UPF2介导八肋游仆虫NMD途径SURF和EJC的耦联
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摘要
通过生物信息学分析发现八肋游仆虫的UPF2(EoUPF2)包含三个串联的MIF4G(middle domain of translation initiation factor 4G)结构域和一个C末端UPF1的结合区域。对24种UPF2氨基酸序列的进化关系分析发现,EoUPF2与酿酒酵母的UPF2(ScUPF2)进化关系较为接近。InterPro database数据库分析结果显示,二者的核心结构域非常相似。酵母双杂交和Pull down实验证实:EoUPF2的C末端与EoUPF1的CH结构域相互作用;EoUPF2在没有UPF3存在的情况下,通过其MIF4G结构与外显子连接复合体(exon-exon junction complex,EJC)核心因子Y14a和Y14b相互作用。β-半乳糖苷酶实验证实EoUPF2与Y14a的作用更强一些。qPCR分析结果排除了EoUPF2与Y14a和Y14b相互作用与Y14a和Y14b本身的拷贝数有关。由此我们推断,在八肋游仆虫的NMD识别无义mRNA过程中,EoUPF2通过与Y14相互作用,介导了UPF1与无义mRNA上的外显子连接复合体的耦联,启动了无义mRNA的识别过程。
Bioinformatics analysis in this paper showed that the EoUPF2 contained three tandem MIF4 G(middle domain of translation initiation factor 4 G)domains and a C-terminal UPF1 binding region.Analysis among 24 UPF2 amino acid sequences indicated that the evolutionary relationship between EoUPF2 and Saccharomyces cerevisiae UPF2 was very close.InterPro database analysis indicated that the core domain of EoUPF2 was similar to that of ScUPF2.Moreover,the yeast two hybrid and pull down experiments were conducted to show the role of EoUPF2 in the initiation of NMD in E.octocarinatus.We demonstrated the interaction between EoUPF1 and EoUPF2 was mediated by CH domain of EoUPF1 and C-terminal domain of EoUPF2.EoUPF2 interacted with Y14 aor Y14 b,core factor of exon-exon junction complex(EJC),by its MIF4 Gstructure in the absence of UPF3.β-galactosidase assay confirmed that Y14 aplayed a significant role.Q-PCR analysis excluded the interaction between EoUPF2 and Y14 aor Y14 bwas related to the copy number of themselves.We concluded that EoUPF2 mediated the coupling of UPF1 with EJC on nonsense mRNA and initiated the recognition of nonsense mRNA by interacting with Y14.
Bioinformatics analysis in this paper showed that the EoUPF2 contained three tandem MIF4 G(middle domain of translation initiation factor 4 G)domains and a C-terminal UPF1 binding region.Analysis among 24 UPF2 amino acid sequences indicated that the evolutionary relationship between EoUPF2 and Saccharomyces cerevisiae UPF2 was very close.InterPro database analysis indicated that the core domain of EoUPF2 was similar to that of ScUPF2.Moreover,the yeast two hybrid and pull down experiments were conducted to show the role of EoUPF2 in the initiation of NMD in E.octocarinatus.We demonstrated the interaction between EoUPF1 and EoUPF2 was mediated by CH domain of EoUPF1 and C-terminal domain of EoUPF2.EoUPF2 interacted with Y14 aor Y14 b,core factor of exon-exon junction complex(EJC),by its MIF4 Gstructure in the absence of UPF3.β-galactosidase assay confirmed that Y14 aplayed a significant role.Q-PCR analysis excluded the interaction between EoUPF2 and Y14 aor Y14 bwas related to the copy number of themselves.We concluded that EoUPF2 mediated the coupling of UPF1 with EJC on nonsense mRNA and initiated the recognition of nonsense mRNA by interacting with Y14.
引文
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[15]Fiorini F,Boudvillain M,Le Hir H.Tight Intramolecular Regulation of the Human Upf1Helicase by its N-and C-terminal Domains[J].Nucleic Acids Res,2013,41(4):2404-2415.DOI:10.1093/nar/gks1320.
[16]Nicholson P,Yepiskoposyan H,Metze S,et al.Nonsense-Mediated mRNA Decay in Human Cells:Mechanistic Insights,Functions beyond Quality Control and the Double-Life of NMD Factors[J].Cell Mol Life Sci,2010,67(5):677-700.DOI:10.1007/s00018-009-0177-1.
[17]Tatsuno T,Nakamura Y,Ma S,et al.Nonsense-mediated mRNA Decay Factor Upf2Exists in both the Nucleoplasm and the Cytoplasm[J].Mol Med Rep,2016,14(1):655-660.DOI:10.3892/mmr.2016.5331.
[18]Aznarez I,Nomakuchi T T,Tetenbaum-Novatt J,et al.Mechanism of Nonsense-Mediated mRNA Decay Stimulation by Splicing Factor SRSF1[J].Cell Rep,2018,23(7):2186-2198.DOI:10.1016/j.celrep.2018.04.039.
[19]Lopez-Perrote A,Castano R,Melero R,et al.Human Nonsense-Mediated mRNA Decay Factor UPF2Interacts Directly with eRF3and the SURF Complex[J].Nucleic Acids Res,2016,44(4):1909-1923.DOI:10.1093/nar/gkv1527.
[20]Fourati Z,Roy B,Millan C,et al.A Highly Conserved Region Essential for NMD in the Upf2N-terminal Domain[J].JMol Biol,2014,426(22):3689-3702.DOI:10.1016/j.jmb.2014.09.015.
[21]Kadlec J,Guilligay D,Ravelli R B,et al.Crystal Structure of the UPF2-Interacting Domain of Nonsense-Mediated mRNADecay Factor UPF1[J].RNA,2006,12(10):1817-1824.DOI:10.1261/rna.177606.
[22]Melero R,Buchwald G,Castano R,et al.The Cryo-EM Structure of the UPF-EJC Complex Shows UPF1Poised toward the RNA 3′end[J].Nat Struct Mol Biol,2012,19(5):498-505,S491-492.DOI:10.1038/nsmb.2287.
[23]Le Hir H,Gatfield D,Izaurralde E,et al.The Exon-exon Junction Complex Provides a Binding Platform for Factors Involved in mRNA Export and Nonsense-Mediated mRNA Decay[J].EMBO J,2001,20(17):4987-4997.DOI:10.1093/emboj/20.17.4987.
[24]Czaplinski K,Ruiz-Echevarria M J,Paushkin S V,et al.The Surveillance Complex Interacts with the Translation Release Factors to Enhance Termination and Degrade Aberrant mRNAs[J].Genes Dev,1998,12(11):1665-1677.
[25]孙泉红,柴宝峰,梁爱华.八肋游仆虫第二类释放因子基因的克隆与序列分析[J].中国生物化学与分子生物学报,2002,18(3):347-351.DOI:10.13865/j.cnki.cjbmb.2002.03.020.Sun Q H,Chai B F,Liang A H,et al.Cloning and Sequence of eRF3Gene from Euplotes Octocarinatus[J].Chin J Biochem Mol Biol,2002,18(3):347-351.DOI:10.13865/j.cnki.cjbmb.2002.03.020.
[26]Kashima I,Yamashita A,Izumi N,et al.Binding of a Novel SMG-1-Upf1-eRF1-eRF3Complex(SURF)to the Exon Junction Complex Triggers Upf1Phosphorylation and Nonsense-Mediated mRNA Decay[J].Genes Dev,2006,20(3):355-367.DOI:10.1101/gad.1389006.
[2]Martinez-Montiel N,Morales-Lara L,Hernandez-Perez J M,et al.In Silico Analysis of the Structural and Biochemical Features of the NMD Factor UPF1in Ustilago Maydis[J].PLoS One,2016,11(2):e0148191.DOI:10.1371/journal.pone.0148191.
[3]Hug N,Longman D,Caceres J F.Mechanism and Regulation of the Nonsense-Mediated Decay Pathway[J].Nucleic Acids Res,2016,44(4):1483-1495.DOI:10.1093/nar/gkw010.
[4]柴宝峰,王美,石文鑫,等.无义mRNA降解途径的机制与进化[J].山西大学学报(自然科学版),2017,40(3):639-644.Chai B F,Wang M,Shi W X,et al.Mechanism and Evolution of Nonsense-Mediated mRNA Decay[J].J Shanxi Univ(Nat Sci Ed),2017,40(3):639-644.DOI:10.13451/j.cnki.shanxi.univ(nat.sci.).2017.03.032.
[5]Tian M,Yang W,Zhang J,et al.Nonsense-Mediated mRNA Decay in Tetrahymena is EJC Independent and Requires a Protozoa-Specific Nuclease[J].Nucleic Acids Res,2017,45(11):6848-6863.DOI:10.1093/nar/gkx256.
[6]Chen Y H,Su L H,Sun C H.Incomplete Nonsense-Mediated mRNA Decay in Giardia Lamblia[J].Int J Parasitol,2008,38(11):1305-1317.DOI:10.1016/j.ijpara.2008.02.006.
[7]王美,吕佳,石文鑫,等.SMG1-UPF1-eRF1-eRF3复合体参与贾第虫无义介导的mRNA降解激活[J].中国生物化学与分子生物学报,2018,34(5):519-529.DOI:10.13865/j.cnki.cjbmb.2018.05.09.Wang M,LüJ,Shi W X,et al.SMG1-UPF1-eRF1-eRF3Complex is Involved in the Nonsense-Mediated mRNA Decay in Protozoan Giardia Lamblia[J].Chin J Biochem Mol Biol,2018,34(5):519-529.DOI:10.13865/j.cnki.cjbmb.2018.05.09.
[8]Contreras J,Begley V,Macias S,et al.An UPF3-Based Nonsense-Mediated Decay in Paramecium[J].Res Microbiol,2014,165(10):841-846.DOI:10.1016/j.resmic.2014.10.008.
[9]Clerici M,Deniaud A,Boehm V,et al.Structural and Functional Analysis of the Three MIF4GDomains of Nonsense-Mediated Decay Factor UPF2[J].Nucleic Acids Res,2014,42(4):2673-2686.DOI:10.1093/nar/gkt1197.
[10]Clerici M,Mourao A,Gutsche I,et al.Unusual Bipartite Mode of Interaction between the Nonsense-mediated Decay Factors,UPF1and UPF2[J].EMBO J,2009,28(15):2293-2306.DOI:10.1038/emboj.2009.175.
[11]石文鑫,王美,柴杨丽,等.八肋游仆虫NMD通路的初步研究[J].中国细胞生物学学报,2018,40(6):984-991.Shi W X,Wang M,Chai Y L,et al.Preliminary Analysis of NMD Pathway in Euplotes Octocarinatus[J].Chin J Cell Biol,2018,40(6):984-991.DOI:10.11844/cjcb.2018.06.0017.
[12]Gupta P,Li Y R.Upf Proteins:Highly Conserved Factors Involved in Nonsense mRNA Mediated Decay[J].Mol Biol Rep,2018,45(1):39-55.DOI:10.1007/s11033-017-4139-7.
[13]Cheng Z,Muhlrad D,Lim M K,et al.Structural and Functional Insights into the Human Upf1Helicase Core[J].EMBOJ,2007,26(1):253-264.DOI:10.1038/sj.emboj.7601464.
[14]Chakrabarti S,Jayachandran U,Bonneau F,et al.Molecular Mechanisms for the RNA-Dependent ATPase Activity of Upf1and its Regulation by Upf2[J].Mol Cell,2011,41(6):693-703.DOI:10.1016/j.molcel.2011.02.010.
[15]Fiorini F,Boudvillain M,Le Hir H.Tight Intramolecular Regulation of the Human Upf1Helicase by its N-and C-terminal Domains[J].Nucleic Acids Res,2013,41(4):2404-2415.DOI:10.1093/nar/gks1320.
[16]Nicholson P,Yepiskoposyan H,Metze S,et al.Nonsense-Mediated mRNA Decay in Human Cells:Mechanistic Insights,Functions beyond Quality Control and the Double-Life of NMD Factors[J].Cell Mol Life Sci,2010,67(5):677-700.DOI:10.1007/s00018-009-0177-1.
[17]Tatsuno T,Nakamura Y,Ma S,et al.Nonsense-mediated mRNA Decay Factor Upf2Exists in both the Nucleoplasm and the Cytoplasm[J].Mol Med Rep,2016,14(1):655-660.DOI:10.3892/mmr.2016.5331.
[18]Aznarez I,Nomakuchi T T,Tetenbaum-Novatt J,et al.Mechanism of Nonsense-Mediated mRNA Decay Stimulation by Splicing Factor SRSF1[J].Cell Rep,2018,23(7):2186-2198.DOI:10.1016/j.celrep.2018.04.039.
[19]Lopez-Perrote A,Castano R,Melero R,et al.Human Nonsense-Mediated mRNA Decay Factor UPF2Interacts Directly with eRF3and the SURF Complex[J].Nucleic Acids Res,2016,44(4):1909-1923.DOI:10.1093/nar/gkv1527.
[20]Fourati Z,Roy B,Millan C,et al.A Highly Conserved Region Essential for NMD in the Upf2N-terminal Domain[J].JMol Biol,2014,426(22):3689-3702.DOI:10.1016/j.jmb.2014.09.015.
[21]Kadlec J,Guilligay D,Ravelli R B,et al.Crystal Structure of the UPF2-Interacting Domain of Nonsense-Mediated mRNADecay Factor UPF1[J].RNA,2006,12(10):1817-1824.DOI:10.1261/rna.177606.
[22]Melero R,Buchwald G,Castano R,et al.The Cryo-EM Structure of the UPF-EJC Complex Shows UPF1Poised toward the RNA 3′end[J].Nat Struct Mol Biol,2012,19(5):498-505,S491-492.DOI:10.1038/nsmb.2287.
[23]Le Hir H,Gatfield D,Izaurralde E,et al.The Exon-exon Junction Complex Provides a Binding Platform for Factors Involved in mRNA Export and Nonsense-Mediated mRNA Decay[J].EMBO J,2001,20(17):4987-4997.DOI:10.1093/emboj/20.17.4987.
[24]Czaplinski K,Ruiz-Echevarria M J,Paushkin S V,et al.The Surveillance Complex Interacts with the Translation Release Factors to Enhance Termination and Degrade Aberrant mRNAs[J].Genes Dev,1998,12(11):1665-1677.
[25]孙泉红,柴宝峰,梁爱华.八肋游仆虫第二类释放因子基因的克隆与序列分析[J].中国生物化学与分子生物学报,2002,18(3):347-351.DOI:10.13865/j.cnki.cjbmb.2002.03.020.Sun Q H,Chai B F,Liang A H,et al.Cloning and Sequence of eRF3Gene from Euplotes Octocarinatus[J].Chin J Biochem Mol Biol,2002,18(3):347-351.DOI:10.13865/j.cnki.cjbmb.2002.03.020.
[26]Kashima I,Yamashita A,Izumi N,et al.Binding of a Novel SMG-1-Upf1-eRF1-eRF3Complex(SURF)to the Exon Junction Complex Triggers Upf1Phosphorylation and Nonsense-Mediated mRNA Decay[J].Genes Dev,2006,20(3):355-367.DOI:10.1101/gad.1389006.
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