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RiboSys, a high-resolution, quantitative approach to measure the in vivo kinetics of pre-mRNA splicing and 3'-end processing in Saccharomyces cerevisiae.
Alexander, Ross D; Barrass, J David; Dichtl, Beatriz; Kos, Martin; Obtulowicz, Tomasz; Robert, Marie-Cecile; Koper, Michal; Karkusiewicz, Iwona; Mariconti, Luisa; Tollervey, David; Dichtl, Bernhard; Kufel, Joanna; Bertrand, Edouard; Beggs, Jean D.
Afiliação
  • Alexander RD; Wellcome Trust Centre for Cell Biology, University of Edinburgh, King's Buildings, Edinburgh EH9 3JR, United Kingdom.
RNA ; 16(12): 2570-80, 2010 Dec.
Article em En | MEDLINE | ID: mdl-20974745
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.
Assuntos

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Saccharomyces cerevisiae / Precursores de RNA / Splicing de RNA / Genes Reporter / Reação em Cadeia da Polimerase Via Transcriptase Reversa / Processamento de Terminações 3' de RNA Tipo de estudo: Diagnostic_studies / Evaluation_studies Idioma: En Revista: RNA Assunto da revista: BIOLOGIA MOLECULAR Ano de publicação: 2010 Tipo de documento: Article País de afiliação: Reino Unido

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Saccharomyces cerevisiae / Precursores de RNA / Splicing de RNA / Genes Reporter / Reação em Cadeia da Polimerase Via Transcriptase Reversa / Processamento de Terminações 3' de RNA Tipo de estudo: Diagnostic_studies / Evaluation_studies Idioma: En Revista: RNA Assunto da revista: BIOLOGIA MOLECULAR Ano de publicação: 2010 Tipo de documento: Article País de afiliação: Reino Unido