运用紫外交联免疫沉淀技术构建RNA结合蛋白的靶基因文库
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摘要
紫外交联免疫沉淀(crosslinking-immunoprecipitation,CLIP)是一种研究RNA结合蛋白的技术,它通过特异性抗体富集靶RNA片段,然后逆转录构建cDNA文库,最后进行高通量测序。它可以在基因组水平上全景式研究靶RNA的特点,为揭示该蛋白生物学功能与分子功能提供新的依据。本研究针对一个已知的RNA结合蛋白MOV10(moloney leukemia virus 10),通过CLIP技术构建cDNA文库,然后小规模克隆后测序,得到小部分靶RNA信息,并利用RNA免疫沉淀技术(RNA-immunoprecipitation,RIP)进行验证。测序提示MOV10结合mRNA,而其中有部分是其已知靶标。说明该文库可以用于后续深度测序,为研究该蛋白在精子发生领域内的功能和机制奠定基础。
Crosslinkin g-Immunoprecipitation(CLIP) is a technology with which could research RNA binding proteins. It enriches target RNA fragments by specific antibody, then constructs cDNA library, and finally carries out high-throughput sequencing. It could accomplish a panoramic research on the features of target RNAs on the genome level, which could pave the way for revealing the biological function and molecular function of this protein. Target libraries of MOV10, a putative RNA binding protein, were constructed by the technology of CLIP.Part of information about bounded RNAs with sub-clone sequencing was acquired, and some of them were submitted to RIP(RNA-Immunoprecipitation) to verify the combination with MOV10. Sub-clone sequencing identified that targets belonged to known m RNAs and some of them had already been bound with MOV10. The cNDA libraries could be applied to deep sequencing, which was expect to lay the foundation for researching the function and mechanism of MOV10 in regulating spermatogenesis.
Crosslinkin g-Immunoprecipitation(CLIP) is a technology with which could research RNA binding proteins. It enriches target RNA fragments by specific antibody, then constructs cDNA library, and finally carries out high-throughput sequencing. It could accomplish a panoramic research on the features of target RNAs on the genome level, which could pave the way for revealing the biological function and molecular function of this protein. Target libraries of MOV10, a putative RNA binding protein, were constructed by the technology of CLIP.Part of information about bounded RNAs with sub-clone sequencing was acquired, and some of them were submitted to RIP(RNA-Immunoprecipitation) to verify the combination with MOV10. Sub-clone sequencing identified that targets belonged to known m RNAs and some of them had already been bound with MOV10. The cNDA libraries could be applied to deep sequencing, which was expect to lay the foundation for researching the function and mechanism of MOV10 in regulating spermatogenesis.
引文
Balakrishnan I.,Yang X.D.,Brown J.,Ramakrishnan A.,Torok-Storb B.,Kabos P.,Hesselberth J.R.,and Pillai M.M.,2014,Genome-wide analysis of mi RNA-m RNA interactions in marrow stromal,Cells,32(3):662-673
Chi S.W.,Zang J.B.,Mele A.,and Darnell R.B.,2009,Argonaute HITS-CLIP decodes micro RNA-m RNA interaction maps,Nature,460(7254):479-486
Darnell R.B.,2010,HITS-CLIP:panoramic views of protein-RNAregulation in living cells,Wiley Interdiscip.Rev.RNA,1(2):266-286
Jankowsky E.,2011,RNA Helicases at work:binding and rearranging,Trends Biochem.Sci.,36(1):19-29
Kenny P.J.,Zhou H.J.,Kim M.,Skariah G.,Khetani R.S.,Drnevich J.,Arcila M.L.,Kosik K.S.,and Ceman S.,2014,MOV10 and FMRP regulate AGO2 association with micro RNA recognition elements,Cell Report,9(5):1729-1741
Liu C.,Zhang X.,Huang F.,Yang B.,Li J.,Liu B.F.,Luo H.H.,Zhang P.,and Zhang H.,2012,APOBEC3G inhibits micro RNA-mediated repression of translation by interfering with the interaction between argonaute-2 and MOV10,The Journal of Biological Chemistry,287(35):29373-29383
Paronetto M.P.,and Sette C.,2009,Role of RNA-binding proteins in mammalian spermatogenesis,Andrology,33(1):2-12
Preitner N.,Quan J.,Nowakowsk D.W.,Hancock M.L.,Shi J.H.,Tcherkezian J.,Young-Pearse T.L.,and Flanagan J.G.,2014,APC is an RNA-binding protein,and its interactome provides a link to neural development and microtubule assembly,Cells,158(2):368-382
Spengler R.M.,Zhang X.M.,Cheng C.S.,Mc Lendon J.M.,Skeie J.M.,Johnson F.L.,Davidson B.L.,and Boudreau R.L.,2016,Elucidation of transcriptome-wide micro RNA binding sites in human cardiac tissues by Ago2 HITS-CLIP,Nucleic.Acids Res.,44(15):7120-7231
Tanaka H.,and Baba T.,2005,Gene expression in spermiogenesis,Cellular and Molecular Life Sciences,62(3):344-354
Wang T.,Xiao G.H.,Chu Y.J.,Zhang M.Q.,Corey D.R.,and Xie Y.,2015,Design and bioinformatics analysis of genome-wide CLIP experiments,Nucleic Acids Res.,43(11):5263-5274
Wilinski D.,Qiu C.,Lapointe C.P.,Nevil M.,Campbell Z.T.,Tanaka Hall T.M.,and Wickens M.,2015,RNA regulatory networks diversified through curvature of the PUF protein scaffold,Nature Communications,6:8213-8224
Zhang J.D.,Eto K.,Honmyou A.,Nakao K.,Kiyonari H.,and AbéS.,2011,Neuregulins are essential for spermatogonial proliferation and meiotic initiation in neonatal mouse testis,Development,138(15):3159-3168
Chi S.W.,Zang J.B.,Mele A.,and Darnell R.B.,2009,Argonaute HITS-CLIP decodes micro RNA-m RNA interaction maps,Nature,460(7254):479-486
Darnell R.B.,2010,HITS-CLIP:panoramic views of protein-RNAregulation in living cells,Wiley Interdiscip.Rev.RNA,1(2):266-286
Jankowsky E.,2011,RNA Helicases at work:binding and rearranging,Trends Biochem.Sci.,36(1):19-29
Kenny P.J.,Zhou H.J.,Kim M.,Skariah G.,Khetani R.S.,Drnevich J.,Arcila M.L.,Kosik K.S.,and Ceman S.,2014,MOV10 and FMRP regulate AGO2 association with micro RNA recognition elements,Cell Report,9(5):1729-1741
Liu C.,Zhang X.,Huang F.,Yang B.,Li J.,Liu B.F.,Luo H.H.,Zhang P.,and Zhang H.,2012,APOBEC3G inhibits micro RNA-mediated repression of translation by interfering with the interaction between argonaute-2 and MOV10,The Journal of Biological Chemistry,287(35):29373-29383
Paronetto M.P.,and Sette C.,2009,Role of RNA-binding proteins in mammalian spermatogenesis,Andrology,33(1):2-12
Preitner N.,Quan J.,Nowakowsk D.W.,Hancock M.L.,Shi J.H.,Tcherkezian J.,Young-Pearse T.L.,and Flanagan J.G.,2014,APC is an RNA-binding protein,and its interactome provides a link to neural development and microtubule assembly,Cells,158(2):368-382
Spengler R.M.,Zhang X.M.,Cheng C.S.,Mc Lendon J.M.,Skeie J.M.,Johnson F.L.,Davidson B.L.,and Boudreau R.L.,2016,Elucidation of transcriptome-wide micro RNA binding sites in human cardiac tissues by Ago2 HITS-CLIP,Nucleic.Acids Res.,44(15):7120-7231
Tanaka H.,and Baba T.,2005,Gene expression in spermiogenesis,Cellular and Molecular Life Sciences,62(3):344-354
Wang T.,Xiao G.H.,Chu Y.J.,Zhang M.Q.,Corey D.R.,and Xie Y.,2015,Design and bioinformatics analysis of genome-wide CLIP experiments,Nucleic Acids Res.,43(11):5263-5274
Wilinski D.,Qiu C.,Lapointe C.P.,Nevil M.,Campbell Z.T.,Tanaka Hall T.M.,and Wickens M.,2015,RNA regulatory networks diversified through curvature of the PUF protein scaffold,Nature Communications,6:8213-8224
Zhang J.D.,Eto K.,Honmyou A.,Nakao K.,Kiyonari H.,and AbéS.,2011,Neuregulins are essential for spermatogonial proliferation and meiotic initiation in neonatal mouse testis,Development,138(15):3159-3168