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

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. 10.1261/rna.2162610 Access the most recent version at doi: published online October 25, 2010 RNA Ross D. Alexander, J. David Barrass, Beatriz Dichtl, et al. Saccharomyces cerevisiae -end processing in ′ vivo kinetics of pre-mRNA splicing and 3 RiboSys, a high-resolution, quantitative approach to measure the in Material Supplemental http://rnajournal.cshlp.org/content/suppl/2010/10/13/rna.2162610.DC1.html P<P Published online October 25, 2010 in advance of the print journal. Open Access Freely available online through the RNA Open Access option. service Email alerting click here top right corner of the article or Receive free email alerts when new articles cite this article sign up in the box at the object identifier (DOIs) and date of initial publication. by PubMed from initial publication. Citations to Advance online articles must include the digital publication). Advance online articles are citable and establish publication priority; they are indexed appeared in the paper journal (edited, typeset versions may be posted when available prior to final Advance online articles have been peer reviewed and accepted for publication but have not yet http://rnajournal.cshlp.org/subscriptions go to: RNA To subscribe to Copyright © 2010 RNA Society Cold Spring Harbor Laboratory Press on November 1, 2010 Published by rnajournal.cshlp.org Downloaded from