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.

[1]  Ross D. Alexander,et al.  Processivity and Coupling in Messenger RNA Transcription , 2010, PloS one.

[2]  J. François,et al.  Validation of reference genes for quantitative expression analysis by real-time RT-PCR in Saccharomyces cerevisiae , 2009, BMC Molecular Biology.

[3]  D. Bentley,et al.  "Cotranscriptionality": the transcription elongation complex as a nexus for nuclear transactions. , 2009, Molecular cell.

[4]  R. Padgett,et al.  Rates of in situ transcription and splicing in large human genes , 2009, Nature Structural &Molecular Biology.

[5]  D. Tollervey,et al.  The Many Pathways of RNA Degradation , 2009, Cell.

[6]  D. Larson,et al.  Single-RNA counting reveals alternative modes of gene expression in yeast , 2008, Nature Structural &Molecular Biology.

[7]  S. Chávez,et al.  Systems for applied gene control in Saccharomyces cerevisiae , 2008, Biotechnology Letters.

[8]  A. Kornblihtt,et al.  The transcriptional cycle of HIV-1 in real-time and live cells. , 2007, The Journal of cell biology.

[9]  Joaquín Moreno,et al.  Genomics and gene transcription kinetics in yeast. , 2007, Trends in genetics : TIG.

[10]  Farren J. Isaacs,et al.  Phenotypic consequences of promoter-mediated transcriptional noise. , 2006, Molecular cell.

[11]  Daniel F Tardiff,et al.  A genome-wide analysis indicates that yeast pre-mRNA splicing is predominantly posttranscriptional. , 2006, Molecular cell.

[12]  Michael J. Osborn,et al.  The relative merits of the tetO2 and tetO7 promoter systems for the functional analysis of heterologous genes in yeast and a compilation of essential yeast genes with tetO2 promoter substitutions , 2006, Yeast.

[13]  S. Lacadie,et al.  Cotranscriptional spliceosome assembly dynamics and the role of U1 snRNA:5'ss base pairing in yeast. , 2005, Molecular cell.

[14]  K. Neugebauer,et al.  Cotranscriptional spliceosome assembly occurs in a stepwise fashion and requires the cap binding complex. , 2005, Molecular cell.

[15]  S. Oliver,et al.  Doxycycline, the drug used to control the tet‐regulatable promoter system, has no effect on global gene expression in Saccharomyces cerevisiae , 2005, Yeast.

[16]  J. Pérez-Ortín,et al.  Genomic run-on evaluates transcription rates for all yeast genes and identifies gene regulatory mechanisms. , 2004, Molecular cell.

[17]  T. Hughes,et al.  Exploration of Essential Gene Functions via Titratable Promoter Alleles , 2004, Cell.

[18]  O. Gadal,et al.  Nuclear Retention of Unspliced mRNAs in Yeast Is Mediated by Perinuclear Mlp1 , 2004, Cell.

[19]  R. Parker,et al.  Cytoplasmic degradation of splice-defective pre-mRNAs and intermediates. , 2003, Molecular cell.

[20]  S. Oliver,et al.  An improved tetO promoter replacement system for regulating the expression of yeast genes , 2003, Yeast.

[21]  Roy Parker,et al.  Computational Modeling and Experimental Analysis of Nonsense-Mediated Decay in Yeast , 2003, Cell.

[22]  M. Holland,et al.  Transcript Abundance in Yeast Varies over Six Orders of Magnitude* , 2002, The Journal of Biological Chemistry.

[23]  R Parker,et al.  Computational modeling of eukaryotic mRNA turnover. , 2001, RNA.

[24]  R. Parker,et al.  The Transcription Factor Associated Ccr4 and Caf1 Proteins Are Components of the Major Cytoplasmic mRNA Deadenylase in Saccharomyces cerevisiae , 2001, Cell.

[25]  D. Tollervey,et al.  Identification of a Regulated Pathway for Nuclear Pre-mRNA Turnover , 2000, Cell.

[26]  L. Minvielle-Sebastia,et al.  mRNA polyadenylation and its coupling to other RNA processing reactions and to transcription. , 1999, Current opinion in cell biology.

[27]  G. Bellí,et al.  Functional analysis of yeast essential genes using a promoter‐substitution cassette and the tetracycline‐regulatable dual expression system , 1998, Yeast.

[28]  F S Fay,et al.  Visualization of single RNA transcripts in situ. , 1998, Science.

[29]  G. Bellí,et al.  An activator/repressor dual system allows tight tetracycline-regulated gene expression in budding yeast. , 1998, Nucleic acids research.

[30]  D. Tollervey,et al.  Lithium toxicity in yeast is due to the inhibition of RNA processing enzymes , 1997, The EMBO journal.

[31]  M Aldea,et al.  A Set of Vectors with a Tetracycline‐Regulatable Promoter System for Modulated Gene Expression in Saccharomyces cerevisiae , 1997, Yeast.

[32]  W. Hess,et al.  Precise branch point mapping and quantification of splicing intermediates. , 1997, Nucleic acids research.

[33]  Wei Zhou,et al.  Characterization of the Yeast Transcriptome , 1997, Cell.

[34]  V. Iyer,et al.  Absolute mRNA levels and transcriptional initiation rates in Saccharomyces cerevisiae. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[35]  M. Gossen,et al.  Transcriptional activation by tetracyclines in mammalian cells. , 1995, Science.

[36]  R. Müller,et al.  Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. , 1995, Gene.

[37]  R. Parker,et al.  Translation and a 42-nucleotide segment within the coding region of the mRNA encoded by the MAT alpha 1 gene are involved in promoting rapid mRNA decay in yeast. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[38]  D. Tranchina,et al.  Mechanistic model of bursts in mRNA synthesis , 2006 .