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

• Published in: RNA (Cambridge) Vol. 16; no. 12; pp. 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: Journal Article
• Language: English
• Published: United States Cold Spring Harbor Laboratory Press 01.12.2010
• 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; Computer-Assisted In Situ Hybridization; Genetic RNA 3' End Processing/genetics/physiology RNA Precursors/analysis/genetics/metabolism RNA Splicing/genetics Reverse Transcriptase Polymerase Chain Reaction/methods Saccharomyces cerevisiae/genetics/metabolism; Fluorescence/methods Kinetics Models; Biological Models; Algorithms Evaluation Studies as Topic Genes; Reporter Image Processing
• ISSN: 1355-8382; 1469-9001
• DOI: 10.1261/rna.2162610

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.