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Identification of RNA-binding Proteins
RNA结合蛋白质的识别

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Abstract

This protocol describes the extraction of RNA-binding proteins (RBPs) from cell lysates. In order to pull down target RBPs, 5-bromo-UTP (BrUTP)-incorporated RNA probes are used, which are generated by in vitro transcription. The schematic diagram (Flowchart) with procedure is indicated (Figure1 and Figure 2).


Figure 1. Schematic diagram of procedure (A-H). Flow chart of experimental procedure is indicated at A-H.


Figure 2. Linearization of plasmids by restricted enzyme. The plasmid is cut at restriction sites adjacent to its cloning element.

Keywords: RNA-protein interactions(RNA和蛋白质的相互作用), 5-bromo-UTP-incorporated RNA(5-bromo-utp-incorporated RNA), Mass spectrometry(质谱法), Pull-down assay(拉法)

Materials and Reagents

  1. Microcentrifuge tubes (1.5 to 2.0 ml)
  2. Desalting MobiSpin columns (MoBiTec, catalog number: M105035F )
  3. DNA template (e.g., pBluescript vector encoding the target sequences such as non-coding elements IL-6 3’UTR and TNF-α 3’UTR)
  4. Reagents for in vitro transcription kits (TAKARA BIO, catalog number: 6140 )
  5. 5-bromouridine 5’-triphosphate (sodium salt) (Cayman Chemical, catalog number: 18140 )
  6. 50 mM ATP solution
  7. 50 mM GTP solution
  8. 50 mM CTP solution
  9. 50 mM UTP solution
  10. RNaseOUTTM recombinant ribonuclease inhibitor (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10777-019 )
  11. T7 RNA polymerase
  12. Dnase I (RNase free) (New England Biolabs, catalog number: M0303S )
  13. Protease inhibitor cocktail (Sigma-Aldrich, catalog number: MSSAFE )
  14. Protease inhibitor cocktail (NACALAI TESQUE, catalog number: 0 4080 )
  15. TRIzol® Reagent (Thermo Fisher Scientific, AmbionTM, catalog number: 15596-026 )
  16. Anti-BrdU antibody (IIB5) (Abcam, catalog number: ab8152 )
  17. Protein G sepharose 4 fast flow (GE Healthcare, catalog number: 17061801 )
  18. Nuclease-free PBS (NACALAI TESQUE, catalog number: 14249 )
  19. Nuclease-free water (Thermo Fisher Scientific, AmbionTM, catalog number: 4387936 )
  20. Dithiothreitol (DTT) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0861 )
  21. Chloroform (NACALAI TESQUE, catalog number: 08401-65 )
  22. DEPC water (Thermo Fisher Scientific, AmbionTM, catalog number: AM9916 )
  23. 96% ethanol or 70% ethanol (NACALAI TESQUE, catalog number: 09666-85 )
  24. NE-PER nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific, catalog number: 78833 )
  25. 0.25% trypsin-EDTA solution (Thermo Fisher Scientific, catalog number: 25200056 )
  26. 1 M Tris-HCl, pH 7.4 (NACALAI TESQUE, Gibco®, catalog number: 35436-01 )
  27. 5 M NaCl (NACALAI TESQUE, catalog number: 31320-05 )
  28. 0.1 M spermidine (Sigma-Aldrich, catalog number: S2626-1G )
  29. 10% Nonidet(R) P-40 (NACALAI TESQUE, catalog number: 25223-04 )
  30. 0.5 M EDTA (NACALAI TESQUE, catalog number: 06894-14 )
  31. 1 M MgCl2 (NACALAI TESQUE, catalog number: 20942-34 )
  32. 1% Tween 20 (NACALAI TESQUE, catalog number: 35624-15 )
  33. PBS, pH 7.0 (NACALAI TESQUE, catalog number: 14249-24)
  34. 1% SDS (NACALAI TESQUE, catalog number: 30562-04 )
  35. Coomassie brilliant blue (CBB) (Thermo Fisher Scientific, catalog number: 20278 )
  36. 10x transcription buffer (see Recipes)
  37. Bead washing buffer (see Recipes)
  38. RNA-binding buffer (see Recipes)
  39. RNA-protein wash buffer (see Recipes)
  40. Lysis buffer (see Recipes)
  41. Elution buffer (see Recipes)

Equipment

  1. MALDI-QIT-TOF (Shimadzu Europa GmbH, model: AXIMA Resonance )
  2. Microcentrifuge capable of reaching up to 16,000 x g
  3. Vortex mixer
  4. Centrifuge capable of reaching up to 2,000 x g

Procedure

  1. In vitro transcription of 5-bromo-UTP (BrUTP)-incorporated RNA probes
    Note: DNA template such as pBluescript vector encoding the target sequences should be made linear DNA by restriction enzyme digestion and purified by phenol/chloroform extraction and ethanol precipitation (Figure 2).
    1. Preparation of DNA template such as plasmids, PCR-generated or synthetic oligonucleotides.
    2. In vitro transcription reaction:
      20 ng-1 μg DNA template
      2 μl 10x transcription buffer
      2 μl 50 mM ATP solution
      2 μl 50 mM GTP solution
      2 μl 50 mM CTP solution
      1 μl 50 mM UTP solution
      1 μl 50 mM 5-bromo-UTP (BrUTP) solution
      0.5 μl RNase inhibitor
      2 μl T7 RNA polymerase
      X μl RNase free ddH2O (up to total 20 μl)
      Mix by pipetting, and centrifuge.
    3. Incubate for 2 h at 42 °C.
    4. Add 10-20 U DNase to 20 μl total solution to digest any DNA. After mixing, incubate for 1-2 h at 37 °C.
    5. The RNA probes are purified using Trizol following standard protocol.
      Optional: If you want to confirm the size of RNA transcripts, agarose gel electrophoresis of RNA in formaldehyde will be performed following standard protocol.

  2. Conjugation of anti-BrdU antibodies with protein G beads
    1. Wash protein G agarose beads three times with an equal volume of nuclease-free PBS (centrifuge at 2,000 x g for 1 min at 4 °C).
    2. Apply 100 μl of the solution (50% beads made up with PBS) to 1.5 ml new microcentrifuge tubes.
    3. Add 500 μl of bead wash buffer to each tube.
    4. Add 50 μl of anti-BrUTP m Ab to each tube.
    5. Incubate the tubes while rotating for at least 1 h at 4 °C. The sample can be incubated at 4 °C overnight.
    6. Wash antibody-conjugated beads once with 1 ml of bead wash buffer (centrifuge at 2,000 x g for 1 min at 4 °C).
    7. Discard the supernatants carefully.

  3. Binding of BrUTP-labbeled RNA to antibody-conjugated beads
    1. Resuspend the antibody-conjugated beads with 500 μl of bead wash buffer.
    2. Add 50 pmol of prepared BrUTP-labeled RNA (prepared in step A), and RNase inhibitor.
    3. Incubate while rotating for 2 to 3 h at 4 °C.
    4. Centrifuge at 2,000 x g for 1 min at 4 °C.
    5. Discard supernatant and wash BrUTP labeled RNA bound to the antibody-conjugated beads with 500 μl of bead wash buffer (centrifuge at 2,000 x g for 1 min at 4 °C) (To step E).

  4. Pre-clearing the protein extraction
    Optional: Cytoplasmic and nuclear protein extract can be separated using NE-PER Nuclear and cytoplasmic extraction reagents. Protein extracts were prepared from at least 1.0 x 108 cells.
    1. The preparation of the protein extracts
      1. Collect the cultured cells into a 1.5 ml new microcentrifuge tube (if dissociation is necessary, Trypsin-EDTA solution can be used).
      2. Centrifuge the tube at 2,000 x g for 3 min at 4 °C.
      3. Discard the supernatants carefully.
      4. Transfer 1 ml lysis buffer into the tube with cell pellet, and resuspend the cells on ice.
      5. Put the tube on ice for 10 min.
      6. Centrifuge the tube at 16,000 x g for 10 min at 4 °C.
      7. Transfer the supernatant (protein extract) carefully into a 1.5 ml new microcentrifuge tube.
      8. Store 50 μl protein extract as input.
    2. The preparation of protein G beads
      1. Wash 50 μl protein G agarose beads three times with an equal volume of nuclease-free PBS (centrifuge at 2,000 x g for 1 min at 4 °C). Discard supernatant gently.
      2. Apply 100 μl of the solution (50% beads made up with PBS) to 1.5 ml new microcentrifuge tubes (50 μl beads per sample).
      3. Add 500 μl of bead wash buffer to each tube.
      4. Centrifuge the tubes at 2,000 x g for 1 min at 4 °C.
      5. Discard the supernatant carefully.
    3. The mixture of the protein extract and 50% bead solution
      1. Transfer the protein extract into the tube with protein G beads.
      2. Incubate the tube while rotating for 1 h at 4 °C.

  5. Binding of proteins to BrUTP-labeled RNA-conjugated beads
    1. Centrifuge the sample tubes containing the protein extract and protein G agarose beads at 2,000 x g for 2 min at 4 °C (save 10 μl of the protein extract as an input).
    2. Transfer the protein extract (optional: either purified cytoplasmic or nuclear protein extract) into the tubes prepared in step C.
    3. Incubate with rotation for 2 h at 4 °C.

  6. Purification of the binding proteins to RNA-conjugated beads
    1. After incubation, centrifuge the samples at 2,000 x g for 1 min at 4 °C. Discard the supernatant carefully.
    2. The protein-RNA complex on the beads is washed three to four times with 1 ml RNA-binding buffer. Centrifuge at 2,000 x g for 2 min at 4 °C.

  7. Elution of proteins binding to BrUTP-labeled RNA-conjugated beads
    1. Resuspend the beads with 200 μl of nuclease-free PBS and transfer the slurry to a bottom-plugged spin column.
    2. Detach the bottom plug from the spin column, and then put the column into a new centrifuge tube. Centrifuge the column at 1,000 x g for 30 sec at 4 °C.
    3. Attach the bottom plug to the spin column, and put the column into a new centrifuge tube. Add 50 μl of elution buffer into the column.
    4. Incubate for 30 min at 4 °C with gentle shaking.
    5. Detach the bottom plug from the spin column, and put the column into a new centrifuge tube.
    6. Elute the protein-BrUTP-labeled RNA complexes by centrifugation.
    7. For enrichment of purified proteins, repeat elution steps G3-6, and collect the eluates into a new tube.

  8. Detection of RNA-binding proteins by LC/MS/MS
    1. The eluted samples are subjected to SDS-PAGE, followed by CBB staining.
    2. Cut the gel on the several compartments, in which some bands were detected.
    3. The samples cut are eluted, and then analyzed by LC/MS/MS.
    4. Target RNA-binding proteins are confirmed by Western blotting (After step G).

Recipes

  1. 10x transcription buffer
    0.4 M Tris-HCl, pH 7.4
    100 mM MgCl2
    0.5 M NaCl
    0.1 M spermidine
  2. Bead washing buffer
    20 mM Tris-HCl, pH 7.4
    137 mM NaCl
    1% NP-40
    2 mM EDTA
    1.5 mM DTT
  3. RNA-binding buffer
    0.2 M Tris-HCl, pH 7.4
    0.5 M NaCl
    20 mM MgCl2
    1% Tween 20
  4. RNA-protein wash buffer
    20 mM Tris-HCl, pH 7.4
    10 mM NaCl
    1% Tween 20
  5. Lysis buffer
    50 mM Tris-HCl, pH 7.4
    150 mM NaCl
    1% NP-40
    1% protease inhibitor cocktail
    1 mM DTT
  6. Elution buffer
    PBS (pH 7.0)
    1% SDS

Acknowledgments

This work was supported by funds of the Japanese Science and Technology Agency and the Japanese Ministry of Education, Culture, Sports, Science and Technology for integrated promotion of social system reform and research and development, and the Kishimoto foundation.

References

  1. Masuda, K., Ripley, B., Nishimura, R., Mino, T., Takeuchi, O., Shioi, G., Kiyonari, H. and Kishimoto, T. (2013). Arid5a controls IL-6 mRNA stability, which contributes to elevation of IL-6 level in vivo. Proc Natl Acad Sci U S A 110(23): 9409-9414.

简介

该协议描述了从细胞裂解物中提取RNA结合蛋白(RBP)。 为了下拉靶标RBP,使用通过体外转录产生的含有5-溴-UTP(BrUTP)的RNA探针。 示意图(流程图)与过程(图1和图2)。


图1.程序示意图(AH)。实验程序的流程图在AH指示。


图2.质粒的线性化 限制性酶。在与其克隆元件相邻的限制性位点切割质粒。


关键字:RNA和蛋白质的相互作用, 5-bromo-utp-incorporated RNA, 质谱法, 拉法

材料和试剂

  1. 微量离心管(1.5〜2.0 ml)
  2. Desalting MobiSpin色谱柱(MoBiTec,目录号:M105035F)
  3. DNA模板(例如编码靶序列的pBluescript载体,例如非编码元件IL-6 3'UTR和TNF-α3'UTR)。
  4. 体外试剂试剂盒(TAKARA BIO,目录号:6140)
  5. 5-溴尿苷5'-三磷酸(钠盐)(Cayman Chemical,目录号:18140)
  6. 50mM ATP溶液
  7. 50mM GTP溶液
  8. 50 mM CTP溶液
  9. 50mM UTP溶液
  10. RNaseOUT TM重组核糖核酸酶抑制剂(Thermo Fisher Scientific,Invitrogen TM ,目录号:10777-019)
  11. T7 RNA聚合酶
  12. Dnase I(不含RNase)(New England Biolabs,目录号:M0303S)
  13. 蛋白酶抑制剂混合物(Sigma-Aldrich,目录号:MSSAFE)
  14. 蛋白酶抑制剂混合物(NACALAI TESQUE,目录号:04080)
  15. TRIzol 试剂(Thermo Fisher Scientific,Ambion TM ,目录号:15596-026)
  16. 抗BrdU抗体(IIB5)(Abcam,目录号:ab8152)
  17. 蛋白G Sepharose 4快速流(GE Healthcare,目录号:17061801)
  18. 无核酸酶的PBS(NACALAI TESQUE,目录号:14249)
  19. 无核酸酶水(Thermo Fisher Scientific,Ambion TM ,目录号:4387936)
  20. 二硫苏糖醇(DTT)(Thermo Fisher Scientific,Thermo Scientific ,目录号:R0861)
  21. 氯仿(NACALAI TESQUE,目录号:08401-65)
  22. DEPC水(Thermo Fisher Scientific,Ambion TM,目录号:AM9916)
  23. 96%乙醇或70%乙醇(NACALAI TESQUE,目录号:09666-85)
  24. NE-PER核和细胞质提取试剂(Thermo Fisher Scientific,目录号:78833)
  25. 0.25%胰蛋白酶-EDTA溶液(Thermo Fisher Scientific,目录号:25200056)
  26. 1M Tris-HCl,pH 7.4(NACALAI TESQUE,Gibco ,目录号:35436-01)
  27. 5 N NaCl(NACALAI TESQUE,目录号:31320-05)
  28. 0.1M亚精胺(Sigma-Aldrich,目录号:S2626-1G)
  29. 10%Nonidet P-40(NACALAI TESQUE,目录号:25223-04)
  30. 0.5 M EDTA(NACALAI TESQUE,目录号:06894-14)
  31. 1 M MgCl 2(NACALAI TESQUE,目录号:20942-34)
  32. 1%Tween 20(NACALAI TESQUE,目录号:35624-15)
  33. PBS,pH7.0(NACALAI TESQUE,目录号:14249-24)
  34. 1%SDS(NACALAI TESQUE,目录号:30562-04)
  35. 考马斯亮蓝(CBB)(Thermo Fisher Scientific,目录号:20278)
  36. 10x转录缓冲液(参见配方)
  37. 珠洗涤缓冲液(见配方)
  38. RNA结合缓冲液(参见配方)
  39. RNA-蛋白洗涤缓冲液(参见配方)
  40. 裂解缓冲液(见配方)
  41. 洗脱缓冲液(参见配方)

设备

  1. MALDI-QIT-TOF(Shimadzu Europa GmbH,型号:AXIMA Resonance)
  2. 微量离心机能够达到16,000 x g
  3. 涡流搅拌器
  4. 离心机能够达到高达2,000×g

程序

  1. 含有5-溴-UTP(BrUTP)的RNA探针的体外转录 注意:编码靶序列的DNA模板如pBluescript载体应通过限制酶消化制成线性DNA,并通过苯酚/氯仿提取和乙醇沉淀纯化(图2)。
    1. DNA模板如质粒,PCR产生或合成寡核苷酸的制备
    2. 体外转录反应:
      20 ng-1μgDNA模板
      2μl10x转录缓冲液
      2μl50mM ATP溶液
      2μl50mM GTP溶液
      2μl50mM CTP溶液
      1μl50mM UTP溶液
      1μl50mM 5-溴-UTP(BrUTP)溶液
      0.5微升RNA酶抑制剂
      2μlT7 RNA聚合酶
      Xμl无RNA酶的ddH 2 O(最多总共20μl) 用移液器混匀,离心
    3. 在42℃孵育2小时。
    4. 加入10-20 U DNase至20μl总溶液消化任何DNA。 混合后,在37℃下孵育1-2小时
    5. 根据标准方案使用Trizol纯化RNA探针 可选:如果要确认RNA转录物的大小,则按照标准方案进行RNA在甲醛中的琼脂糖凝胶电泳。

  2. 抗-BrdU抗体与蛋白G小珠的结合
    1. 用等体积的无核酸酶的PBS洗涤蛋白G琼脂糖珠三次(在4℃下在2,000×g离心1分钟)。
    2. 应用100微升的溶液(50%珠用PBS制成)到1.5毫升新的微量离心管。
    3. 每管加入500μl珠洗涤缓冲液
    4. 每管加入50μl的抗BrUTP m Ab
    5. 孵育管,同时在4℃下旋转至少1小时。 样品可以在4℃下孵育过夜
    6. 用1ml珠洗涤缓冲液洗涤抗体偶联的珠一次(在4℃下以2,000xg离心1分钟)。
    7. 小心弃去上清液。

  3. BrUTP-labbeled RNA与抗体缀合的珠的结合
    1. 用500μl珠洗涤缓冲液重悬抗体偶联的珠子
    2. 加入50 pmol制备的BrUTP标记的RNA(在步骤A中制备)和RNA酶抑制剂
    3. 孵育,同时在4℃下旋转2至3小时
    4. 在4℃下以2,000xg离心1分钟。
    5. 弃去上清液,并用500μl珠洗涤缓冲液(在2,000xg下在4℃离心1分钟)洗涤与抗体缀合的珠结合的BrUTP标记的RNA(至步骤E)。 >
  4. 预清除蛋白质提取
    可选:细胞质和核蛋白提取物可以使用NE-PER核和细胞质提取试剂分离。 从至少1.0×10 8个细胞制备蛋白质提取物。
    1. 蛋白质提取物的制备
      1. 将培养的细胞收集到1.5ml新的微量离心管中(如果需要分离,可以使用胰蛋白酶-EDTA溶液)。
      2. 在4℃下以2,000×g离心管3分钟。
      3. 小心弃去上清液。
      4. 转移1毫升裂解缓冲液到细胞沉淀管,并重悬在冰上的细胞
      5. 将管在冰上10分钟。
      6. 在4℃下以16,000×g离心管10分钟。
      7. 将上清液(蛋白质提取物)小心地转移到1.5 ml新的微量离心管中
      8. 存储50μl蛋白提取物作为输入
    2. 蛋白G珠的制备
      1. 用等体积的无核酸酶的PBS洗涤50μl蛋白G琼脂糖珠三次(在4℃下在2,000×g离心1分钟)。 轻轻丢弃上清液。
      2. 应用100微升的溶液(50%珠用PBS制成)到1.5毫升新的微量离心管(50微升珠 每个样本)。
      3. 每管加入500μl珠洗涤缓冲液
      4. 在4℃下以2,000×g离心管1分钟。
      5. 小心弃去上清液。
    3. 蛋白质提取物和50%珠溶液的混合物
      1. 用蛋白G珠将蛋白提取物转移到管中
      2. 孵育管,同时在4℃下旋转1小时
  5. 蛋白质与BrUTP标记的RNA缀合的珠的结合
    1. 在4℃下将含有蛋白质提取物和蛋白G琼脂糖珠的样品管在2,000×g离心2分钟(保存10μl蛋白质提取物作为输入)。
    2. 将蛋白质提取物(可选:纯化的细胞质或核蛋白提取物)转移到步骤C中制备的试管中。
    3. 在4℃下旋转孵育2小时。

  6. 将结合蛋白纯化至RNA缀合的珠子
    1. 孵育后,在4℃下以2,000xg离心样品1分钟。 小心弃去上清液。
    2. 珠子上的蛋白质-RNA复合物用1ml RNA结合缓冲液洗涤三至四次。 在4℃下以2,000xg离心2分钟。

  7. 洗脱结合BrUTP-标记的RNA-缀合的珠的蛋白质
    1. 用200μl不含核酸酶的PBS重悬珠子,并将浆液转移到底部封闭的离心柱中。
    2. 从旋转柱上分离底部塞子,然后将柱子放入新的离心管中。 在4℃下,将柱子以1,000×g离心30秒
    3. 将底部塞子连接到旋转柱上,并将柱子放入新的离心管中。 向柱中加入50μl洗脱缓冲液。
    4. 在4℃下温和振荡孵育30分钟
    5. 从旋转柱上分离底部塞子, 并将柱子放入新的离心管中
    6. 通过离心洗脱蛋白质-BrUTP标记的RNA复合物。
    7. 为了富集纯化的蛋白质,重复洗脱步骤G3-6,并将洗脱液收集到新管中
  8. 通过LC/MS/MS检测RNA结合蛋白质
    1. 将洗脱的样品进行SDS-PAGE,然后进行CBB染色
    2. 在几个隔室切割凝胶,其中检测到一些条带
    3. 将切割的样品洗脱,然后通过LC/MS/MS分析。
    4. 通过Western印迹证实靶RNA结合蛋白(步骤G之后)

食谱

  1. 10x转录缓冲液
    0.4M Tris-HCl,pH7.4 100mM MgCl 2/v/v 0.5 M NaCl
    0.1 M亚精胺
  2. 珠洗涤缓冲液
    20mM Tris-HCl,pH7.4 137 mM NaCl 1%NP-40
    2mM EDTA 1.5 mM DTT
  3. RNA结合缓冲液
    0.2M Tris-HCl,pH7.4 0.5 M NaCl
    20mM MgCl 2/
    1%Tween 20
  4. RNA-蛋白洗涤缓冲液
    20mM Tris-HCl,pH7.4 10mM NaCl 1%Tween 20
  5. 裂解缓冲液
    50mM Tris-HCl,pH7.4 150mM NaCl 1%NP-40
    1%蛋白酶抑制剂混合物
    1 mM DTT
  6. 洗脱缓冲液
    PBS(pH 7.0)
    1%SDS

致谢

这项工作由日本科技局和日本教育,文化,体育,科学和技术部的资金支持,用于综合促进社会制度改革和研究与开发,以及基本地基金。

参考文献

  1. Masuda,K.,Ripley,B.,Nishimura,R.,Mino,T.,Takeuchi,O.,Shioi,G.,Kiyonari,H.and Kishimoto,T.(2013)。  Arid5a控制IL-6 mRNA稳定性,其有助于提高 IL-6水平体内。 Proc Natl Acad Sci U S A 110(23):9409-9414。
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引用:Masuda, K. and Kishimoto, T. (2016). Identification of RNA-binding Proteins. Bio-protocol 6(17): e1920. DOI: 10.21769/BioProtoc.1920.
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