Permanent URL to this publication: http://dx.doi.org/10.5167/uzh-36719
Alexander, R D; Barrass, J D; Dichtl, B; Kos, M; Obtulowicz, T; Robert, M C; Koper, M; Karkusiewicz, I; Mariconti, L; Tollervey, D; Dichtl, B; Kufel, J; Bertrand, E; Beggs, J D (2010). RiboSys, a high-resolution, quantitative approach to measure the in vivo kinetics of pre-mRNA splicing and 3'-end processing in Saccharomyces cerevisiae. RNA, 16:2570-2580.
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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.
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|Item Type:||Journal Article, refereed, original work|
|Communities & Collections:||07 Faculty of Science > Institute of Molecular Life Sciences|
|Dewey Decimal Classification:||570 Life sciences; biology|
|Date:||25 October 2010|
|Deposited On:||09 Nov 2010 17:10|
|Last Modified:||21 Jul 2014 06:35|
|Publisher:||Cold Spring Harbor Laboratory Press|
|Additional Information:||Open Access article|
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