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 journalArticlepeer-review

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 39-end cleavage and polyadenylation. This system permits pre-mRNA production, splicing, 39-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 39-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

  • single-molecule FISH
  • RNA quantification
  • splicing
  • transcription
  • yeast

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 <em>Saccharomyces cerevisiae</em>'. Together they form a unique fingerprint.

Cite this