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

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

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Active Transport, Cell Nucleus
  • Capsid Proteins / genetics
  • Capsid Proteins / metabolism
  • Gene Expression Regulation, Fungal*
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Microscopy, Fluorescence / methods
  • Protein Biosynthesis
  • RNA Stability
  • RNA Transport
  • RNA, Fungal / chemistry*
  • RNA, Fungal / genetics
  • RNA, Fungal / metabolism
  • RNA, Messenger / chemistry*
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae / ultrastructure*
  • Single Molecule Imaging / methods*
  • Staining and Labeling / methods*
  • Transcription, Genetic

Substances

  • Capsid Proteins
  • RNA, Fungal
  • RNA, Messenger
  • Recombinant Fusion Proteins
  • Green Fluorescent Proteins