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

R. D. Alexander , J. D. Barrass , B. Dichtl , M. Kos , T. Obtulowicz , M. C. Robert , M. Koper , I. Karkusiewicz , L. Mariconti , D. Tollervey , J. Kufel , E. Bertrand (1) , J. D. Beggs

1 ERA 6 Risque sismique - Equipe recherche associée au LCPC - ERA 6 Risque sismique

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.

Mots clés: Algorithms Evaluation Studies as Topic *Genes; Biological Models; Computer-Assisted In Situ Hybridization; Fluorescence/methods Kinetics Models; 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; Reporter Image Processing

Domaines: Biochimie, Biologie Moléculaire

R. D. Alexander, J. D. Barrass, B. Dichtl, M. Kos, T. Obtulowicz, et al.. RiboSys, a high-resolution, quantitative approach to measure the in vivo kinetics of pre-mRNA splicing and 3'-end processing in Saccharomyces cerevisiae. RNA, 2010, 16 (12), pp.2570--80. ⟨10.1261/rna.2162610⟩. ⟨hal-02193430⟩