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RNA. 2010 Dec;16(12):2570-80. doi: 10.1261/rna.2162610. Epub 2010 Oct 25.

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

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  • 1Wellcome Trust Centre for Cell Biology, University of Edinburgh, King's Buildings, Edinburgh EH9 3JR, United Kingdom.

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.

PMID:
20974745
PMCID:
PMC2995417
DOI:
10.1261/rna.2162610
[PubMed - indexed for MEDLINE]
Free PMC Article
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