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

Ross D. Alexander, J. David Barrass, Beatriz Dichtl, Martin Kos, Tomasz Obtulowicz, Marie-Cecile Robert, Michal Koper, Iwona Karkusiewicz, Luisa Mariconti, David Tollervey, Bernhard Dichtl, Joanna Kufel, Edouard Bertrand, Jean D. Beggs

Research output: Contribution to journalArticle

40 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)2570-2580
Number of pages11
JournalRNA
Volume16
Issue number12
DOIs
Publication statusPublished - Dec 2010

Keywords

  • Algorithms
  • Evaluation Studies as Topic
  • Genes, Reporter
  • Image Processing, Computer-Assisted
  • In Situ Hybridization, Fluorescence/methods
  • Kinetics
  • Models, Biological
  • Models, Genetic
  • RNA 3' End Processing/genetics
  • RNA Precursors/analysis
  • RNA Splicing/genetics
  • Reverse Transcriptase Polymerase Chain Reaction/methods
  • Saccharomyces cerevisiae/genetics

Fingerprint 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.

Cite this