RIP-Seq技术寻找与DNA-PKcs蛋白结合的RNA
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摘要
目的利用RNA免疫沉淀技术和深度测序方法寻找人骨肉瘤细胞U2OS中与DNA依赖蛋白激酶催化亚单位(DNA-PKcs)蛋白结合的RNA,进一步研究特定RNA的功能。方法提取对照组及8 Gy剂量照射组的U2OS细胞核蛋白,选择DNA-PKcs蛋白进行免疫沉淀,将复合体中RNA分离纯化并进行鉴定,行高通量测序分析。通过生物信息学分析得到与DNA-PKcs蛋白结合的所有RNA种类和染色体分布,KEGG和GO分析得到RNA的功能分类。最后选取特定RNA分子,利用qRT-PCR方法分别对U2OS细胞总RNA及RIP提取的RNA进行表达验证。结果U2OS细胞经8 Gy照射后DNA-PKcs蛋白结合的RNA与对照组相比,与细胞黏附相关的基因:整合素α3(integrinα3,ITGA3)、α5(ITGA5)和αV(ITGAV),以及白细胞分化抗原分化簇44(cluster of differentiation44,CD44)和多配体聚糖4(syndecan 4,SDC4)均有更强的富集。结论成功获得了与U2OS细胞DNA-PKcs蛋白结合的RNA高通量测序库;通过测序寻找到了DNA-PKcs蛋白结合的特定RNA分子,为下一步研究其功能奠定了基础。
Objective To search for RNAs binding to DNA dependent protein kinase catalytic subunit( DNA-PKcs) in human osteosarcoma cells U2 OS using the RNA-immunoprecipitation and deep sequencing methods in order to further study the function of specific RNAs. Methods Nuclear protein of U2 OS was extracted from the control group and 8 Gy irradiation group. The DNA-PKcs protein was immunoprecipitated and the RNA in the complex was purified and identified. Then RNAs were processed for deep sequencing. Bioinformatics analysis was performed to discover the RNA classification and chromosomal location. KEGG and GO analysis were used to categorize the RNAs based on their functions. Finally,specific RNA molecules were selected and qRT-PCR was used to verify their expression in U2 OS total RNA and RIP extracted RNA. Results Both KEGG and GO analysis indicated that mRNAs involved in cell adhesion were mostly abundant in the DNA-PKcs RIP. The qRT-PCR results showed that the enrichment of the mRNAs related to cell adhesion,including integrinα3( ITGA3),ITGA5 and ITGAV,and also cluster of differentiation44( CD44) and syndecan 4( SDC4),was increased in the ionizing radiation treated cells. Conclusion The RNAs in the DNA-PK complex are successfully obtained with RIP-Seq technology. More study is needed to elucidate the functions of the DNA-PKcs-RNA complexes.
Objective To search for RNAs binding to DNA dependent protein kinase catalytic subunit( DNA-PKcs) in human osteosarcoma cells U2 OS using the RNA-immunoprecipitation and deep sequencing methods in order to further study the function of specific RNAs. Methods Nuclear protein of U2 OS was extracted from the control group and 8 Gy irradiation group. The DNA-PKcs protein was immunoprecipitated and the RNA in the complex was purified and identified. Then RNAs were processed for deep sequencing. Bioinformatics analysis was performed to discover the RNA classification and chromosomal location. KEGG and GO analysis were used to categorize the RNAs based on their functions. Finally,specific RNA molecules were selected and qRT-PCR was used to verify their expression in U2 OS total RNA and RIP extracted RNA. Results Both KEGG and GO analysis indicated that mRNAs involved in cell adhesion were mostly abundant in the DNA-PKcs RIP. The qRT-PCR results showed that the enrichment of the mRNAs related to cell adhesion,including integrinα3( ITGA3),ITGA5 and ITGAV,and also cluster of differentiation44( CD44) and syndecan 4( SDC4),was increased in the ionizing radiation treated cells. Conclusion The RNAs in the DNA-PK complex are successfully obtained with RIP-Seq technology. More study is needed to elucidate the functions of the DNA-PKcs-RNA complexes.
引文
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[2]Mahaney BL,Meek K,Lees-Miller SP.Repair of ionizing radiation-induced DNA double-strand breaks by non-homologous endjoining[J].Biochem J,2009,417(3):639-650.
[3]Jackson SP,Bartek J.The DNA-damage response in human biology and disease[J].Nature,2009,461(7267):1071-1078.
[4]Postel-Vinay S,Vanhecke E,Olaussen KA,et al.The potential of exploiting DNA-repair defects for optimizing lung cancer treatment[J].Nat Rev Clin Oncol,2012,9(3):144-155.
[5]Ceccaldi R,Rondinelli B,D'Andrea AD.Repair pathway choices and consequences at the double-strand break[J].Trends Biol,2016,26(1):52-64.
[6]Arlander SJ,Greene BT,Innes CL,et al.DNA protein kinasedependent G2checkpoint revealed following knockdown of ataxiatelangiectasia mutated in human mammary epithelial cells[J].Cancer Res,2008,68(1):89-97.
[7]Tu W,Dong C,Konishi T,et al.G2-M Phase-correlative bystander effects are co-mediated by DNA-PKcs and ATM after carbon ion irradiation[J].Mutat Res Genet Toxicol Environ Mutagn,2016,795:1-6.
[8]Stamm S,Benari S,Rafalska I,et al.Function of alternative splicing[J].Gene,2005,344(1):1-20.
[9]Matlin AJ,Clark F,Smith CW.Understanding alternative splicing:towards a cellular code[J].Nat Rev Mol Cell Biol,2005,6(5):386-398.
[10]Li H,Durbin R.Fast and accurate short read alignment with Burrows-Wheeler transform[J].Bioinformatics,2009,25(14):1754-1760.
[11]Ashburner M,Ball CA,Blake JA,et al.Gene ontology:tool for the unification of biology[J].Nat Genet,2000,25(1):25-29.
[12]Kanehisa M,Goto S.KEGG:Kyoto Encyclopedia of Genes and Genomes[J].Nucleic Acids Res,2000,28(1):27-30.
[13]An J,Huang YC,Xu QZ,et al.DNA-PKcs plays a dominant role in the regulation of H2AX phosphorylation in response to DNA damage and cell cycle progregression[J].BMC Mol Biol,2010,11:18.
[14]Hung JH,Weng Z.Identifying regions enriched in a Ch IP-seq data set(peak finding)[J].Cold Spring Harb Protoc,2017,2017(2):pdb.prot093187.
[15]Gatewood ML,Bralley P,Weil MR,et al.RNA-Seq and RNA immunoprecipitation analyses ofthe transcriptome of Streptomyces coelicolor identify substrates for RNaseⅢ[J].J Bacteriol,2012,194(9):2228-2237.
[16]Head SR,Komori HK,La Mere SA,et al.Library construction fornext-generation sequencing:overviews and challenges[J].Biotechniques,2014,56(2):61-64,66,68,passim.
[17]Cho J,Chang H,Kwon SC,et al.LIN28A is a suppressor of ER-associated translation in embryonic stem cells[J].Cell,2012,151(4):765-777.