@article {46716, title = {Single-molecule analysis of gene expression using two-color RNA labeling in live yeast.}, journal = {Nature Methods}, volume = {10}, year = {2013}, month = {2013 Feb}, pages = {119-21}, abstract = {

Live-cell imaging of mRNA yields important insights into gene expression, but it has generally been limited to the labeling of one RNA species and has never been used to count single mRNAs over time in yeast. We demonstrate a two-color imaging system with single-molecule resolution using MS2 and PP7 RNA labeling. We use this methodology to measure intrinsic noise in mRNA levels and RNA polymerase II kinetics at a single gene.

}, keywords = {Adenosine Triphosphatases, Gene Expression, In Situ Hybridization, Fluorescence, Levivirus, RNA Polymerase II, RNA, Fungal, RNA, Messenger, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins}, issn = {1548-7105}, doi = {10.1038/nmeth.2305}, author = {Hocine, Sami and Raymond, Pascal and Zenklusen, Daniel and Chao, Jeffrey A and Singer, Robert H} } @article {46725, title = {Single-mRNA counting using fluorescent in situ hybridization in budding yeast.}, journal = {Nature Protocols}, volume = {7}, year = {2012}, month = {2012 Feb 2}, pages = {408-19}, abstract = {

Fluorescent in situ hybridization (FISH) allows the quantification of single mRNAs in budding yeast using fluorescently labeled single-stranded DNA probes, a wide-field epifluorescence microscope and a spot-detection algorithm. Fixed yeast cells are attached to coverslips and hybridized with a mixture of FISH probes, each conjugated to several fluorescent dyes. Images of cells are acquired in 3D and maximally projected for single-molecule analysis. Diffraction-limited labeled mRNAs are observed as bright fluorescent spots and can be quantified using a spot-detection algorithm. FISH preserves the spatial distribution of cellular RNA distribution within the cell and the stochastic fluctuations in individual cells that can lead to phenotypic differences within a clonal population. This information, however, is lost if the RNA content is measured on a population of cells by using reverse transcriptase PCR, microarrays or high-throughput sequencing. The FISH procedure and image acquisition described here can be completed in 3 d.

}, keywords = {Algorithms, Antisense Elements (Genetics), In Situ Hybridization, Fluorescence, RNA, Fungal, RNA, Messenger, Saccharomyces cerevisiae}, issn = {1750-2799}, doi = {10.1038/nprot.2011.451}, author = {Trcek, Tatjana and Chao, Jeffrey A and Larson, Daniel R and Park, Hye Yoon and Zenklusen, Daniel and Shenoy, Shailesh M and Singer, Robert H} } @article {46741, title = {A nucleoporin, Nup60p, affects the nuclear and cytoplasmic localization of ASH1 mRNA in S. cerevisiae.}, journal = {RNA}, volume = {17}, year = {2011}, month = {2011 Jan}, pages = {134-44}, abstract = {

The biogenesis of a localization-competent mRNP begins in the nucleus. It is thought that the coordinated action of nuclear and cytoplasmic components of the localization machinery is required for the efficient export and subsequent subcellular localization of these mRNAs in the cytoplasm. Using quantitative poly(A)(+) and transcript-specific fluorescent in situ hybridization, we analyzed different nonessential nucleoporins and nuclear pore-associated proteins for their potential role in mRNA export and localization. We found that Nup60p, a nuclear pore protein located on the nucleoplasmic side of the nuclear pore complex, was required for the mRNA localization pathway. In a Δnup60 background, localized mRNAs were preferentially retained within the nucleus compared to nonlocalized transcripts. However, the export block was only partial and some transcripts could still reach the cytoplasm. Importantly, downstream processes were also affected. Localization of ASH1 and IST2 mRNAs to the bud was impaired in the Δnup60 background, suggesting that the assembly of a localization competent mRNP ("locasome") was inhibited when NUP60 was deleted. These results demonstrate transcript specificity of a nuclear mRNA retention defect and identify a specific nucleoporin as a functional component of the localization pathway in budding yeast.

}, keywords = {Cell Nucleus, Cytoplasm, Gene Deletion, In Situ Hybridization, Fluorescence, Nuclear Pore Complex Proteins, Poly A, Repressor Proteins, RNA Transport, RNA, Fungal, RNA, Messenger, RNA-Binding Proteins, Saccharomyces cerevisiae Proteins, Transcription, Genetic}, issn = {1469-9001}, doi = {10.1261/rna.1210411}, author = {Powrie, Erin A and Zenklusen, Daniel and Singer, Robert H} } @article {6596, title = {Real-time observation of transcription initiation and elongation on an endogenous yeast gene.}, journal = {Science}, volume = {332}, year = {2011}, month = {2011 Apr 22}, pages = {475-8}, abstract = {

Cellular messenger RNA levels are achieved by the combinatorial complexity of factors controlling transcription, yet the small number of molecules involved in these pathways fluctuates stochastically. It has not yet been experimentally possible to observe the activity of single polymerases on an endogenous gene to elucidate how these events occur in vivo. Here, we describe a method of fluctuation analysis of fluorescently labeled RNA to measure dynamics of nascent RNA{\textendash}including initiation, elongation, and termination{\textendash}at an active yeast locus. We find no transcriptional memory between initiation events, and elongation speed can vary by threefold throughout the cell cycle. By measuring the abundance and intranuclear mobility of an upstream transcription factor, we observe that the gene firing rate is directly determined by trans-activating factor search times.

}, keywords = {Adenosine Triphosphatases, Cell Cycle, Cell Nucleus, DNA Polymerase I, Facilitated Diffusion, Fluorescence, Fungal, Genes, Genetic, Glutamate Synthase, Green Fluorescent Proteins, Kinetics, Messenger, Microscopy, Models, Promoter Regions, RNA, RNA Polymerase II, RNA Precursors, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Spectrometry, Transcription, Transcription Factors, Transcriptional Activation}, issn = {1095-9203}, doi = {10.1126/science.1202142}, author = {Larson, Daniel R and Zenklusen, Daniel and Wu, Bin and Chao, Jeffrey A and Singer, Robert H} } @article {46740, title = {Transcription of functionally related constitutive genes is not coordinated.}, journal = {Nature Structural \& Molecular Biology}, volume = {18}, year = {2011}, month = {2011 Jan}, pages = {27-34}, abstract = {

Expression of an individual gene can vary considerably among genetically identical cells because of stochastic fluctuations in transcription. However, proteins comprising essential complexes or pathways have similar abundances and lower variability. It is not known whether coordination in the expression of subunits of essential complexes occurs at the level of transcription, mRNA abundance or protein expression. To directly measure the level of coordination in the expression of genes, we used highly sensitive fluorescence in situ hybridization (FISH) to count individual mRNAs of functionally related and unrelated genes within single Saccharomyces cerevisiae cells. Our results revealed that transcript levels of temporally induced genes are highly correlated in individual cells. In contrast, transcription of constitutive genes encoding essential subunits of complexes is not coordinated because of stochastic fluctuations. The coordination of these functional complexes therefore must occur post-transcriptionally, and likely post-translationally.

}, keywords = {Gene Expression Regulation, In Situ Hybridization, Fluorescence, Models, Genetic, RNA, Messenger, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Transcription, Genetic}, issn = {1545-9985}, doi = {10.1038/nsmb.1934}, author = {Gandhi, Saumil J and Zenklusen, Daniel and Lionnet, Timoth{\'e}e and Singer, Robert H} } @article {2390, title = {Imaging transcription in living cells.}, journal = {Annual Review of Biophysics}, volume = {38}, year = {2009}, month = {2009 Feb 3}, pages = {173-96}, abstract = {

The advent of new technologies for the imaging of living cells has made it possible to determine the properties of transcription, the kinetics of polymerase movement, the association of transcription factors, and the progression of the polymerase on the gene. We report here the current state of the field and the progress necessary to achieve a more complete understanding of the various steps in transcription. Our Consortium is dedicated to developing and implementing the technology to further this understanding.

}, keywords = {Computer-Assisted, Fluorescence, Gene Expression Profiling, Image Interpretation, Microscopy, Transcription Factors, Transcriptional Activation}, issn = {1936-122X}, doi = {10.1073/pnas.1100640108}, author = {Darzacq, Xavier and Yao, Jie and Larson, Daniel R and Causse, Sebastien Z and Bosanac, Lana and de Turris, Valeria and Ruda, Vera M and Lionnet, Timoth{\'e}e and Zenklusen, Daniel and Guglielmi, Benjamin and Tjian, Robert and Singer, Robert H} }