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

Alexander, Ross D., Barrass, J. David, Dichtl, Beatriz, Kos, Martin, Obtulowicz, Tomasz, Robert, Marie-Cecile, Koper, Michal, Karkusiewicz, Iwona, Mariconti, Luisa, Tollervey, David, Dichtl, Bernhard, Kufel, Joanna, Bertrand, Edouard and Beggs, Jean 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, vol. 16, no. 12, pp. 2570-2580, doi: 10.1261/rna.2162610.

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Title RiboSys, a high-resolution, quantitative approach to measure the in vivo kinetics of pre-mRNA splicing and 3'-end processing in Saccharomyces cerevisiae
Author(s) Alexander, Ross D.
Barrass, J. David
Dichtl, Beatriz
Kos, Martin
Obtulowicz, Tomasz
Robert, Marie-Cecile
Koper, Michal
Karkusiewicz, Iwona
Mariconti, Luisa
Tollervey, David
Dichtl, BernhardORCID iD for Dichtl, Bernhard orcid.org/0000-0001-5514-4982
Kufel, Joanna
Bertrand, Edouard
Beggs, Jean D.
Journal name RNA
Volume number 16
Issue number 12
Start page 2570
End page 2580
Total pages 11
Publisher Cold Spring Harbor Laboratory Press
Place of publication Woodbury, N.Y.
Publication date 2010
ISSN 1355-8382
1469-9001
Keyword(s) single-molecule
yeast
transcription
splicing
RNA quantification
FISH
Summary 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.
Language eng
DOI 10.1261/rna.2162610
Field of Research 060199 Biochemistry and Cell Biology not elsewhere classified
Socio Economic Objective 970106 Expanding Knowledge in the Biological Sciences
HERDC Research category C1.1 Refereed article in a scholarly journal
Copyright notice ©2010, Cold Spring Harbor Laboratory Press
Persistent URL http://hdl.handle.net/10536/DRO/DU:30039344

Document type: Journal Article
Collection: School of Life and Environmental Sciences
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