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Approaches for measuring the dynamics of RNA–protein interactions

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Abstract RNA–protein interactions are pivotal for the regulation of gene expression from bacteria to human. RNA–protein interactions are dynamic; they change over biologically relevant timescales. Understanding the regulation of gene expression at the RNA level therefore requires knowledge of the dynamics of RNA–protein interactions. Here, we discuss the main experimental approaches to measure dynamic aspects of RNA–protein interactions. We cover techniques that assess dynamics of cellular RNA–protein interactions that accompany biological processes over timescales of hours or longer and techniques measuring the kinetic dynamics of RNA–protein interactions in vitro. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein–RNA Interactions: Functional Implications RNA Interactions with Proteins and Other Molecules > Protein–RNA Recognition RNA Interactions with Proteins and Other Molecules > RNA–Protein Complexes RNA Evolution and Genomics > Ribonomics
Classification of approaches to measure dynamics of RNA–protein interactions
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RNA‐MaP. Schematic depiction of the method. DNA is shown in black, RNA in blue. The gray circle represents the RNA polymerase. The yellow circle represents the RBP, the star the fluorophor
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HiTS‐kin. Schematic depiction of the method as applied to a reaction involves cleavage of the RNA substrates. The cleavage reaction is followed over time. Separated substrate pools are isolated from the PAGE and a cDNA library is generated and analyzed for each timepoint
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Measuring RNA–protein interactions with optical tweezers. (a) Measuring an RNA–protein interaction that is associated with RNA unwinding. The schematic setup shows two laser traps, but is possible to perform the measurements with a single trap and another fixed point. A force is applied to one of the laser traps and leads to a small displacement of one bead in the trap. The protein interaction causes partial unwinding of the RNA helix, which causes an extension of the RNA and a movement of the bead in the trap, which can be measured with high sensitivity. (b) Schematic timetrace of bead position of an individual molecule. Rate constants are calculated from histograms (at varying force) of many bead displacement events
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Single molecule FRET to measure RNA–protein interactions. (a) Principle of smFRET. (b) Protein binding measured with an RNA construct bearing both, donor and acceptor labels. (c) Schematic timetraces of donor and acceptor fluorescence and the corresponding FRET trace for an individual molecule. Rate constants are calculated from histograms of many binding and dissociation events
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Surface Plasmon resonance for measuring RNA–protein interactions. (a) Principle of SPR. (b) Surface immobilization of RNA. (c) Schematic timecourses of the respective signals for association and dissociation reactions
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Ensemble fluorescence techniques to measure RNA–protein interactions. (a) Fluorescence anisotropy, (b) fluorescence quenching, (c) FRET: Schematic reactions and timecourses of the respective signals for association and dissociation reactions
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Measuring association and dissociation rates with pulse‐chase regimes. Upper panel: Basic reaction scheme of protein binding to a single RNA‐site (kbind: Association rate costant, kdiss: Dissociation rate constant). Left panel: Reaction scheme to measure association rates. Separation refers to any technique that separates unbound RNA (or protein) from the RNA‐protein complex. Right panel: Reaction scheme to measure dissociation rates
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Kinetics of RNA–protein interactions. The reaction of the protein interaction with a given RNA site is marked RNA–protein interaction. RNA* indicates chemically or physically altered RNA. Differences in the reactions between RNA‐binding proteins and enzymes that interact with RNA are marked. Cellular parameters that can impact a given reaction state are marked as biological context. The arrows between the parameters indicate their potential interdependence
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Classification of approaches to measure the dynamics of RNA–protein interactions in cells. For abbreviations of methods, see text. The asterisk denotes that CLIP encompasses multiple specialized techniques. For details, see text
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RNA Interactions with Proteins and Other Molecules > Protein–RNA Interactions: Functional Implications
RNA Evolution and Genomics > Ribonomics
RNA Interactions with Proteins and Other Molecules > Protein–RNA Recognition
RNA Interactions with Proteins and Other Molecules > RNA–Protein Complexes

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