Imaging single mRNAs to study dynamics of mRNA export in the yeast Saccharomyces cerevisiae

Methods : a Companion to Methods in Enzymology
Pierre BensidounDaniel Zenklusen

Abstract

Regulation of mRNA and protein expression occurs at many levels, initiated at transcription and followed by mRNA processing, export, localization, translation and mRNA degradation. The ability to study mRNAs in living cells has become a critical tool to study and analyze how the various steps of the gene expression pathway are carried out. Here we describe a detailed protocol for real time fluorescent RNA imaging using the PP7 bacteriophage coat protein, which allows mRNA detection with high spatial and temporal resolution in the yeast Saccharomyces cerevisiae, and can be applied to study various stages of mRNA metabolism. We describe the different parameters required for quantitative single molecule imaging in yeast, including strategies for genomic integration, expression of a PP7 coat protein GFP fusion protein, microscope setup and analysis strategies. We illustrate the method's use by analyzing the behavior of nuclear mRNA in yeast and the role of the nuclear basket in mRNA export.

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Citations

Mar 2, 2016·Methods : a Companion to Methods in Enzymology·Yaron Shav-Tal
Dec 30, 2017·Nucleus·Evgeny SmirnovIvan Raška
Feb 13, 2018·Biochemical Society Transactions·Neal K Williams, Bernhard Dichtl
Mar 3, 2020·PloS One·Evgeny SmirnovDušan Cmarko
Jun 17, 2020·Wiley Interdisciplinary Reviews. RNA·Fatu Badiane MarkeyMona Batish
Sep 20, 2021·Journal of Molecular Biology·Nitesh Kumar PodhGunjan Mehta

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