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

Mohamed Fareh; Chirlmin Joo

doi:10.1007/978-1-4939-8591-3_16

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

Mohamed Fareh, Chirlmin Joo

Department of Physics

Research output: Chapter in Book/Report/Conference proceeding › Chapter › peer-review

2 Scopus citations

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 language	English
Title of host publication	Methods in Molecular Biology
Publisher	Humana Press Inc.
Pages	267-285
Number of pages	19
DOIs	https://doi.org/10.1007/978-1-4939-8591-3_16
State	Published - 2018

Publication series

Name	Methods in Molecular Biology
Volume	1814
ISSN (Print)	1064-3745

Bibliographical note

Funding Information:
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].

Publisher Copyright:
© 2018, Springer Science+Business Media, LLC, part of Springer Nature.

Keywords

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

Access to Document

10.1007/978-1-4939-8591-3_16

Cite this

@inbook{c87eafb0e22e4b8596a0c5839582782a,

title = "Probing RNA–protein interactions with single-molecule pull-down assays",

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.",

keywords = "Protein complex, RNA labeling, RNA ligation, Single protein pull-down, Single-molecule fluorescence",

author = "Mohamed Fareh and Chirlmin Joo",

note = "Funding Information: 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{\textquoteright}s Seventh Framework Programme [FP7/2007–2013/ERC grant 309509 to C.J]; and the Fondation pour la Recherche Medicale [SPE20120523964 to M.F]. Publisher Copyright: {\textcopyright} 2018, Springer Science+Business Media, LLC, part of Springer Nature.",

year = "2018",

doi = "10.1007/978-1-4939-8591-3_16",

language = "English",

series = "Methods in Molecular Biology",

publisher = "Humana Press Inc.",

pages = "267--285",

booktitle = "Methods in Molecular Biology",

}

TY - CHAP

T1 - Probing RNA–protein interactions with single-molecule pull-down assays

AU - Fareh, Mohamed

AU - Joo, Chirlmin

N1 - Funding Information: 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]. Publisher Copyright: © 2018, Springer Science+Business Media, LLC, part of Springer Nature.

PY - 2018

Y1 - 2018

N2 - 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.

AB - 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.

KW - Protein complex

KW - RNA labeling

KW - RNA ligation

KW - Single protein pull-down

KW - Single-molecule fluorescence

UR - http://www.scopus.com/inward/record.url?scp=85049354465&partnerID=8YFLogxK

U2 - 10.1007/978-1-4939-8591-3_16

DO - 10.1007/978-1-4939-8591-3_16

M3 - Chapter

C2 - 29956238

AN - SCOPUS:85049354465

T3 - Methods in Molecular Biology

SP - 267

EP - 285

BT - Methods in Molecular Biology

PB - Humana Press Inc.

ER -

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

Abstract

Publication series

Bibliographical note

Keywords

Access to Document

Fingerprint

Cite this