Single-mRNA detection in living S. cerevisiae using a re-engineered MS2 system.

Area of Science:

• Molecular Biology
• Cell Biology
• Genetics

Background:

• The MS2 system is crucial for imaging single mRNAs in various organisms.
• Previous MS2 systems caused mRNA degradation, leading to inaccurate expression and localization data.
• Accurate mRNA detection is vital for understanding gene regulation.

Purpose of the Study:

• To describe a protocol for a re-engineered MS2 system (MBSV6) for detecting endogenous mRNAs in living yeast.
• To provide a method for accurate single mRNA imaging without affecting mRNA stability.
• To enable the study of all mRNA expression phases in Saccharomyces cerevisiae.

Main Methods:

• Endogenous gene tagging with the MBSV6 system.
• Two-color single-molecule RNA fluorescent in situ hybridization (smFISH) to validate mRNA behavior.
• Live imaging using wide-field microscopy for single mRNA quantification.

Main Results:

• The MBSV6 system allows for precise imaging of single mRNAs in living yeast.
• The re-engineered system does not significantly affect mRNA degradation.
• The protocol enables quantitative assessment of mRNA dynamics.

Conclusions:

• The MBSV6 system offers a robust method for studying mRNA expression in yeast.
• This approach can be extended to other organisms like E. coli, Drosophila, C. elegans, and mammalian cells.
• Accurate mRNA imaging facilitates a comprehensive understanding of gene expression regulation.