Establishing MS2-MCP-based single-molecule RNA visualization in Schizosaccharomyces pombe.

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.