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UV Cross-linking Assay and Competition Assay
RNA与RNA结合蛋白互作研究-紫外交联联合探针竞争   

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Abstract

UV cross-linking assay is a standard method used to detect protein-RNA interaction. This method takes advantage of UV irradiation to trigger the formation of the covalently bonded RNP (ribonucleoprotein) complex that is more stable and makes it possible to be isolated in the denaturing conditions. Briefly, 32P-labeled RNA probe and proteins are incubated to form RNP complexes spontaneously; the mixture is then exposed to UV irradiation, followed by treatment with ribonuclease to remove RNA fragments not covalently bound to protein. The oligoribonucleotide-protein complexes are analyzed by SDS-PAGE, and the signals visualized by phosphorimaging. Competitive UV cross-linking assay is a method to determine the protein binding sites and specificity on the RNA substrate. In this assay, the excessive amounts of unlabeled competitor RNA are pre-incubated with the proteins prior to the addition of 32P-labeled RNA probe. If the competitor RNA comprises the protein binding region, the bindings between proteins and RNA probes will be competed and the radioactive binding signals will be reduced.

Keywords: UV cross-linking assay(紫外交联分析), Competition assay(竞争性测定), Protein-RNA interaction(蛋白质与RNA的相互作用)

Materials and Reagents

  1. Purified recombinant protein or cell extract
  2. RNase inhibitor (Promega Corporation, catalog number: N251B )
  3. BSA (Sigma-Aldrich, catalog number: A7906 )
  4. Yeast total RNA (Life Technologies, Ambion®, catalog number: AM9260G )
  5. Tween 20 (AM7118)
  6. RNase A (Sigma-Aldrich, catalog number: R4875 )
  7. RNase T1 (Epicentre Biotechnologies, catalog number: NT09100K )
  8. Materials for SDS-PAGE
  9. Transcription optimized 5x buffer (Promega Corporation, catalog number: P118B )
  10. Recombinant RNasin® Ribonuclease Inhibitor (20 unit) (Promega Corporation, catalog number: N251B )
  11. RNase-Free DNase (1 unit) (Promega Corporation, catalog number: M610A )
  12. [α-32P] UTP (50 μCi at 10mCi/ml) (IZOTOP)
  13. Illustra MicroSpin G-50 Column (GE Healthcare Life Sciences, catalog number: GE-27-5330-02 )
  14. T7 RNA Polymerase (20 unit) (Promega Corporation, catalog number: P207B )
  15. DTT (Promega Corporation, catalog number: P117B )
  16. Bromphenol blue
  17. β-Mercaptoethanol
  18. 32P-labeled RNA probe (see Recipes)
  19. 4x binding buffer (see Recipes)
  20. 5x protein sample buffer (see Recipes)
  21. Competitor RNA transcripts (see Recipes)

Equipment

  1. 37 °C and 100 °C water baths
  2. 254-nm UV light source (Stratagene, UV stratalikerTM, model: 1800 )
  3. Equipments for SDS-PAGE
  4. Gel Dryer (Bio-Rad Laboratories, model: 583 )
  5. Imaging plate (Fujifilm Corporation)
  6. Fujifilm BAS-1500 Image Analyzer (Fujifilm Corporation, Multi Gauge)

Procedure

  1. UV cross-linking assay
    1. Prepare the following mixture on ice:
      2.5 μl 4x binding buffer
      0.5 μl RNase inhibitor (10 units/μl)
      1.0 μl BSA (1 μg/μl)
      1.0 μl Yeast total RNA (1 μg/μl)
      0.5 μg purified protein
      25 fmol 32P-labeled RNA probe
      Adjust the final volume to 10 μl with Nuclease-free water and mix the reaction.
      Note: The amount of protein used in the reaction depends on the RNA binding affinity. Therefore the amount of protein, either purified one or from cell extract, needs to be tested.
    2. Incubate at room temperature (RT) for 10 min.
    3. Place the cap-opened tube containing the reaction mixture on ice, and illuminate under a UV lamp at 254 nm wavelength for 20 min.
      Note: The distance between the sample and UV source is about 10 cm.
    4. Add 1 μl of 10 mg/ml protease-free RNase A and 1 μl of 0.5 unit/μl RNaseT1 to the reaction mixture. Incubate at 37 °C for 30 min.
      Note: A reaction (as shown in procedure 1a) excluding the protein addition needs to be done as the negative control to make sure the unbound RNA probe is complete digested.
    5. Add 3 μl of 5x protein sample buffer to the reaction mixture. Incubate the sample in a boiling water bath for 5 min.
    6. Analyze the sample by electrophoresis with a standard SDS-PAGE gel.
    7. Dry the gel by the using of Bio-Rad model 583 Gel dryer and place an imaging plate over the dried gel. Expose overnight to visualize the radioactive signals of cross-linked proteins.
    8. Scan the radioactive images by the using of Fujifilm BAS-1500 Image Analyzer.

  2. Competition assay
    1. The procedures are the same as UV cross-linking assay described above except that the unlabeled competitor RNA is pre-incubated with the protein at RT for 10 min prior to the addition of 32P-labeled RNA probe.
    2. Prepare the following mixture on ice:
      2.5 μl 4x binding buffer
      0.5 μl RNase inhibitor (10 units/μl)
      1.0 μl BSA (1 μg/μl)
      1.0 μl Yeast total RNA (1 μg/μl)
      0.5 μg purified protein
      50 fmole (2x), 250 fmole (10x), or 1,250 fmole (50x) competitor RNA.
      Adjust the final volume to 10 μl with Nuclease-free water and mix the reaction.
    3. Incubate at RT for 10 min.
    4. Add 1 μl 32P-labeled RNA probe (25 fmole) and Incubate at RT for 10 min.
    5. The following steps are the same as UV cross-linking assay described above (from procedure 1c-1 h).

Recipes

  1. 32P-labeled RNA probe preparation
    In vitro transcription is performed with linearized DNA template and T7 RNA, polymerase in the presence of [α-32P] UTP.
    1. Prepare the following mixture on ice:
      5 μl Transcription optimized 5x buffer
      2.5 μl 100 mM DTT
      5 μl rNTP mix (10 mM each of ATP, CTP, and GTP, and 20 μM of UTP)
      0.5 μl recombinant RNasin® Ribonuclease Inhibitor (20 unit)
      1 μl T7 RNA polymerase (20 unit)
      5 μl [α-32P] UTP (50 μCi at 10 mCi/ml)
      Linearized DNA template (1 μg)
      Adjust the final volume to 25 μl with Nuclease-free water and mix the reaction.
    2. Incubate at 37 °C water bath for 1 h.
    3. Add 1 μl RQ1 RNase-free DNase (1 unit) and Incubate at 37 °C water bath for 15 min.
    4. Load the mixture into illustra MicroSpin G-50 Column and spin 2 min at 735 x g for elution of labeled transcripts and sequestering from free [α-32P] UTP.
  2. 4x binding buffer
    80 mM Tris-Cl (pH 8.0)
    12 mM MgCl2
    40 mM KCl
    8 mM DTT
    16 % (v/v) glycerol
  3. 5x protein sample buffer
    312.5 mM Tris-Cl (pH 6.8)
    50% (v/v) glycerol
    10% (w/v) SDS
    0.1% (w/v) bromphenol blue
    12.5% (v/v) β-Mercaptoethanol
  4. Competitor RNA transcripts
    In vitro transcription is performed with linearized DNA template and T7 RNA polymerase.
    1. Prepare the following mixture on ice:
      10 μl Transcription Optimized 5x buffer
      5 μl 100 mM DTT
      8 μl rNTP mix (4 mM each of ATP, CTP, GTP, and UTP)
      0.5 μl Recombinant RNasin® Ribonuclease Inhibitor (20 unit)
      1 μl T7 RNA Polymerase (20 unit)
      Linearized DNA template (1 μg)
      Adjust the final volume to 50 μl with Nuclease-free water and mix the reaction.
    2. Incubate at 37 °C water bath for 2 h.
    3. Add 1 μl RQ1 RNase-Free DNase (1 unit) and Incubate at 37 °C water bath for 15 min.
    4. Purification of RNA transcripts with Phenol/Chloroform/Isoamyl Alcohol (25:24:1) and then ethanol precipitation of RNA.

Acknowledgments

This protocol was adapted from our previous publication: Huang et al. (2012). This research was supported by the Ministry of Science and Technology, Taiwan (grants NSC-98-2628-B-005-020-MY3 and NSC-98-2811-B-005-030).

References

  1. Huang, Y. W., Hu, C. C., Liou, M. R., Chang, B. Y., Tsai, C. H., Meng, M., Lin, N. S. and Hsu, Y. H. (2012). Hsp90 interacts specifically with viral RNA and differentially regulates replication initiation of Bamboo mosaic virus and associated satellite RNA. PLoS Pathog 8(5): e1002726.

简介

UV交联测定是用于检测蛋白-RNA相互作用的标准方法。该方法利用UV辐射来触发更稳定的共价结合的RNP(核糖核蛋白)复合物的形成,并且使得可以在变性条件下分离。简言之,将32 P标记的RNA探针和蛋白质自发孵育以形成RNP复合物;然后将混合物暴露于UV辐射,随后用核糖核酸酶处理以除去未与蛋白质共价结合的RNA片段。通过SDS-PAGE分析寡核糖核苷酸 - 蛋白复合物,并通过磷光成像显现信号。竞争性UV交联测定是确定蛋白质结合位点和对RNA底物的特异性的方法。在该测定中,将过量的未标记的竞争剂RNA与蛋白质预孵育,然后加入32 P标记的RNA探针。如果竞争剂RNA包含蛋白质结合区,则蛋白质和RNA探针之间的结合将竞争并且放射性结合信号将降低。

关键字:紫外交联分析, 竞争性测定, 蛋白质与RNA的相互作用

材料和试剂

  1. 纯化的重组蛋白或细胞提取物
  2. RNase抑制剂(Promega Corporation,目录号:N251B)
  3. BSA(Sigma-Aldrich,目录号:A7906)
  4. 酵母总RNA(Life Technologies,Ambion ,目录号:AM9260G)
  5. Tween 20(AM7118)
  6. RNA酶A(Sigma-Aldrich,目录号:R4875)
  7. RNase T1(Epicentre Biotechnologies,目录号:NT09100K)
  8. SDS-PAGE材料
  9. 转录优化的5x缓冲液(Promega Corporation,目录号:P118B)
  10. 重组RNasin核糖核酸酶抑制剂(20单位)(Promega Corporation,目录号:N251B)
  11. RNase-Free DNase(1单位)(Promega Corporation,目录号:M610A)
  12. [α- 32 P] UTP(50mCi,10mCi/ml)(IZOTOP)
  13. Illustra MicroSpin G-50柱(GE Healthcare Life Sciences,目录号:GE-27-5330-02)
  14. T7 RNA聚合酶(20单位)(Promega Corporation,目录号:P207B)
  15. DTT(Promega公司,目录号:P117B)
  16. 溴酚蓝
  17. β-巯基乙醇
  18. 32标记的RNA探针(参见Recipes)
  19. 4x绑定缓冲区(参见配方)
  20. 5x蛋白质样品缓冲液(见配方)
  21. 竞争对手RNA转录物(参见配方)

设备

  1. 37℃和100℃水浴
  2. 254-nm UV光源(Stratagene,UV stratoniker TM ,型号:1800)
  3. SDS-PAGE设备
  4. 凝胶干燥器(Bio-Rad Laboratories,型号:583)
  5. 成像板(Fujifilm Corporation)
  6. Fujifilm BAS-1500图像分析仪(Fujifilm Corporation,Multi Gauge)

程序

  1. UV交联测定
    1. 在冰上制备以下混合物:
      2.5μl4x结合缓冲液
      0.5μlRNA酶抑制剂(10单位/μl)
      1.0μlBSA(1μg/μl)
      1.0μl酵母总RNA(1μg/μl)
      0.5μg纯化的蛋白质 25fmol <32> P标记的RNA探针
      用无核酸酶水将最终体积调节至10μl,并混合反应 注意:反应中使用的蛋白质的量取决于RNA结合亲和力。 因此,需要测试蛋白质(纯化的或细胞提取物)的量。
    2. 在室温(RT)下孵育10分钟
    3. 将含有反应混合物的开盖管置于冰上,在UV灯下以254nm波长照射20分钟。
      注意:样品和紫外线源之间的距离约为10厘米。
    4. 向反应混合物中加入1μl的10mg/ml的无蛋白酶的RNA酶A和1μl的0.5单位/μlRNaseT1。在37℃孵育30分钟。
      注意:不包括蛋白质添加的反应(如过程1a所示)需要作为阴性对照进行,以确保未结合的RNA探针完全消化。
    5. 向反应混合物中加入3μl5x蛋白质样品缓冲液。将样品在沸水浴中孵育5分钟。
    6. 用标准SDS-PAGE凝胶电泳分析样品
    7. 通过使用Bio-Rad 583型凝胶干燥器干燥凝胶并将成像板置于干燥的凝胶上。暴露一夜,以显示交联蛋白的放射性信号
    8. 使用Fujifilm BAS-1500图像分析仪扫描放射性图像。

  2. 竞争测定
    1. 该程序与上述的UV交联测定相同,除了在加入32 P标记的RNA探针之前将未标记的竞争剂RNA与蛋白质在RT预温育10分钟。
    2. 在冰上制备以下混合物:
      2.5μl4x结合缓冲液
      0.5μlRNA酶抑制剂(10单位/μl)
      1.0μlBSA(1μg/μl)
      1.0μl酵母总RNA(1μg/μl)
      0.5μg纯化的蛋白质 50fmole(2x),250fmole(10x)或1,250fmole(50x)竞争RNA。
      用无核酸酶水将最终体积调节至10μl,并混合反应
    3. 在RT孵育10分钟。
    4. 加入1μl32 sup-标记的RNA探针(25fmol),并在RT孵育10分钟。
    5. 以下步骤与上述(来自步骤1c-1h)的UV交联测定相同

食谱

  1. 32 P标记的RNA探针制备
    在[α-32 P] UTP存在下,用线性化的DNA模板和T7RNA,聚合酶进行体外转录。
    1. 在冰上制备以下混合物:
      5μl转录优化的5x缓冲液
      2.5μl100mM DTT
      5μlrNTP混合物(各为10mM的ATP,CTP和GTP,和20μM的UTP)
      0.5μl重组RNasin 核糖核酸酶抑制剂(20单位)
      1μlT7 RNA聚合酶(20单位)
      5μl[α-32 P] UTP(50mCi,10mCi/ml)
      线性化DNA模板(1μg)
      用无核酸酶水将最终体积调节至25μl,并混合反应
    2. 在37℃水浴中孵育1小时
    3. 加入1μlRQ1 RNase-free DNase(1个单位),并在37℃水浴孵育15分钟。
    4. 将混合物装入示意图MicroSpin G-50柱中并在735×g离心2分钟以洗脱标记的转录物并从游离的[α-32 P] UTP螯合。 />
  2. 4x结合缓冲液
    80mM Tris-Cl(pH8.0) 12mM MgCl 2/
    40 mM KCl
    8 mM DTT
    16%(v/v)甘油
  3. 5x蛋白质样品缓冲液
    312.5mM Tris-Cl(pH6.8) 50%(v/v)甘油 10%(w/v)SDS
    0.1%(w/v)溴酚蓝
    12.5%(v/v)β-巯基乙醇
  4. 竞争对手RNA转录本
    使用线性化的DNA模板和T7RNA聚合酶进行体外转录。
    1. 在冰上制备以下混合物:
      10μl转录优化的5x缓冲液
      5μl100 mM DTT
      8μlrNTP混合物(ATP,CTP,GTP和UTP各4mM)
      0.5μl重组RNasin 核糖核酸酶抑制剂(20单位)
      1μlT7 RNA聚合酶(20单位)
      线性化DNA模板(1μg)
      用无核酸酶水将最终体积调节至50μl,并混合反应
    2. 在37℃水浴中孵育2小时
    3. 加入1μlRQ1 RNase-Free脱氧核糖核酸酶(1个单位),并在37℃水浴中孵育15分钟。 />
    4. 用苯酚/氯仿/异戊醇(25:24:1)纯化RNA转录物,然后用乙醇沉淀RNA。

致谢

该协议改编自我们先前的出版物:Huang等人(2012)。 这项研究得到了台湾科学技术部(授予NSC-98-2628-B-005-020-MY3和NSC-98-2811-B-005-030)的支持。

参考文献

  1. Huang,Y.W.,Hu,C.C.,Liou,M.R.,Chang,B.Y.,Tsai,C.H.,Meng,M.,Lin,N.S.and Hsu,Y.H。 Hsp90与病毒RNA特异性相互作用,差异调节竹花叶病毒和相关卫星RNA的复制起始。/a> PLoS Pathog 8(5):e1002726。
  • English
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Huang, Y. and Hsu, Y. (2012). UV Cross-linking Assay and Competition Assay. Bio-protocol 2(24): e310. DOI: 10.21769/BioProtoc.310.
  2. Huang, Y. W., Hu, C. C., Liou, M. R., Chang, B. Y., Tsai, C. H., Meng, M., Lin, N. S. and Hsu, Y. H. (2012). Hsp90 interacts specifically with viral RNA and differentially regulates replication initiation of Bamboo mosaic virus and associated satellite RNA. PLoS Pathog 8(5): e1002726.
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Ant Br
Universtity of New Mexico
do you let hot nucleotides made by the RNAse digest run off gel and contaminate the running buffer?
12/20/2013 3:31:53 PM Reply