RiboSys, a high-resolution, quantitative approach to measure the in vivo kinetics of pre-mRNA splicing and 3'-end processing in Saccharomyces cerevisiae
Data(s) |
01/01/2010
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Resumo |
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. <br /> |
Identificador | |
Idioma(s) |
eng |
Publicador |
Cold Spring Harbor Laboratory Press |
Relação |
http://dro.deakin.edu.au/eserv/DU:30039344/dichtl-ribosys-2010.pdf http://dx.doi.org/10.1261/rna.2162610 |
Direitos |
2010, Cold Spring Harbor Laboratory Press |
Palavras-Chave | #single-molecule #yeast #transcription #splicing #RNA quantification #FISH |
Tipo |
Journal Article |