Quick Search:

uzh logo
Browse by:
bullet
bullet
bullet
bullet

Zurich Open Repository and Archive

Permanent URL to this publication: http://dx.doi.org/10.5167/uzh-36719

Alexander, R D; Barrass, J D; Dichtl, B; Kos, M; Obtulowicz, T; Robert, M C; Koper, M; Karkusiewicz, I; Mariconti, L; Tollervey, D; Dichtl, B; Kufel, J; Bertrand, E; Beggs, J 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, 16:2570-2580.

[img]
Preview
PDF
1MB

View at publisher

Abstract

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.

Citations

17 citations in Web of Science®
19 citations in Scopus®
Google Scholar™

Altmetrics

Downloads

34 downloads since deposited on 09 Nov 2010
18 downloads since 12 months

Detailed statistics

Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:07 Faculty of Science > Institute of Molecular Life Sciences
DDC:570 Life sciences; biology
Language:English
Date:25 October 2010
Deposited On:09 Nov 2010 17:10
Last Modified:21 Jul 2014 06:35
Publisher:Cold Spring Harbor Laboratory Press
ISSN:1355-8382
Additional Information:Open Access article
Publisher DOI:10.1261/rna.2162610
PubMed ID:20974745

Users (please log in): suggest update or correction for this item

Repository Staff Only: item control page