Probing RNA–Protein Interactions with Single-Molecule Pull-Down Assays
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Recent advances in single-molecule techniques allow for dynamic observations of the interactions between various protein assemblies and RNA molecules with high spatiotemporal resolution. However, it remains challenging to obtain functional eukaryotic protein complexes and cost-effective fluorescently labeled RNAs to study their interactions at the single-molecule level. Here, we describe protocols combining single-molecule fluorescence with various protein complex pull-down techniques to determine the function of RNA-interacting protein complexes of interest. We provide step-by-step guidance for using novel single-molecule techniques including RNA labeling, protein complexes purification, and single-molecule imaging. As a proof-of-concept of the utility of our single-molecule approaches, we show how human Dicer and its cofactor TRBP orchestrate the biogenesis of microRNA in real time. These single-molecule pull-down and fluorescence assays provide sub-second time resolution and can be applied to various ribonucleoprotein complexes that are essential for cellular processes.
Key wordsSingle-molecule fluorescence Single protein pull-down Protein complex RNA ligation RNA labeling
We thank C.J. lab members for technical help and discussions. We thank Luuk Loeff, Malwina Szczepaniak, Anna C. Haagsma, Kyu-Hyeon Yeom for their help and support throughout the development of our single-molecule pulldown technique. This work was supported by a European Research Council Starting Grant under the European Union’s Seventh Framework Programme [FP7/2007–2013/ERC grant 309509 to C.J]; and the Fondation pour la Recherche Medicale [SPE20120523964 to M.F].
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