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.
RiboSys, a high-resolution, quantitative approach to measure the in vivo kinetics of pre-mRNA splicing and 3′-end processing in Saccharomyces cerevisiae
Alexander, R. D.; Barrass, J. D.; Dichtl, B.; Kos, M.; Obtulowicz, T.; Robert, M. C.; Koper, M.; Karkusiewicz, I.; Mariconti, L.; Tollervey, D.; Kufel, J.; Bertrand, E.; Beggs, J. D.
2010-12 / vol 16 / pages 2570-80
1469-9001 (Electronic) 1355-8382 (Linking)
Models, Biological; Image Processing, Computer-Assisted; Models, Genetic; RNA Splicing/*genetics; Algorithms; Kinetics; *Genes, Reporter; *Saccharomyces cerevisiae/genetics/metabolism; Evaluation Studies as Topic; In Situ Hybridization, Fluorescence/methods; Reverse Transcriptase Polymerase Chain Reaction/*methods; RNA 3' End Processing/*genetics/physiology; RNA Precursors/analysis/genetics/*metabolism