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Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae
Published on: June 14, 2013
Establishing MS2-MCP-based single-molecule RNA visualization in Schizosaccharomyces pombe
Douglas E Weidemann1,2, Sarah C Turner1,2, Silke Hauf1,2
1Department of Biological Sciences, Virginia Tech, Blacksburg, VA 24061, USA.
Abstract:
Single-molecule RNA imaging using the MS2-MCP system has transformed the study of RNA biology across model organisms. However, this technology has remained unavailable for fission yeast (Schizosaccharomyces pombe), even though fission yeast is a central model for eukaryotic gene expression. Achieving single-molecule sensitivity requires identifying a narrow optimum where RNA labels are sufficiently bright while background fluorescence remains minimal. We have now accomplished this for S. pombe by systematically optimizing MCP expression and localization-screening a panel of constitutive S. pombe promoters and evaluating combinations of nuclear localization and export signals (NLSs and NESs). The resulting, successful constructs use tandem StayGold as the MCP fluorescent tag, taking advantage of its superior photostability. Together with optimized vectors for MS2 stem-loop tagging of endogenous transcripts, these tools enable single-molecule RNA imaging in fission yeast, opening the door to quantitative analyses of RNA dynamics in this core genetic model.
Insights
Researchers developed single-molecule RNA imaging for fission yeast using the MS2-MCP system. This breakthrough enables quantitative analysis of RNA dynamics in this key model organism.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- The MS2-MCP system revolutionized RNA biology by enabling single-molecule RNA imaging.
- Fission yeast (Schizosaccharomyces pombe) is a crucial model for eukaryotic gene expression but lacked this technology.
- Optimizing RNA labeling for brightness and minimal background is essential for single-molecule sensitivity.
Purpose of the Study:
- To adapt and optimize the MS2-MCP system for single-molecule RNA imaging in fission yeast.
- To enable quantitative analyses of RNA dynamics in Schizosaccharomyces pombe.
- To overcome technical challenges in achieving single-molecule sensitivity in fission yeast.
Main Methods:
- Systematic optimization of MCP (MS2 coat protein) expression and localization in S. pombe.
- Screening of constitutive S. pombe promoters for optimal MCP expression.
- Evaluation of nuclear localization signals (NLSs) and nuclear export signals (NESs) for MCP.
- Utilizing tandem StayGold as a photostable fluorescent tag for MCP.
Main Results:
- Successful adaptation of the MS2-MCP system for single-molecule RNA imaging in fission yeast.
- Identification of optimal MCP expression and localization strategies.
- Development of constructs utilizing tandem StayGold for enhanced signal stability.
- Creation of optimized vectors for MS2 stem-loop tagging of endogenous transcripts.
Conclusions:
- Single-molecule RNA imaging is now feasible in fission yeast (Schizosaccharomyces pombe).
- The developed tools facilitate quantitative studies of RNA dynamics in this model system.
- This advancement opens new avenues for understanding gene expression regulation in eukaryotes.
