超分辨显微技术结合smFISH方法在E.coli sRNA SgrS定位中的应用
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摘要
细菌调节小RNA通常与靶mRNA结合,影响翻译和mRNA降解过程.了解细菌小RNA的定量和定位信息,将有助于揭示细菌转录后水平的调控机制.小RNA SgrS通过抑制ptsG mRNA翻译起始,参与细菌磷酸葡萄糖代谢的应激过程.本研究应用单分子荧光原位杂交(smFISH)方法和超分辨显微技术可视化定位大肠杆菌细胞内小RNA SgrS,并初步验证伴侣分子Hfq蛋白和RNaseE降解酶对小RNA SgrS定位的影响.选取大肠杆菌模式菌MG1655 (野生株)、sgrS敲除株(△sgrS)和过表达株(△sgrS-pBAD-SgrS),使用RNA印迹和smFISH方法验证SgrS的过表达.应用smFISH方法分别在野生菌株、hfq敲除株(△hfq)和rne敲除株(△rne-710)中定位小RNA SgrS和ptsG mRNA,超分辨成像观察.与野生株相比,△hfq和△rne-710中SgrS主要定位于近膜区域,ptsG mRNA定位于细菌胞浆中,并且这两种RNA拷贝数均极显著增加.以上结果表明,分别敲除大肠杆菌hfq和rne-710基因导致SgrS和ptsG mRNA的表达量增加. smFISH方法和超分辨技术的结合应用为细菌RNA的直观定量和定位提供了高敏感性的检测方法,可用于基因调控的功能研究.
Bacterial small regulatory RNAs could influence the translation and/or mRNA degradation by binding with target mRNA. It is helpful to reveal the regulatory mechanism of bacterial post-transcriptional level for knowing quantitative and localization information of bacterial small RNA(sRNA). Small RNA SgrS is participated in the stress process of bacterial glucose phosphate metabolism by inhibiting ptsG mRNA translation initiation. In this study, Escherichia coli intracellular sRNA SgrS was located visually by smFISH method and super-resolution microscopy technology, and the effects of chaperone Hfq protein and RNase E degrading enzyme on the localization of sRNA SgrS were verified preliminarily. Over-expression of SgrS in E. coli model strain MG1655(wild strain), sgrS knockout strain( △sgrS) and over-expression strain( △sgrS-pBAD-SgrS) were validated by Northern blot and smFISH methods. sRNA SgrS and ptsG mRNA were located respectively in the wild-type strain, the hfq knockout strain( △hfq) and the rne knockout strain( △rne-710) by smFISH method.Comparing with the wild-type strain, SgrS was mainly located nearby cell membrane, ptsG mRNA was located in bacterial cytoplasm in △hfq and △rne-710 strains by super-resolution imaging, and the copy numbers of both RNA were significantly increased(P<0.01). These results suggest that the expression levels of SgrS and ptsG mRNA were increased significantly in hfq knockout E. coli and rne-710 knockout E. coli. Integrated application of smFISH method and super-resolution technology found a highly sensitive detection method for the intuitive quantification and localization of bacterial RNA, which can be used for the research of gene regulation functional.
Bacterial small regulatory RNAs could influence the translation and/or mRNA degradation by binding with target mRNA. It is helpful to reveal the regulatory mechanism of bacterial post-transcriptional level for knowing quantitative and localization information of bacterial small RNA(sRNA). Small RNA SgrS is participated in the stress process of bacterial glucose phosphate metabolism by inhibiting ptsG mRNA translation initiation. In this study, Escherichia coli intracellular sRNA SgrS was located visually by smFISH method and super-resolution microscopy technology, and the effects of chaperone Hfq protein and RNase E degrading enzyme on the localization of sRNA SgrS were verified preliminarily. Over-expression of SgrS in E. coli model strain MG1655(wild strain), sgrS knockout strain( △sgrS) and over-expression strain( △sgrS-pBAD-SgrS) were validated by Northern blot and smFISH methods. sRNA SgrS and ptsG mRNA were located respectively in the wild-type strain, the hfq knockout strain( △hfq) and the rne knockout strain( △rne-710) by smFISH method.Comparing with the wild-type strain, SgrS was mainly located nearby cell membrane, ptsG mRNA was located in bacterial cytoplasm in △hfq and △rne-710 strains by super-resolution imaging, and the copy numbers of both RNA were significantly increased(P<0.01). These results suggest that the expression levels of SgrS and ptsG mRNA were increased significantly in hfq knockout E. coli and rne-710 knockout E. coli. Integrated application of smFISH method and super-resolution technology found a highly sensitive detection method for the intuitive quantification and localization of bacterial RNA, which can be used for the research of gene regulation functional.
引文
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[4]Singer Z S,Yong J,Tischler J,et al.Dynamic heterogeneity and DNA methylation in embryonic stem cells.Molecular Cell,2014,55(2):319-331
[5]Sinsimer K S,Lee J J,Thiberge S Y,et al.Germ plasm anchoring is a dynamic state that requires persistent trafficking.Cell Reports,2013,5(5):1169-1177
[6]Arraiano C M,Maquat L E.Post-transcriptional control of gene expression:effectors of mRNA decay.Molecular Microbiology,2003,49(1):267-276
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[8]Teppei M,Kimika M,Hiroji A.RNase E-based ribonucleoprotein complexes:mechanical basis of mRNA destabilization mediated by bacterial noncoding RNAs.Genes&Development,2006,19(18):2176-2186
[9]Fei J,Singh D,Zhang Q,et al.RNA biochemistry.Determination of in vivo target search kinetics of regulatory noncoding RNA.Science,2015,347(6228):1371-1374
[10]Hourvitz A,Gershon E,Hennebold J D,et al.Ovulation-selective genes:the generation and characterization of an ovulatoryselective cDNA library.Journal of Endocrinology,2006,188(3):531-548
[11]Li W,Zhang Z,Liu X,et al.The FOXN3-NEAT1-SIN3A repressor complex promotes progression of hormonally responsive breast cancer.Journal of Clinical Investigation,2017,127(9):3421-3440
[12]Lubeck E,Cai L.Single cell systems biology by super-resolution imaging and combinatorial labeling.Nature Methods,2012,9(7):743-748
[13]Skinner S O,Sepulveda L A,Xu H,et al.Measuring mRNA copy number in individual Escherichia coli cells using single-molecule fluorescent in situ hybridization.Nat Protoc,2013,8(6):1100-1113
[14]Vanderpool C K,Gottesman S.Noncoding RNAs at the membrane.Nature Structural&Molecular Biology,2005,12(12):285-286
[15]Tsui H C,Leung H C,Winkler M E.Characterization of broadly pleiotropic phenotypes caused by an hfq insertion mutation in Escherichia coli K-12.Molecular Microbiology,1994,13(1):35-49
[16]Mathew S,Christian L,Federico G D L,et al.Live-cell superresolution microscopy reveals the organization of RNApolymerase in the bacterial nucleoid.Proc Natl Acad Sci USA,2015,112(32):E4390
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[20]Mcclure L V,Lin Y T,Sullivan C S.Detection of viral microRNAs by Northern blot analysis.Methods in Molecular Biology,2011,721:153-171
[21]ChmyrovA,ChmyrovA,Keller J,et al.Nanoscopy with more than100,000'doughnuts'.Nature Methods,2013,10(8):737-740
[22]Chen X,Xi P.Hundred-Thousand light holes push nanoscopy to go parallel.Microscopy Research&Technique,2015,78(1):8-10
[23]Bi-Chang C,Legant W R,Kai W,et al.Lattice light-sheet microscopy:imaging molecules to embryos at high spatiotemporal resolution.Science,2014,346(6208):1257998