New Generations of MS2 Variants and MCP Fusions to Detect Single mRNAs in Living Eukaryotic Cells

Xavier Pichon; Marie Cécile Robert; Edouard Bertrand; Robert H. Singer; Evelina Tutucci

doi:10.1007/978-1-0716-0712-1_7

New Generations of MS2 Variants and MCP Fusions to Detect Single mRNAs in Living Eukaryotic Cells

Xavier Pichon, Marie Cécile Robert, Edouard Bertrand, Robert H. Singer, Evelina Tutucci

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

Abstract

Live imaging of single RNA from birth to death brought important advances in our understanding of the spatiotemporal regulation of gene expression. These studies have provided a comprehensive understanding of RNA metabolism by describing the process step by step. Most of these studies used for live imaging a genetically encoded RNA-tagging system fused to fluorescent proteins. One of the best characterized RNA-tagging systems is derived from the bacteriophage MS2 and it allows single RNA imaging in real-time and live cells. This system has been successfully used to track the different steps of mRNA processing in many living organisms. The recent development of optimized MS2 and MCP variants now allows the labeling of endogenous RNAs and their imaging without modifying their behavior. In this chapter, we discuss the improvements in detecting single mRNAs with different variants of MCP and fluorescent proteins that we tested in yeast and mammalian cells. Moreover, we describe protocols using MS2-MCP systems improved for real-time imaging of single mRNAs and transcription dynamics in S. cerevisiae and mammalian cells, respectively.

Original language	English
Title of host publication	RNA Tagging
Subtitle of host publication	Methods and Protocols
Editors	Manfred Heinlein
Place of Publication	New York, NY
Publisher	Humana
Pages	121-144
Number of pages	24
ISBN (Electronic)	9781071607121
ISBN (Print)	9781071607114, 9781071607145
DOIs	https://doi.org/10.1007/978-1-0716-0712-1_7
Publication status	Published - 2020

Publication series

Name	Methods in molecular biology (MIMB)
Volume	2166
ISSN (Print)	1064-3745
ISSN (Electronic)	1940-6029

Funding

This work was supported by NIH Grant GM57071 to R.H.S., and by an ANRS Grant to E.B. E.T. was supported by Swiss National Science Foundation Fellowships P2GEP3_155692 and P300PA_164717. X.P. was supported by a fellowship from the Labex EpiGenMed Montpellier/Université de Montpellier, and E.B. had a travel grant from the Philippe Foundation.

Funders	Funder number
Labex EpiGenMed Montpellier/Université de Montpellier
National Institutes of Health	GM57071
Philippe Foundation
Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung	P300PA_164717, P2GEP3_155692
Agence Nationale de Recherches sur le Sida et les Hépatites Virales

Keywords

Gene expression
Mammalian cells
mRNA labeling
mRNA localization
MS2-MCP system
S. cerevisiae
Single cell
Single molecule
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10.1007/978-1-0716-0712-1_7

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abstract = "Live imaging of single RNA from birth to death brought important advances in our understanding of the spatiotemporal regulation of gene expression. These studies have provided a comprehensive understanding of RNA metabolism by describing the process step by step. Most of these studies used for live imaging a genetically encoded RNA-tagging system fused to fluorescent proteins. One of the best characterized RNA-tagging systems is derived from the bacteriophage MS2 and it allows single RNA imaging in real-time and live cells. This system has been successfully used to track the different steps of mRNA processing in many living organisms. The recent development of optimized MS2 and MCP variants now allows the labeling of endogenous RNAs and their imaging without modifying their behavior. In this chapter, we discuss the improvements in detecting single mRNAs with different variants of MCP and fluorescent proteins that we tested in yeast and mammalian cells. Moreover, we describe protocols using MS2-MCP systems improved for real-time imaging of single mRNAs and transcription dynamics in S. cerevisiae and mammalian cells, respectively.",

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New Generations of MS2 Variants and MCP Fusions to Detect Single mRNAs in Living Eukaryotic Cells. / Pichon, Xavier; Robert, Marie Cécile; Bertrand, Edouard et al.
RNA Tagging: Methods and Protocols. ed. / Manfred Heinlein. New York, NY: Humana, 2020. p. 121-144 (Methods in molecular biology (MIMB); Vol. 2166).

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

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New Generations of MS2 Variants and MCP Fusions to Detect Single mRNAs in Living Eukaryotic Cells

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