Study of RNA–Protein Interactions and RNA Structure in Ribonucleoprotein Particles

Marchand, Virginie; Mougin, Annie; Mreau, Agns; Branlant, Christiane

doi:10.1002/9783527619504.ch11

Cited by 2 publications

(2 citation statements)

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“…As binding competitors can also reduce the amount of specific binding, it is best to test a range of competitor concentrations, and to optimize the discrimination of specific and nonspecific binding. In this case, useful competitors include heparin, poly d(A) and poly d(T) DNAs, poly (U) RNA and mixed tRNAs (these are useful when the proteins in question are not specifically tRNA-binding proteins) [51]. The key point here is that the competitor should have structural or sequence attributes that allow nonspecific binding, but it should lack the sequence and/or secondary structures needed for specific binding by the protein(s) of interest (see also Chapter 32 Branlant for a discussion of inhibitors).…”

Section: Competing Nucleic Acids and Polyanionsmentioning

confidence: 99%

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Detection of RNA–Protein Complexes by Electrophoretic Mobility Shift Assay

Fried

2012

Alternative Pre‐mRNA Splicing

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The electrophoretic mobility shift assay (EMSA) is commonly used to study proteinnucleic acid interactions. The technique is based on the observation that proteinbound nucleic acid molecules migrate more slowly than free nucleic acid molecules when subjected to native polyacrylamide or agarose gel electrophoresis. After electrophoresis, the distribution of species containing nucleic acid is determined by autoradiography or other sensitive imaging technique. Under appropriate conditions, the method can be used for quantitative binding analysis, but it is most widely used as a qualitative means of detecting protein-RNA complexes. In this chapter, features relevant to the design of EMSA experiments are identified, and the technical factors found to be important for the success of the assay are outlined. Theoretical BackgroundThe electrophoretic mobility shift assay (EMSA) is a simple and powerful method for detecting interactions between nucleic acids and proteins [1][2][3][4][5][6]. Although the simplicity of the method accounts for its popularity, it also conceals technical subtleties. In this chapter, the aim is to help select the most convenient EMSA variants for experimental purposes, and to avoid the most important pitfalls associated with the technique.The EMSA is based on the observation that the gel-electrophoretic mobility of a protein-nucleic acid complex is often less than that of the corresponding free nucleic acid. Current versions of the assay differ little from those originally described by Garner and Revzin [7] and Fried and Crothers [8], although precursors can be found in earlier reports [9][10][11]. Although originally developed to analyze DNA-protein complexes, the method is also useful for the characterization of RNA-protein interactions [12][13][14].The electrophoretic mobilities of RNA molecules in polyacrylamide or agarose gels depend on: (i) the sizes, shapes, and charges of the molecules; (ii) the conductivities of the gel and sample buffers; and (iii) the concentrations of the gel polymer and its degree of crosslinking [15]. Protein binding can result in complexes that differ from the parent RNA in terms of charge, size or shape; changes in any or all of these factors may produce a difference in gel-mobility that allows the detection of binding. Changes in the type of gel matrix (e.g., agarose versus polyacrylamide), its concentration, and changes in the composition of the gel buffer, can each affect electrophoretic resolution and, just as importantly, the stabilities of the protein-nucleic acid complexes within the gel [16][17][18][19]. Optimization of these factors is straightforward and can significantly improve the results of an EMSA.Alternative pre-mRNA Splicing: Theory and Protocols, First Edition. EditedThe selection of a binding substrate requires assumptions to be made about the RNA structure(s) needed for appropriate protein binding. If the structure of the RNA probe differs significantly from the native cellular binding target, the behavior of the assay system may not be repre...

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Section: Competing Nucleic Acids and Polyanionsmentioning

confidence: 99%

“…Current versions of the EMSA provide very little direct information about the location(s) of the binding site(s) that are occupied in protein-nucleic acid complexes. However, footprinting assays [50,51,60,61] carried out in parallel with EMSA provide an effective approach to identifying the occupied sequences.…”

Section: Example Experimentsmentioning

confidence: 99%

Detection of RNA–Protein Complexes by Electrophoretic Mobility Shift Assay

Fried

2012

Alternative Pre‐mRNA Splicing

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Probing mRNA Structure and sRNA–mRNA Interactions in Bacteria Using Enzymes and Lead(II)

Chevalier¹,

Geissmann²,

Helfer³

et al. 2009

Methods in Molecular Biology

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Enzymatic probing and lead(II)-induced cleavages have been developed to study the secondary structure of RNA molecules either free or engaged in complex with different ligands. Using a combination of probes with different specificities (unpaired vs. paired regions), it is possible to get information on the accessibility of each nucleotide, on the binding site of a ligand (noncoding RNAs, protein, metabolites), and on RNA conformational changes that accompanied ligand binding or environmental conditions (temperature, pH, ions, etc.). The detection of the cleavages can be conducted by two different ways, which are chosen according to the length of the studied RNA. The first method uses end-labeled RNA molecules and the second one involves primer extension by reverse transcriptase. We provide here an experimental procedure that was designed to map the structure of mRNA and mRNA-sRNA interaction in vitro.

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Study of RNA–Protein Interactions and RNA Structure in Ribonucleoprotein Particles

Cited by 2 publications

References 59 publications

Detection of RNA–Protein Complexes by Electrophoretic Mobility Shift Assay

Detection of RNA–Protein Complexes by Electrophoretic Mobility Shift Assay

Probing mRNA Structure and sRNA–mRNA Interactions in Bacteria Using Enzymes and Lead(II)

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