@article {46699, title = {Dynamic visualization of transcription and RNA subcellular localization in zebrafish.}, journal = {Development}, volume = {142}, year = {2015}, month = {2015 Apr 1}, pages = {1368-74}, abstract = {

Live imaging of transcription and RNA dynamics has been successful in cultured cells and tissues of vertebrates but is challenging to accomplish in vivo. The zebrafish offers important advantages to study these processes--optical transparency during embryogenesis, genetic tractability and rapid development. Therefore, to study transcription and RNA dynamics in an intact vertebrate organism, we have adapted the MS2 RNA-labeling system to zebrafish. By using this binary system to coexpress a fluorescent MS2 bacteriophage coat protein (MCP) and an RNA of interest tagged with multiple copies of the RNA hairpin MS2-binding site (MBS), live-cell imaging of RNA dynamics at single RNA molecule resolution has been achieved in other organisms. Here, using a Gateway-compatible MS2 labeling system, we generated stable transgenic zebrafish lines expressing MCP, validated the MBS-MCP interaction and applied the system to investigate zygotic genome activation (ZGA) and RNA localization in primordial germ cells (PGCs) in zebrafish. Although cleavage stage cells are initially transcriptionally silent, we detect transcription of MS2-tagged transcripts driven by the βactin promoter at \~{} 3-3.5 h post-fertilization, consistent with the previously reported ZGA. Furthermore, we show that MS2-tagged nanos3 3{\textquoteright}UTR transcripts localize to PGCs, where they are diffusely cytoplasmic and within larger cytoplasmic accumulations reminiscent of those displayed by endogenous nanos3. These tools provide a new avenue for live-cell imaging of RNA molecules in an intact vertebrate. Together with new techniques for targeted genome editing, this system will be a valuable tool to tag and study the dynamics of endogenous RNAs during zebrafish developmental processes.

}, issn = {1477-9129}, doi = {10.1242/dev.118968}, author = {Campbell, Philip D and Chao, Jeffrey A and Singer, Robert H and Marlow, Florence L} } @article {46533, title = {Translation. An RNA biosensor for imaging the first round of translation from single cells to living animals.}, journal = {Science}, volume = {347}, year = {2015}, month = {2015 Mar 20}, pages = {1367-671}, abstract = {

Analysis of single molecules in living cells has provided quantitative insights into the kinetics of fundamental biological processes; however, the dynamics of messenger RNA (mRNA) translation have yet to be addressed. We have developed a fluorescence microscopy technique that reports on the first translation events of individual mRNA molecules. This allowed us to examine the spatiotemporal regulation of translation during normal growth and stress and during Drosophila oocyte development. We have shown that mRNAs are not translated in the nucleus but translate within minutes after export, that sequestration within P-bodies regulates translation, and that oskar mRNA is not translated until it reaches the posterior pole of the oocyte. This methodology provides a framework for studying initiation of protein synthesis on single mRNAs in living cells.

}, issn = {1095-9203}, doi = {10.1126/science.aaa3380}, author = {Halstead, James M and Lionnet, Timoth{\'e}e and Wilbertz, Johannes H and Wippich, Frank and Ephrussi, Anne and Singer, Robert H and Chao, Jeffrey A} } @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 {46723, title = {Fluorescence fluctuation spectroscopy enables quantitative imaging of single mRNAs in living cells.}, journal = {Biophysical Journal}, volume = {102}, year = {2012}, month = {2012 Jun 20}, pages = {2936-44}, abstract = {

Imaging mRNA with single-molecule sensitivity in live cells has become an indispensable tool for quantitatively studying RNA biology. The MS2 system has been extensively used due to its unique simplicity and sensitivity. However, the levels of the coat protein needed for consistent labeling of mRNAs limits the sensitivity and quantitation of this technology. Here, we applied fluorescence fluctuation spectroscopy to quantitatively characterize and enhance the MS2 system. Surprisingly, we found that a high fluorescence background resulted from inefficient dimerization of fluorescent protein (FP)-labeled MS2 coat protein (MCP). To mitigate this problem, we used a single-chain tandem dimer of MCP (tdMCP) that significantly increased the uniformity and sensitivity of mRNA labeling. Furthermore, we characterized the PP7 coat protein and the binding to its respective RNA stem loop. We conclude that the PP7 system performs better for RNA labeling. Finally, we used these improvements to study endogenous β-actin mRNA, which has 24xMS2 binding sites inserted into the 3{\textquoteright} untranslated region. The tdMCP-FP allowed uniform RNA labeling and provided quantitative measurements of endogenous mRNA concentration and diffusion. This work provides a foundation for quantitative spectroscopy and imaging of single mRNAs directly in live cells.

}, keywords = {Actins, Animals, Capsid Proteins, Cell Line, Cell Nucleus, Cell Survival, Diffusion, Mice, Molecular Imaging, Protein Multimerization, Protein Structure, Quaternary, RNA, Messenger, Spectrometry, Fluorescence}, issn = {1542-0086}, doi = {10.1016/j.bpj.2012.05.017}, author = {Wu, Bin and Chao, Jeffrey A and Singer, Robert H} } @article {46720, title = {Imaging translation in single cells using fluorescent microscopy.}, journal = {Cold Spring Harbor Perspectives in Biology}, volume = {4}, year = {2012}, month = {2012 Nov}, abstract = {

The regulation of translation provides a mechanism to control not only the abundance of proteins, but also the precise time and subcellular location that they are synthesized. Much of what is known concerning the molecular basis for translational control has been gleaned from experiments (e.g., luciferase assays and polysome analysis) that measure average changes in the protein synthesis of a population of cells, however, mechanistic insights can be obscured in ensemble measurements. The development of fluorescent microscopy techniques and reagents has allowed translation to be studied within its cellular context. Here we highlight recent methodologies that can be used to study global changes in protein synthesis or regulation of specific mRNAs in single cells. Imaging of translation has provided direct evidence for local translation of mRNAs at synapses in neurons and will become an important tool for studying translational control.

}, keywords = {Animals, Gene Expression Regulation, Humans, Microscopy, Fluorescence, Neurons, Protein Biosynthesis, Puromycin, Synapses}, issn = {1943-0264}, doi = {10.1101/cshperspect.a012310}, author = {Chao, Jeffrey A and Yoon, Young J 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 {46732, title = {Spatial arrangement of an RNA zipcode identifies mRNAs under post-transcriptional control.}, journal = {Genes \& Development}, volume = {26}, year = {2012}, month = {2012 Jan 1}, pages = {43-53}, abstract = {

How RNA-binding proteins recognize specific sets of target mRNAs remains poorly understood because current approaches depend primarily on sequence information. In this study, we demonstrate that specific recognition of messenger RNAs (mRNAs) by RNA-binding proteins requires the correct spatial positioning of these sequences. We characterized both the cis-acting sequence elements and the spatial restraints that define the mode of RNA binding of the zipcode-binding protein 1 (ZBP1/IMP1/IGF2BP1) to the β-actin zipcode. The third and fourth KH (hnRNP K homology) domains of ZBP1 specifically recognize a bipartite RNA element comprised of a 5{\textquoteright} element (CGGAC) followed by a variable 3{\textquoteright} element (C/A-CA-C/U) that must be appropriately spaced. Remarkably, the orientation of these elements is interchangeable within target transcripts bound by ZBP1. The spatial relationship of this consensus binding site identified conserved transcripts that were verified to associate with ZBP1 in vivo. The dendritic localization of one of these transcripts, spinophilin, was found to be dependent on both ZBP1 and the RNA elements recognized by ZBP1 KH34.

}, keywords = {Actins, Animals, Base Sequence, Cells, Cultured, Consensus Sequence, Dendrites, Gene Expression Regulation, Glycoproteins, Humans, Ligands, Mice, Microfilament Proteins, Models, Molecular, Molecular Sequence Data, Nerve Tissue Proteins, Protein Binding, Protein Structure, Tertiary, RNA, Messenger}, issn = {1549-5477}, doi = {10.1101/gad.177428.111}, author = {Patel, Vivek L and Mitra, Somdeb and Harris, Richard and Buxbaum, Adina R and Lionnet, Timoth{\'e}e and Brenowitz, Michael and Girvin, Mark and Levy, Matthew and Almo, Steven C and Singer, Robert H and Chao, Jeffrey A} } @article {46722, title = {An unbiased analysis method to quantify mRNA localization reveals its correlation with cell motility.}, journal = {Cell Reports}, volume = {1}, year = {2012}, month = {2012 Feb 23}, pages = {179-84}, abstract = {

Localization of mRNA is a critical mechanism used by a large fraction of transcripts to restrict its translation to specific cellular regions. Although current high-resolution imaging techniques provide ample information, the analysis methods for localization have either been qualitative or employed quantification in nonrandomly selected regions of interest. Here, we describe an analytical method for objective quantification of mRNA localization using a combination of two characteristics of its molecular distribution, polarization and dispersion. The validity of the method is demonstrated using single-molecule FISH images of budding yeast and fibroblasts. Live-cell analysis of endogenous β-actin mRNA in mouse fibroblasts reveals that mRNA polarization has a half-life of ~16 min and is cross-correlated with directed cell migration. This novel approach provides insights into the dynamic regulation of mRNA localization and its physiological roles.

}, keywords = {Actins, Animals, Cell Movement, Chick Embryo, Computer Simulation, Embryo, Mammalian, Fibroblasts, Green Fluorescent Proteins, Imaging, Three-Dimensional, In Situ Hybridization, Mice, Monte Carlo Method, RNA Transport, RNA, Messenger, Saccharomyces cerevisiae}, issn = {2211-1247}, doi = {10.1016/j.celrep.2011.12.009}, author = {Park, Hye Yoon and Trcek, Tatjana and Wells, Amber L and Chao, Jeffrey A 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 {6601, title = {A transgenic mouse for in vivo detection of endogenous labeled mRNA.}, journal = {Nature Methods}, volume = {8}, year = {2011}, month = {2011 Feb}, pages = {165-70}, abstract = {

Live-cell single mRNA imaging is a powerful tool but has been restricted in higher eukaryotes to artificial cell lines and reporter genes. We describe an approach that enables live-cell imaging of single endogenous labeled mRNA molecules transcribed in primary mammalian cells and tissue. We generated a knock-in mouse line with an MS2 binding site (MBS) cassette targeted to the 3{\textquoteright} untranslated region of the essential β-actin gene. As β-actin-MBS was ubiquitously expressed, we could uniquely address endogenous mRNA regulation in any tissue or cell type. We simultaneously followed transcription from the β-actin alleles in real time and observed transcriptional bursting in response to serum stimulation with precise temporal resolution. We tracked single endogenous labeled mRNA particles being transported in primary hippocampal neurons. The MBS cassette also enabled high-sensitivity fluorescence in situ hybridization (FISH), allowing detection and localization of single β-actin mRNA molecules in various mouse tissues.

}, keywords = {Animals, Cell Survival, Cells, Cultured, Fluorescence, Genetic, In Situ Hybridization, Messenger, Mice, RNA, Transcription, Transgenic}, issn = {1548-7105}, doi = {10.1038/nmeth.1551}, author = {Lionnet, Timoth{\'e}e and Czaplinski, Kevin and Darzacq, Xavier and Shav-Tal, Yaron and Wells, Amber L and Chao, Jeffrey A and Park, Hye Yoon and de Turris, Valeria and Lopez-Jones, Melissa and Singer, Robert H} }