression and its connection with the TNF-α-NF-κB axis in human hepatocellular carcinoma.Molecular Cancer. 2010; 9 (20356387): 7110.1186/1476-4598-9-71Crossref PubMed Scopus (95) Google Scholar, 19Ptaszynska M.M. Pendrak M.L. Bandle R.W. Stracke M.L. Roberts D.D. Positive feedback between vascular endothelial growth factor-A and autotaxin in ovarian cancer cells.Mol. Cancer Res. 2008; 6 (18337445): 352-36310.1158/1541-7786.MCR-07-0143Crossref PubMed Scopus (60) Google Scholar, 20Castelino F.V. Bain G. Pace V.A. Black K.E. George L. Probst C.K. Goulet L. Lafyatis R. Tager A.M. An autotaxin/lysophosphatidic acid/interleukin-6 amplification loop drives scleroderma fibrosis.Arthritis & rheumatology. 2016; 68 (27390295): 2964-297410.1002/art.39797Crossref PubMed Scopus (41) Google Scholar). Regulation of ATX expression at the level of transcription has been intensively reported. For examples, ATX can be regulated at the transcriptional level by transcriptional factors Stat3, AP-1, and SP (21Azare J. Doane A. Leslie K. Chang Q. Berishaj M. Nnoli J. Mark K. Al-Ahmadie H. Gerald W. Hassimi M. Viale A. Stracke M. Lyden D. Bromberg J. Stat3 mediates expression of autotaxin in breast cancer.PLoS ONE. 2011; 6 (22140473)e2785110.1371/journal.pone.0027851Crossref PubMed Scopus (49) Google Scholar, 22Farina A.R. Cappabianca L. Ruggeri P. Di Ianni N. Ragone M. Merolle S. Sano K. Stracke M.L. Horowitz J.M. Gulino A. Mackay A.R. Constitutive autotaxin transcription by Nmyc-amplified and non-amplified neuroblastoma cells is regulated by a novel AP-1 and SP-mediated mechanism and abrogated by curcumin.FEBS Lett. 2012; 586 (22975311): 3681-369110.1016/j.febslet.2012.08.026Crossref PubMed Scopus (13) Google Scholar, 23Chen M. O'Connor K.L. Integrin α6β4 promotes expression of autotaxin/ENPP2 autocrine motility factor in breast carcinoma cells.Oncogene. 2005; 24 (15897878): 5125-513010.1038/sj.onc.1208729Crossref PubMed Scopus (92) Google Scholar), as well as histone deacetylases (24Li S. Wang B.L. Xu Y. Zhang J.J. Autotaxin is induced by TSA through HDAC3 and HDAC7 inhibition and antagonizes the TSA-induced cell apoptosis.Mol. Cancer. 2011; 10 (21314984): 1810.1186/1476-4598-10-18Crossref PubMed Scopus (14) Google Scholar). However, whether mRNA methylation is involved in the regulation of ATX is still unknown. In the present study, we show that RNA methyltransferase NSun2 catalyzes the methylation of ATX mRNA 3′UTR at C2756. Methylation by NSun2 promotes the export of ATX mRNA from nucleus to cytoplasm in an ALYREF-dependent manner and enhances the translation of ATX. This NSun2-ATX regulatory process affects cancer cell migration. N6-methyladenosine (m6A) and m5C are two major types of mRNA methylation. To test whether m6A or m5C modification exists in ATX mRNA, ribonucleoprotein-immunoprecipitation (RNP-IP) assays were performed by using anti-m5C or -m6A antibody. CDK1 and cyclin A were the positive control and negative control of m5C IP, respectively, whereas MYC and HIF-1α were the counterparts of m6A IP (8Xing J. Yi J. Cai X. Tang H. Liu Z. Zhang X. Martindale J.L. Yang X. Jiang B. Gorospe M. Wang W. NSun2 promotes cell growth via elevating cyclin-dependent kinase 1 translation.Mol. Cell. Biol. 2015; 35 (26391950): 4043-405210.1128/MCB.00742-15Crossref PubMed Scopus (45) Google Scholar, 25Dominissini D. Moshitch-Moshkovitz S. Schwartz S. Salmon-Divon M. Ungar L. Osenberg S. Cesarkas K. Jacob-Hirsch J. Amariglio N. Kupiec M. Sorek R. Rechavi G. Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq.Nature. 2012; 485 (22575960): 201-20610.1038/nature11112Crossref PubMed Scopus (1829) Google Scholar). As shown in Fig. 1A, ATX mRNA could be effectively enriched by anti-m5C antibody but not by anti-m6A antibody, suggesting that m5C modification may occur in ATX mRNA. Because NSun2 is a main mRNA methyltransferase catalyzing m5C formation, we further tested whether NSun2 could interact with ATX mRNA. To this end, a vector expressing FLAG-tagged NSun2 (C271A), which could tightly bind to target RNAs, was transfected into U87 cells. It was found by immunoprecipitation assays using anti-FLAG antibody that FLAG-tagged NSun2 (C271A) could enrich ATX mRNA, suggesting that NSun2 may directly bind to ATX mRNA (Fig. 1B). We further tested the protein levels of ATX from the cells with silenced NSun2 or overexpressed NSun2. As shown in Fig. 1C, knockdown of NSun2 decreased the protein levels of ATX. On the other hand, ectopic expression of NSun2, but not that of the RNA methyltransferase enzyme-dead mutant NSun2 (C321A), elevated the protein levels of ATX (Fig. 1D). The effect of NSun2 knockdown or overexpression on ATX protein levels was further confirmed by ELISA (Fig. 1, E and F). However, neither knockdown nor overexpression of NSun2 could alter the mRNA levels of ATX (Fig. 1, G and H). Apart from U87 cells, the effects of NSun2 intervention on ATX expression were also confirmed in colon cancer cells Colo320 and prostate cancer cells DU145 (Fig. S1). Therefore, NSun2 may regulate ATX expression at the post-transcriptional level. To test whether NSun2 was capable of catalyzing ATX mRNA methylation, we used 3H-labeled SAM as methyl donor and performed in vitro methylation assays. The in vitro transcribed ATX mRNA fragments depicted in Fig. 2A were incubated with His-NSun2 protein and 3H-labeled SAM. ATX cDNA served as the negative control, whereas bacterial tRNA molecules were used as the positive control. As shown in Fig. 2, B and C, the methylation levels of ATX 3′UTR and 3′UTR-A were significantly higher than those of 5′UTR, coding sequence (CDS), 3′UTR-B, 3′UTR-C, and 3′UTR-D fragments. Furthermore, methylated and unmethylated 3′UTR-A fragments were subjected to HPLC-MS assays to determine the formation of m5C in ATX 3′UTR-A. As shown in Fig. 2D, m5C, but not m6A modification, was detected in the methylated ATX mRNA 3′UTR-A. These results indicate that ATX mRNA can be methylated by NSun2 in vitro and that the m5C methylation site is located in the ATX mRNA 3′UTR-A. To further identify the methylation site in the 3′UTR of ATX mRNA, the methylated ATX 3′UTR-A fragment was subjected to bisulfite RNA-Seq (Fig. 3A). As shown in Fig. 3B, three m5C sites were identified (C2756, 81%; C2757, 17%; and C2792, 4%), and C2756 was identified as the site with the highest methylation efficiency. By using in vitro methylation assays, mutation of C2756 in 3′UTR and 3′UTR-A fragments (3′UTR-M and 3′UTR-A-M with the C2756T mutation) greatly reduced their methylation rates (Fig. 3C). Furthermore, as shown in Fig. 3D, by bisulfite sequencing analysis, U87 cells silenced with NSun2 exhibited a much lower methylation rate at C2756 (17.5%) compared with that observed in control cells (70%). Although C2757 was mildly methylated by NSun2 in vitro, knockdown of NSun2 had no significant effect on the methylation of C2757 in cells (Fig. 3D). Therefore, C2756 in ATX 3′UTR was identified as the major methylation site by NSun2. Next, we tested the role of NSun2-mediated ATX mRNA 3′UTR methylation in the regulation of protein expression. To this end, a series of pGL3-derived reporter vectors bearing different fragments of ATX cDNA were constructed (Fig. 4A). By using reporter gene assays, we found that the luciferase activity of pGL3-derived reporter bearing ATX 3′UTR, but not that bearing ATX 5′UTR, CDS, or 3′UTR-M (3′UTR with the C2756T mutation), was reduced in cells with silenced NSun2 and increased in cells with overexpressed NSun2 (Fig. 4, B and C). However, the luciferase mRNA levels were not changed (Fig. S2). These data suggest that methylation by NSun2 in ATX 3′UTR may have a positive effect on the expression of ATX at the post-transcriptional level. Because intervention of NSun2 expression in cells did not alter the levels of ATX mRNA (Fig. 1, G and H and Fig. S1, B and D), we performed in vitro translation assays to confirm whether NSun2-mediated ATX mRNA 3′UTR methylation could regulate protein expression at the translational level. The transcripts transcribed from pGL3, pGL3-3′UTR, and pGL3-3′UTR-M (C2756T) were methylated by NSun2 in vitro or kept unmethylated. These transcripts then were used for in vitro translation assays. The luciferase activity was quantified as the readout of translation efficiency. As shown in Fig. 4D, the luciferase activity observed from the methylated pGL3-3′UTR transcripts was significantly higher than that from the unmethylated pGL3-3′UTR transcripts. Mutation of C2756 in 3′UTR abolished the effect of methylation in promoting the reporter activity (Fig. 4D). Furthermore, knockdown of NSun2 reduced the recruitment of ATX mRNA to polysomes (Fig. 4E). Together, by methylating ATX mRNA at the 3′UTR, NSun2 may enhance the expression of ATX at the translational level. It has been reported that m5C formation promotes the nuclear export of mRNA (26Yang X. Yang Y. Sun B.F. Chen Y.S. Xu J.W. Lai W.Y. Li A. Wang X. Bhattarai D.P. Xiao W. Sun H.Y. Zhu Q. Ma H.L. Adhikari S. Sun M. et al.5-methylcytosine promotes mRNA export - NSUN2 as the methyltransferase and ALYREF as an m(5)C reader.Cell Res. 2017; 27 (28418038): 606-62510.1038/cr.2017.55Crossref PubMed Scopus (247) Google Scholar). To validate whether NSun2-mediated mRNA methylation promotes the nuclear export of ATX mRNA, the relative abundance of cytoplasmic and nuclear ATX mRNA in cells with silenced or overexpressed NSun2 was tested. As shown in Fig. 5A, knockdown of NSun2 in U87 cells significantly decreased the levels of ATX mRNA in cytoplasm and increased the levels of ATX mRNA in nucleus. On the other hand, overexpression of NSun2 increased the presence of ATX mRNA in cytoplasm but reduced its presence in nucleus. Similar results were obtained in Colo320 and DU145 cells (Fig. S3). Because RNA binding protein ALYREF is necessary for the nuclear export of m5C-modified mRNAs (26Yang X. Yang Y. Sun B.F. Chen Y.S. Xu J.W. Lai W.Y. Li A. Wang X. Bhattarai D.P. Xiao W. Sun H.Y. Zhu Q. Ma H.L. Adhikari S. Sun M. et al.5-methylcytosine promotes mRNA export - NSUN2 as the methyltransferase and ALYREF as an m(5)C reader.Cell Res. 2017; 27 (28418038): 606-62510.1038/cr.2017.55Crossref PubMed Scopus (247) Google Scholar), the role of ALYREF in NSun2-regulated nuclear export of ATX mRNA was further examined. Interestingly, ALYREF could interact with ATX mRNA in cells (Fig. 5B) and was prone to associate with methylated ATX 3′UTR fragment (Fig. 5C). The cytoplasmic ATX mRNA levels were decreased in U87 cells with silenced ALYREF (Fig. 5D). Knockdown of NSun2 decreased the association of ATX mRNA with ALYREF (Fig. 5E). These data indicate that NSun2-mediated m5C methylation promotes the export of ATX mRNA from nucleus to cytoplasm in an ALYREF-dependent manner. NSun2 is highly expressed in various cancer cells. Our results suggest that m5C methylation of ATX mRNA by NSun2 increases the expression of ATX, the key enzyme for extracellular LPA production. In addition, it has been reported that the ATX-LPA axis can promote cell migration (15Liu S. Umezu-Goto M. Murph M. Lu Y. Liu W. Zhang F. Yu S. Stephens L.C. Cui X. Murrow G. Coombes K. Muller W. Hung M.C. Perou C.M. Lee A.V. et al.Expression of autotaxin and lysophosphatidic acid receptors increases mammary tumorigenesis, invasion, and metastases.Cancer Cell. 2009; 15 (19477432): 539-55010.1016/j.ccr.2009.03.027Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar, 16Nam S.W. Clair T. Campo C.K. Lee H.Y. Liotta L.A. Stracke M.L. Autotaxin (ATX), a potent tumor motogen, augments invasive and metastatic potential of ras-transformed cells.Oncogene. 2000; 19 (10645002): 241-24710.1038/sj.onc.1203263Crossref PubMed Scopus (157) Google Scholar). To illustrate the effects of the NSun2-ATX regulatory process on cell migration, wound-healing and transwell assays were performed. 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Fujii M. Qi G. Ogawa I. Takata T. Shimamoto F. Tatsuka M. Frequent increased gene copy number and high protein expression of tRNA (cytosine-5-)-methyltransferase (NSUN2) in human cancers.DNA Cell Biol. 2012; 31 (22136356): 660-67110.1089/dna.2011.1446Crossref PubMed Scopus (47) Google Scholar). However, the role of NSUN2 in cancers and the mechanisms underlying are largely unknown. ATX is a secreted glycoprotein that catalyzes the conversion of lysophosphatidylcholine into LPA. The ATX-LPA pathway plays a vital role in cancer development, especially in cancer cell migration. In the present study, we demonstrate that NSun2 is able to methylate ATX mRNA in 3′UTR to enhance ATX expression at the post-transcriptional level. Knockdown of NSun2 led to the suppression of cell migration, which could be recovered by the addition of LPA. These data indicate that NSun2 participates in cell migration through regulating the ATX-LPA axis. ALYREF can specifically recognize m5C on mRNAs and facilitate the nuclear export of target mRNAs as an adaptor (26Yang X. Yang Y. Sun B.F. Chen Y.S. Xu J.W. Lai W.Y. Li A. Wang X. Bhattarai D.P. Xiao W. Sun H.Y. Zhu Q. Ma H.L. Adhikari S. Sun M. et al.5-methylcytosine promotes mRNA export - NSUN2 as the methyltransferase and ALYREF as an m(5)C reader.Cell Res. 2017; 27 (28418038): 606-62510.1038/cr.2017.55Crossref PubMed Scopus (247) Google Scholar). Our findings indicate that ALYREF is prone to associate with the methylated ATX 3′UTR fragment, in turn enhancing ATX translation through promoting the nuclear export of ATX mRNA. Knockdown of NSun2 and ALYREF together exhibited a stronger effect than knockdown NSun2 along in reducing the presence of ATX mRNA in polysomes (Fig. S4), suggesting that ALYREF may contribute to the assembly of ATX mRNA in polysomes. However, whether ALYREF is directly involved in the promotion of ATX protein translation in cytoplasm is still unknown and remains to be further explored. In addition to ALYREF, YBX1 is an m5C reader protein of methylated mRNA that has been reported (13Chen X. Li A. Sun B.-F. Yang Y. Han Y.-N. Yuan X. Chen R.-X. Wei W.-S. Liu Y. Gao C.-C. Chen Y.-S. Zhang M. Ma X.-D. Liu Z.-W. Luo J.-H. et al.5-methylcytosine promotes pathogenesis of bladder cancer through stabilizing mRNAs.Nat. Cell Biol. 2019; 21 (31358969): 978-99010.1038/s41556-019-0361-yCrossref PubMed Scopus (96) Google Scholar). We explored the effects of YBX1 to ATX expression. However, we did not detect any notably difference in ATX protein or mRNA levels after knocking down YBX1 compared with control ones (Fig. S5, A–C). RNP-IP assays demonstrated that YBX1 may not interact with ATX mRNA (Fig. S5D). These results indicate that YBX1 is not the reader protein of methylated ATX mRNA and that other m5C reader proteins of methylated ATX mRNA may exist. NSun2 is highly expressed in neuroepithelial progenitors in the neural tube (31Flores J.V. Cordero-Espinoza L. Oeztuerk-Winder F. Andersson-Rolf A. Selmi T. Blanco S. Tailor J. Dietmann S. Frye M. Cytosine-5 RNA Methylation Regulates Neural Stem Cell Differentiation and Motility.Stem Cell Reports. 2017; 8 (28041877): 112-12410.1016/j.stemcr.2016.11.014Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Loss of function of the NSun2 gene impairs brain development during early embryo formation stage and causes microcephaly in mouse and human (32Blanco S. Dietmann S. Flores J.V. Hussain S. Kutter C. Humphreys P. Lukk M. Lombard P. Treps L. Popis M. Kellner S. Hölter S.M. Garrett L. Wurst W. 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Neurosci. 2015; 9 (25788872): 5310.3389/fnins.2015.00053Crossref PubMed Scopus (15) Google Scholar). NSun2 and ATX coexist in the neuronal progenitors, and both participate in the development of the brain. Therefore, the NSun2-ATX regulatory process may be also involved in the aforementioned processes. In addition, NSun2 may take part in other biological processes through modulating the ATX-LPA axis, which remains to be explored in the future. The NSun2 antibody, cyclin A antibody, β-actin antibody, and GAPDH antibody were all purchased from Santa Cruz Biotechnology. The CDK1 antibody and FLAG antibody were purchased from Cell Signaling Technology. The ALYREF antibody, YBX1 antibody, IgG antibody, anti-m5C antibody, and anti-m6A antibody were purchased from Abcam. The ATX primary antibody was generated as previously described (35Li S. Zhang J. Lipopolysaccharide induces autotaxin expression in human monocytic THP-1 cells.Biochem. Biophys. Res. Commun. 2009; 378 (19027716): 264-26810.1016/j.bbrc.2008.11.047Crossref PubMed Scopus (30) Google Scholar). U87, DU145, and HEK293T cells were cultured in DMEM (Macgene, Beijing, China). Colo320 cells were maintained in RPMI 1640 medium (Macgene, Beijing, China). The medium was supplemented with 10% FBS (Biowest, Riverside, MO), 100 units/ml penicillin, and 0.1 mg/ml streptomycin (Thermo Fisher Scientific). All cells were cultured at 37 °C in 5% CO2. The plasmids and siRNAs were transfected using Lipofectamine 2000 (Thermo Fisher Scientific) following the manufacturer's instructions. The siRNAs used were 5′-GAGAUCCUCUUCUAUGAUCTT-3′ for siNSun2-1; 5′-GGAGAACAAGCUGUUCGAGTT-3′ for siNSun2-2; 5′-GAGGUGGCAUGACUAGAAATT-3′ for siALYREF; and 5′-GGAACGGAUAUGGUUUCAUTT-3′ for siYBX1. Two NSun2 siRNAs were used in Fig. 1, and siNSun2-1 w
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