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

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RNA Tagging

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2166))

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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Acknowledgments

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.

Contributions: X.P. performed the experiments and wrote the protocol for mammalian cells. M.C.R. performed the experiments testing different FP in yeast. E.T. performed experiments and designed the protocol for yeast. E.T. wrote the manuscript with inputs from E.B. and R.H.S.

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Correspondence to Evelina Tutucci .

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MDN1 mRNAs tagged with 24xMBSV6 in the 3′UTR and labeled with MCP-NLS-2xyeGFP are shown in gray; Nup49-tdTomato is shown in red. One single Z-plane was streamed at 100 ms intervals for a total of 100 frames. The white arrowhead indicates a transcription site in the nucleus, brighter than single spots in the cytoplasm. The blue arrowheads indicate single mRNAs in the cytoplasm. Scale bar 3 μm (MP4 136 kb)

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Pichon, X., Robert, MC., Bertrand, E., Singer, R.H., Tutucci, E. (2020). New Generations of MS2 Variants and MCP Fusions to Detect Single mRNAs in Living Eukaryotic Cells. In: Heinlein, M. (eds) RNA Tagging. Methods in Molecular Biology, vol 2166. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0712-1_7

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  • DOI: https://doi.org/10.1007/978-1-0716-0712-1_7

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0711-4

  • Online ISBN: 978-1-0716-0712-1

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