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 re...
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Published in | RNA (Cambridge) Vol. 16; no. 12; pp. 2570 - 2580 |
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Main Authors | , , , , , , , , , , , , , |
Format | Journal Article |
Language | English |
Published |
United States
Cold Spring Harbor Laboratory Press
01.12.2010
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Subjects | |
Online Access | Get full text |
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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. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 ObjectType-Article-2 ObjectType-Feature-1 PMCID: PMC2995417 These authors contributed equally to this work. Present address: Biochemie-Zentrum der Universität Heidelberg (BZH), Im Neuenheimer Feld 328, 69120 Heidelberg, Germany. Reprint requests to: Jean D. Beggs, Wellcome Trust Centre for Cell Biology, University of Edinburgh, King's Buildings, Edinburgh EH9 3JR, UK; e-mail: jbeggs@ed.ac.uk; fax: 44 131 6508650. |
ISSN: | 1355-8382 1469-9001 |
DOI: | 10.1261/rna.2162610 |