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Updated: Mar 27, 2026
Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae
Published on: June 14, 2013
Imaging single mRNAs to study dynamics of mRNA export in the yeast Saccharomyces cerevisiae
Pierre Bensidoun1, Pascal Raymond2, Marlene Oeffinger3
1Département 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.
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.
Insights
This study introduces a real-time fluorescent RNA imaging technique in yeast using PP7 bacteriophage coat protein for high-resolution mRNA analysis. This method enables detailed studies of gene expression and mRNA metabolism in living cells.
Area of Science:
- Molecular Biology
- Cell Biology
- Biophysics
Background:
- Gene expression regulation involves multiple steps from transcription to degradation.
- Studying messenger RNA (mRNA) in living cells is crucial for understanding gene expression dynamics.
- Existing methods may lack the spatial and temporal resolution needed for detailed mRNA metabolism studies.
Purpose of the Study:
- To present a detailed protocol for real-time fluorescent RNA imaging in yeast.
- To enable high-resolution, quantitative analysis of mRNA in living cells.
- To facilitate the study of various mRNA metabolism stages.
Main Methods:
- Utilized the PP7 bacteriophage coat protein system for mRNA detection.
- Developed strategies for genomic integration and expression of PP7 coat protein-GFP fusion proteins.
- Optimized microscopy setup and data analysis for quantitative single-molecule imaging in yeast (Saccharomyces cerevisiae).
Main Results:
- Established a robust protocol for real-time fluorescent RNA imaging in yeast.
- Achieved high spatial and temporal resolution for mRNA detection.
- Demonstrated the method's utility by analyzing nuclear mRNA behavior and its role in export.
Conclusions:
- The PP7 system provides a powerful tool for studying mRNA metabolism in living yeast.
- This technique offers new avenues for quantitative analysis of gene expression pathways.
- The method is applicable to diverse research questions in molecular and cell biology.
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