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RNA-protein UV-crosslinking Assay
紫外交联实验检测RNA-蛋白的相互作用   

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Abstract

RNA-protein interactions play a crucial role in every aspect of RNA metabolism, and also plays a major role in post-transcriptional gene regulation. RNA-binding proteins have been implicated in viral gene expression (Ray and Das, 2002) and microRNA-mediated gene regulation (Poria et al., 2016). Here we have described the protocol which (1) covalently links transiently interacting RNA-protein complexes by UV crosslinking, (2) removes the unprotected RNA by RNase digestion and (3) detects the RNA-protein complexes by SDS-PAGE analysis. This protocol provides a rapid and reliable means to directly assay RNA-protein interactions and their kinetics using purified proteins and also help in identifying novel RNA-protein interactions

Keywords: RNA-protein interaction(RNA-蛋白质相互作用), UV-crosslinking(紫外交联), RNA-binding proteins(RNA结合蛋白)

Background

RNA-protein interactions are mediated by transient non-covalent interactions such as electrostatic interactions and hydrogen bonds between specific residues in RNA and protein molecules. Short wave UV radiation can induce covalent bond formation between two closely placed aromatic rings. Aromatic ring structures are found in several amino acids in proteins and in nitrogenous bases in nucleic acids. Therefore, UV irradiation is used to covalently link RNA and interacting proteins, whereby the RNA-protein complex can be further analysed by SDS-Polyacrylamide gel electrophoresis. This protocol describes a simple and rapid assay system that can assay RNA-protein interactions and their binding kinetics in vitro. Also, mass spectrometric anaylsis of the fluorescently-labeled RNA-protein complexes obtained by this method can lead to identification of novel RNA-protein interactions.

Materials and Reagents

  1. 1.5 ml RNase, DNase free microcentrifuge tube (Corning, Axygen®, catalog number: MCT-150-C or equivalent)
  2. 96-well round-bottomed plate (Greiner Bio one International, catalog number: 650101 )
  3. Agarose (Lonza, catalog number: 50004 )
  4. Transcription Kit (MAXIscript® Kit) (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM1312 or any equivalent)
  5. 10 µCi/µl α-P32 UTP (BRIT, catalog number: PLC 108 or PerkinElmer, catalog number: BLU007H250UC ) (Alternatively, Cy5-UTP can be used to generate fluorescently labelled RNA [GE Healthcare, catalog number: PA55026 ])
  6. DNase I (optional) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: EN0521 )
  7. 100% ethanol (EMD Millipore, catalog number: 100983 )
  8. Nuclease free water
  9. Ammonium acetate (Thermo Fisher Scientific, Affymetrix, catalog number: 75901 )
  10. Glycerol (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15514 )
  11. Urea-polyacrylamide gel
  12. Yeast tRNA (Sigma-Aldrich, catalog number: R8759 )
  13. RNase inhibitor (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: EO0381 )
  14. RNase A (Sigma-Aldrich, catalog number: R6513 )
  15. 10% SOD-PAGE
  16. Pre-stained protein markers or radiolabeled protein markers
  17. HEPES (pH 7.4) (Thermo Fisher Scientific, Affymetrix, catalog number: 16926 )
  18. Potassium chloride (KCl) (AMRESCO, catalog number: 0395 )
  19. Magnesium chloride hexahydrate (MgCl2·6H2O) (AMRESCO, catalog number: 0288 )
  20. Dithiothreitol (DTT) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0861 )
  21. EDTA (AMRESCO, catalog number: 0105 )
  22. ATP (Sigma-Aldrich, catalog number: A8937 )
  23. SDS
  24. Tris-Cl (pH 6.8)
  25. Bromophenol blue
  26. 2x RNA binding buffer (see Recipes)
  27. 2x denaturing protein loading buffer (see Recipes)

Equipment

  1. Refrigerated centrifuge (Eppendorf, model: 5418 R )
  2. UV cross-linker or UV torch with 254 nm wavelength UV (UVP, model: CL1000 )
  3. Vertical gel electrophoretic system (Bio-Rad Laboratories, model: Mini-PROTEAN Tetra Cell , catalog number: 1658000EDU)
  4. Scintillation counter (Hidex, model: Triathler or any equivalent model)
  5. Phosphorimager (GE Healthcare, model: Typhoon Trio or any equivalent model)

Procedure

  1. Template preparation for in vitro transcription
    1. Clone the DNA template encoding the RNA of interest derived by a T7 promoter in a plasmid vector and linearize the plasmid at a site downstream of the DNA template using specific restriction enzyme.
    2. Run the linearized plasmid DNA on a 0.8% agarose gel, excise the band and extract the linearized DNA for in vitro transcription.
    3. Alternatively use a T7 promoter-adapter containing forward primer and gene specific reverse primer to amplify target DNA incorporating T7 promoter for in vitro transcription (Figure 1).


      Figure 1. Preparation of template DNA for transcription using T7 promoter

    4. For oligo driven transcription of short RNAs, commercially synthesized template DNA oligo with T7 promoter site is annealed with a T7 promoter-adapter oligo to generate the template for in vitro transcription (Figure 2).


      Figure 2. Preparation of DNA template for oligo-driven transcription

  2. P32UTP body-labelled RNA preparation (proper biosafety protocol should be followed)
    1. Setup the following in vitro transcription reaction using α-P32UTP as radiolabel
      10x transcription buffer
      2 μl
      10 mM ATP
      1 μl
      10 mM CTP
      1 μl
      10 mM GTP
      1 μl
      100 μM UTP
      1 μl
      α-P32 UTP
      2 μl
      Linearized DNA template
      10 μl (~1 µg)
      T7 RNA polymerase
      2 μl
      Incubate for 1 h 30 min at 37 °C.
      Add 1 μl DNase I (optional) to remove the DNA template. Incubate at 37 °C for 15 min.
    2. Remove unincorporated nucleotides either by ethanol precipitation or column purification. For ethanol precipitation, add 29 μl nuclease free water to the transcription reaction to bring the volume to 50 μl. Add 5 μl 5 M ammonium acetate, 2 μg glycogen and 2 volumes of 100% ethanol, and precipitate at -80 °C for at least 2 h. Spin for 20 min at maximum speed in a 4 °C centrifuge and wash the pellet once with cold 70% ethanol before drying.
    3. Dissolve the dried RNA pellet in 20 μl nuclease free water. Take 1 μl of labelled RNA and count the specific activity with a scintillation counter or run on an 8 M urea-polyacrylamide gel at the percentage appropriate for the size of the RNA.

  3. RNA-protein binding and UV crosslinking
    1. Setup the following binding reaction for each sample in 1.5 ml microcentrifuge tubes on ice
      2x RNA binding buffer (with 3 mM ATP)
      6 μl
      10 mg/ml yeast tRNA
      1 μl
      Radiolabeled RNA
      x μl (~100,000 cpm)
      RNase inhibitor (40 U/μl)
      0.5 μl
      Purified protein/cell lysate
      x μl (minimum 50 ng)
      Nuclease free water
      to make up the volume to 12 µl
    2. Incubate on ice for 30 min.
    3. Carefully transfer to a pre cooled 96-well round-bottomed plate placed on ice.
    4. Place the plate containing the reaction mixture under the UV light source of UV crosslinker or UV torch and apply 500 mJ/cm2 radiation for 10 min on ice.
    5. Transfer the reaction to 1.5 ml tubes.
    6. Add 2 µl RNase A (10 μg/μl) and incubate the tubes for 30 min at 37 °C.

  4. Separation of RNA-protein complex and visualization (Figure 3)
    1. Add 13 μl of 2x denaturing protein loading buffer.
    2. Boil the samples for 5 min at 100 °C and resolve in 10% SOD-PAGE with pre-stained protein markers or radiolabeled protein markers.
    3. Dry the gel and expose overnight to a phosphorimager screen.
    4. Scan the screen in a phosphorimager.
    5. Alternatively expose the dried gel to X-ray film for 24 h and develop the film.


      Figure 3. UV-crosslinking of 32P labelled PDCD4-3’UTR RNA with 500 ng of purified 6x His-tagged HuR protein (lane 1) and in presence of 10x (lane 2) and 100x unlabelled PDCD4 3’UTR RNA. RNA-protein complexes were digested with RNase A and resolved in 10% SDS-PAGE and exposed for phosphorimaging. The HuR-32P PDCD4-3’UTR RNA complexes are indicated by the arrow.

Recipes

  1. 2x RNA binding buffer
    10 mM HEPES (pH 7.4)
    50 mM KCl
    4 mM MgCl2
    4 mM dithiothreitol (DTT)
    0.2 mM EDTA
    7.6% glycerol
    3 mM ATP
  2. 2x denaturing protein loading buffer
    4 ml 10% (w/v) SDS (final concentration 4%)
    2 ml glycerol (final concentration 20%)
    1.2 ml 1 M Tris-Cl (pH 6.8) (120 mM)
    2.8 ml DEPC-H2O (to make volume upto 10 ml)
    Add bromophenol blue to a final concentration of 0.02% (w/v)

Acknowledgments

We thank members of our laboratory for trying out and standardizing this protocol. Research which led to the development of these protocols was funded by a Wellcome Trust-DBT India Alliance Intermediate fellowship (WT500139/Z/09/Z) to PSR and a CSIR, India Senior Research Fellowship to DKP. This protocol was adapted from the protocol described in Ray and Das (2002).

References

  1. Poria, D. K., Guha, A., Nandi, I. and Ray, P. S. (2016). RNA-binding protein HuR sequesters microRNA-21 to prevent translation repression of proinflammatory tumor suppressor gene programmed cell death 4. Oncogene 35(13): 1703-1715.
  2. Ray, P. S. and Das, S. (2002). La autoantigen is required for the internal ribosome entry site- mediated translation of Coxsackievirus B3 RNA. Nucleic Acids Res 30(20): 4500-4508.

简介

RNA-蛋白质的相互作用在RNA代谢的各个方面发挥着至关重要的作用,并且在转录后基因调控中起重要作用。 RNA结合蛋白涉及病毒基因表达(Ray和Das,2002)和微小RNA介导的基因调控(Poria等人,2016)。这里我们描述了(1)通过紫外线交联共价连接瞬时相互作用的RNA蛋白复合物的方案,(2)通过RNA酶消化去除未保护的RNA,(3)通过SDS-PAGE分析检测RNA-蛋白复合物。该方案提供了一种快速可靠的手段来直接测定RNA-蛋白质相互作用及其使用纯化蛋白质的动力学,也有助于鉴定新的RNA-蛋白质相互作用

背景 RNA-蛋白质相互作用由瞬时非共价相互作用介导,例如RNA和蛋白质分子中特异残基之间的静电相互作用和氢键。短波UV辐射可以诱导两个紧密放置的芳环之间的共价键形成。在蛋白质和核酸的含氮碱基中的几个氨基酸中发现芳环结构。因此,使用紫外线照射共价连接RNA和相互作用的蛋白质,从而可以通过SDS-聚丙烯酰胺凝胶电泳进一步分析RNA蛋白复合物。该协议描述了一种简单快速的测定系统,可以在体外测定RNA-蛋白质相互作用及其结合动力学。此外,通过该方法获得的荧光标记的RNA-蛋白复合物的质谱分析可以导致新型RNA-蛋白质相互作用的鉴定。

关键字:RNA-蛋白质相互作用, 紫外交联, RNA结合蛋白

材料和试剂

  1. 1.5ml RNA酶,无DNA酶的微量离心管(Corning,Axygen,目录号:MCT-150-C或等同物)
  2. 96孔圆底板(Greiner Bio one International,目录号:650101)
  3. 琼脂糖(Lonza,目录号:50004)
  4. 转录试剂盒(MAXIscript ® Kit)(Thermo Fisher Scientific,Invitrogen TM,目录号:AM1312或任何等同物)
  5. 10μCi/μlα-P 32 UTP(BRIT,目录号:PLC 108或PerkinElmer,目录号:BLU007H250UC)(或者,Cy5-UTP可用于产生荧光标记的RNA [GE Healthcare,目录号:PA55026])
  6. DNase I(可选)(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:EN0521)
  7. 100%乙醇(EMD Millipore,目录号:100983)
  8. 核酸酶免费水
  9. 乙酸铵(Thermo Fisher Scientific,Affymetrix,目录号:75901)
  10. 甘油(Thermo Fisher Scientific,Invitrogen TM,目录号:15514)
  11. 尿素 - 聚丙烯酰胺凝胶
  12. 酵母tRNA(Sigma-Aldrich,目录号:R8759)
  13. 核糖核酸酶抑制剂(Thermo Fisher Scientific,Thermo Scientific TM,目录号:EO0381)
  14. RNase A(Sigma-Aldrich,目录号:R6513)
  15. 10%SOD-PAGE
  16. 预先染色的蛋白质标记物或放射性标记的蛋白质标记物
  17. HEPES(pH 7.4)(Thermo Fisher Scientific,Affymetrix,目录号:16926)
  18. 氯化钾(KCl)(AMRESCO,目录号:0395)
  19. 氯化镁六水合物(MgCl 2·6H 2 O)(AMRESCO,目录号:0288)
  20. 二硫苏糖醇(DTT)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:R0861)
  21. EDTA(AMRESCO,目录号:0105)
  22. ATP(Sigma-Aldrich,目录号:A8937)
  23. SDS
  24. Tris-Cl(pH 6.8)
  25. 溴酚蓝
  26. 2x RNA结合缓冲液(参见食谱)
  27. 2x变性蛋白加载缓冲液(参见食谱)

设备

  1. 冷冻离心机(Eppendorf,型号:5418 R)
  2. 紫外线交联剂或紫外线手电筒,具有254 nm波长的紫外线(UVP,型号:CL1000)
  3. 立式凝胶电泳系统(Bio-Rad Laboratories,型号:Mini-PROTEAN Tetra Cell,目录号:1658000EDU)
  4. 闪烁计数器(Hidex,型号:Triathler或任何等效型号)
  5. 磷光仪(GE Healthcare,型号:Typhoon Trio或任何等效型号)

程序

  1. 模板准备用于体外转录
    1. 克隆编码由质粒载体中T7启动子衍生的目标RNA的DNA模板,并使用特异性限制性内切酶在DNA模板下游的位点进行线性化。
    2. 在0.8%琼脂糖凝胶上运行线性化质粒DNA,切除条带并提取线性化的DNA,以进行体外转录。
    3. 或者使用含有正向引物和基因特异性反向引物的T7启动子 - 衔接子扩增体外转录中结合T7启动子的靶DNA(图1)。


      图1.使用T7启动子进行转录的模板DNA的制备

    4. 对于短RNA的寡核苷酸驱动的转录,将商业合成的具有T7启动子位点的模板DNA寡核苷酸与T7启动子 - 衔接子寡核苷酸退火以产生用于体外转录的模板(图2)。


      图2.寡核苷酸转录的DNA模板的制备

  2. UTP体标记的RNA制剂(应遵循适当的生物安全方案)
    1. 使用α-P 32 UTP作为放射性标签设置以下体外转录反应
      10x转录缓冲液
      2μl
      10 mM ATP
      1μl
      10 mM CTP
      1μl
      10 mM GTP
      1μl
      100μMUTP
      1μl
      α-P32 UTP
      2μl
      线性化DNA模板
      10μl(〜1μg)
      T7 RNA聚合酶
      2μl
      在37°C孵育1小时30分钟。
      加入1μlDNase I(可选)去除DNA模板。在37℃孵育15分钟。
    2. 通过乙醇沉淀或柱纯化除去未掺入的核苷酸。对于乙醇沉淀,向转录反应中加入29μl无核酸酶的水,使体积达到50μl。加入5μl5M乙酸铵,2μg糖原和2体积的100%乙醇,并在-80℃下沉淀至少2小时。在4℃离心机中以最大速度旋转20分钟,然后用冷70%乙醇洗涤沉淀一次,然后干燥。
    3. 将干燥的RNA颗粒溶解在20μl无核酸酶的水中。取1μl标记的RNA,用闪烁计数器计数比活性,或以8 M尿素 - 聚丙烯酰胺凝胶的速度,以适合RNA大小的百分比运行。

  3. RNA蛋白结合和紫外线交联
    1. 在冰上的1.5ml微量离心管中为每个样品设置以下结合反应
      2x RNA结合缓冲液(含3mM ATP)
      6μl
      10毫克/毫升酵母tRNA
      1μl
      放射性标记的RNA
      xμl(〜100,000 cpm)
      RNase抑制剂(40 U /μl)
      0.5μl
      纯化蛋白/细胞裂解物 xμl(最小50 ng)
      核酸酶免费水
      将体积补充至12μl
    2. 在冰上孵育30分钟。
    3. 仔细转移到放置在冰上的预冷96孔圆底板。
    4. 将含有反应混合物的板放在UV交联剂或UV手电筒的UV光源下,并在冰上施加500mJ/cm 2的辐射10分钟。
    5. 将反应物转移至1.5 ml管
    6. 加入2μlRNase A(10μg/μl),37℃孵育30 min
  4. 分离RNA蛋白复合物和可视化(图3)
    1. 加入13μl2x变性蛋白加载缓冲液
    2. 在100℃下将样品煮沸5分钟,并在10%SOD-PAGE中用预先染色的蛋白质标记物或放射性标记的蛋白质标记物进行分析。
    3. 将凝胶干燥并暴露于荧光显微镜屏幕上
    4. 扫描荧光屏中的屏幕。
    5. 或者将干燥的凝胶暴露于X射线胶片中24小时并显影。


      图3.具有500ng纯化的6x His标记的HuR蛋白(泳道1)和10倍(泳道2)存在的标记的PDCD4-3'UTR RNA的紫外 - 交联和100x未标记的PDCD4 3'UTR RNA。将RNA蛋白复合物用RNase A消化,并在10%SDS-PAGE中分解并暴露用于磷酸成像。 HuR- 32 P PDCD4-3'UTR RNA复合物用箭头表示。

食谱

  1. 2x RNA结合缓冲液
    10 mM HEPES(pH 7.4)
    50 mM KCl
    4mM MgCl 2
    4mM二硫苏糖醇(DTT)
    0.2 mM EDTA
    7.6%甘油
    3 mM ATP
  2. 2x变性蛋白加载缓冲液
    4 ml 10%(w/v)SDS(终浓度4%)
    2毫升甘油(终浓度20%)
    1.2ml 1M Tris-Cl(pH6.8)(120mM)
    2.8ml DEPC-H 2 O(使体积高达10毫升)
    加入溴酚蓝至终浓度为0.02%(w/v)

致谢

我们感谢我们实验室的成员尝试和规范这个协议。导致这些协议发展的研究由惠康信托 - 印度联盟中级联盟(WT500139/Z/09/Z)向PSR和CSIR,印度DKP高级研究奖学金资助。该协议根据Ray和Das(2002)中描述的协议进行了修改。

参考文献

  1. Poria,DK,Guha,A.,Nandi,I. and Ray,PS(2016)。  RNA结合蛋白HuR螯合微小RNA-21以防止炎症性肿瘤抑制基因程序性细胞死亡的翻译抑制4. 致癌基因 35(13):1703 -1715。
  2. Ray,PS and Das,S。(2002)。  La自身抗原是内部核糖体进入位点介导的柯萨奇病毒B3 RNA的翻译所必需的。核酸Res 30/20(20):4500-4508。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Poria, D. K. and Ray, P. S. (2017). RNA-protein UV-crosslinking Assay. Bio-protocol 7(6): e2193. DOI: 10.21769/BioProtoc.2193.
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