Imaging single mRNAs to study dynamics of mRNA export in the yeast Saccharomyces cerevisiae
| Autor: | Pierre Bensidoun, Daniel Zenklusen, Pascal Raymond, Marlene Oeffinger |
|---|---|
| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Transcription Genetic RNA Stability Recombinant Fusion Proteins Green Fluorescent Proteins Saccharomyces cerevisiae Active Transport Cell Nucleus Biology RNA Transport General Biochemistry Genetics and Molecular Biology 03 medical and health sciences Transcription (biology) Gene Expression Regulation Fungal Gene expression P-bodies Protein biosynthesis RNA Messenger Molecular Biology Messenger RNA Staining and Labeling RNA RNA Fungal biology.organism_classification Molecular biology Single Molecule Imaging Yeast Cell biology 030104 developmental biology Microscopy Fluorescence Protein Biosynthesis Capsid Proteins |
| Zdroj: | Methods. 98:104-114 |
| ISSN: | 1046-2023 |
| DOI: | 10.1016/j.ymeth.2016.01.006 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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