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Methods. 2016 Apr 1;98:104-114. doi: 10.1016/j.ymeth.2016.01.006. Epub 2016 Jan 16.

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

Author information

1
Département de Biochimieet médecine moléculaire, Faculté de médecine, Université de Montréal, Montréal, Québec H3C 3J7, ​Canada; Institut de recherches cliniques de Montréal, 110 Avenue des Pins Ouest, Montréal, Québec H2W 1R7, Canada.
2
Département de Biochimieet médecine moléculaire, Faculté de médecine, Université de Montréal, Montréal, Québec H3C 3J7, ​Canada.
3
Département de Biochimieet médecine moléculaire, Faculté de médecine, Université de Montréal, Montréal, Québec H3C 3J7, ​Canada; Institut de recherches cliniques de Montréal, 110 Avenue des Pins Ouest, Montréal, Québec H2W 1R7, Canada; Faculty of Medicine, Division of Experimental Medicine, McGill University, Montréal, Québec H3A 1A3, Canada.
4
Département de Biochimieet médecine moléculaire, Faculté de médecine, Université de Montréal, Montréal, Québec H3C 3J7, ​Canada. Electronic address: daniel.r.zenklusen@umontreal.ca.

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.

KEYWORDS:

PP7; Single molecule imaging; Yeast; mRNA; mRNA export

PMID:
26784711
DOI:
10.1016/j.ymeth.2016.01.006
[Indexed for MEDLINE]

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