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In vitro RNA-protein Binding Assay by UV Crosslinking
采用紫外交联进行体外RNA-蛋白质结合试验   

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Abstract

Because covalent bond can form between RNA and its binding proteins after UV irradiation, UV cross-linking is widely used to identify the specific RNA binding proteins. This protocol is described in details as follows.

Materials and Reagents

  1. Linearized DNA template for transcription of the RNA of interest
  2. MAXIscript in vitro Transcription Kit (Applied Biosciences, catalog number: AM1308M ) (This kit contain ATP, CTP, GTP, α-P32UTP and DNase I)
  3. RNasin (Promega Corporation, catalog number: N2611 )
  4. RNase T1 (Applied Biosciences, catalog number: 10109193001 )
  5. 0.5 M EDTA (Life Technologies, Gibco®, catalog number: 15575-038 )
  6. ddH2O (GeneMate Loyalty, catalog number: UPW-1000 )
  7. HEPES (Sigma-Aldrich, catalog number: H3375 )
  8. MgCl2 (Sigma-Aldrich, catalog number: M-2393 )
  9. Glycerol (Sigma-Aldrich, catalog number: G7893-1L )
  10. DTT (Sigma-Aldrich, catalog number: D9779-5G )
  11. Tris-Base (Thermo Fisher Scientific, catalog number: BP152-1 )
  12. SDS (Sigma-Aldrich, catalog number: L-4390 )
  13. Bromphenol blue (Sigma-Aldrich, catalog number: B5525 )
  14. TEMED
  15. Ammoniampersulfate
  16. 5x binding buffer (see Recipes)
  17. 2x SDS loading buffer (see Recipes)
  18. 10x TGE buffer (see Recipes)
  19. 6% TGE gel (see Recipes)

Equipment

  1. SPIN-PureTM column (G-50) (PireBiotech SCW50-50 DEPC-water)
  2. SDS-PAGE system (Bio-Rad, catalog number: 165-1802 )
  3. UV Stratalinker 1800 (Stratagene, catalog number: 474645 )
  4. Film processor (Konica Minolta, model: SRX-101A )
  5. Centrifuges (Eppendorf, catalog number: 5810R )
  6. Scintillation Counter (Beckman, catalog number: LS6500 )
  7. Bench top Radiation Shield with 6-1/4" base (Cole-Parmer, catalog number: WU-36218-00 )
  8. 37 °C water bath

Procedure

  1. In vitro transcription of 32P-labled RNA probes
    1. DNA templates for synthesis of the RNA probe are generated by PCR using specific primers.
    2. Thaw frozen reagents, place RNA polymerase on ice and vortex the 10x Transcription buffer and keep the buffer at room temperature. All reagents should be centrifuged briefly before opening.
    3. Assemble transcription reaction at room temperature as the following sequence:
      1 μg DNA template
      ddH2O to 20 μl
      2 μl 10x transcription buffer (after addition of water and DNA)
      1 μl 10 mM ATP
      1 μl 10 mM CTP
      1 μl 10 mM GTP
      5 μl [α-32P]UTP
      2 μl T7 enzyme mix
      2 μl RNasin (40 U/μl)
      Mix thoroughly.
    4. Incubate 1 h at 37 °C.
    5. Add 1 μl of 5 mg/ml DNase I to digest the rest template DNA and incubate 10 min in 37 °C water bath.
    6. Add 1 μl of 0.5 M EDTA, mix and add 78 μl ddH2O to adjust final volume to 100 μl and mix.

  2. Purification of 32P-labled RNA probes with SPIN-Pure column (G-50)
    1. Allow a minimum of 30 min of room temperature to warm the columns before the following steps.
    2. Gently invert the column several times to suspend the column buffer (DEPC-Water).
    3. Remove the top cap from the column, and then remove the bottom tip.
    4. Allow the column buffer to drain by gravity before proceeding.
    5. Place this column/tube apparatus into an adaptor tube. Centrifuge at 1,100 x g for 2 min at room temperature.
    6. Repeat centrifugation again to let the column dry completely and discard the collection tube and the eluted buffer.
    7. Put the column in a second collection tube in upright position and apply the RNA sample (20 to 50 μl) to the center of the column gel very slowly and carefully (the rest RNA sample can be stored at -80 °C for one week).
    8. Centrifuge at 1,100 x g for 4 min. The purified 32P-labled RNA probe is collected in the bottom of the collection tube. Discard the spin column and continue with the following procedure.
    9. Pick 1 μl of the RNA sample and count the cpm value.

  3. RNA and protein binding reaction
    1. Prepare the following mixture on ice:
      32P-labled RNA probe (2 x 105 cpm)
      1 μl 12.5 mM ATP
      3μl 5x binding buffer
      3 μl 0.5 M KCl
      Protein (100 ng) (this protein can be a GST fusion protein purified according to the protocol prepared by GE Healthcare Life Sciences)
      H2O to final volume of 15 μl.
    2. Mix gently by pipetting up and down for 10 times, avoiding vortex.
    3. Place the uncovered tube containing the reaction mixture on ice, directly underneath the bulb (about 10 cm from the surface) of a 254-nm UV light source, irradiation with 4 x 105 μJ/cm2 energy.
    4. Incubate at room temperature for 30 min then keep on ice for another 3 h.

  4. Remove the non-incorporated 32P
    1. Add 1 μl of RNase T1 (1 U/μl) to each tube and incubate for additional 10 min at 37 °C to degrade the free RNA.
    2. Add an equal volume (16 μl) of 2x SDS loading dye andseparate the sample in TGE-gel or SDS-PAGE (no boiling need).
    3. Autoradiography for 12 h to 7 days for visualization.


Recipes

  1. 5x binding buffer
    50 mM HEPES (pH 7.2)
    15 mM MgCl2
    25% glycerol
    5 mM DTT
    Stored at -20 °C
  2. 2x SDS loading buffer
    125 mM Tris-Cl (pH 7.6)
    20% (v/v) glycerol
    4% (w/v) SDS
    0.008% (w/v) bromphenol blue
    20 mM DTT
  3. 10x TGE buffer
    Tris 100 mM
    Glycine 1 M
    EDTA 10 mM
    pH 8.3
  4. 6% TGE gel
    6 ml 30% acrylamide
    5 ml 10x TGE buffer
    35 ml H2O
    300 μl ammoniampersulfate
    22 μl TEMED
    Notes:
    1. Avoiding RNase contamination is the key for successful.
    2. As 32P is radioactive, this experiment should be performed under the protection of Benchtop Radiation Shield.

Acknowledgments

This work was supported by the National Institutes of Health (R01 CA123490 and R01CA143107 to MZ) and CURE (MZ and LG).

References

  1. Gu, L., Zhang, H., He, J., Li, J., Huang, M. and Zhou, M. (2012). MDM2 regulates MYCN mRNA stabilization and translation in human neuroblastoma cells. Oncogene 31(11): 1342-1353.
  2. Walker, J., de Melo Neto, O. and Standart, N. (1998). Gel retardation and UV-crosslinking assays to detect specific RNA-protein interactions in the 5' or 3' UTRs of translationally regulated mRNAs. Methods Mol Biol 77: 365-378.

简介

因为紫外线照射后可以在RNA及其结合蛋白之间形成共价键,所以紫外交联被广泛用于鉴定特异性RNA结合蛋白。 该协议详细描述如下。

材料和试剂

  1. 用于感兴趣的RNA的转录的线性化DNA模板
  2. (Applied Biosciences,目录号:AM1308M)(此试剂盒含有ATP,CTP,GTP,α-P32UTP和DNase I)。
  3. RNasin(Promega Corporation,目录号:N2611)
  4. RNase T1(Applied Biosciences,目录号:10109193001)
  5. 0.5 M EDTA(Life Technologies,Gibco ,目录号:15575-038)
  6. ddH 2 O(GeneMate Loyalty,目录号:UPW-1000)
  7. HEPES(Sigma-Aldrich,目录号:H3375)
  8. MgCl 2(Sigma-Aldrich,目录号:M-2393)
  9. 甘油(Sigma-Aldrich,目录号:G7893-1L)
  10. DTT(Sigma-Aldrich,目录号:D9779-5G)
  11. Tris-Base(Thermo Fisher Scientific,目录号:BP152-1)
  12. SDS(Sigma-Aldrich,目录号:L-4390)
  13. 溴酚蓝(Sigma-Aldrich,目录号:B5525)
  14. TEMED
  15. 二氨基硫酸盐
  16. 5x绑定缓冲区(参见配方)
  17. 2x SDS加样缓冲液(见配方)
  18. 10x TGE缓冲区(参见配方)
  19. 6%TGE凝胶(见配方)

设备

  1. SPIN-PureTM柱(G-50)(PireBiotech SCW50-50DEPC-水)
  2. SDS-PAGE系统(Bio-Rad,目录号:165-1802)
  3. UV Stratalinker 1800(Stratagene,目录号:474645)
  4. 电影处理器(Konica Minolta,型号:SRX-101A)
  5. 离心机(Eppendorf,目录号:5810R)
  6. 闪烁计数器(Beckman,目录号:LS6500)
  7. 带6-1/4"底座的台式辐射屏蔽(Cole-Parmer,目录号:WU-36218-00)
  8. 37°C水浴

程序

  1. 32转录的RNA标记物的体外转录
    1. 用于合成RNA探针的DNA模板通过使用特异性引物的PCR产生
    2. 解冻冷冻的试剂,将RNA聚合酶置于冰上,并涡旋10x转录缓冲液,并将缓冲液保持在室温。 所有试剂应在开启前短暂离心
    3. 在室温下按如下顺序组装转录反应:
      1μgDNA模板
      ddH 2 2至20μl
      2μl10×转录缓冲液(加入水和DNA后)
      1μl10 mM ATP
      1μl10 mM CTP
      1μl10 mM GTP
      5μl[α- 32 P] UTP
      2μlT7酶混合物
      2μlRNasin(40U /μl)
      彻底混合。
    4. 在37℃孵育1小时。
    5. 加入1微升5毫克/毫升DNase I消化其余模板DNA,并在37℃水浴中孵育10分钟。
    6. 加入1μl0.5M EDTA,混合并加入78μlddH 2 O以将终体积调整至100μl并混合。

  2. 用SPIN-Pure柱(G-50)纯化32P-标记的RNA探针,
    1. 在进行以下步骤之前,至少等待30分钟的室温对色谱柱进行加温。
    2. 轻轻倒置几次柱子以悬浮柱缓冲液(DEPC-Water)。
    3. 从柱中取出顶盖,然后取下底部端
    4. 在继续之前,允许柱缓冲液通过重力排出。
    5. 将此柱/管装置放入适配器管中。 在室温下以1,100×g离心2分钟。
    6. 再次重复离心使柱子完全干燥,弃去收集管和洗脱缓冲液
    7. 将柱置于直立的第二个收集管中,非常缓慢和仔细地将RNA样品(20至50μl)应用于柱凝胶的中心(其余的RNA样品可以储存在-80° C一个星期)。
    8. 以1,100×g离心4分钟。 将纯化的32 P标记的RNA探针收集在收集管的底部。 丢弃旋转柱并继续以下过程。
    9. 选择1μlRNA样品并计数cpm值。

  3. RNA和蛋白结合反应
    1. 在冰上制备以下混合物:
      32-标记的RNA探针(2×10 5次/cpm)
      1μl12.5 mM ATP
      3μl5×结合缓冲液
      3μl0.5M KCl
      蛋白(100ng)(该蛋白可以是根据GE Healthcare Life Sciences制备的方案纯化的GST融合蛋白)
      H 2 O至最终体积为15μl
    2. 通过上下吹打10次轻轻混匀,避免涡旋
    3. 将含有反应混合物的未覆盖管置于冰上,直接在254-nm UV光源的灯泡下方(距离表面约10cm),用4×10 5μJ/cm 2 > 2 能量。
    4. 在室温下孵育30分钟,然后在冰上再保持3小时

  4. 删除未并入的 32 P
    1. 加入1微升的核糖核酸酶T1(1 U /微升)到每个管,在37℃下孵育另外10分钟,以降解游离RNA。
    2. 加入等体积(16μl)2x SDS上样染料,并将样品在TGE凝胶或SDS-PAGE(无需煮沸)中分离。
    3. 放射自显影12小时至7天可视化。


食谱

  1. 5x结合缓冲液
    50mM HEPES(pH 7.2)
    15mM MgCl 2·h/v 25%甘油 5 mM DTT
    储存于-20°C
  2. 2x SDS上样缓冲液
    125mM Tris-Cl(pH7.6) 20%(v/v)甘油 4%(w/v)SDS
    0.008%(w/v)溴酚蓝
    20 mM DTT
  3. 10x TGE缓冲区
    Tris 100mM
    甘氨酸1 mM / EDTA 10mM
    pH 8.3
  4. 6%TGE凝胶
    6ml 30%丙烯酰胺 5 ml 10x TGE缓冲液
    35ml H 2 O 2 / 300μl氨基硫酸盐
    22μlTEMED
    注意:
    1. 避免RNA酶污染是成功的关键。
    2. 因为 32 P是放射性的,所以该实验应在保护台式辐射屏蔽下进行。

致谢

这项工作由国家卫生研究院(R01 CA123490和R01CA143107到MZ)和CURE(MZ和LG)支持。

参考文献

  1. Gu,L.,Zhang,H.,He,J.,Li,J.,Huang,M.and Zhou,M.(2012)。 MDM2调节MYCN mRNA在人神经母细胞瘤细胞中的稳定和翻译。癌基因 31(11):1342-1353。
  2. Walker,J.,de Melo Neto,O.and Standart,N。(1998)。 凝胶阻滞和紫外线交联试验检测特异性RNA-蛋白相互作用的5'或3' UTRs of translationally regulated mRNAs。 Methods Mol Biol 77:365-378。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Zhang, H. and Zhou, M. (2012). In vitro RNA-protein Binding Assay by UV Crosslinking. Bio-protocol 2(21): e287. DOI: 10.21769/BioProtoc.287.
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Guosheng Qu
UAlbany
1. I notice that ATP is added in the RNA-protein binding mixture before UV-crosslinking. Is it necessary for UV-crosslinking? If yes, why?

2. I did iCLIP by mixing RNA with protein in a different buffer (50 mM Tris-HCl, pH 6.8; 140 mM NaCl; 0.05% Triton-X100) and UV-cross-linking with 400 mJ/cm2. Under this condition, IP works and UV caused RNA damage is seen but no UV-crosslinks between RNA and protein have been detected (I use reverse transcription to detect peptidyl ligands left on the RNA after proteinase K digestion). Do you think what my troubles could be?
1/21/2016 7:08:48 AM Reply