RNA-protein UV-crosslinking Assay
紫外交联实验检测RNA-蛋白的相互作用
发布: 2017年03月20日第7卷第6期 DOI: 10.21769/BioProtoc.2193 浏览次数: 17435
评审: HongLok LungAnonymous reviewer(s)
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The authors used this protocol in:
Mar 2016
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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 analysis of the fluorescently-labeled RNA-protein complexes obtained by this method can lead to identification of novel RNA-protein interactions.
Materials and Reagents
- 1.5 ml RNase, DNase free microcentrifuge tube (Corning, Axygen®, catalog number: MCT-150-C or equivalent)
- 96-well round-bottomed plate (Greiner Bio one International, catalog number: 650101 )
- Agarose (Lonza, catalog number: 50004 )
- Transcription Kit (MAXIscript® Kit) (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM1312 or any equivalent)
- 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 ])
- DNase I (optional) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: EN0521 )
- 100% ethanol (EMD Millipore, catalog number: 100983 )
- Nuclease free water
- Ammonium acetate (Thermo Fisher Scientific, Affymetrix, catalog number: 75901 )
- Glycerol (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15514 )
- Urea-polyacrylamide gel
- Yeast tRNA (Sigma-Aldrich, catalog number: R8759 )
- RNase inhibitor (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: EO0381 )
- RNase A (Sigma-Aldrich, catalog number: R6513 )
- 10% SOD-PAGE
- Pre-stained protein markers or radiolabeled protein markers
- HEPES (pH 7.4) (Thermo Fisher Scientific, Affymetrix, catalog number: 16926 )
- Potassium chloride (KCl) (AMRESCO, catalog number: 0395 )
- Magnesium chloride hexahydrate (MgCl2·6H2O) (AMRESCO, catalog number: 0288 )
- Dithiothreitol (DTT) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0861 )
- EDTA (AMRESCO, catalog number: 0105 )
- ATP (Sigma-Aldrich, catalog number: A8937 )
- SDS
- Tris-Cl (pH 6.8)
- Bromophenol blue
- 2x RNA binding buffer (see Recipes)
- 2x denaturing protein loading buffer (see Recipes)
Equipment
- Refrigerated centrifuge (Eppendorf, model: 5418 R )
- UV cross-linker or UV torch with 254 nm wavelength UV (UVP, model: CL1000 )
- Vertical gel electrophoretic system (Bio-Rad Laboratories, model: Mini-PROTEAN Tetra Cell , catalog number: 1658000EDU)
- Scintillation counter (Hidex, model: Triathler or any equivalent model)
- Phosphorimager (GE Healthcare, model: Typhoon Trio or any equivalent model)
Procedure
文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
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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分类
癌症生物学 > 癌症生物化学 > 蛋白质
分子生物学 > RNA > RNA-蛋白质相互作用
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