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 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.
Alexander
Ross D
RD
Wellcome Trust Centre for Cell Biology, University of Edinburgh, King's Buildings, Edinburgh EH9 3JR, United Kingdom.
Barrass
J David
JD
Dichtl
Beatriz
B
Kos
Martin
M
Obtulowicz
Tomasz
T
Robert
Marie-Cecile
MC
Koper
Michal
M
Karkusiewicz
Iwona
I
Mariconti
Luisa
L
Tollervey
David
D
Dichtl
Bernhard
B
Kufel
Joanna
J
Bertrand
Edouard
E
Beggs
Jean D
JD
D000465:Algorithms; D005069:Evaluation Studies as Topic; D017930:Genes, Reporter; D007091:Image Processing, Computer-Assisted; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D007700:Kinetics; D008954:Models, Biological; D008957:Models, Genetic; D026721:RNA 3' End Processing / Q000235:genetics* / Q000502:physiology; D012322:RNA Precursors / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D012326:RNA Splicing / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism
D012322:RNA Precursors