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Affinity Pulldown of Biotinylated RNA for Detection of Protein-RNA Complexes
Pulldown生物素化的RNA以检测蛋白质-RNA复合物   

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本实验方案简略版
Nucleic Acids Research
Mar 2016

Abstract

RNA-binding proteins (RBPs) have recently emerged as crucial players in the regulation of gene expression. The interactions of RBPs with target mRNAs control the levels of gene products by altering different regulatory steps, including pre-mRNA splicing and maturation, nuclear mRNA export, and mRNA stability and translation (Glisovic et al., 2008). There are several methodologies available today to identify RNAs bound to specific RBPs; some detect only recombinant molecules in vitro, others detect recombinant and endogenous molecules, while others detect only endogenous molecules. Examples include systematic evolution of ligands by exponential enrichment (SELEX), biotinylated RNA pulldown assay, RNA immunoprecipitation (RIP) assay, electrophoretic mobility shift assay (EMSA), RNA footprinting analysis, and various UV crosslinking and immunoprecipitation (CLIP) methods such as CLIP, PAR-CLIP, and iCLIP (Popova et al., 2015). Here, we describe a simple and informative method to study and identify the RNA region of interaction between an RBP and its target transcript (Panda et al., 2014 and 2016). Its reproducibility and ease of use make this protocol a fast and useful method to identify interactions between RBPs and specific RNAs.

Keywords: Tagged RNA (标记的RNA), RNA-binding proteins (RNA结合蛋白), Ribonucleoprotein complex (核糖核蛋白复合物), Biotin pulldown (生物素pull-down), in vitro transcription

Background

RNA-protein interactions critically influence gene expression patterns. The identification of these ribonucleoprotein (RNP) complexes is essential for understanding the regulatory mechanisms governed by RNA-binding proteins (RBPs). Recently, extensive efforts have led to the development of methods for systematic analysis of RNA-protein interactions. Highly informative methods to identify RNP complexes include a number of different types of RNP immunoprecipitation (IP) analyses. RIP methods involve RNP IP without crosslinking, while CLIP methods involve crosslinking of the RNP before IP. While RIP is fast, inexpensive, and capable of assessing many endogenous RBPs and RNAs, it does not typically permit the identification of the precise RNA region that interacts with the RBP. CLIP analysis (including its variant forms HITS-CLIP, PAR-CLIP, and iCLIP) does allow the discovery of the precise RNA sequences that interact with an RBP, as it includes an RNase step that digests all unprotected RNA and yields the RNA bound to the RBP. However, CLIP analysis is costly, time-consuming, and technically challenging (Panda et al., 2016). Therefore, alternatives to testing the binding of endogenous proteins to RNAs of interest are needed.

The biotinylated RNA-pulldown method described here theoretically works for all RBPs, as this assay is performed in a cell-free system. The method involves the in vitro synthesis of RNAs of interest in the presence of biotinylated CTP; the RNA tagged in this manner is then incubated with a cell-free system to allow RBPs to recognize RNA regions to which it has affinity, while regions without affinity do not interact with RBPs. After the binding is complete, the biotinylated RNA is pulled down using streptavidin-coated beads and the RBPs are typically detected by Western blot analysis. This method can be used to map the RNA sequence with which the RBP interacts if the user tests progressively smaller RNA fragments in a systematic fashion, as described here. Furthermore, this method allows for the identification of all of the proteins that interact with the RNA of interest if the biotin-RNA pulldown is followed by mass spectroscopy. In summary, this approach can successfully identify the interaction of an endogenous (or recombinant) RBP with in vitro-synthesized RNAs of interest.

Materials and Reagents

  1. ThermoGridTM rigid strip 0.2-ml PCR tubes (Denville Scientific, catalog number: C18064 [1000859])
  2. Posi-Click 1.7-ml microcentrifuge tube (Denville Scientific, catalog number: C2171 )
  3. 1.5-ml tubes
  4. 10-cm dishes
  5. NucAway spin columns (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM10070 )
  6. Disposable cuvettes, 1.5-ml (Stockwell Scientific, catalog number: 2410 )
  7. Nuclease-free water (Thermo Fisher Scientific, AmbionTM, catalog number: AM9930 )
  8. cDNA prepared from total RNA
  9. DreamTaq DNA polymerase (5 U/µl) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: EP0701 )
  10. dNTP mix (10 mM each) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0193 )
  11. Agarose LE (Denville Scientific, catalog number: CA3510-8 )
  12. QIAquick Gel Extraction Kit (50) (QIAGEN, catalog number: 28704 )
  13. MEGAshortscriptTM T7 Kit with manual (for RNA shorter than 0.5 Kb) (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM1354 )
  14. RiboLock RNase inhibitor (40 U/µl) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: EO0381 )
  15. Biotin-14-CTP (Thermo Fisher Scientific, InvitrogenTM, catalog number: 19519-016 )
  16. MEGAscript® T7 Transcription Kit (*for RNA longer than 0.5 Kb) (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM1333 )
  17. Novex® TBE-urea gels, 6% (Thermo Fisher Scientific, InvitrogenTM, catalog number: EC6865BOX )
  18. 1x TBE buffer
  19. Dulbecco’s phosphate-buffered saline (DPBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 14040-133 )
  20. cOmplete protease inhibitor cocktail (Sigma-Aldrich, catalog number: 11697498001 )
  21. 2x Laemmli sample buffer (Bio-Rad Laboratories, catalog number: 1610737 )
  22. Dynabeads® M-280 streptavidin (Thermo Fisher Scientific, InvitrogenTM, catalog number: 11205D )
  23. 2-mercaptoethanol (β-mercaptoethanol/BME)
  24. 10x Tris/glycine/SDS running buffer (Bio-Rad Laboratories, catalog number: 1610732 )
  25. 4-20% Mini-PROTEAN® TGX Stain-FreeTM protein gels (Bio-Rad Laboratories, catalog number: 4568094 )
  26. Ethidium bromide solution (Sigma-Aldrich, catalog number: E1510 )
  27. SpectraTM multicolor broad range protein ladder (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 26634 )
  28. Bio-Rad protein assay dye reagent concentrate (Bradford reagent) (Bio-Rad Laboratories, catalog number: 500-0006 )
  29. Tris-HCl (pH 8.0)
  30. KCl
  31. MgCl2
  32. Nonidet P-40
  33. EDTA
  34. NaCl
  35. Triton X-100
  36. Polysome extraction buffer (PEB) (see Recipes)
  37. 2x Tris, EDTA, NaCl, Triton (TENT) buffer (see Recipes)
  38. 1x TENT (see Recipes)

Equipment

  1. PCR strip tube rotor, mini centrifuge C1201 (Denville Scientific, catalog number: C1201-S [1000806])
  2. Veriti® 96-Well thermal cycler (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4375786 )
  3. Ultraviolet transilluminator
  4. NanoDrop One spectrophotometer (Thermo Fisher Scientific, Thermo Scientific, catalog number: ND-ONE-W )
  5. Eppendorf Thermomixer® R (Eppendorf, catalog number: 022670581 )
  6. Incubator
  7. Vortexer
  8. Cell scrapers
  9. Refrigerated centrifuge (Eppendorf, model: 5430R )
  10. SmartSpec Plus spectrophotometer (Bio-Rad Laboratories, catalog number: 1702525 ) or other spectrophotometer with 595 nm wavelength
  11. MagneSphere(R) stand (Promega, catalog number: Z5342 )
  12. Trans-Blot® TurboTM transfer starter system (Bio-Rad Laboratories, catalog number: 1704155 )
  13. Mini-PROTEAN® Tetra vertical electrophoresis cell (Bio-Rad Laboratories, catalog number: 1658004 )

Procedure

  1. Primer design and template generation for in vitro transcription
    1. The forward and reverse primers with a length of 20-25 nt and melting temperature of ~60 °C are designed using Primer 3 online tool (http://bioinfo.ut.ee/primer3-0.4.0/) (Untergasser et al., 2012) (Note 1).
    2. As shown in Figure 1, add the T7 RNA polymerase promoter sequence (T7) [5’AGTAATACGACTCACTATAGGG] (red lines) upstream of the actual forward primer sequence (Figure 1).
    3. Dissolve the primers in nuclease-free water to a final concentration of 100 µM, then prepare a primer mix of forward and reverse primers at a final concentration of 1 µM each in nuclease-free water (e.g., 10 µl of each primer from the 100 µM stock into 980 µl of nuclease-free water and mix well).
    4. For one PCR reaction of 40 µl in a 0.2-ml PCR tube, add 4 µl of 10x DreamTaq DNA polymerase buffer, 10 µl of 1 µM primer mix, 1 µl DreamTaq DNA polymerase, 1 µl of 10 mM dNTP mix, 0.2 µl (~2 ng) cDNA, and water to make the final volume 40 µl. (The cDNA was prepared from total RNA as described previously [Panda et al., 2016].)
    5. Mix the reaction by vortexing for 5 sec and centrifuge the PCR tube strip for 30 sec using minicentrifuge to settle the reactions at the bottom of the wells.
    6. Set up the Veriti® 96-Well Thermal Cycler program as follows: 5 min at 95 °C and 40 cycles of 5 sec at 95 °C and 60 sec at 60 °C.
    7. Resolve the PCR products on an ethidium bromide stained 2% agarose gel for 1 h at 100 volts and visualize with an ultraviolet transilluminator.
    8. Cut the desired bands from the gel. Purify the PCR products corresponding to each fragment using the QIAquick Gel Extraction Kit and dissolve in elution buffer provided with the kit.
    9. Measure the concentration of the PCR products/templates with a NanoDrop spectrophotometer.
    10. The PCR product (T7 DNA template) can be stored at -20 °C or -80 °C, or used immediately for in vitro transcription.

  2. In vitro transcription of biotinylated RNA fragments
    1. The biotinylated RNA can be prepared using the MEGAshortscriptTM T7 Kit following the manufacturer’s protocol.


      Figure 1. Schematic of biotinylated RNA pulldown assay

    2. In vitro transcription reaction for each DNA template can be prepared in a 1.7-ml PCR tube containing 2 µl of 10x MEGAshortscriptTM T7 buffer, 2 µl of MEGAshortscriptTM T7 enzyme mix, 1 µl of RiboLock, 1 µl of 75 mM rATP, 1 µl of 75 mM rUTP, 1 µl of 75 mM rGTP, 0.8 µl of 75 mM rCTP, 1.5 µl of 10 mM Biotin-14-CTP, 100 ng of T7 DNA template and water to make the final volume 20 µl. (For longer RNA fragments [> 500 nt] the user should employ the MEGAscriptTM T7 Kit for in vitro RNA preparation) (Notes 5 and 6)
    3. Mix and centrifuge for ~5 sec at 500 x g to settle the reaction mixture at the bottom of the tube.
    4. Incubate the reaction at 37 °C for 4 h in a Thermomixer (Note 2).
    5. Add 1 µl of DNase provided in the kit and mix well followed by incubation for 15 min at 37 °C.
    6. Store the samples at -20 °C or -80 °C or proceed immediately to RNA purification.

  3. Purification and quality check of in vitro-transcribed biotinylated RNA fragments
    1. Dilute the entire 20 µl in vitro transcribed RNA by adding 50 µl of nuclease-free water and keep on ice.
    2. Use one column (NucAway Spin Columns Kit) for each RNA fragment.
    3. Tap the powders to the bottom of columns and add 650 µl of nuclease-free water followed by vortexing at the highest speed for 10 sec.
    4. Incubate the column for 15 min at room temperature followed by centrifugation for 2 min at 750 x g.
    5. Pipet the in vitro-transcribed RNA onto the top of the column and put the column in a fresh 1.5-ml tube.
    6. Centrifuge the column for 2 min at 750 x g to collect the flow-through containing biotinylated RNA.
    7. The RNA concentration can be determined using the NanoDrop spectrophotometer.
    8. Store the purified samples at -20 °C or -80 °C until the quality of RNA is checked.
    9. To check the quality of the RNA, transfer 1 µg of transcribed RNA to a fresh tube and denature for 5 min at 70 °C with RNA sample preparation buffer provided in the MEGAshortscript kit.
    10. Resolve on a 6% TBE-Urea gel with 1x TBE buffer and visualize the RNAs on an ultraviolet transilluminator after staining with ethidium bromide.
    11. All RNA fragments should show single bands of the expected size.
    12. Store the RNA samples at -20 °C or -80 °C or use in the pulldown reaction.

  4. Preparation of cell lysate
    1. Wash two 10-cm dishes of ~70% confluent cells of interest three times with PBS and collect them using a cell scraper. As the protein yield varies with the cell line, the user should adjust the number of cells needed to extract at least 500 µg protein for each pulldown assay.
    2. Pellet the cells in a 1.7-ml tube by centrifuging for 5 min at 500 x g.
    3. Disrupt the cell pellet by pipetting 10-20 times with 500 µl of polysome extraction buffer (PEB) containing protease and RNase inhibitors. Incubate for 10 min on ice.
    4. Collect the supernatant after centrifugation at 15,000 x g for 10 min at 4 °C using a refrigerated centrifuge.
    5. Determine protein concentrations by the Bradford protein assay following the manufacturer’s instructions using disposable cuvettes and a spectrophotometer at 595 nm wavelength.
    6. Store protein samples at -20 °C or -80 °C or use directly in pulldown reaction.

  5. Pulldown of interacting RNA-binding protein
    1. Prepare the pulldown reaction in 1.5-ml tube containing 500 µg of cell lysate, 1 µg of purified biotinylated RNA, 1x protease inhibitor (add fresh), 5 µl of RiboLock, 250 µl of 2x TENT buffer (Tris, EDTA, NaCl, Triton; see Recipes) and PEB in 500 µl final volume.
    2. Mix the reaction by pipetting and incubate at room temperature for 30 min.
    3. In the meantime, wash 50 µl streptavidin-coupled Dynabeads for each reaction three times with 1x TENT buffer using the MagneSphere stand.
    4. Add 50 µl washed beads to the RNA-protein mixture prepared in step E1 (Note 7).
    5. Mix the beads by pipetting up and down.
    6. Incubate the reaction at room temperature for another 30 min with intermittent mixing by tapping the tube every ~5 min.
    7. Incubate the tube on the magnetic stand for 1 min to allow the magnetic beads to settle to one side of the tube.
    8. Discard the supernatant and mix the magnetic beads with 1 ml of ice-cold 1x TENT buffer.
    9. Repeat steps E7 and E8 3 more times.
    10. Add 40 µl of 1x Laemmli sample buffer supplemented with β-mercaptoethanol to the magnetic beads.
    11. Heat the samples for 5 min at 95 °C and proceed to Western blotting.

  6. Detection of interacting RNA-binding proteins (RBPs)
    1. Use Western blotting techniques to detect the RBP interest in the pulldown. Briefly, load at least three lanes on a 4-20% SDS-PAGE gel: (1) 10 µg (or 2% of the protein used for pulldown) of cell lysate as input, (2) the entire sample from the pulldown assay, and (3) a pre-stained protein ladder. Resolve these samples by electrophoresis following the manufacturer’s protocol.
    2. Transfer the protein to a nitrocellulose membrane using a Trans-Blot transfer system following the manufacturer’s instructions.
    3. Use standard Western blotting techniques to detect the RBP of interest using primary antibodies recognizing that RBP (Mahmood and Yang, 2012).
    4. Incubate with the appropriate secondary antibody to recognize the primary antibody and detect the signals using Enhanced Chemiluminescence (ECL).

Data analysis

As shown in Figure 1, Western blot analysis reveals the presence of the RBP in the input lysate (positive control) and in the pulldown material using biotinylated fragment C, suggesting that the sequence in fragment C includes a region with which this RBP interacts. This analysis includes negative control samples ‘Beads only’ and ‘Neg. control biot-RNA’, which do not pulldown the RBP, and shows that fragments A and B do not bind the RBP, supporting the conclusion that binding of the RBP to fragment C is specific. The experiment should be repeated at least three times to conclude the specificity of the RNA-RBP interaction.

Notes

  1. The size of the RNA fragment can be decided by the researcher, but an overlap of at least 20 bp between adjacent fragments is recommended.
  2. Longer (more than 4 h) incubation of in vitro transcription reaction may lead to degradation of the in vitro-transcribed RNA.
  3. As a control, use a ‘beads only’ sample without any biotinylated RNA to see if the magnetic beads alone will bind to RBP of interest in this pulldown condition.
  4. Use a negative control (NC) biotinylated RNA such as GAPDH/ACTB that does not bind the RBP studied in order to check the specificity of interaction between the RBP and the mRNA of interest.
  5. This protocol uses a ratio of 1 biotin-CTP:4 unlabeled CTP (i.e., 1 in 5 CTP is biotin-labeled). The ratio between biotin-CTP and unlabeled CTP is adjusted, and the user has to be mindful of the number of Cs present in the RNA product. For long transcripts, the biotin-CTP ratio can be lowered to 1 biotin-CTP:10 unlabeled CTP.
  6. Labeling the RNA at higher than 1:4 ratio of biotinylated to non-biotinylated CTP may inhibit RBP binding. The user must ensure that each RNA fragment has at least 1 or 2 biotinylated Cs in its sequence.
  7. Using higher amount of biotin-labeled RNA does not help and may be detrimental to the pulldown assay, as 50 µl of streptavidin magnetic beads can bind ~100 pmol of biotin-labeled RNA.
  8. Longer RNA may result in false positive pulldown result as the RBP of interest may interact with the RNA bait indirectly through another RBP.
  9. Lysis of cells in PEB isolates cytoplasmic fractions effectively. Thus, this protocol is best suited for studying the interactions of cytoplasmic proteins with RNAs of interest. RBPs interacting with the RNA bait in vitro may or may not interact in the cells due to differential subcellular localization of RNA or protein of interest.

Recipes

  1. Polysome extraction buffer (PEB) in nuclease-free water
    20 mM Tris-HCl (pH 7.5)
    100 mM KCl
    5 mM MgCl2
    0.5% Nonidet P-40
  2. 2x Tris, EDTA, NaCl, Triton (TENT) buffer in nuclease-free water
    20 mM Tris-HCl (pH 8.0)
    2 mM EDTA (pH 8.0)
    500 mM NaCl
    1% (v/v) Triton X-100
  3. 1x TENT
    Equal volumes of 2x TENT and nuclease-free water mixed together

Acknowledgments

This work was supported by the National Institute on Aging Intramural Research Program, National Institutes of Health.

References

  1. Glisovic, T., Bachorik, J. L., Yong, J. and Dreyfuss, G. (2008). RNA-binding proteins and post-transcriptional gene regulation. FEBS Lett 582(14): 1977-1986.
  2. Mahmood, T. and Yang, P. C. (2012). Western blot: technique, theory, and trouble shooting. N Am J Med Sci 4(9): 429-34.
  3. Panda, A. C., Abdelmohsen, K., Martindale, J. L., Di Germanio, C., Yang, X., Grammatikakis, I., Noh, J. H., Zhang, Y., Lehrmann, E., Dudekula, D. B., De, S., Becker, K. G., White, E. J., Wilson, G. M., de Cabo, R. and Gorospe, M. (2016). Novel RNA-binding activity of MYF5 enhances Ccnd1/Cyclin D1 mRNA translation during myogenesis. Nucleic Acids Res 44(5): 2393-2408.
  4. Panda, A. C., Abdelmohsen, K., Yoon, J. H., Martindale, J. L., Yang, X., Curtis, J., Mercken, E. M., Chenette, D. M., Zhang, Y., Schneider, R. J., Becker, K. G., de Cabo, R. and Gorospe, M. (2014). RNA-binding protein AUF1 promotes myogenesis by regulating MEF2C expression levels. Mol Cell Biol 34(16): 3106-3119.
  5. Popova, V. V., Kurshakova, M. M. and Kopytova, D. V. (2015). [Methods to study the RNA-protein interactions]. Mol Biol (Mosk) 49(3): 472-481.
  6. Untergasser, A., Cutcutache, I., Koressaar, T., Ye, J., Faircloth, B. C., Remm, M. and Rozen, S. G. (2012). Primer3—new capabilities and interfaces. Nucleic Acids Res 40(15): e115-e115.

简介

RNA结合蛋白(RBP)近来已经成为调控基因表达的关键因素。 RBP与靶mRNA的相互作用通过改变不同的调节步骤来控制基因产物的水平,包括mRNA前体剪接和成熟,核mRNA输出和mRNA稳定性和翻译(Glisovic et al。,2008) )。目前有几种方法可用于鉴定与特定RBP结合的RNA;一些仅在体外检测重组分子,其他检测重组和内源性分子,而其他检测仅内源性分子。实例包括通过指数富集(SELEX),生物素化RNA下拉测定,RNA免疫沉淀(RIP)测定,电泳迁移率变动测定(EMSA),RNA足迹分析和各种UV交联和免疫沉淀(CLIP)方法如CLIP ,PAR-CLIP和iCLIP(Popova等人,2015)。在这里,我们描述了一种简单而有信息的方法来研究和鉴定RBP与其目标转录物之间相互作用的RNA区域(Panda等人,2014和2016)。其重现性和易用性使得该方案成为识别RBP与特异性RNA之间相互作用的快速有效的方法。

背景 RNA蛋白相互作用严重影响基因表达模式。这些核糖核蛋白(RNP)复合物的鉴定对于理解由RNA结合蛋白(RBP)控制的调控机制是必不可少的。最近,广泛的努力导致了开发用于系统分析RNA-蛋白质相互作用的方法。识别RNP复合物的高度信息化方法包括许多不同类型的RNP免疫沉淀(IP)分析。 RIP方法涉及无交联的RNP IP,而CLIP方法涉及IP之前的RNP交联。虽然RIP快速,便宜,并且能够评估许多内源RBP和RNA,但它通常不允许鉴定与RBP相互作用的精确RNA区域。 CLIP分析(包括其变体形式HITS-CLIP,PAR-CLIP和iCLIP)确实允许发现与RBP相互作用的精确RNA序列,因为它包括RNase步骤,其消化所有未受保护的RNA并产生结合到RBP。然而,CLIP分析是昂贵的,耗时的和技术上的挑战(熊猫等人,2016)。因此,需要将内源蛋白与目的RNA的结合进行测试的替代方法。
  这里描述的生物素化的RNA下拉法在理论上适用于所有RBP,因为该测定法在无细胞系统中进行。该方法涉及在生物素化CTP存在下的目标RNA的体外合成;然后以这种方式标记的RNA与无细胞系统一起孵育,以允许RBP识别具有亲和力的RNA区域,而没有亲和力的区域不与RBP相互作用。结合完成后,使用链霉抗生物素蛋白包被的珠子将生物素化的RNA拉下,通常通过蛋白质印迹分析来检测RBP。如本文所述,如果用户以系统方式测试逐渐变小的RNA片段,则该方法可用于映射与其相关的RNA序列。此外,如果生物素-RNA下拉之后是质谱法,则该方法允许鉴定与感兴趣的RNA相互作用的所有蛋白质。总之,这种方法可以成功地鉴定出一种内源(或重组)RBP与体外合成的感兴趣的RNA之间的相互作用。

关键字:标记的RNA, RNA结合蛋白, 核糖核蛋白复合物, 生物素pull-down

材料和试剂

  1. ThermoGrid TM 刚性条带0.2-ml PCR管(Denville Scientific,目录号:C18064 [1000859])
  2. Posi-Click 1.7-ml微量离心管(Denville Scientific,目录号:C2171)
  3. 1.5 ml管子
  4. 10厘米的菜肴
  5. NucAway旋转柱(Thermo Fisher Scientific,Invitrogen TM,目录号:AM10070)
  6. 一次性比色皿,1.5毫升(Stockwell Scientific,目录号:2410)
  7. 无核酸酶水(Thermo Fisher Scientific,Ambion TM ,目录号:AM9930)
  8. 从总RNA制备的cDNA
  9. DreamTaq DNA聚合酶(5U /μl)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:EP0701)
  10. dNTP混合物(每种10mM)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:R0193)
  11. Agarose LE(Denville Scientific,目录号:CA3510-8)
  12. QIAquick凝胶提取试剂盒(50)(QIAGEN,目录号:28704)
  13. MEGAshortscriptTM T7试剂盒,手册(RNA短于0.5 Kb)(Thermo Fisher Scientific,Invitrogen TM,目录号:AM1354)
  14. RiboLock RNase抑制剂(40U /μl)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:EO0381)
  15. 生物素-14-CTP(Thermo Fisher Scientific,Invitrogen TM,目录号:19519-016)
  16. MEGAscript ® T7转录试剂盒(*用于长于0.5 Kb的RNA)(Thermo Fisher Scientific,Invitrogen TM,目录号:AM1333)
  17. Novex ® TBE-尿素凝胶,6%(Thermo Fisher Scientific,Invitrogen TM,目录号:EC6865BOX)
  18. 1x TBE缓冲区
  19. Dulbecco的磷酸盐缓冲盐水(DPBS)(Thermo Fisher Scientific,Gibco TM,目录号:14040-133)
  20. 完全蛋白酶抑制剂混合物(Sigma-Aldrich,目录号:11697498001)
  21. 2x Laemmli样品缓冲液(Bio-Rad Laboratories,目录号:1610737)
  22. Dynabeads M-280链霉抗生物素蛋白(Thermo Fisher Scientific,Invitrogen TM,目录号:11205D)
  23. 2-巯基乙醇(β-巯基乙醇/BME)
  24. 10x Tris /甘氨酸/SDS运行缓冲液(Bio-Rad Laboratories,目录号:1610732)
  25. 4-20%Mini-PROTEAN ® TGX无污染蛋白凝胶(Bio-Rad Laboratories,目录号:4568094)
  26. 溴化乙锭溶液(Sigma-Aldrich,目录号:E1510)
  27. Spectra TM 多色宽范围蛋白梯(Thermo Fisher Scientific,Thermo Scientific TM,目录号:26634)
  28. Bio-Rad蛋白测定染料试剂浓缩物(Bradford试剂)(Bio-Rad Laboratories,目录号:500-0006)
  29. Tris-HCl(pH8.0)
  30. KCl
  31. MgCl 2
  32. Nonidet P-40
  33. EDTA
  34. NaCl
  35. Triton X-100
  36. 多聚赖氨酸提取缓冲液(PEB)(参见食谱)
  37. 2x Tris,EDTA,NaCl,Triton(TENT)缓冲液(参见食谱)
  38. 1x帐篷(见配方)

设备

  1. PCR带管转子,微型离心机C1201(Denville Scientific,目录号:C1201-S [1000806])
  2. Veriti ® 96孔热循环仪(Thermo Fisher Scientific,Applied Biosystems TM,目录号:4375786)
  3. 紫外线透照仪
  4. NanoDrop One分光光度计(Thermo Fisher Scientific,Thermo Scientific,目录号:ND-ONE-W)
  5. Eppendorf Thermomixer ® R(Eppendorf,目录号:022670581)
  6. 孵化器
  7. Vortexer
  8. 电池刮刀
  9. 冷冻离心机(Eppendorf,型号:5430R)
  10. SmartSpec Plus分光光度计(Bio-Rad Laboratories,目录号:1702525)或其他具有595 nm波长的分光光度计
  11. MagneSphere(R)支架(Promega,目录号:Z5342)
  12. Trans-Blot ® Turbo TM 转移起动器系统(Bio-Rad Laboratories,目录号:1704155)
  13. Mini-PROTEAN ®四立式电泳池(Bio-Rad Laboratories,目录号:1658004)

程序

  1. 用于体外转录的引物设计和模板生成
    1. 使用Primer 3在线工具( http://bioinfo.ut.ee/primer3-0.4.0/)(Untergasser 等人,2012) (注1)
    2. 如图1所示,加入实际正向引物序列上游的T7 RNA聚合酶启动子序列(T7)[5'AGTAATACGACTCACTATAGGG](红线)(图1)。
    3. 将引物溶解于无核酸酶的水中至终浓度为100μM,然后在无核酸酶的水(例如10天)中制备终浓度为1μM的正向引物和反向引物的引物混合物将每个引物从100μM储备液中加入980μl不含核酸酶的水中并充分混合)
    4. 对于在0.2-ml PCR管中40μl的一次PCR反应,加入4μl10x DreamTaq DNA聚合酶缓冲液,10μl1μM引物混合物,1μlDreamTaq DNA聚合酶,1μl10mM dNTP混合物,0.2μl( 〜2ng)cDNA和水,使最终体积为40μl。 (如前所述[Panda等人,2016],由总RNA制备cDNA。)
    5. 通过涡旋混合反应5秒,并使用微型离心机离心PCR管条30秒,以沉淀孔底部的反应。
    6. 设置Veriti ® 96孔热循环程序如下:95℃5分钟,95℃5秒,60℃60秒60次循环。
    7. 在溴化乙锭染色的2%琼脂糖凝胶上在100伏下解析PCR产物1小时,并用紫外线透射仪观察。
    8. 从凝胶中切下所需的条带。使用QIAquick Gel Extraction Kit纯化与每个片段相对应的PCR产物,并溶解于试剂盒提供的洗脱缓冲液中。
    9. 用NanoDrop分光光度计测量PCR产物/模板的浓度
    10. PCR产物(T7 DNA模板)可以在-20°C或-80°C保存,或立即用于体外转录。

  2. 生物素化RNA片段的体外转录
    1. 生物素化的RNA可以按照制造商的方案使用MEGAshortscript TM T7试剂盒制备。


      图1.生物素化RNA下拉测定示意图

    2. 每个DNA模板的体外转录反应可以在含有2μl10×MEGAshortscript T7缓冲液,2μlMEGAshortscript TM的1.7ml PCR管中制备 T7酶混合物,1μlRiboLock,1μl75mM rATP,1μl75mM rUTP,1μl75mM rGTP,0.8μl75mM rCTP,1.5μl10mM生物素-14- CTP,100ng T7 DNA模板和水,使最终体积为20μl。 (对于较长的RNA片段[500nt],用户应该在体外使用MEGAscript TM T7试剂盒进行RNA制备)(注5和6)
    3. 混合并在500 x g下离心约5秒,以使管底部的反应混合物沉降。
    4. 在Thermomixer(注2)中,将反应在37℃下孵育4小时
    5. 加入试剂盒中提供的1μlDNA酶,并充分混匀,然后在37°C孵育15分钟
    6. 将样品储存在-20°C或-80°C或立即进行RNA纯化。

  3. 体外转录的生物素化RNA片段的纯化和质量检查
    1. 通过加入50μl无核酸酶的水稀释整个20μl体外转录的RNA,并保持在冰上。
    2. 每个RNA片段使用一个柱(NucAway Spin Columns Kit)。
    3. 点击粉末到柱底,加入650μl无核酸酶水,然后以最高速度涡旋10秒。
    4. 在室温下孵育柱15分钟,然后以750×g离心2分钟。
    5. 将体外转录的RNA吸入柱顶部,并将柱放入新鲜的1.5 ml管中。
    6. 以750×g离心柱离心2分钟以收集包含生物素化RNA的流通。
    7. RNA浓度可以使用NanoDrop分光光度计测定
    8. 将纯化的样品储存在-20°C或-80°C,直到检查RNA的质量
    9. 为了检查RNA的质量,将1μg转录的RNA转移到新鲜管中,并在70℃下用MEGAshortscript试剂盒中提供的RNA样品制备缓冲液变性5分钟。
    10. 用1×TBE缓冲液溶解在6%TBE-尿素凝胶上,并用溴化乙锭染色后在紫外线透射仪上显现RNA。
    11. 所有RNA片段应显示预期大小的单一条带。
    12. 将RNA样品储存在-20°C或-80°C或用于下拉反应。

  4. 细胞裂解物的制备
    1. 用PBS洗涤两次10厘米浓度的约70%融合细胞,并用细胞刮刀收集。随着蛋白质产量随细胞系而变化,用户应调整每次下拉测定法提取至少500μg蛋白质所需的细胞数量。
    2. 通过以500×g离心5分钟将细胞置于1.7ml管中。
    3. 通过用500μl含有蛋白酶和RNA酶抑制剂的多聚糖提取缓冲液(PEB)移液10-20次来破坏细胞沉淀。在冰上孵育10分钟。
    4. 使用冷冻离心机在4℃下以15,000×g离心10分钟收集上清液。
    5. 根据制造商的说明书使用一次性比色皿和595 nm波长的分光光度计,通过Bradford蛋白测定法确定蛋白质浓度。
    6. 将蛋白质样品储存于-20°C或-80°C或直接用于下拉反应。

  5. 相互作用的RNA结合蛋白的下调
    1. 在含有500μg细胞裂解物,1μg纯化的生物素化RNA,1x蛋白酶抑制剂(加入新鲜),5μlRiboLock,250μl2xTent缓冲液(Tris,EDTA,NaCl,Triton)的1.5ml管中制备下拉反应;见食谱)和PEB,最终体积为500μl
    2. 通过移液混合反应,并在室温下孵育30分钟
    3. 同时,使用MagneSphere支架,用1x TENT缓冲液洗涤每次反应的50μl链霉亲和素偶联的dynabeads。
    4. 向步骤E1(注7)中制备的RNA-蛋白混合物中加入50μl洗涤的珠。
    5. 通过上下移动来混合珠子。
    6. 将反应在室温下孵育另外30分钟,间歇混合,每隔5分钟点击管子。
    7. 在磁架上孵育管1分钟,使磁珠沉入管的一侧
    8. 弃去上清液,并用1ml冰冷的1x TENT缓冲液混合磁珠
    9. 再次重复步骤E7和E8 3次。
    10. 向磁珠中加入40μl含有β-巯基乙醇的1x Laemmli样品缓冲液
    11. 在95℃加热样品5分钟,并进行Western印迹
  6. 检测相互作用的RNA结合蛋白(RBPs)
    1. 使用Western印迹技术检测下拉中的RBP兴趣。简言之,在4-20%SDS-PAGE凝胶上载入至少三条泳道:(1)10μg(或用于下拉的蛋白质的2%)细胞裂解物作为输入,(2)来自下拉试验的全部样品,和(3)预染色蛋白梯。通过制造商的方案电泳解决这些样品。
    2. 使用Trans-Blot转移系统按照制造商的说明书将蛋白转移到硝酸纤维素膜上。
    3. 使用标准的Western印迹技术使用识别RBP的一级抗体(Mahmood和Yang,2012)来检测感兴趣的RBP。
    4. 与适当的二次抗体孵育以识别一抗,并使用增强化学发光(ECL)检测信号。

数据分析

如图1所示,蛋白质印迹分析揭示了输入裂解物(阳性对照)和使用生物素化片段C的下拉材料中RBP的存在,表明片段C中的序列包含与该RBP相互作用的区域。该分析包括阴性对照样品"仅珠子"和"控制生物RNA",其不下拉RBP,并且显示片段A和B不结合RBP,支持RBP与片段C的结合是特异性的结论。应重复实验至少三次以得出RNA-RBP相互作用的特异性。

笔记

  1. RNA片段的大小可以由研究人员决定,但建议在相邻片段之间重叠至少20bp。
  2. 体外转录反应的长时间(超过4小时)的孵育可能导致体外转录的RNA的降解。
  3. 作为对照,使用没有任何生物素化RNA的"仅珠子"样品,以查看在下拉条件下单独的磁珠是否与感兴趣的RBP结合。
  4. 使用不结合所研究的RBP的阴性对照(NC)生物素化RNA,例如GAPDH /ACTB ,以检查RBP与mRNA之间的相互作用的特异性兴趣。
  5. 该方案使用1个生物素-CTP:4个未标记的CTP(即,即5个CTP中的1/5是生物素标记的)的比率。调整生物素-CTP与未标记的CTP之间的比例,用户必须注意RNA产物中存在的Cs的数目。对于长转录物,生物素-CTP比可降低至1个生物素-CTP:10个未标记的CTP。
  6. 标记生物素化至非生物素化CTP比例高于1:4的RNA可抑制RBP结合。用户必须确保每个RNA片段在其序列中具有至少1或2个生物素化的Cs。
  7. 使用较高量的生物素标记的RNA不能帮助并且可能不利于下拉式测定,因为50μl链霉抗生物素蛋白磁珠可结合〜100 pmol生物素标记的RNA。
  8. 较长的RNA可能导致假阳性下拉结果,因为感兴趣的RBP可以通过另一个RBP间接与RNA诱饵相互作用。
  9. PEB细胞裂解有效分离细胞质级分。因此,该方案最适用于研究细胞质蛋白与感兴趣的RNA的相互作用。由于与感兴趣的RNA或蛋白质的差异亚细胞定位,在细胞中与RNA诱饵相互作用的RBP可能或可能不与细胞相互作用。

食谱

  1. 不含核酸酶的水中的多聚糖提取缓冲液(PEB)
    20mM Tris-HCl(pH7.5)
    100 mM KCl
    5mM MgCl 2
    0.5%Nonidet P-40
  2. 2x Tris,EDTA,NaCl,Triton(TENT)缓冲液在无核酸酶的水中 20mM Tris-HCl(pH8.0)
    2mM EDTA(pH8.0)
    500 mM NaCl
    1%(v/v)Triton X-100
  3. 1x TENT
    等量的2xTENT和无核酸酶的水混合在一起

致谢

这项工作得到了国立卫生研究院老龄化研究方案的支持。

参考文献

  1. Glisovic,T.,Bachorik,JL,Yong,J.and Dreyfuss,G。(2008)。 RNA结合蛋白和转录后基因调控。 FEBS Lett 582(14):1977-1986。
  2. Mahmood,T.and Yang,PC(2012)。< a class ="ke-insertfile"href ="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3456489/"target =" _blank">蛋白质印迹:技术,理论和麻烦。 N Am J Med Sci 4(9):429-34。
  3. 熊猫,AC,Abdelmohsen,K.,Martindale,JL,Di Germanio,C.,Yang,X.,Grammatikakis,I.,Noh,JH,Zhang,Y.,Lehrmann,E.,Dudekula,DB,De,S 。,Becker,KG,White,EJ,Wilson,GM,de Cabo,R.and Gorospe,M。(2016)。 MYF5的新型RNA结合活性增强了肌发生期间的Ccnd1 /细胞周期蛋白D1 mRNA翻译。核酸抗体 44(5):2393-2408。
  4. 熊猫,AC,Abdelmohsen,K.,Yoon,JH,Martindale,JL,Yang,X.,Curtis,J.,Mercken,EM,Chenette,DM,Zhang,Y.,Schneider,RJ,Becker,KG,de Cabo R.和Gorospe,M.(2014)。 RNA结合蛋白AUF1通过调节MEF2C表达水平来促进肌细胞生成。分子细胞生物学34(16):3106-3119。
  5. Popova,VV,Kurshakova,MM和Kopytova,DV(2015)。  [研究RNA-蛋白质相互作用的方法] Mol Biol(Mosk) 49(3):472-481。
  6. Untergasser,A.,Cutcutache,I.,Koressaar,T.,Ye,J.,Faircloth,BC,Remm,M.and Rozen,SG(2012)。< a class ="ke-insertfile"href = http://nar.oxfordjournals.org/content/40/15/e115.short"target ="_ blank"> Primer3-新的功能和界面。 核酸研究组40(15 ):e115-e115。
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引用:Panda, A. C., Martindale, J. L. and Gorospe, M. (2016). Affinity Pulldown of Biotinylated RNA for Detection of Protein-RNA Complexes. Bio-protocol 6(24): e2062. DOI: 10.21769/BioProtoc.2062.
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