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Abstract / Description of output
We describe methods for obtaining a quantitative description of RNA processing at high resolution in budding yeast. As a model gene expression system, we constructed tetON (for induction studies) and tetOFF (for repression, derepression, and RNA degradation studies) yeast strains with a series of reporter genes integrated in the genome under the control of a tetO7 promoter. Reverse transcription and quantitative real-time-PCR (RT-qPCR) methods were adapted to allow the determination of mRNA abundance as the average number of copies per cell in a population. Fluorescence in situ hybridization (FISH) measurements of transcript numbers in individual cells validated the RT-qPCR approach for the average copy-number determination despite the broad distribution of transcript levels within a population of cells. In addition, RT-qPCR was used to distinguish the products of the different steps in splicing of the reporter transcripts, and methods were developed to map and quantify 39-end cleavage and polyadenylation. This system permits pre-mRNA production, splicing, 39-end maturation and degradation to be quantitatively monitored with unprecedented kinetic detail, suitable for mathematical modeling. Using this approach, we demonstrate that reporter transcripts are spliced prior to their 39-end cleavage and polyadenylation, that is, cotranscriptionally.
Original language | English |
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Pages (from-to) | 2570-2580 |
Number of pages | 11 |
Journal | RNA |
Volume | 16 |
Issue number | 12 |
DOIs | |
Publication status | Published - Dec 2010 |
Keywords / Materials (for Non-textual outputs)
- single-molecule FISH
- RNA quantification
- splicing
- transcription
- yeast
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Dive into the research topics of 'RiboSys, a high-resolution, quantitative approach to measure the in vivo kinetics of pre-mRNA splicing and 3 '-end processing in Saccharomyces cerevisiae'. Together they form a unique fingerprint.Projects
- 1 Finished
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SynthSys; formerly CSBE: Centre for Systems Biology at Edinburgh
Millar, A., Beggs, J., Ghazal, P., Goryanin, I., Hillston, J., Plotkin, G., Tollervey, D., Walton, A. & Robertson, K.
8/01/07 → 31/12/12
Project: Research