PMID- 20974745
OWN - NLM
STAT- MEDLINE
DCOM- 20101213
LR  - 20230525
IS  - 1469-9001 (Electronic)
IS  - 1355-8382 (Print)
IS  - 1355-8382 (Linking)
VI  - 16
IP  - 12
DP  - 2010 Dec
TI  - RiboSys, a high-resolution, quantitative approach to measure the in vivo kinetics 
      of pre-mRNA splicing and 3'-end processing in Saccharomyces cerevisiae.
PG  - 2570-80
LID - 10.1261/rna.2162610 [doi]
AB  - 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.
FAU - Alexander, Ross D
AU  - Alexander RD
AD  - Wellcome Trust Centre for Cell Biology, University of Edinburgh, King's 
      Buildings, Edinburgh EH9 3JR, United Kingdom.
FAU - Barrass, J David
AU  - Barrass JD
FAU - Dichtl, Beatriz
AU  - Dichtl B
FAU - Kos, Martin
AU  - Kos M
FAU - Obtulowicz, Tomasz
AU  - Obtulowicz T
FAU - Robert, Marie-Cecile
AU  - Robert MC
FAU - Koper, Michal
AU  - Koper M
FAU - Karkusiewicz, Iwona
AU  - Karkusiewicz I
FAU - Mariconti, Luisa
AU  - Mariconti L
FAU - Tollervey, David
AU  - Tollervey D
FAU - Dichtl, Bernhard
AU  - Dichtl B
FAU - Kufel, Joanna
AU  - Kufel J
FAU - Bertrand, Edouard
AU  - Bertrand E
FAU - Beggs, Jean D
AU  - Beggs JD
LA  - eng
GR  - WT_/Wellcome Trust/United Kingdom
GR  - BB/D019621/1/BB_/Biotechnology and Biological Sciences Research Council/United 
      Kingdom
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20101025
PL  - United States
TA  - RNA
JT  - RNA (New York, N.Y.)
JID - 9509184
RN  - 0 (RNA Precursors)
SB  - IM
MH  - Algorithms
MH  - Evaluation Studies as Topic
MH  - *Genes, Reporter
MH  - Image Processing, Computer-Assisted
MH  - In Situ Hybridization, Fluorescence/methods
MH  - Kinetics
MH  - Models, Biological
MH  - Models, Genetic
MH  - RNA 3' End Processing/*genetics/physiology
MH  - RNA Precursors/analysis/genetics/*metabolism
MH  - RNA Splicing/*genetics
MH  - Reverse Transcriptase Polymerase Chain Reaction/*methods
MH  - *Saccharomyces cerevisiae/genetics/metabolism
PMC - PMC2995417
EDAT- 2010/10/27 06:00
MHDA- 2010/12/14 06:00
PMCR- 2010/12/01
CRDT- 2010/10/27 06:00
PHST- 2010/10/27 06:00 [entrez]
PHST- 2010/10/27 06:00 [pubmed]
PHST- 2010/12/14 06:00 [medline]
PHST- 2010/12/01 00:00 [pmc-release]
AID - rna.2162610 [pii]
AID - RA [pii]
AID - 10.1261/rna.2162610 [doi]
PST - ppublish
SO  - RNA. 2010 Dec;16(12):2570-80. doi: 10.1261/rna.2162610. Epub 2010 Oct 25.