RiboSys, a high-resolution, quantitative approach to measure the in vivo kinetics of pre-mRNA splicing and 3'-end processing in Saccharomyces cerevisiae

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 re...

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Bibliographic Details
Published in:RNA (Cambridge) 2010-12, Vol.16 (12), p.2570-2580
Main Authors: Alexander, Ross D, Barrass, J David, Dichtl, Beatriz, Kos, Martin, Obtulowicz, Tomasz, Robert, Marie-Cecile, Koper, Michal, Karkusiewicz, Iwona, Mariconti, Luisa, Tollervey, David, Dichtl, Bernhard, Kufel, Joanna, Bertrand, Edouard, Beggs, Jean D
Format: Article
Language:English
Subjects:
Algorithms
Biochemistry, Molecular Biology
Evaluation Studies as Topic
Genes, Reporter
Image Processing, Computer-Assisted
In Situ Hybridization, Fluorescence - methods
Kinetics
Life Sciences
Method
Models, Biological
Models, Genetic
Reverse Transcriptase Polymerase Chain Reaction - methods
RNA 3' End Processing - genetics
RNA 3' End Processing - physiology
RNA Precursors - analysis
RNA Precursors - genetics
RNA Precursors - metabolism
RNA Splicing - genetics
Saccharomyces cerevisiae
Saccharomyces cerevisiae - genetics
Saccharomyces cerevisiae - metabolism
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Summary: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 3'-end cleavage and polyadenylation. This system permits pre-mRNA production, splicing, 3'-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 3'-end cleavage and polyadenylation, that is, cotranscriptionally.
ISSN:1355-8382
1469-9001
DOI:10.1261/rna.2162610