Transcript counting in single cells reveals dynamics of rDNA transcription

Most eukaryotes contain many tandem repeats of ribosomal RNA genes of which only a subset is transcribed at any given time. Current biochemical methods allow for the determination of the fraction of transcribing repeats (ON) versus non‐transcribing repeats (OFF) but do not provide any dynamical information and obscure any transcription activity at the single‐cell level. Here, we use a fluorescence in situ hybridization (FISH) technique that allows the detection of single‐RNA molecules in individual yeast cells. We use this method complemented with theoretical modeling to determine the rate of switching from OFF to ON (activation rate) and the average number of RNA molecules produced during each transcriptional burst (burst size). We explore how these two variables change in mutants and different growth conditions, and show that this method resolves changes in these two variables even when the average rDNA expression is unaltered. These phenotypic changes could not have been detected by traditional biochemical assays.

[1]  M. Nomura,et al.  In Exponentially Growing Saccharomyces cerevisiae Cells, rRNA Synthesis Is Determined by the Summed RNA Polymerase I Loading Rate Rather than by the Number of Active Genes , 2003, Molecular and Cellular Biology.

[2]  J. Sogo,et al.  Replication of transcriptionally active chromatin , 1995, Nature.

[3]  Barbara L. Billington,et al.  Position effect at S. cerevisiae telomeres: Reversible repression of Pol II transcription , 1990, Cell.

[4]  I. Grummt.  Life on a planet of its own: regulation of RNA polymerase I transcription in the nucleolus. , 2003, Genes & development.

[5]  J. Warner,et al.  The economics of ribosome biosynthesis in yeast. , 1999, Trends in biochemical sciences.

[6]  Christopher M. Gallo,et al.  RPD3 is required for the inactivation of yeast ribosomal DNA genes in stationary phase , 2002, The EMBO journal.

[7]  R. Morse,et al.  Direct Role for the Rpd3 Complex in Transcriptional Induction of the Anaerobic DAN/TIR Genes in Yeast , 2007, Molecular and Cellular Biology.

[8]  O. Miller,et al.  Visualization of Nucleolar Genes , 1969, Science.

[9]  R. Dammann,et al.  Chromatin structures and transcription of rDNA in yeast Saccharomyces cerevisiae. , 1993, Nucleic acids research.

[10]  J. Sogo,et al.  Two different chromatin structures coexist in ribosomal RNA genes throughout the cell cycle , 1989, Cell.

[11]  D. Tranchina,et al.  Stochastic mRNA Synthesis in Mammalian Cells , 2006, PLoS biology.

[12]  K. Struhl,et al.  Targeted Recruitment of Rpd3 Histone Deacetylase Represses Transcription by Inhibiting Recruitment of Swi/Snf, SAGA, and TATA Binding Protein , 2002, Molecular and Cellular Biology.

[13]  James R Broach,et al.  Single-cell observations reveal intermediate transcriptional silencing states. , 2006, Molecular cell.

[14]  Han-kuei Huang,et al.  RNA Polymerase I-Promoted HIS4Expression Yields Uncapped, Polyadenylated mRNA That Is Unstable and Inefficiently Translated in Saccharomyces cerevisiae , 1998, Molecular and Cellular Biology.

[15]  N. Proudfoot,et al.  RNA polymerase I in yeast transcribes dynamic nucleosomal rDNA , 2007, Nature Structural &Molecular Biology.

[16]  M. Smerdon,et al.  Transcription-coupled repair in RNA polymerase I-transcribed genes of yeast , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[17]  Y. Nogi,et al.  Multiprotein transcription factor UAF interacts with the upstream element of the yeast RNA polymerase I promoter and forms a stable preinitiation complex. , 1996, Genes & development.

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

[19]  R. Dammann,et al.  Transcription in the yeast rRNA gene locus: distribution of the active gene copies and chromatin structure of their flanking regulatory sequences , 1995, Molecular and cellular biology.

[20]  C. Cadwell,et al.  The yeast nucleolar protein Cbf5p is involved in rRNA biosynthesis and interacts genetically with the RNA polymerase I transcription factor RRN3 , 1997, Molecular and cellular biology.