Chapter 7
New Generations of MS2 Variants and MCP Fusions
to Detect Single mRNAs in Living Eukaryotic Cells
Xavier Pichon, Marie-Ce ´ cile Robert, Edouard Bertrand,
Robert H. Singer, and Evelina Tutucci
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 realtime 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.
Key words MS2-MCP system, mRNA labeling, Single molecule, Single cell, S. cerevisiae, Mammalian
cells, Gene expression, mRNA localization, Transcription
1 Introduction
Cells are the basic unit of life. Within a single cell, networks of
molecules control how the environment is sensed through signaling and how cells adapt via metabolic changes and modulation of
gene expression. Quantitative methods are required to model these
fundamental processes, which often involve only tens or less molecules [1–3]. To detect variations in gene expression, one approach
is to measure mRNA levels. Even though these are not always a
proxy for protein expression, i.e., when a delay exists between
Manfred Heinlein (ed.), RNA Tagging: Methods and Protocols, Methods in Molecular Biology, vol. 2166,
https://doi.org/10.1007/978-1-0716-0712-1_7, © Springer Science+Business Media, LLC, part of Springer Nature 2020
Electronic supplementary material: The online version of this chapter (https://doi.org/10.1007/978-1-07160712-1_7) contains supplementary material, which is available to authorized users.
121
New Generations of MS2 Variants and MCP Fusions
to Detect Single mRNAs in Living Eukaryotic Cells
Xavier Pichon, Marie-Ce ´ cile Robert, Edouard Bertrand,
Robert H. Singer, and Evelina Tutucci
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 realtime 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.
Key words MS2-MCP system, mRNA labeling, Single molecule, Single cell, S. cerevisiae, Mammalian
cells, Gene expression, mRNA localization, Transcription
1 Introduction
Cells are the basic unit of life. Within a single cell, networks of
molecules control how the environment is sensed through signaling and how cells adapt via metabolic changes and modulation of
gene expression. Quantitative methods are required to model these
fundamental processes, which often involve only tens or less molecules [1–3]. To detect variations in gene expression, one approach
is to measure mRNA levels. Even though these are not always a
proxy for protein expression, i.e., when a delay exists between
Manfred Heinlein (ed.), RNA Tagging: Methods and Protocols, Methods in Molecular Biology, vol. 2166,
https://doi.org/10.1007/978-1-0716-0712-1_7, © Springer Science+Business Media, LLC, part of Springer Nature 2020
Electronic supplementary material: The online version of this chapter (https://doi.org/10.1007/978-1-07160712-1_7) contains supplementary material, which is available to authorized users.
121