Probing RNA–protein interactions with single-molecule pull-down assays

Mohamed Fareh, Chirlmin Joo

Research output: Chapter in Book/Conference proceedings/Edited volumeChapterScientificpeer-review

Abstract

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.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherSpringer
Pages267-285
Volume1814
DOIs
Publication statusPublished - 1 Jan 2018

Publication series

NameMethods in Molecular Biology
Volume1814
ISSN (Print)1064-3745

Keywords

  • Protein complex
  • RNA labeling
  • RNA ligation
  • Single protein pull-down
  • Single-molecule fluorescence

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  • Cite this

    Fareh, M., & Joo, C. (2018). Probing RNA–protein interactions with single-molecule pull-down assays. In Methods in Molecular Biology (Vol. 1814, pp. 267-285). (Methods in Molecular Biology; Vol. 1814). Springer. https://doi.org/10.1007/978-1-4939-8591-3_16