多能干细胞关键因子Oct4 RNA结合蛋白的分离与鉴定
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摘要
目的·分离多能干细胞关键因子Oct4的RNA结合蛋白。方法·培养小鼠胚胎干细胞(R1),从中提取细胞总RNA,反转录为cDNA,PCR扩增Oct4并转录为m RNA。采用链霉素亲和层析和质谱技术筛选候选RNA结合蛋白,采用RNA免疫沉淀技术鉴定候选RNA结合蛋白。结果·经高效液相色谱-质谱法鉴定和筛选出121个能与Oct4 3'非翻译区(untranslated region,UTR)和Oct45'UTR相结合的RNA结合蛋白,肽段图谱匹配数≥2的有11个。并对其中的7个候选RNA结合蛋白进行了进一步验证,结果显示Oct4可与DSP、SOD1、LMNA、NPM1、PSIP1、NCL和HDGF蛋白发生相互作用,其中HDGF与Oct4结合能力最强。结论·Oct4RNA结合蛋白的鉴定将为Oct4的调控机制提供一定理论依据,为后续其功能研究提供基础。
Objective · To identify the RNA binding proteins of Oct4, a key factor of embryonic stem cells. Methods · Total RNA was extracted from mouse embryonic stem cells(R1), which was reverse-transcribed into cDNA. Then PCR product of Oct4 mRNA were transcribed into Oct4 mRNA.Finally the candidate RNA-binding proteins were eluted applying the streptomycin affinity chromatography, and submitted for high-performance liquid chromatography combined with the mass spectrometry. The identified RNA binding proteins were further confirmed by RNA immunoprecipitation(RIP). Results · 121 RNA binding proteins of Oct4 3' untranslated region(UTR) and Oct4 5' UTR were identified with liquid chromatography/mass spectrometry. There were 11 proteins with the number of peptide spectrum matches more or equal to 2, and 7 of them were selected for additional confirmation using RIP method. RIP results showed that Oct4 interacted with DSP, SOD1, LMNA, NPM1, PSIP1, NCL and HDGF. Among them, HDGF had the strongest binding ability to Oct4 mRNA. Conclusion · Identification of Oct4 RNA binding proteins will provide a theoretical basis for the regulation mechanism of Oct4, and will be a basis for the further study of its function.
Objective · To identify the RNA binding proteins of Oct4, a key factor of embryonic stem cells. Methods · Total RNA was extracted from mouse embryonic stem cells(R1), which was reverse-transcribed into cDNA. Then PCR product of Oct4 mRNA were transcribed into Oct4 mRNA.Finally the candidate RNA-binding proteins were eluted applying the streptomycin affinity chromatography, and submitted for high-performance liquid chromatography combined with the mass spectrometry. The identified RNA binding proteins were further confirmed by RNA immunoprecipitation(RIP). Results · 121 RNA binding proteins of Oct4 3' untranslated region(UTR) and Oct4 5' UTR were identified with liquid chromatography/mass spectrometry. There were 11 proteins with the number of peptide spectrum matches more or equal to 2, and 7 of them were selected for additional confirmation using RIP method. RIP results showed that Oct4 interacted with DSP, SOD1, LMNA, NPM1, PSIP1, NCL and HDGF. Among them, HDGF had the strongest binding ability to Oct4 mRNA. Conclusion · Identification of Oct4 RNA binding proteins will provide a theoretical basis for the regulation mechanism of Oct4, and will be a basis for the further study of its function.
引文
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[26]Ma N,Matsunaga S,Takata H,et al.Nucleolin functions in nucleolus formation and chromosome congression[J].J Cell Sci,2007,120(Pt 12):2091-2105.
[27]Yang A,Shi G,Zhou C,et al.Nucleolin maintains embryonic stem cell selfrenewal by suppression of p53 protein-dependent pathway[J].J Biol Chem,2011,286(50):43370-43382.
[28]Johansson H,Svensson F,Runnberg R,et al.Phosphorylated nucleolin interacts with translationally controlled tumor protein during mitosis and with Oct4during interphase in ES cells[J].PLoS One,2010,5(10):e13678.
[2]Whitmill A,Liu Y,Timani KA,et al.Tip110 deletion impaired embryonic and stem cell development involving downregulation of stem cell factors Nanog,Oct4,and Sox2[J].Stem Cells,2017,35(7):1674-1686.
[3]Wang H,Wang X,Xu X,et al.Germ cell nuclear factor(GCNF)represses Oct4expression and globally modulates gene expression in human embryonic stem(hES)cells[J].J Biol Chem,2016,291(16):8644-8652.
[4]Xiao L,Song Y,Huang W,et al.Expression of SOX2,NANOG and OCT4in a mouse model of lipopolysaccharide-induced acute uterine injury and intrauterine adhesions[J].Reprod Biol Endocrinol,2017,15(1):14.
[5]Lunde BM,Moore C,Varani G.RNA-binding proteins:modular design for efficient function[J].Nat Rev Mol Cell Biol,2007,8(6):479-490.
[6]Hentze MW,Castello A,Schwarzl T,et al.A brave new world of RNA-binding proteins[J].Nat Rev Mol Cell Biol,2018,19(5):327-341.
[7]Brinegar AE,Cooper TA.Roles for RNA-binding proteins in development and disease[J].Brain Res,2016,1647:1-8.
[8]Windbichler N,Schroeder R.Isolation of specific RNA-binding proteins using the streptomycin-binding RNA aptamer[J].Nat Protoc,2006,1(2):637-640.
[9]Milne TA,Zhao K,Hess JL.Chromatin immunoprecipitation(ChIP)for analysis of histone modifications and chromatin-associated proteins[J].Methods Mol Biol,2009,538:409-423.
[10]Niwa H,Miyazaki J,Smith AG.Quantitative expression of Oct-3/4 defines differentiation,dedifferentiation or self-renewal of ES cells[J].Nat Genet,2000,24(4):372-376.
[11]Frum T,Halbisen MA,Wang C,et al.Oct4 cell-autonomously promotes primitive endoderm development in the mouse blastocyst[J].Dev Cell,2013,25(6):610-622.
[12]Tanimura N,Saito M,Ebisuya M,et al.Stemness-related factor Sall4 interacts with transcription factors Oct-3/4 and Sox2 and occupies Oct-Sox elements in mouse embryonic stem cells[J].J Biol Chem,2013,288(7):5027-5038.
[13]Shu J,Wu C,Wu Y,et al.Induction of pluripotency in mouse somatic cells with lineage specifiers[J].Cell,2015,161(5):1229.
[14]Bao C,Wang J,Ma W,et al.HDGF:a novel jack-of-all-trades in cancer[J].Future Oncol,2014,10(16):2675-2685.
[15]Xiao Q,Qu K,Wang C,et al.HDGF-related protein-3 is required for anchorage-independent survival and chemoresistance in hepatocellular carcinomas[J].Gut,2013,62(3):440-451.
[16]Nakamura H,Enomoto H,Kishima Y,et al.Hepatoma-derived growth factor(HDGF)and HDGF family[J].Nihon Rinsho,2001,59(Suppl 6):834-838.
[17]Luhrig S,Siamishi I,Tesmer-Wolf M,et al.LRRC34,a novel nucleolar protein,interacts with NPM1 and NCL and has an impact on pluripotent stem cells[J].Stem Cells Dev,2014,23(23):2862-2874.
[18]Johansson H,Simonsson S.Core transcription factors,Oct4,Sox2 and Nanog,individually form complexes with nucleophosmin(Npm1)to control embryonic stem(ES)cell fate determination[J].Aging,2010,2(11):815-822.
[19]Wang BB,Lu R,Wang WC,et al.Inducible and reversible suppression of Npm1 gene expression using stably integrated small interfering RNA vector in mouse embryonic stem cells[J].Biochem Biophys Res Commun,2006,347(4):1129-1137.
[20]Johansson H,Vizlin-Hodzic D,Simonsson T,et al.Translationally controlled tumor protein interacts with nucleophosmin during mitosis in ES cells[J].Cell cycle,2010,9(11):2160-2169.
[21]Qing Y,Yingmao G,Lujun B,et al.Role of Npm1 in proliferation,apoptosis and differentiation of neural stem cells[J].J Neurol Sci,2008,266(1/2):131-137.
[22]Ugrinova I,Monier K,Ivaldi C,et al.Inactivation of nucleolin leads to nucleolar disruption,cell cycle arrest and defects in centrosome duplication[J].BMC Mol Biol,2007,8:66.
[23]Olson MO,Kirstein MN,Wallace MO.Limited proteolysis as a probe of the conformation and nucleic acid binding regions of nucleolin[J].Biochemistry,1990,29(24):5682-5686.
[24]Ginisty H,Amalric F,Bouvet P.Nucleolin functions in the first step of ribosomal RNA processing[J].EMBO J,1998,17(5):1476-1486.
[25]Bugler B,Caizergues-Ferrer M,Bouche G,et al.Detection and localization of a class of proteins immunologically related to a 100-kDa nucleolar protein[J].Eur J Biochem,1982,128(2/3):475-480.
[26]Ma N,Matsunaga S,Takata H,et al.Nucleolin functions in nucleolus formation and chromosome congression[J].J Cell Sci,2007,120(Pt 12):2091-2105.
[27]Yang A,Shi G,Zhou C,et al.Nucleolin maintains embryonic stem cell selfrenewal by suppression of p53 protein-dependent pathway[J].J Biol Chem,2011,286(50):43370-43382.
[28]Johansson H,Svensson F,Runnberg R,et al.Phosphorylated nucleolin interacts with translationally controlled tumor protein during mitosis and with Oct4during interphase in ES cells[J].PLoS One,2010,5(10):e13678.