@article {70179, title = {Secretome translation shaped by lysosomes and lunapark-marked ER junctions.}, journal = {Nature}, year = {2025}, month = {2025 Nov 05}, abstract = {

The endoplasmic reticulum (ER) is a highly interconnected membrane network that serves as a central site for protein synthesis and maturation. A crucial subset of ER-associated transcripts, termed secretome mRNAs, encode secretory, lumenal and integral membrane proteins, representing nearly one-third of human protein-coding genes. Unlike cytosolic mRNAs, secretome mRNAs undergo co-translational translocation, and thus require precise coordination between translation and protein insertion. Disruption of this process, such as through altered elongation rates, activates stress response pathways that impede cellular growth, raising the question of whether secretome translation is spatially organized to ensure fidelity. Here, using live-cell single-molecule imaging, we demonstrate that secretome mRNA translation is preferentially localized to ER junctions that are enriched with the structural protein lunapark and in close proximity to lysosomes. Lunapark depletion reduced ribosome density and translation efficiency of secretome mRNAs near lysosomes, an effect that was dependent on eIF2-mediated initiation and was reversed by the integrated stress response inhibitor ISRIB. Lysosome-associated translation was further modulated by nutrient status: amino acid deprivation enhanced lysosome-proximal\ translation, whereas lysosomal pH neutralization suppressed it. These findings identify a mechanism by which ER junctional proteins and lysosomal activity cooperatively pattern secretome mRNA translation, linking ER architecture and nutrient sensing to the production of secretory and membrane proteins.

bioRxiv preprint:\ https://doi.org/10.1101/2024.11.21.624573

}, issn = {1476-4687}, doi = {10.1038/s41586-025-09718-0}, url = {https://doi.org/10.1038/s41586-025-09718-0}, author = {Choi, Heejun and Liao, Ya-Cheng and Yoon, Young J and Grimm, Jonathan and Wang, Nan and Lavis, Luke D and Singer, Robert H and Lippincott-Schwartz, Jennifer} } @article {68935, title = {Lysosomal release of amino acids at ER three-way junctions regulates transmembrane and secretory protein mRNA translation.}, journal = {bioRxiv}, year = {2023}, month = {2023 Aug 03}, abstract = {

One-third of the mammalian proteome is comprised of transmembrane and secretory proteins that are synthesized on endoplasmic reticulum (ER). Here, we investigate the spatial distribution and regulation of mRNAs encoding these membrane and secretory proteins (termed "secretome" mRNAs) through live cell, single molecule tracking to directly monitor the position and translation states of secretome mRNAs on ER and their relationship to other organelles. Notably, translation of secretome mRNAs occurred preferentially near lysosomes on ER marked by the ER junction-associated protein, Lunapark. Knockdown of Lunapark reduced the extent of secretome mRNA translation without affecting translation of other mRNAs. Less secretome mRNA translation also occurred when lysosome function was perturbed by raising lysosomal pH or inhibiting lysosomal proteases. Secretome mRNA translation near lysosomes was enhanced during amino acid deprivation. Addition of the integrated stress response inhibitor, ISRIB, reversed the translation inhibition seen in Lunapark knockdown cells, implying an eIF2 dependency. Altogether, these findings uncover a novel coordination between ER and lysosomes, in which local release of amino acids and other factors from ER-associated lysosomes patterns and regulates translation of mRNAs encoding secretory and membrane proteins.

}, doi = {10.1101/2023.08.01.551382}, author = {Choi, Heejun and Liao, Ya-Cheng and Yoon, Young J and Grimm, Jonathan and Lavis, Luke D and Singer, Robert H and Lippincott-Schwartz, Jennifer} } @article {67992, title = {Bromodomains regulate dynamic targeting of the PBAF chromatin remodeling complex to chromatin hubs.}, journal = {Biophysical Journal}, volume = {121}, year = {2022}, month = {2022 May 3}, pages = {1738-1752}, abstract = {

Chromatin remodelers actively target arrays of acetylated nucleosomes at select enhancers and promoters to facilitate or shut down the repeated recruitment of RNA Pol II during transcriptional bursting. It is poorly understood how chromatin remodelers such as PBAF dynamically target different chromatin states inside a live cell. Our live-cell single molecule fluorescence microscopy study reveals chromatin hubs throughout the nucleus where PBAF rapidly cycles on and off the genome. Deletion of PBAF{\textquoteright}s bromodomains impairs targeting and stable engagement of chromatin in hubs. Dual color imaging reveals that PBAF targets both euchromatic and heterochromatic hubs with distinct genome binding kinetic profiles that mimic chromatin stability. Removal of PBAF{\textquoteright}s bromodomains stabilizes H3.3 binding within chromatin indicating that bromodomains may play a direct role in remodeling of the nucleosome. Our data suggests that PBAF{\textquoteright}s dynamic bromodomain mediated engagement of a nucleosome may reflect the chromatin remodeling potential of differentially bound chromatin states.

}, issn = {1542-0086}, doi = {10.1016/j.bpj.2022.03.027}, author = {Kenworthy, Charles A and Haque, Nayem and Liou, Shu-Hao and Chandris, Panagiotis and Wong, Vincent and Dziuba, Patrycja and Lavis, Luke D and Liu, Wei-Li and Singer, Robert H and Coleman, Robert A} } @article {68462, title = {Cap-dependent translation initiation monitored in living cells.}, journal = {Nature Communications}, volume = {13}, year = {2022}, month = {2022 Nov 02}, pages = {6558}, abstract = {

mRNA translation is tightly regulated to preserve cellular homeostasis. Despite extensive biochemical, genetic, and structural studies, a detailed understanding of mRNA translation regulation is lacking. Imaging methodologies able to resolve the binding dynamics of translation factors at single-cell and single-mRNA resolution were necessary to fully elucidate regulation of this paramount process. Here live-cell spectroscopy and single-particle tracking were combined to interrogate the binding dynamics of endogenous initiation factors to the 5{\textquoteright}cap. The diffusion of initiation factors (IFs) changed markedly upon their association with mRNA. Quantifying their diffusion characteristics revealed the sequence of IFs assembly and disassembly in cell lines and the clustering of translation in neurons. This approach revealed translation regulation at high spatial and temporal resolution that can be applied to the formation of any endogenous complex that results in a measurable shift in diffusion.

}, keywords = {Peptide Chain Initiation, Translational, Peptide Initiation Factors, Protein Biosynthesis, RNA Caps, RNA, Messenger}, issn = {2041-1723}, doi = {10.1038/s41467-022-34052-8}, author = {Gandin, Valentina and English, Brian P and Freeman, Melanie and Leroux, Louis-Philippe and Preibisch, Stephan and Walpita, Deepika and Jaramillo, Maritza and Singer, Robert H} } @article {68556, title = {Inhibition of coronavirus HCoV-OC43 by targeting the eIF4F complex.}, journal = {Frontiers in Pharmacology}, volume = {13}, year = {2022}, month = {2022 Dec 01}, pages = {1029093}, abstract = {

The translation initiation complex 4F (eIF4F) is a rate-limiting factor in protein synthesis. Alterations in eIF4F activity are linked to several diseases, including cancer and infectious diseases. To this end, coronaviruses require eIF4F complex activity to produce proteins essential for their life cycle. Efforts to target coronaviruses by abrogating translation have been largely limited to repurposing existing eIF4F complex inhibitors. Here, we report the results of a high throughput screen to identify small molecules that disrupt eIF4F complex formation and inhibit coronavirus RNA and protein levels. Of 338,000 small molecules screened for inhibition of the eIF4F-driven, CAP-dependent translation, we identified SBI-1232 and two structurally related analogs, SBI-5844 and SBI-0498, that inhibit human coronavirus OC43 (HCoV-OC43; OC43) with minimal cell toxicity. Notably, gene expression changes after OC43 infection of Vero E6 or A549 cells were effectively reverted upon treatment with SBI-5844 or SBI-0498. Moreover, SBI-5844 or SBI-0498 treatment effectively impeded the eIF4F complex assembly, with concomitant inhibition of newly synthesized OC43 nucleocapsid protein and OC43 RNA and protein levels. Overall, we identify SBI-5844 and SBI-0498 as small molecules targeting the eIF4F complex that may limit coronavirus transcripts and proteins, thereby representing a basis for developing novel therapeutic modalities against coronaviruses.

}, issn = {1663-9812}, doi = {10.3389/fphar.2022.1029093}, author = {Feng, Yongmei and Grotegut, Stefan and Jovanovic, Predrag and Gandin, Valentina and Olson, Steven H and Murad, Rabi and Beall, Anne and Colayco, Sharon and De-Jesus, Paul and Chanda, Sumit and English, Brian P and Singer, Robert H and Jackson, Michael and Topisirovic, Ivan and Ronai, Ze{\textquoteright}ev A} } @article {68297, title = {A single-cell transcriptomic atlas of complete insect nervous systems across multiple life stages.}, journal = {Neural Development}, volume = {17}, year = {2022}, month = {2022 Aug 24}, pages = {8}, abstract = {

Molecular profiles of neurons influence neural development and function but bridging the gap between genes, circuits, and behavior has been very difficult. Here we used single cell RNAseq to generate a complete gene expression atlas of the Drosophila larval central nervous system composed of 131,077 single cells across three developmental stages (1\ h, 24\ h and 48\ h after hatching). We identify 67 distinct cell clusters based on the patterns of gene expression. These include 31 functional mature larval neuron clusters, 1 ring gland cluster, 8 glial clusters, 6 neural precursor clusters, and 13 developing immature adult neuron clusters. Some clusters are present across all stages of larval development, while others are stage specific (such as developing adult neurons). We identify genes that are differentially expressed in each cluster, as well as genes that are differentially expressed at distinct stages of larval life. These differentially expressed genes provide promising candidates for regulating the function of specific neuronal and glial types in the larval nervous system, or the specification and differentiation of adult neurons. The cell transcriptome Atlas of the Drosophila larval nervous system is a valuable resource for developmental biology and systems neuroscience and provides a basis for elucidating how genes regulate neural development and function.

}, keywords = {Animals, Drosophila, Gene Expression Regulation, Developmental, Larva, Neuroglia, Neurons, Transcriptome}, issn = {1749-8104}, doi = {10.1186/s13064-022-00164-6}, author = {Corrales, Marc and Cocanougher, Benjamin T and Kohn, Andrea B and Wittenbach, Jason D and Long, Xi S and Lemire, Andrew and Cardona, Albert and Singer, Robert H and Moroz, Leonid L and Zlatic, Marta} } @article {67210, title = {Intracellular mRNA transport and localized translation.}, journal = {Nature Reviews Molecular Cell Biology}, volume = {22}, year = {2021}, month = {2021 Jul 1}, pages = {483-504}, abstract = {

Fine-tuning cellular physiology in response to intracellular and environmental cues requires precise temporal and spatial control of gene expression. High-resolution imaging technologies to detect mRNAs and their translation state have revealed that all living organisms localize mRNAs in subcellular compartments and create translation hotspots, enabling cells to tune gene expression locally. Therefore, mRNA localization is a conserved and integral part of gene expression regulation from prokaryotic to eukaryotic cells. In this Review, we discuss the mechanisms of mRNA transport and local mRNA translation across the kingdoms of life and at organellar, subcellular and multicellular resolution. We also discuss the properties of messenger ribonucleoprotein and higher order RNA granules and how they may influence mRNA transport and local protein synthesis. Finally, we summarize the technological developments that allow us to study mRNA localization and local translation through the simultaneous detection of mRNAs and proteins in single cells, mRNA and nascent protein single-molecule imaging, and bulk RNA and protein detection methods.

}, issn = {1471-0080}, doi = {10.1038/s41580-021-00356-8}, author = {Das, Sulagna and Vera, Maria and Gandin, Valentina and Singer, Robert H and Tutucci, Evelina} } @article {65160, title = {Mamo decodes hierarchical temporal gradients into terminal neuronal fate.}, journal = {Elife}, volume = {8}, year = {2019}, month = {2019 Sep 23}, abstract = {

Temporal patterning is a seminal method of expanding neuronal diversity. Here we unravel a mechanism decoding neural stem cell temporal gene expression and transforming it into discrete neuronal fates. This mechanism is characterized by hierarchical gene expression. First, neuroblasts express opposing temporal gradients of RNA-binding proteins, Imp and Syp. These proteins promote or inhibit translation, yielding a descending neuronal gradient. Together, first and second-layer temporal factors define a temporal expression window of BTB-zinc finger nuclear protein, Mamo. The precise temporal induction of Mamo is achieved via both transcriptional and post-transcriptional regulation. Finally, Mamo is essential for the temporally defined, terminal identity of α{\textquoteright}/β{\textquoteright} mushroom body neurons and identity maintenance. We describe a straightforward paradigm of temporal fate specification where diverse neuronal fates are defined via integrating multiple layers of gene regulation. The neurodevelopmental roles of orthologous/related mammalian genes suggest a fundamental conservation of this mechanism in brain development.

}, issn = {2050-084X}, doi = {10.7554/eLife.48056}, author = {Liu, Ling-Yu and Long, Xi and Yang, Ching-Po and Miyares, Rosa L and Sugino, Ken and Singer, Robert H and Lee, Tzumin} } @article {64849, title = {Neurotransmitter identity is acquired in a lineage-restricted manner in the Drosophila CNS.}, journal = {Elife}, volume = {8}, year = {2019}, month = {2019 Mar 26}, abstract = {

The vast majority of the adult fly ventral nerve cord is composed of 34 hemilineages, which are clusters of lineally related neurons. Neurons in these hemilineages use one of the three fast-acting neurotransmitters (acetylcholine, GABA, or glutamate) for communication. We generated a comprehensive neurotransmitter usage map for the entire ventral nerve cord. We did not find any cases of neurons using more than one neurotransmitter, but found that the acetylcholine specific gene ChAT is transcribed in many glutamatergic and GABAergic neurons, but these transcripts typically do not leave the nucleus and are not translated. Importantly, our work uncovered a simple rule: All neurons within a hemilineage use the same neurotransmitter. Thus, neurotransmitter identity is acquired at the stem cell level. Our detailed transmitter- usage/lineage identity map will be a great resource for studying the developmental basis of behavior and deciphering how neuronal circuits function to regulate behavior.

}, issn = {2050-084X}, doi = {10.7554/eLife.43701}, author = {Lacin, Haluk and Chen, Hui-Min and Long, Xi and Singer, Robert H and Lee, Tzumin and Truman, James W} } @article {65176, title = {Rational design of fluorogenic and spontaneously blinking labels for super-resolution imaging.}, journal = {ACS Central Science}, volume = {5}, year = {2019}, month = {2019 Sep 25}, pages = {1602-1613}, abstract = {

Rhodamine dyes exist in equilibrium between a fluorescent zwitterion and a nonfluorescent lactone. Tuning this equilibrium toward the nonfluorescent lactone form can improve cell-permeability and allow creation of "fluorogenic" compounds-ligands that shift to the fluorescent zwitterion upon binding a biomolecular target. An archetype fluorogenic dye is the far-red tetramethyl-Si-rhodamine (SiR), which has been used to create exceptionally useful labels for advanced microscopy. Here, we develop a quantitative framework for the development of new fluorogenic dyes, determining that the lactone-zwitterion equilibrium constant () is sufficient to predict fluorogenicity. This rubric emerged from our analysis of known fluorophores and yielded new fluorescent and fluorogenic labels with improved performance in cellular imaging experiments. We then designed a novel fluorophore-Janelia Fluor 526 (JF)-with SiR-like properties but shorter fluorescence excitation and emission wavelengths. JF is a versatile scaffold for fluorogenic probes including ligands for self-labeling tags, stains for endogenous structures, and spontaneously blinking labels for super-resolution immunofluorescence. JF constitutes a new label for advanced microscopy experiments, and our quantitative framework will enable the rational design of other fluorogenic probes for bioimaging.

}, issn = {2374-7943}, doi = {10.1021/acscentsci.9b00676}, author = {Zheng, Qinsi and Ayala, Anthony X and Chung, Inhee and Weigel, Aubrey V and Ranjan, Anand and Falco, Natalie and Grimm, Jonathan B and Tkachuk, Ariana N and Wu, Carl and Lippincott-Schwartz, Jennifer and Singer, Robert H and Lavis, Luke D} } @article {49245, title = {Imaging mRNA in vivo, from birth to death.}, journal = {Annual Review of Biophysics}, volume = {47}, year = {2018}, month = {2018 May 20}, pages = {85-106}, abstract = {

RNA is the fundamental information transfer system in the cell. The ability to follow single messenger RNAs (mRNAs) from transcription to degradation with fluorescent probes gives quantitative information about how the information is transferred from DNA to proteins. This review focuses on the latest technological developments in the field of single-mRNA detection and their usage to study gene expression in both fixed and live cells. By describing the application of these imaging tools, we follow the journey of mRNA from transcription to decay in single cells, with single-molecule resolution. We review current theoretical models for describing transcription and translation that were generated by single-molecule and single-cell studies. These methods provide a basis to study how single-molecule interactions generate phenotypes, fundamentally changing our understating of gene expression regulation.

}, issn = {1936-1238}, doi = {10.1146/annurev-biophys-070317-033037}, author = {Tutucci, Evelina and Livingston, Nathan M and Singer, Robert H and Wu, Bin} } @article {49587, title = {Mapping Neurotransmitter Identity in the Whole-Mount Brain Using Multiplex High-Throughput Fluorescence Hybridization.}, journal = {Genetics}, volume = {211}, year = {2018}, month = {2018 Dec 18}, pages = {473-82}, abstract = {

Identifying the neurotransmitters used by specific neurons is a critical step in understanding the function of neural circuits. However, methods for the consistent and efficient detection of neurotransmitter markers remain limited. Fluorescence hybridization (FISH) enables direct labeling of type-specific mRNA in neurons. Recent advances in FISH allow this technique to be carried out in intact tissue samples such as whole-mount brains. Here, we present a FISH platform for high-throughput detection of eight common neurotransmitter phenotypes in brains. We greatly increase FISH throughput by processing samples mounted on coverslips and optimizing fluorophore choice for each probe to facilitate multiplexing. As application examples, we demonstrate cases of neurotransmitter co-expression, reveal neurotransmitter phenotypes of specific cell types and explore the onset of neurotransmitter expression in the developing optic lobe. Beyond neurotransmitter markers, our protocols can in principle be used for large scale FISH detection of any mRNA in whole-mount fly brains.

}, issn = {1943-2631}, doi = {10.1534/genetics.118.301749}, author = {Meissner, Geoffrey W and Nern, Aljoscha and Singer, Robert H and Wong, Allan M and Malkesman, Oz and Long, Xi} } @article {49289, title = {A transgenic mouse for imaging activity-dependent dynamics of endogenous Arc mRNA in live neurons.}, journal = {Science Advances}, volume = {4}, year = {2018}, month = {2018 Jun}, pages = {eaar3448}, abstract = {

Localized translation plays a crucial role in synaptic plasticity and memory consolidation. However, it has not been possible to follow the dynamics of memory-associated mRNAs in living neurons in response to neuronal activity in real time. We have generated a novel mouse model where the endogenous Arc/Arg3.1 gene is tagged in its 3{\textquoteright} untranslated region with stem-loops that bind a bacteriophage PP7 coat protein (PCP), allowing visualization of individual mRNAs in real time. The physiological response of the tagged gene to neuronal activity is identical to endogenous Arc and reports the true dynamics of Arc mRNA from transcription to degradation. The transcription dynamics of Arc in cultured hippocampal neurons revealed two novel results: (i) A robust transcriptional burst with prolonged ON state occurs after stimulation, and (ii) transcription cycles continue even after initial stimulation is removed. The correlation of stimulation with Arc transcription and mRNA transport in individual neurons revealed that stimulus-induced Ca activity was necessary but not sufficient for triggering Arc transcription and that blocking neuronal activity did not affect the dendritic transport of newly synthesized Arc mRNAs. This mouse will provide an important reagent to investigate how individual neurons transduce activity into spatiotemporal regulation of gene expression at the synapse.

}, issn = {2375-2548}, doi = {10.1126/sciadv.aar3448}, author = {Das, Sulagna and Moon, Hyungseok C and Singer, Robert H and Park, Hye Yoon} } @article {49182, title = {Transvection Goes Live-Visualizing Enhancer-Promoter Communication between Chromosomes.}, journal = {Molecular Cell}, volume = {70}, year = {2018}, month = {2018 Apr 19}, pages = {195-196}, abstract = {

Lim et\ al. (2018) use live imaging in Drosophila embryos to show that enhancers can drive transcription from promoters on another chromosome when they are in close proximity. In addition, they show that multiple promoters can access the same enhancer without competition, potentially sharing a pool of factors in a transcriptional "hub."

}, issn = {1097-4164}, doi = {10.1016/j.molcel.2018.04.004}, author = {Tsai, Albert and Singer, Robert H and Crocker, Justin} } @article {48860, title = {Genomic probes.}, volume = {A1}, number = {US20170268035 }, year = {2017}, month = {2017 Sep 21}, edition = {C12Q 1/68 20060101 C12Q001/68; C12N 15/09 20060101 C12N015/09; C12P 19/34 20060101 C12P019/34; C07H 21/04 20060101 C07H021/04}, abstract = {Labeled probes, and methods of use thereof, comprise a Cas polypeptide conjugated to gRNA that is specific for target nucleic acid sequences, including genomic DNA sequences. The probes and methods can be used to label nucleic acid sequences without global DNA denaturation. The presently-disclosed subject matter meets some or all of the above identified needs, as will become evident to those of ordinary skill in the art after a study of information provided in this document.}, url = {http://appft.uspto.gov/netacgi/nph-Parser?Sect1=PTO1\&Sect2=HITOFF\&d=PG01\&p=1\&u=\%2Fnetahtml\%2FPTO\%2Fsrchnum.html\&r=1\&f=G\&l=50\&s1=\%2220170268035\%22.PGNR.\&OS=DN/20170268035\&RS=DN/20170268035}, author = {Singer, Robert H and Deng, Wulan and Lionnet, Timoth{\'e}e} } @article {48318, title = {Imaging mRNA and protein interactions within neurons.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {114}, year = {2017}, month = {2017 Mar 07}, pages = {E1875-E1884}, abstract = {

RNA-protein interactions are essential for proper gene expression regulation, particularly in neurons with unique spatial constraints. Currently, these interactions are defined biochemically, but a method is needed to evaluate them quantitatively within morphological context. Colocalization of two-color labels using wide-field microscopy is a method to infer these interactions. However, because of chromatic aberrations in the objective lens, this approach lacks the resolution to determine whether two molecules are physically in contact or simply nearby by chance. Here, we developed a robust super registration methodology that corrected the chromatic aberration across the entire image field to within 10 nm, which is capable of determining whether two molecules are physically interacting or simply in proximity by random chance. We applied this approach to image single-molecule FISH in combination with immunofluorescence (smFISH-IF) and determined whether the association between an mRNA and binding protein(s) within a neuron was significant or accidental. We evaluated several mRNA-binding proteins identified from RNA pulldown assays to determine which of these exhibit bona fide interactions. Surprisingly, many known mRNA-binding proteins did not bind the mRNA in situ, indicating that adventitious interactions are significant using existing technology. This method provides an ability to evaluate two-color registration compatible with the scale of molecular interactions.

}, issn = {1091-6490}, doi = {10.1073/pnas.1621440114}, author = {Eliscovich, Carolina and Shenoy, Shailesh M and Singer, Robert H} } @article {48907, title = {Intercellular mRNA trafficking via membrane nanotube-like extensions in mammalian cells.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {114}, year = {2017}, month = {2017 Oct 24}, pages = {E9873-E9882}, abstract = {

RNAs have been shown to undergo transfer between mammalian cells, although the mechanism behind this phenomenon and its overall importance to cell physiology is not well understood. Numerous publications have suggested that RNAs (microRNAs and incomplete mRNAs) undergo transfer via extracellular vesicles (e.g., exosomes). However, in contrast to a diffusion-based transfer mechanism, we find that full-length mRNAs undergo direct cell-cell transfer via cytoplasmic extensions characteristic of membrane nanotubes (mNTs), which connect donor and acceptor cells. By employing a simple coculture experimental model and using single-molecule imaging, we provide quantitative data showing that mRNAs are transferred between cells in contact. Examples of mRNAs that undergo transfer include those encoding GFP, mouse β-actin, and human Cyclin D1, BRCA1, MT2A, and HER2. We show that intercellular mRNA transfer occurs in all coculture models tested (e.g., between primary cells, immortalized cells, and in cocultures of immortalized human and murine cells). Rapid mRNA transfer is dependent upon actin but is independent of de novo protein synthesis and is modulated by stress conditions and gene-expression levels. Hence, this work supports the hypothesis that full-length mRNAs undergo transfer between cells through a refined structural connection. Importantly, unlike the transfer of miRNA or RNA fragments, this process of communication transfers genetic information that could potentially alter the acceptor cell proteome. This phenomenon may prove important for the proper development and functioning of tissues as well as for host-parasite or symbiotic interactions.

}, issn = {1091-6490}, doi = {10.1073/pnas.1706365114}, author = {Haimovich, Gal and Ecker, Christopher M and Dunagin, Margaret C and Eggan, Elliott and Raj, Arjun and Gerst, Jeffrey E and Singer, Robert H} } @article {48920, title = {Nuclear microenvironments modulate transcription from low-affinity enhancers.}, journal = {eLife}, volume = {6}, year = {2017}, month = {2017 Nov 02}, abstract = {

Transcription factors bind low-affinity DNA sequences for only short durations. It is not clear how brief, low-affinity interactions can drive efficient transcription. Here we report that the transcription factor Ultrabithorax (Ubx) utilizes low-affinity binding sites in the Drosophila melanogastershavenbaby (svb) locus and related enhancers in nuclear microenvironments of high Ubx concentrations. Related enhancers colocalize to the same microenvironments independently of their chromosomal location, suggesting that microenvironments are highly differentiated transcription domains. Manipulating the affinity of svb enhancers revealed an inverse relationship between enhancer affinity and Ubx concentration required for transcriptional activation. The Ubx cofactor, Homothorax (Hth), was co-enriched with Ubx near enhancers that require Hth, even though Ubx and Hth did not co-localize throughout the nucleus. Thus, microenvironments of high local transcription factor and cofactor concentrations could help low-affinity sites overcome their kinetic inefficiency. Mechanisms that generate these microenvironments could be a general feature of eukaryotic transcriptional regulation.

}, issn = {2050-084X}, doi = {10.7554/eLife.28975}, author = {Tsai, Albert and Muthusamy, Anand K and Alves, Mariana Rp and Lavis, Luke D and Singer, Robert H and Stern, David L and Crocker, Justin} } @article {48559, title = {Quantitative mRNA imaging throughout the entire Drosophila brain.}, journal = {Nature Methods}, volume = {14}, year = {2017}, month = {2017 Jun 05}, pages = {703-6}, abstract = {

We describe a fluorescence in situ hybridization method that permits detection of the localization and abundance of single mRNAs (smFISH) in cleared whole-mount adult Drosophila brains. The approach is rapid and multiplexable and does not require molecular amplification; it allows facile quantification of mRNA expression with subcellular resolution on a standard confocal microscope. We further demonstrate single-mRNA detection across the entire brain using a custom Bessel beam structured illumination microscope (BB-SIM).

}, issn = {1548-7105}, doi = {10.1038/nmeth.4309}, author = {Long, Xi and Colonell, Jennifer and Wong, Allan M and Singer, Robert H and Lionnet, Timoth{\'e}e} } @article {48303, title = {RNP transport in cell biology: the long and winding road.}, journal = {Current Opinion in Cell Biology}, volume = {45}, year = {2017}, month = {2017 Feb 28}, pages = {38-46}, abstract = {

Regulation of gene expression is key determinant to cell structure and function. RNA localization, where specific mRNAs are transported to subcellular regions and then translated, is highly conserved in eukaryotes ranging from yeast to extremely specialized and polarized cells such as neurons. Messenger RNA and associated proteins (mRNP) move from the site of transcription in the nucleus to their final destination in the cytoplasm both passively through diffusion and actively via directed transport. Dysfunction of RNA localization, transport and translation machinery can lead to pathology. Single-molecule live-cell imaging techniques have revealed unique features of this journey with unprecedented resolution. In this review, we highlight key recent findings that have been made using these approaches and possible implications for spatial control of gene function.

}, issn = {1879-0410}, doi = {10.1016/j.ceb.2017.02.008}, author = {Eliscovich, Carolina and Singer, Robert H} } @article {47967, title = {Glutamate-induced RNA localization and translation in neurons.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, year = {2016}, month = {2016 Sep 13}, abstract = {

Localization of mRNA is required for protein synthesis to occur within discrete intracellular compartments. Neurons represent an ideal system for studying the precision of mRNA trafficking because of their polarized structure and the need for synapse-specific targeting. To investigate this targeting, we derived a quantitative and analytical approach. Dendritic spines were stimulated by glutamate uncaging at a diffraction-limited spot, and the localization of single β-actin mRNAs was measured in space and time. Localization required NMDA receptor activity, a dynamic actin cytoskeleton, and the transacting RNA-binding protein, Zipcode-binding protein 1 (ZBP1). The ability of the mRNA to direct newly synthesized proteins to the site of localization was evaluated using a Halo-actin reporter so that RNA and protein were detected simultaneously. Newly synthesized Halo-actin was enriched at the site of stimulation, required NMDA receptor activity, and localized preferentially at the periphery of spines. This work demonstrates that synaptic activity can induce mRNA localization and local translation of β-actin where the new actin participates in stabilizing the expanding synapse in dendritic spines.

}, doi = {10.1073/pnas.1614267113}, author = {Yoon, Young J and Wu, Bin and Buxbaum, Adina R and Das, Sulagna and Tsai, Albert and English, Brian P and Grimm, Jonathan B and Lavis, Luke D and Singer, Robert H} } @article {47398, title = {Mapping translation {\textquoteright}hot-spots{\textquoteright} in live cells by tracking single molecules of mRNA and ribosomes.}, journal = {eLife}, volume = {5}, year = {2016}, month = {2016}, abstract = {

Messenger RNA localization is important for cell motility by local protein translation. However, while single mRNAs can be imaged and their movements tracked in single cells, it has not yet been possible to determine whether these mRNAs are actively translating. Therefore, we imaged single β-actin mRNAs tagged with MS2 stem loops colocalizing with labeled ribosomes to determine when polysomes formed. A dataset of tracking information consisting of thousands of trajectories per cell demonstrated that mRNAs co-moving with ribosomes have significantly different diffusion properties from non-translating mRNAs that were exposed to translation inhibitors. These data indicate that ribosome load changes mRNA movement and therefore highly translating mRNAs move slower. Importantly, β-actin mRNA near focal adhesions exhibited sub-diffusive corralled movement characteristic of increased translation. This method can identify where ribosomes become engaged for local protein production and how spatial regulation of mRNA-protein interactions mediates cell directionality.

}, issn = {2050-084X}, doi = {10.7554/eLife.10415}, author = {Katz, Zachary B and English, Brian P and Lionnet, Timoth{\'e}e and Yoon, Young J and Monnier, Nilah and Ovryn, Ben and Bathe, Mark and Singer, Robert H} } @article {47521, title = {Translation dynamics of single mRNAs in live cells and neurons.}, journal = {Science (New York, N.Y.)}, volume = {352}, year = {2016}, month = {2016 May 05}, pages = {1430-5}, abstract = {

Translation is the fundamental biological process converting mRNA information into proteins. Single molecule imaging in live cells has illuminated the dynamics of RNA transcription; however, it is not yet applicable to translation. Here we report Single molecule Imaging of NAscent PeptideS (SINAPS) to assess translation in live cells. The approach provides direct readout of initiation, elongation, and location of translation. We show that mRNAs coding for endoplasmic reticulum (ER) proteins are translated when they encounter the ER membrane. Single molecule fluorescence recovery after photobleaching provides direct measurement of elongation speed (5 AA/s). In primary neurons mRNAs are translated in proximal dendrites but repressed in distal dendrites and display {\textquotedblleft}bursting{\textquotedblright} translation. This technology provides a tool to address the spatiotemporal translation mechanism of single mRNAs in living cells.

}, doi = {10.1126/science.aaf1084}, author = {Wu, Bin and Eliscovich, Carolina and Yoon, Young J and Singer, Robert H} } @article {47032, title = {CASFISH: CRISPR/Cas9-mediated in situ labeling of genomic loci in fixed cells.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {112}, year = {2015}, month = {2015 Aug 31}, pages = {11870-5}, abstract = {

Direct visualization of genomic loci in the 3D nucleus is important for understanding the spatial organization of the genome and its association with gene expression. Various DNA FISH methods have been developed in the past decades, all involving denaturing dsDNA and hybridizing fluorescent nucleic acid probes. Here we report a novel approach that uses in vitro constituted nuclease-deficient clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated caspase 9 (Cas9) complexes as probes to label sequence-specific genomic loci fluorescently without global DNA denaturation (Cas9-mediated fluorescence in situ hybridization, CASFISH). Using fluorescently labeled nuclease-deficient Cas9 (dCas9) protein assembled with various single-guide RNA (sgRNA), we demonstrated rapid and robust labeling of repetitive DNA elements in pericentromere, centromere, G-rich telomere, and coding gene loci. Assembling dCas9 with an array of sgRNAs tiling arbitrary target loci, we were able to visualize nonrepetitive genomic sequences. The dCas9/sgRNA binary complex is stable and binds its target DNA with high affinity, allowing sequential or simultaneous probing of multiple targets. CASFISH assays using differently colored dCas9/sgRNA complexes allow multicolor labeling of target loci in cells. In addition, the CASFISH assay is remarkably rapid under optimal conditions and is applicable for detection in primary tissue sections. This rapid, robust, less disruptive, and cost-effective technology adds a valuable tool for basic research and genetic diagnosis.

}, issn = {1091-6490}, doi = {10.1073/pnas.1515692112}, author = {Deng, Wulan and Shi, Xinghua and Tjian, Robert and Lionnet, Timoth{\'e}e and Singer, Robert H} } @article {46546, title = {Cellular levels of signaling factors are sensed by β-actin alleles to modulate transcriptional pulse intensity.}, journal = {Cell Reports}, volume = {11}, year = {2015}, month = {2015 Apr 7}, pages = {419-32}, abstract = {

The transcriptional response of β-actin to extra-cellular stimuli is a paradigm for transcription factor complex assembly and regulation. Serum induction leads to a precisely timed pulse of β-actin transcription in the cell population. Actin protein is proposed to be involved in this response, but it is not known whether cellular actin levels affect nuclear β-actin transcription. We perturbed the levels of key signaling factors and examined the effect on the induced transcriptional pulse by following endogenous β-actin alleles in single living cells. Lowering serum response factor (SRF) protein levels leads to loss of pulse integrity, whereas reducing actin protein levels reveals positive feedback regulation, resulting in elevated gene activation and a prolonged transcriptional response. Thus, transcriptional pulse fidelity requires regulated amounts of signaling proteins, and perturbations in factor levels eliminate the physiological response, resulting in either tuning down or exaggeration of the transcriptional pulse.

}, issn = {2211-1247}, doi = {10.1016/j.celrep.2015.03.039}, author = {Kalo, Alon and Kanter, Itamar and Shraga, Amit and Sheinberger, Jonathan and Tzemach, Hadar and Kinor, Noa and Singer, Robert H and Lionnet, Timoth{\'e}e and Shav-Tal, Yaron} } @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 {46267, title = {A general method to improve fluorophores for live-cell and single-molecule microscopy.}, journal = {Nature Methods}, volume = {12}, year = {2015}, month = {2015 Jan 19}, pages = {244-50}, abstract = {

Specific labeling of biomolecules with bright fluorophores is the keystone of fluorescence microscopy. Genetically encoded self-labeling tag proteins can be coupled to synthetic dyes inside living cells, resulting in brighter reporters than fluorescent proteins. Intracellular labeling using these techniques requires cell-permeable fluorescent ligands, however, limiting utility to a small number of classic fluorophores. Here we describe a simple structural modification that improves the brightness and photostability of dyes while preserving spectral properties and cell permeability. Inspired by molecular modeling, we replaced the N,N-dimethylamino substituents in tetramethylrhodamine with four-membered azetidine rings. This addition of two carbon atoms doubles the quantum efficiency and improves the photon yield of the dye in applications ranging from in vitro single-molecule measurements to super-resolution imaging. The novel substitution is generalizable, yielding a palette of chemical dyes with improved quantum efficiencies that spans the UV and visible range.

}, issn = {1548-7105}, doi = {10.1038/nmeth.3256}, author = {Grimm, Jonathan B and English, Brian P and Chen, Jiji and Slaughter, Joel P and Zhang, Zhengjian and Revyakin, Andrey and Patel, Ronak and Macklin, John J and Normanno, Davide and Singer, Robert H and Lionnet, Timoth{\'e}e and Lavis, Luke D} } @article {47734, title = {Imaging transcription: past, present, and future.}, journal = {Cold Spring Harbor Symposia on Quantitative Biology}, volume = {80}, year = {2015}, month = {2015}, pages = {1-8}, abstract = {

Transcription, the first step of gene expression, is exquisitely regulated in higher eukaryotes to ensure correct development and homeostasis. Traditional biochemical, genetic, and genomic approaches have proved successful at identifying factors, regulatory sequences, and potential pathways that modulate transcription. However, they typically only provide snapshots or population averages of the highly dynamic, stochastic biochemical processes involved in transcriptional regulation. Single-molecule live-cell imaging has, therefore, emerged as a complementary approach capable of circumventing these limitations. By observing sequences of molecular events in real time as they occur in their native context, imaging has the power to derive cause-and-effect relationships and quantitative kinetics to build predictive models of transcription. Ongoing progress in fluorescence imaging technology has brought new microscopes and labeling technologies that now make it possible to visualize and quantify the transcription process with single-molecule resolution in living cells and animals. Here we provide an overview of the evolution and current state of transcription imaging technologies. We discuss some of the important concepts they uncovered and present possible future developments that might solve long-standing questions in transcriptional regulation.

}, issn = {1943-4456}, doi = {10.1101/sqb.2015.80.027201}, author = {Coleman, Robert A and Liu, Zhe and Darzacq, Xavier and Tjian, Robert and Singer, Robert H and Lionnet, Timoth{\'e}e} } @article {46701, title = {In the right place at the right time: visualizing and understanding mRNA localization.}, journal = {Nature Reviews Molecular Cell Biology}, volume = {16}, year = {2015}, month = {2015 Feb}, pages = {95-109}, abstract = {

The spatial regulation of protein translation is an efficient way to create functional and structural asymmetries in cells. Recent research has furthered our understanding of how individual cells spatially organize protein synthesis, by applying innovative technology to characterize the relationship between mRNAs and their regulatory proteins, single-mRNA trafficking dynamics, physiological effects of abrogating mRNA localization in vivo and for endogenous mRNA labelling. The implementation of new imaging technologies has yielded valuable information on mRNA localization, for example, by observing single molecules in tissues. The emerging movements and localization patterns of mRNAs in morphologically distinct unicellular organisms and in neurons have illuminated shared and specialized mechanisms of mRNA localization, and this information is complemented by transgenic and biochemical techniques that reveal the biological consequences of mRNA mislocalization.

}, keywords = {Animals, Biological Transport, Humans, Neurons, Protein Biosynthesis, Protein Transport, RNA, Messenger}, issn = {1471-0080}, doi = {10.1038/nrm3918}, author = {Buxbaum, Adina R and Haimovich, Gal and Singer, Robert H} } @article {46929, title = {Inferring transient particle transport dynamics in live cells.}, journal = {Nature Methods}, volume = {12}, year = {2015}, month = {2015 Jul 20}, pages = {838-40}, abstract = {

Live-cell imaging and particle tracking provide rich information on mechanisms of intracellular transport. However, trajectory analysis procedures to infer complex transport dynamics involving stochastic switching between active transport and diffusive motion are lacking. We applied Bayesian model selection to hidden Markov modeling to infer transient transport states from trajectories of mRNA-protein complexes in live mouse hippocampal neurons and metaphase kinetochores in dividing human cells. The software is available at http://hmm-bayes.org/.

}, issn = {1548-7105}, doi = {10.1038/nmeth.3483}, author = {Monnier, Nilah and Barry, Zachary and Park, Hye Yoon and Su, Kuan-Chung and Katz, Zachary and English, Brian P and Dey, Arkajit and Pan, Keyao and Cheeseman, Iain M and Singer, Robert H and Bathe, Mark} } @article {46925, title = {Nuclear accessibility of β-actin mRNA is measured by 3D single-molecule real-time tracking.}, journal = {Journal of Cell Biology}, volume = {209}, year = {2015}, month = {2015 May 25}, pages = {609-19}, abstract = {

Imaging single proteins or RNAs allows direct visualization of the inner workings of the cell. Typically, three-dimensional (3D) images are acquired by sequentially capturing a series of 2D sections. The time required to step through the sample often impedes imaging of large numbers of rapidly moving molecules. Here we applied multifocus microscopy (MFM) to instantaneously capture 3D single-molecule real-time images in live cells, visualizing cell nuclei at 10 volumes per second. We developed image analysis techniques to analyze messenger RNA (mRNA) diffusion in the entire volume of the nucleus. Combining MFM with precise registration between fluorescently labeled mRNA, nuclear pore complexes, and chromatin, we obtained globally optimal image alignment within 80-nm precision using transformation models. We show that β-actin mRNAs freely access the entire nucleus and fewer than 60\% of mRNAs are more than 0.5 {\textmu}m away from a nuclear pore, and we do so for the first time accounting for spatial inhomogeneity of nuclear organization.

}, issn = {1540-8140}, doi = {10.1083/jcb.201411032}, author = {Smith, Carlas S and Preibisch, Stephan and Joseph, Aviva and Abrahamsson, Sara and Rieger, Bernd and Myers, Eugene and Singer, Robert H and Grunwald, David} } @article {46698, title = {Reminiscences on my life with RNA: a self-indulgent perspective.}, journal = {RNA}, volume = {21}, year = {2015}, month = {2015 Apr}, pages = {508-9}, keywords = {Humans, Narration, RNA}, issn = {1469-9001}, doi = {10.1261/rna.050922.115}, author = {Singer, Robert H} } @article {48315, title = {Single-molecule insights into mRNA dynamics in neurons.}, journal = {Trends in Cell Biology}, volume = {25}, year = {2015}, month = {2015 Aug}, pages = {468-75}, abstract = {

Targeting of mRNAs to neuronal dendrites and axons plays an integral role in intracellular signaling, development, and synaptic plasticity. Single-molecule imaging of mRNAs in neurons and brain tissue has led to enhanced understanding of mRNA dynamics. Here we discuss aspects of mRNA regulation as revealed by single-molecule detection, which has led to quantitative analyses of mRNA diversity, localization, transport, and translation. These exciting new discoveries propel our understanding of the life of an mRNA in a neuron and how its activity is regulated at the single-molecule level.

}, keywords = {Animals, Humans, In Situ Hybridization, Fluorescence, Intracellular Membranes, Neuronal Plasticity, Neurons, Protein Biosynthesis, RNA, Messenger, Signal Transduction}, issn = {1879-3088}, doi = {10.1016/j.tcb.2015.05.005}, author = {Buxbaum, Adina R and Yoon, Young J and Singer, Robert H and Park, Hye Yoon} } @article {46700, title = {Specific interaction of KIF11 with ZBP1 regulates the transport of β-actin mRNA and cell motility.}, journal = {Journal of Cell Science}, volume = {128}, year = {2015}, month = {2015 Mar 1}, pages = {1001-10}, abstract = {

ZBP1-modulated localization of β-actin mRNA enables a cell to establish polarity and structural asymmetry. Although the mechanism of β-actin mRNA localization has been well established, the underlying mechanism of how a specific molecular motor contributes to the transport of the ZBP1 (also known as IGF2BP1) complex in non-neuronal cells remains elusive. In this study, we report the isolation and identification of KIF11, a microtubule motor, which physically interacts with ZBP1 and is a component of β-actin messenger ribonucleoprotein particles (mRNPs). We show that KIF11 colocalizes with the β-actin mRNA, and the ability of KIF11 to transport β-actin mRNA is dependent on ZBP1. We characterize the corresponding regions of ZBP1 and KIF11 that mediate the interaction of the two proteins in vitro and in vivo. Disruption of the in vivo interaction of KIF11 with ZBP1 delocalizes β-actin mRNA and affects cell migration. Our study reveals a molecular mechanism by which a particular microtubule motor mediates the transport of an mRNP through direct interaction with an mRNA-binding protein.

}, issn = {1477-9137}, doi = {10.1242/jcs.161679}, author = {Song, Tingting and Zheng, Yi and Wang, Yarong and Katz, Zachary and Liu, Xin and Chen, Shaoying and Singer, Robert H and Gu, Wei} } @article {46697, title = {Synonymous modification results in high-fidelity gene expression of repetitive protein and nucleotide sequences.}, journal = {Genes \& Development}, volume = {29}, year = {2015}, month = {2015 Apr 15}, pages = {876-86}, abstract = {

Repetitive nucleotide or amino acid sequences are often engineered into probes and biosensors to achieve functional readouts and robust signal amplification. However, these repeated sequences are notoriously prone to aberrant deletion and degradation, impacting the ability to correctly detect and interpret biological functions. Here, we introduce a facile and generalizable approach to solve this often unappreciated problem by modifying the nucleotide sequences of the target mRNA to make them nonrepetitive but still functional ("synonymous"). We first demonstrated the procedure by designing a cassette of synonymous MS2 RNA motifs and tandem coat proteins for RNA imaging and showed a dramatic improvement in signal and reproducibility in single-RNA detection in live cells. The same approach was extended to enhancing the stability of engineered fluorescent biosensors containing a fluorescent resonance energy transfer (FRET) pair of fluorescent proteins on which a great majority of systems thus far in the field are based. Using the synonymous modification to FRET biosensors, we achieved correct expression of full-length sensors, eliminating the aberrant truncation products that often were assumed to be due to nonspecific proteolytic cleavages. Importantly, the biological interpretations of the sensor are significantly different when a correct, full-length biosensor is expressed. Thus, we show here a useful and generally applicable method to maintain the integrity of expressed genes, critical for the correct interpretation of probe readouts.

}, issn = {1549-5477}, doi = {10.1101/gad.259358.115}, author = {Wu, Bin and Miskolci, Veronika and Sato, Hanae and Tutucci, Evelina and Kenworthy, Charles A and Donnelly, Sara K and Yoon, Young J and Cox, Dianne and Singer, Robert H and Hodgson, Louis} } @article {47266, title = {A three-camera imaging microscope for high-speed single-molecule tracking and super-resolution imaging in living cells.}, journal = {Proceedings of SPIE}, volume = {9550}, year = {2015}, month = {2015 Aug 21}, pages = {955008 }, abstract = {

Our aim is to develop quantitative single-molecule assays to study when and where molecules are interacting inside living cells and where enzymes are active. To this end we present a three-camera imaging microscope for fast tracking of multiple interacting molecules simultaneously, with high spatiotemporal resolution. The system was designed around an ASI RAMM frame using three separate tube lenses and custom multi-band dichroics to allow for enhanced detection efficiency. The frame times of the three Andor iXon Ultra EMCCD cameras are hardware synchronized to the laser excitation pulses of the three excitation lasers, such that the fluorophores are effectively immobilized during frame acquisitions and do not yield detections that are motion-blurred. Stroboscopic illumination allows robust detection from even rapidly moving molecules while minimizing bleaching, and since snapshots can be spaced out with varying time intervals, stroboscopic illumination enables a direct comparison to be made between fast and slow molecules under identical light dosage. We have developed algorithms that accurately track and co-localize multiple interacting biomolecules. The three-color microscope combined with our co-movement algorithms have made it possible for instance to simultaneously image and track how the chromosome environment affects diffusion kinetics or determine how mRNAs diffuse during translation. Such multiplexed single-molecule measurements at a high spatiotemporal resolution inside living cells will provide a major tool for testing models relating molecular architecture and biological dynamics.

}, doi = {10.1117/12.2190246}, author = {English, Brian P and Singer, Robert H} } @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 {46706, title = {Background free imaging of single mRNAs in live cells using split fluorescent proteins.}, journal = {Scientific Reports}, volume = {4}, year = {2014}, month = {2014 Jan 9}, pages = {3615}, abstract = {

We describe a technique for imaging single mRNAs in living cells based on fluorescent protein (FP) complementation. We employ the high affinity interaction between the bacterial phage MS2/PP7 coat proteins and their respective RNA binding motifs as an RNA scaffold to bring two halves of a split-FP together to image single reporter mRNAs without background fluorescence.

}, keywords = {Bacteriophages, Fluorescence, Luminescent Proteins, RNA, Messenger}, issn = {2045-2322}, doi = {10.1038/srep03615}, author = {Wu, Bin and Chen, Jiahao and Singer, Robert H} } @article {46708, title = {Dynamics of survival of motor neuron (SMN) protein interaction with the mRNA-binding protein IMP1 facilitates its trafficking into motor neuron axons.}, journal = {Developmental Neurobiology}, volume = {74}, year = {2014}, month = {2014 Mar}, pages = {319-32}, abstract = {

Spinal muscular atrophy (SMA) is a lethal neurodegenerative disease specifically affecting spinal motor neurons. SMA is caused by the homozygous deletion or mutation of the survival of motor neuron 1 (SMN1) gene. The SMN protein plays an essential role in the assembly of spliceosomal ribonucleoproteins. However, it is still unclear how low levels of the ubiquitously expressed SMN protein lead to the selective degeneration of motor neurons. An additional role for SMN in the regulation of the axonal transport of mRNA-binding proteins (mRBPs) and their target mRNAs has been proposed. Indeed, several mRBPs have been shown to interact with SMN, and the axonal levels of few mRNAs, such as the β-actin mRNA, are reduced in SMA motor neurons. In this study we have identified the β-actin mRNA-binding protein IMP1/ZBP1 as a novel SMN-interacting protein. Using a combination of biochemical assays and quantitative imaging techniques in primary motor neurons, we show that IMP1 associates with SMN in individual granules that are actively transported in motor neuron axons. Furthermore, we demonstrate that IMP1 axonal localization depends on SMN levels, and that SMN deficiency in SMA motor neurons leads to a dramatic reduction of IMP1 protein levels. In contrast, no difference in IMP1 protein levels was detected in whole brain lysates from SMA mice, further suggesting neuron specific roles of SMN in IMP1 expression and localization. Taken together, our data support a role for SMN in the regulation of mRNA localization and axonal transport through its interaction with mRBPs such as IMP1.

}, keywords = {Animals, Axonal Transport, Axons, Biological Transport, Active, Brain, Cells, Cultured, Chromaffin Granules, Humans, Mice, Mice, Transgenic, Motor Neurons, Protein Interaction Domains and Motifs, Rats, RNA-Binding Proteins, Survival of Motor Neuron 1 Protein, Survival of Motor Neuron 2 Protein}, issn = {1932-846X}, doi = {10.1002/dneu.22111}, author = {Fallini, Claudia and Rouanet, Jeremy P and Donlin-Asp, Paul G and Guo, Peng and Zhang, Honglai and Singer, Robert H and Rossoll, Wilfried and Bassell, Gary J} } @article {6962, title = {Efficient Bayesian-based multiview deconvolution.}, journal = {Nature Methods}, volume = {11}, year = {2014}, month = {2014 Apr 20}, pages = {645-8}, abstract = {

Light-sheet fluorescence microscopy is able to image large specimens with high resolution by capturing the samples from multiple angles. Multiview deconvolution can substantially improve the resolution and contrast of the images, but its application has been limited owing to the large size of the data sets. Here we present a Bayesian-based derivation of multiview deconvolution that drastically improves the convergence time, and we provide a fast implementation using graphics hardware.

}, issn = {1548-7105}, doi = {10.1038/nmeth.2929}, author = {Preibisch, Stephan and Amat, Fernando and Stamataki, Evangelia and Sarov, Mihail and Singer, Robert H and Myers, Eugene and Tomancak, Pavel} } @article {46703, title = {Gene regulation: the HSP70 gene jumps when shocked.}, journal = {Current Biology}, volume = {24}, year = {2014}, month = {2014 May 19}, pages = {R396-8}, abstract = {

Limited chromosome mobility has been observed in mammalian interphase nuclei. Live imaging shows unidirectional and actin-dependent movement of HSP70 loci towards speckles upon heat shock, resulting in enhanced transcription. This adds further impetus to understanding compartmentalization of function in the nucleus.

}, keywords = {Animals, Heat-Shock Response, Humans, Transcriptional Activation, Transgenes}, issn = {1879-0445}, doi = {10.1016/j.cub.2014.03.070}, author = {Vera, Maria and Singer, Robert H} } @article {45699, title = {Photoswitchable red fluorescent protein with a large stokes shift.}, journal = {Chemistry \& Biology}, volume = {21}, year = {2014}, month = {2014 Oct 23}, pages = {1402-14}, abstract = {

A subclass of fluorescent proteins (FPs), large Stokes shift (LSS) FP, are characterized by increased spread between excitation and emission maxima. We report a photoswitchable variant of a red FP with an LSS, PSLSSmKate, which initially exhibits excitation and emission at 445 and 622\ nm, but violet irradiation photoswitches PSLSSmKate into a common red form with excitation and emission at 573 and 621\ nm. We characterize spectral, photophysical, and biochemical properties of PSLSSmKate in\ vitro and in mammalian cells and determine its crystal structure in the LSS form. Mass spectrometry, mutagenesis, and spectroscopy of PSLSSmKate allow us\ to propose molecular mechanisms for the LSS, pH dependence, and light-induced chromophore transformation. We demonstrate the applicability of PSLSSmKate to superresolution photoactivated localization microscopy and protein dynamics in live cells. Given its promising properties, we expect that PSLSSmKate-like phenotype will be further used for photoactivatable imaging and tracking multiple populations of intracellular objects.

}, issn = {1879-1301}, doi = {10.1016/j.chembiol.2014.08.010}, author = {Piatkevich, Kiryl D and English, Brian P and Malashkevich, Vladimir N and Xiao, Hui and Almo, Steven C and Singer, Robert H and Verkhusha, Vladislav V} } @article {46705, title = {Single β-actin mRNA detection in neurons reveals a mechanism for regulating its translatability.}, journal = {Science}, volume = {343}, year = {2014}, month = {2014 Jan 24}, pages = {419-22}, abstract = {

The physical manifestation of learning and memory formation in the brain can be expressed by strengthening or weakening of synaptic connections through morphological changes. Local actin remodeling underlies some forms of plasticity and may be facilitated by local β-actin synthesis, but dynamic information is lacking. In this work, we use single-molecule in situ hybridization to demonstrate that dendritic β-actin messenger RNA (mRNA) and ribosomes are in a masked, neuron-specific form. Chemically induced long-term potentiation prompts transient mRNA unmasking, which depends on factors active during synaptic activity. Ribosomes and single β-actin mRNA motility increase after stimulation, indicative of release from complexes. Hence, the single-molecule assays we developed allow for the quantification of activity-induced unmasking and availability for active translation. Further, our work demonstrates that β-actin mRNA and ribosomes are in a masked state that is alleviated by stimulation.

}, keywords = {Actins, Animals, Cells, Cultured, Dendrites, In Situ Hybridization, Fluorescence, Long-Term Potentiation, Memory, Mice, Mice, Transgenic, Neuronal Plasticity, Neurons, Protein Biosynthesis, Ribosomes, RNA, Messenger, RNA, Ribosomal, Synapses}, issn = {1095-9203}, doi = {10.1126/science.1242939}, author = {Buxbaum, Adina R and Wu, Bin and Singer, Robert H} } @article {46702, title = {The translation elongation factor eEF1A1 couples transcription to translation during heat shock response.}, journal = {eLife}, volume = {3}, year = {2014}, month = {2014 Sep 16}, pages = {e03164}, abstract = {

Translation elongation factor eEF1A has a well-defined role in protein synthesis. In this study, we demonstrate a new role for eEF1A: it participates in the entire process of the heat shock response (HSR) in mammalian cells from transcription through translation. Upon stress, isoform 1 of eEF1A rapidly activates transcription of HSP70 by recruiting the master regulator HSF1 to its promoter. eEF1A1 then associates with elongating RNA polymerase II and the 3{\textquoteright}UTR of HSP70 mRNA, stabilizing it and facilitating its transport from the nucleus to active ribosomes. eEF1A1-depleted cells exhibit severely impaired HSR and compromised thermotolerance. In contrast, tissue-specific isoform 2 of eEF1A does not support HSR. By adjusting transcriptional yield to translational needs, eEF1A1 renders HSR rapid, robust, and highly selective; thus, representing an attractive therapeutic target for numerous conditions associated with disrupted protein homeostasis, ranging from neurodegeneration to cancer.

}, issn = {2050-084X}, doi = {10.7554/eLife.03164}, author = {Vera, Maria and Pani, Bibhusita and Griffiths, Lowri A and Muchardt, Christian and Abbott, Catherine M and Singer, Robert H and Nudler, Evgeny} } @article {46676, title = {Visualization of dynamics of single endogenous mRNA labeled in live mouse.}, journal = {Science}, volume = {343}, year = {2014}, month = {2014 Jan 24}, pages = {422-4}, abstract = {

The transcription and transport of messenger RNA (mRNA) are critical steps in regulating the spatial and temporal components of gene expression, but it has not been possible to observe the dynamics of endogenous mRNA in primary mammalian tissues. We have developed a transgenic mouse in which all β-actin mRNA is fluorescently labeled. We found that β-actin mRNA in primary fibroblasts localizes predominantly by diffusion and trapping as single mRNAs. In cultured neurons and acute brain slices, we found that multiple β-actin mRNAs can assemble together, travel by active transport, and disassemble upon depolarization by potassium chloride. Imaging of brain slices revealed immediate early induction of β-actin transcription after depolarization. Studying endogenous mRNA in live mouse tissues provides insight into its dynamic regulation within the context of the cellular and tissue microenvironment.

}, keywords = {Actins, Animals, Brain, Cells, Cultured, Fibroblasts, Fluorescent Dyes, Mice, Mice, Transgenic, Neuroimaging, Neurons, Protein Biosynthesis, RNA, Messenger, Staining and Labeling}, issn = {1095-9203}, doi = {10.1126/science.1239200}, author = {Park, Hye Yoon and Lim, Hyungsik and Yoon, Young J and Follenzi, Antonia and Nwokafor, Chiso and Lopez-Jones, Melissa and Meng, Xiuhua and Singer, Robert H} } @article {46712, title = {Colocalization of different influenza viral RNA segments in the cytoplasm before viral budding as shown by single-molecule sensitivity FISH analysis.}, journal = {PLoS Pathogens}, volume = {9}, year = {2013}, month = {2013}, pages = {e1003358}, abstract = {

The Influenza A virus genome consists of eight negative sense, single-stranded RNA segments. Although it has been established that most virus particles contain a single copy of each of the eight viral RNAs, the packaging selection mechanism remains poorly understood. Influenza viral RNAs are synthesized in the nucleus, exported into the cytoplasm and travel to the plasma membrane where viral budding and genome packaging occurs. Due to the difficulties in analyzing associated vRNPs while preserving information about their positions within the cell, it has remained unclear how and where during cellular trafficking the viral RNAs of different segments encounter each other. Using a multicolor single-molecule sensitivity fluorescence in situ hybridization (smFISH) approach, we have quantitatively monitored the colocalization of pairs of influenza viral RNAs in infected cells. We found that upon infection, the viral RNAs from the incoming particles travel together until they reach the nucleus. The viral RNAs were then detected in distinct locations in the nucleus; they are then exported individually and initially remain separated in the cytoplasm. At later time points, the different viral RNA segments gather together in the cytoplasm in a microtubule independent manner. Viral RNAs of different identities colocalize at a high frequency when they are associated with Rab11 positive vesicles, suggesting that Rab11 positive organelles may facilitate the association of different viral RNAs. Using engineered influenza viruses lacking the expression of HA or M2 protein, we showed that these viral proteins are not essential for the colocalization of two different viral RNAs in the cytoplasm. In sum, our smFISH results reveal that the viral RNAs travel together in the cytoplasm before their arrival at the plasma membrane budding sites. This newly characterized step of the genome packaging process demonstrates the precise spatiotemporal regulation of the infection cycle.

}, keywords = {Active Transport, Cell Nucleus, Animals, Cell Nucleus, Dogs, Hemagglutinin Glycoproteins, Influenza Virus, Humans, In Situ Hybridization, Fluorescence, Influenza A virus, Madin Darby Canine Kidney Cells, rab GTP-Binding Proteins, RNA, Viral, Viral Matrix Proteins, Virus Assembly}, issn = {1553-7374}, doi = {10.1371/journal.ppat.1003358}, author = {Chou, Yi-ying and Heaton, Nicholas S and Gao, Qinshan and Palese, Peter and Singer, Robert H and Singer, Robert and Lionnet, Timoth{\'e}e} } @article {46707, title = {Direct observation of frequency modulated transcription in single cells using light activation.}, journal = {eLife}, volume = {2}, year = {2013}, month = {2013 Sep 24}, pages = {e00750}, abstract = {

Single-cell analysis has revealed that transcription is dynamic and stochastic, but tools are lacking that can determine the mechanism operating at a single gene. Here we utilize single-molecule observations of RNA in fixed and living cells to develop a single-cell model of steroid-receptor mediated gene activation. We determine that steroids drive mRNA synthesis by frequency modulation of transcription. This digital behavior in single cells gives rise to the well-known analog dose response across the population. To test this model, we developed a light-activation technology to turn on a single steroid-responsive gene and follow dynamic synthesis of RNA from the activated locus. DOI:http://dx.doi.org/10.7554/eLife.00750.001.

}, issn = {2050-084X}, doi = {10.7554/eLife.00750}, author = {Larson, Daniel R and Fritzsch, Christoph and Sun, Liang and Meng, Xiuhau and Lawrence, David S and Singer, Robert H} } @article {46709, title = {Eukaryotic transcriptional dynamics: from single molecules to cell populations.}, journal = {Nature Reviews Genetics}, volume = {14}, year = {2013}, month = {2013 Aug}, pages = {572-84}, abstract = {

Transcriptional regulation is achieved through combinatorial interactions between regulatory elements in the human genome and a vast range of factors that modulate the recruitment and activity of RNA polymerase. Experimental approaches for studying transcription in vivo now extend from single-molecule techniques to genome-wide measurements. Parallel to these developments is the need for testable quantitative and predictive models for understanding gene regulation. These conceptual models must also provide insight into the dynamics of transcription and the variability that is observed at the single-cell level. In this Review, we discuss recent results on transcriptional regulation and also the models those results engender. We show how a non-equilibrium description informs our view of transcription by explicitly considering time- and energy-dependence at the molecular level.

}, keywords = {DNA-Directed RNA Polymerases, Gene Expression Regulation, Genome, Human, Humans, Transcription Factors, Transcription, Genetic}, issn = {1471-0064}, doi = {10.1038/nrg3484}, author = {Coulon, Antoine and Chow, Carson C and Singer, Robert H and Larson, Daniel R} } @article {46714, title = {The fate of the messenger is pre-determined: a new model for regulation of gene expression.}, journal = {Biochimica et Biophysica Acta (BBA)}, volume = {1829}, year = {2013}, month = {2013 Jun-Jul}, pages = {643-53}, abstract = {

Recent years have seen a rise in publications demonstrating coupling between transcription and mRNA decay. This coupling most often accompanies cellular processes that involve transitions in gene expression patterns, for example during mitotic division and cellular differentiation and in response to cellular stress. Transcription can affect the mRNA fate by multiple mechanisms. The most novel finding is the process of co-transcriptional imprinting of mRNAs with proteins, which in turn regulate cytoplasmic mRNA stability. Transcription therefore is not only a catalyst of mRNA synthesis but also provides a platform that enables imprinting, which coordinates between transcription and mRNA decay. Here we present an overview of the literature, which provides the evidence of coupling between transcription and decay, review the mechanisms and regulators by which the two processes are coupled, discuss why such coupling is beneficial and present a new model for regulation of gene expression. This article is part of a Special Issue entitled: RNA Decay mechanisms.

}, keywords = {Cytoplasm, Gene Expression Regulation, Fungal, Genomic Imprinting, Humans, Poly(A)-Binding Proteins, Ribonucleases, RNA Polymerase II, RNA Stability, RNA, Messenger, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Transcription, Genetic}, issn = {0006-3002}, doi = {10.1016/j.bbagrm.2013.01.004}, author = {Haimovich, Gal and Choder, Mordechai and Singer, Robert H and Trcek, Tatjana} } @article {46710, title = {mRNA on the move: the road to its biological destiny.}, journal = {The Journal of Biological Chemistry}, volume = {288}, year = {2013}, month = {2013 Jul 12}, pages = {20361-8}, abstract = {

Cells have evolved to regulate the asymmetric distribution of specific mRNA targets to institute spatial and temporal control over gene expression. Over the last few decades, evidence has mounted as to the importance of localization elements in the mRNA sequence and their respective RNA-binding proteins. Live imaging methodologies have shown mechanistic details of this phenomenon. In this minireview, we focus on the advanced biochemical and cell imaging techniques used to tweeze out the finer aspects of mechanisms of mRNA movement.

}, keywords = {Animals, Cytoplasm, Humans, In Situ Hybridization, Microscopy, Fluorescence, Models, Genetic, RNA Precursors, RNA Transport, RNA, Messenger, RNA-Binding Proteins}, issn = {1083-351X}, doi = {10.1074/jbc.R113.452094}, author = {Eliscovich, Carolina and Buxbaum, Adina R and Katz, Zachary B and Singer, Robert H} } @article {46711, title = {Single cell analysis of RNA-mediated histone H3.3 recruitment to a cytomegalovirus promoter-regulated transcription site.}, journal = {The Journal of Biological Chemistry}, volume = {288}, year = {2013}, month = {2013 Jul 5}, pages = {19882-99}, abstract = {

Unlike the core histones, which are incorporated into nucleosomes concomitant with DNA replication, histone H3.3 is synthesized throughout the cell cycle and utilized for replication-independent (RI) chromatin assembly. The RI incorporation of H3.3 into nucleosomes is highly conserved and occurs at both euchromatin and heterochromatin. However, neither the mechanism of H3.3 recruitment nor its essential function is well understood. Several different chaperones regulate H3.3 assembly at distinct sites. The H3.3 chaperone, Daxx, and the chromatin-remodeling factor, ATRX, are required for H3.3 incorporation and heterochromatic silencing at telomeres, pericentromeres, and the cytomegalovirus (CMV) promoter. By evaluating H3.3 dynamics at a CMV promoter-regulated transcription site in a genetic background in which RI chromatin assembly is blocked, we have been able to decipher the regulatory events upstream of RI nucleosomal deposition. We find that at the activated transcription site, H3.3 accumulates with sense and antisense RNA, suggesting that it is recruited through an RNA-mediated mechanism. Sense and antisense transcription also increases after H3.3 knockdown, suggesting that the RNA signal is amplified when chromatin assembly is blocked and attenuated by nucleosomal deposition. Additionally, we find that H3.3 is still recruited after Daxx knockdown, supporting a chaperone-independent recruitment mechanism. Sequences in the H3.3 N-terminal tail and αN helix mediate both its recruitment to RNA at the activated transcription site and its interaction with double-stranded RNA in vitro. Interestingly, the H3.3 gain-of-function pediatric glioblastoma mutations, G34R and K27M, differentially affect H3.3 affinity in these assays, suggesting that disruption of an RNA-mediated regulatory event could drive malignant transformation.

}, keywords = {Cell Line, Chromatin Assembly and Disassembly, Cytomegalovirus, Histones, Humans, Nucleosomes, Promoter Regions, Genetic, Protein Structure, Secondary, RNA, Viral, Transcription, Genetic}, issn = {1083-351X}, doi = {10.1074/jbc.M113.473181}, author = {Newhart, Alyshia and Rafalska-Metcalf, Ilona U and Yang, Tian and Joo, Lucy M and Powers, Sara Lawrence and Kossenkov, Andrew V and Lopez-Jones, Melissa and Singer, Robert H and Showe, Louise C and Skordalakes, Emmanuel and Janicki, Susan M} } @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 {46713, title = {Temporal and spatial characterization of nonsense-mediated mRNA decay.}, journal = {Genes \& Development}, volume = {27}, year = {2013}, month = {2013 Mar 1}, pages = {541-51}, abstract = {

Nonsense-mediated mRNA decay (NMD) is a quality control mechanism responsible for "surveying" mRNAs during translation and degrading those that harbor a premature termination codon (PTC). Currently the intracellular spatial location of NMD and the kinetics of its decay step in mammalian cells are under debate. To address these issues, we used single-RNA fluorescent in situ hybridization (FISH) and measured the NMD of PTC-containing β-globin mRNA in intact single cells after the induction of β-globin gene transcription. This approach preserves temporal and spatial information of the NMD process, both of which would be lost in an ensemble study. We determined that decay of the majority of PTC-containing β-globin mRNA occurs soon after its export into the cytoplasm, with a half-life of \<1 min; the remainder is degraded with a half-life of \>12 h, similar to the half-life of normal PTC-free β-globin mRNA, indicating that it had evaded NMD. Importantly, NMD does not occur within the nucleoplasm, thus countering the long-debated idea of nuclear degradation of PTC-containing transcripts. We provide a spatial and temporal model for the biphasic decay of NMD targets.

}, keywords = {Animals, beta-Globins, Cell Line, Cell Nucleus, Cytoplasm, Humans, In Situ Hybridization, Fluorescence, Nonsense Mediated mRNA Decay, RNA, Messenger, Time Factors}, issn = {1549-5477}, doi = {10.1101/gad.209635.112}, author = {Trcek, Tatjana and Sato, Hanae and Singer, Robert H and Maquat, Lynne E} } @article {46715, title = {Time-integrated fluorescence cumulant analysis and its application in living cells.}, journal = {Methods in Enzymology}, volume = {518}, year = {2013}, month = {2013}, pages = {99-119}, abstract = {

Time-integrated fluorescence cumulant analysis (TIFCA) is a data analysis technique for fluorescence fluctuation spectroscopy (FFS) that extracts information from the cumulants of the integrated fluorescence intensity. It is the first exact theory that describes the effect of sampling time on FFS experiment. Rebinning of data to longer sampling times helps to increase the signal/noise ratio of the experimental cumulants of the photon counts. The sampling time dependence of the cumulants encodes both brightness and diffusion information of the sample. TIFCA analysis extracts this information by fitting the cumulants to model functions. Generalization of TIFCA to multicolor FFS experiment is straightforward. Here, we present an overview of the theory, its implementation, as well as the benefits and requirements of TIFCA. The questions of why, when, and how to use TIFCA will be discussed. We give several examples of practical applications of TIFCA, particularly focused on measuring molecular interaction in living cells.

}, keywords = {Cell Line, Cells, Humans, Protein Binding, Proteins, RNA, Messenger, Spectrometry, Fluorescence}, issn = {1557-7988}, doi = {10.1016/B978-0-12-388422-0.00005-4}, author = {Wu, Bin and Singer, Robert H and Mueller, Joachim D} } @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 {46726, title = {IGF2BP1 promotes cell migration by regulating MK5 and PTEN signaling.}, journal = {Genes \& Development}, volume = {26}, year = {2012}, month = {2012 Jan 15}, pages = {176-89}, abstract = {

In primary neurons, the oncofetal RNA-binding protein IGF2BP1 (IGF2 mRNA-binding protein 1) controls spatially restricted β-actin (ACTB) mRNA translation and modulates growth cone guidance. In cultured tumor-derived cells, IGF2BP1 was shown to regulate the formation of lamellipodia and invadopodia. However, how and via which target mRNAs IGF2BP1 controls the motility of tumor-derived cells has remained elusive. In this study, we reveal that IGF2BP1 promotes the velocity and directionality of tumor-derived cell migration by determining the cytoplasmic fate of two novel target mRNAs: MAPK4 and PTEN. Inhibition of MAPK4 mRNA translation by IGF2BP1 antagonizes MK5 activation and prevents phosphorylation of HSP27, which sequesters actin monomers available for F-actin polymerization. Consequently, HSP27-ACTB association is reduced, mobilizing cellular G-actin for polymerization in order to promote the velocity of cell migration. At the same time, stabilization of the PTEN mRNA by IGF2BP1 enhances PTEN expression and antagonizes PIP(3)-directed signaling. This enforces the directionality of cell migration in a RAC1-dependent manner by preventing additional lamellipodia from forming and sustaining cell polarization intrinsically. IGF2BP1 thus promotes the velocity and persistence of tumor cell migration by controlling the expression of signaling proteins. This fine-tunes and connects intracellular signaling networks in order to enhance actin dynamics and cell polarization.

}, keywords = {Actins, Cell Line, Tumor, Cell Movement, Cell Polarity, Enzyme Activation, Extracellular Signal-Regulated MAP Kinases, HSP27 Heat-Shock Proteins, Humans, Intracellular Signaling Peptides and Proteins, Phosphorylation, Protein-Serine-Threonine Kinases, PTEN Phosphohydrolase, RNA, Messenger, Signal Transduction}, issn = {1549-5477}, doi = {10.1101/gad.177642.111}, author = {St{\"o}hr, Nadine and K{\"o}hn, Marcel and Lederer, Marcell and Glass, Markus and Reinke, Claudia and Singer, Robert H and H{\"u}ttelmaier, Stefan} } @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 {46733, title = {Multiscale dynamics in nucleocytoplasmic transport.}, journal = {Current Opinion in Cell Biology}, volume = {24}, year = {2012}, month = {2012 Feb}, pages = {100-6}, abstract = {

The nuclear pore complex (NPC) has long been viewed as a point-like entry and exit channel between the nucleus and the cytoplasm. New data support a different view whereby the complex displays distinct spatial dynamics of variable duration ranging from milliseconds to events spanning the entire cell cycle. Discrete interaction sites outside the central channel become apparent, and transport regulation at these sites seems to be of greater importance than currently thought. Nuclear pore components are highly active outside the NPC or impact the fate of cargo transport away from the nuclear pore. The NPC is a highly dynamic, crowded environment-constantly loaded with cargo while providing selectivity based on unfolded proteins. Taken together, this comprises a new paradigm in how we view import/export dynamics and emphasizes the multiscale nature of NPC-mediated cellular transport.

}, keywords = {Active Transport, Cell Nucleus, Animals, Biological Transport, Cell Nucleus, Cytoplasm, Eukaryotic Cells, Models, Biological, Nuclear Pore, Nuclear Pore Complex Proteins}, issn = {1879-0410}, doi = {10.1016/j.ceb.2011.11.011}, author = {Grunwald, David and Singer, Robert H} } @article {46719, title = {Nuclear pore component Nup98 is a potential tumor suppressor and regulates posttranscriptional expression of select p53 target genes.}, journal = {Molecular Cell}, volume = {48}, year = {2012}, month = {2012 Dec 14}, pages = {799-810}, abstract = {

The p53 tumor suppressor utilizes multiple mechanisms to selectively regulate its myriad target genes, which in turn mediate diverse cellular processes. Here, using conventional and single-molecule mRNA analyses, we demonstrate that the nucleoporin Nup98 is required for full expression of p21, a key effector of the p53 pathway, but not several other p53 target genes. Nup98 regulates p21 mRNA levels by a posttranscriptional mechanism in which a complex containing Nup98 and the p21 mRNA 3{\textquoteright}UTR protects p21 mRNA from degradation by the exosome. An in silico approach revealed another p53 target (14-3-3σ) to be similarly regulated by Nup98. The expression of Nup98 is reduced in murine and human hepatocellular carcinomas (HCCs) and correlates with p21 expression in HCC patients. Our study elucidates a previously unrecognized function of wild-type Nup98 in regulating select p53 target genes that is distinct from the well-characterized oncogenic properties of Nup98 fusion proteins.

}, keywords = {14-3-3 Proteins, 3{\textquoteright} Untranslated Regions, Animals, Antineoplastic Agents, Phytogenic, Apoptosis, Binding Sites, Camptothecin, Carcinoma, Hepatocellular, Cell Aging, Cyclin-Dependent Kinase Inhibitor p21, Exosomes, Gene Expression Regulation, Neoplastic, Hep G2 Cells, Humans, Liver Neoplasms, Male, Mice, Mice, Knockout, Nuclear Pore Complex Proteins, P-Glycoproteins, RNA Interference, RNA Processing, Post-Transcriptional, RNA Stability, RNA, Messenger, Time Factors, Transfection, Tumor Suppressor Protein p53}, issn = {1097-4164}, doi = {10.1016/j.molcel.2012.09.020}, author = {Singer, Stephan and Zhao, Ruiying and Barsotti, Anthony M and Ouwehand, Anette and Fazollahi, Mina and Coutavas, Elias and Breuhahn, Kai and Neumann, Olaf and Longerich, Thomas and Pusterla, Tobias and Powers, Maureen A and Giles, Keith M and Leedman, Peter J and Hess, Jochen and Grunwald, David and Bussemaker, Harmen J and Singer, Robert H and Schirmacher, Peter and Prives, Carol} } @article {46730, title = {Regulation of local expression of cell adhesion and motility-related mRNAs in breast cancer cells by IMP1/ZBP1.}, journal = {The Journal of Cell Science}, volume = {125}, year = {2012}, month = {2012 Jan 1}, pages = {81-91}, abstract = {

Metastasis involves tumor cell detachment from the primary tumor, and acquisition of migratory and invasive capabilities. These capabilities are mediated by multiple events, including loss of cell-cell contact, an increase in focal adhesion turnover and failure to maintain a normal cell polarity. We have previously reported that silencing of the expression of the zipcode-binding protein IMP1/ZBP1 in breast tumor patients is associated with metastasis. IMP1/ZBP1 selectively binds to a group of mRNAs that encode important mediators for cell adhesion and motility. Here, we show that in both T47D and MDA231 human breast carcinoma cells IMP1/ZBP1 functions to suppress cell invasion. Binding of ZBP1 to the mRNAs encoding E-cadherin, β-actin, α-actinin and the Arp2/3 complex facilitates localization of the mRNAs, which stabilizes cell-cell connections and focal adhesions. Our studies suggest a novel mechanism through which IMP1/ZBP1 simultaneously regulates the local expression of many cell-motility-related mRNAs to maintain cell adherence and polarity, decrease focal adhesion turnover and maintain a persistent and directional motility.

}, keywords = {Actin-Related Protein 2-3 Complex, Actinin, Actins, Breast Neoplasms, Cadherins, Cell Adhesion, Cell Line, Tumor, Cell Movement, Focal Adhesions, Gene Expression Regulation, Neoplastic, Humans, Neoplasm Invasiveness, Neoplasm Metastasis, RNA, Messenger, RNA, Neoplasm, RNA-Binding Proteins}, issn = {1477-9137}, doi = {10.1242/jcs.086132}, author = {Gu, Wei and Katz, Zachary and Wu, Bin and Park, Hye Yoon and Li, Deling and Lin, Stanley and Wells, Amber L 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 {46724, title = {Transcription goes digital.}, journal = {EMBO Reports}, volume = {13}, year = {2012}, month = {2012 Apr 2}, pages = {313-21}, abstract = {

Transcription is a complex process that integrates the state of the cell and its environment to generate adequate responses for cell fitness and survival. Recent microscopy experiments have been able to monitor transcription from single genes in individual cells. These observations have revealed two striking features: transcriptional activity can vary markedly from one cell to another, and is subject to large changes over time, sometimes within minutes. How the chromatin structure, transcription machinery assembly and signalling networks generate such patterns is still unclear. In this review, we present the techniques used to investigate transcription from single genes, introduce quantitative modelling tools, and discuss transcription mechanisms and their implications for gene expression regulation.

}, keywords = {Animals, Gene Expression Regulation, Genes, Humans, Models, Genetic, Stochastic Processes, Transcription, Genetic}, issn = {1469-3178}, doi = {10.1038/embor.2012.31}, author = {Lionnet, Timoth{\'e}e and Singer, Robert H} } @article {46731, title = {Transgenic expression of ZBP1 in neurons suppresses cocaine-associated conditioning.}, journal = {Learning \& Memory}, volume = {19}, year = {2012}, month = {2012 Feb}, pages = {35-42}, abstract = {

To directly address whether regulating mRNA localization can influence animal behavior, we created transgenic mice that conditionally express Zipcode Binding Protein 1 (ZBP1) in a subset of neurons in the brain. ZBP1 is an RNA-binding protein that regulates the localization, as well as translation and stability of target mRNAs in the cytoplasm. We took advantage of the absence of ZBP1 expression in the mature brain to examine the effect of expressing ZBP1 on animal behavior. We constructed a transgene conditionally expressing a GFP-ZBP1 fusion protein in a subset of forebrain neurons and compared cocaine-cued place conditioning in these mice versus noninduced littermates. Transgenic ZBP1 expression resulted in impaired place conditioning relative to nonexpressing littermates, and acutely repressing expression of the transgene restored normal cocaine conditioning. To gain insight into the molecular changes that accounted for this change in behavior, we identified mRNAs that specifically immunoprecipitated with transgenic ZBP1 protein from the brains of these mice. These data suggest that RNA-binding proteins can be used as a tool to identify the post-transcriptional regulation of gene expression in the establishment and function of neural circuits involved in addiction behaviors.

}, keywords = {Animals, Brain, Cocaine, Conditioning (Psychology), Cues, DNA-Binding Proteins, Dopamine Uptake Inhibitors, Doxycycline, Gene Expression Regulation, Green Fluorescent Proteins, Immunoprecipitation, Male, Mice, Mice, Transgenic, Microarray Analysis, Neurons, RNA, Messenger, RNA-Binding Proteins}, issn = {1549-5485}, doi = {10.1101/lm.024471.111}, author = {Lapidus, Kyle A B and Nwokafor, Chiso and Scott, Daniel and Baroni, Timothy E and Tenenbaum, Scott A and Hiroi, Noboru and Singer, Robert H and Czaplinski, Kevin} } @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 {46721, title = {β-Actin mRNA compartmentalization enhances focal adhesion stability and directs cell migration.}, journal = {Genes \& Development}, volume = {26}, year = {2012}, month = {2012 Sep 1}, pages = {1885-90}, abstract = {

Directed cell motility is at the basis of biological phenomena such as development, wound healing, and metastasis. It has been shown that substrate attachments mediate motility by coupling the cell{\textquoteright}s cytoskeleton with force generation. However, it has been unclear how the persistence of cell directionality is facilitated. We show that mRNA localization plays an important role in this process, but the mechanism of action is still unknown. In this study, we show that the zipcode-binding protein 1 transports β-actin mRNA to the focal adhesion compartment, where it dwells for minutes, suggesting a means for associating its localization with motility through the formation of stable connections between adhesions and newly synthesized actin filaments. In order to demonstrate this, we developed an approach for assessing the functional consequences of β-actin mRNA and protein localization by tethering the mRNA to a specific location-in this case, the focal adhesion complex. This approach will have a significant impact on cell biology because it is now possible to forcibly direct any mRNA and its cognate protein to specific locations in the cell. This will reveal the importance of localized protein translation on various cellular processes.

}, keywords = {Actins, Animals, Cell Compartmentation, Cell Line, Cell Movement, Cells, Cultured, Fibroblasts, Focal Adhesions, Glycoproteins, Mice, Mice, Knockout, RNA, Messenger}, issn = {1549-5477}, doi = {10.1101/gad.190413.112}, author = {Katz, Zachary B and Wells, Amber L and Park, Hye Yoon and Wu, Bin and Shenoy, Shailesh M and Singer, Robert H} } @article {46736, title = {Cotranscriptional effect of a premature termination codon revealed by live-cell imaging.}, journal = {RNA}, volume = {17}, year = {2011}, month = {2011 Dec}, pages = {2094-107}, abstract = {

Aberrant mRNAs with premature translation termination codons (PTCs) are recognized and eliminated by the nonsense-mediated mRNA decay (NMD) pathway in eukaryotes. We employed a novel live-cell imaging approach to investigate the kinetics of mRNA synthesis and release at the transcription site of PTC-containing (PTC+) and PTC-free (PTC-) immunoglobulin-μ reporter genes. Fluorescence recovery after photobleaching (FRAP) and photoconversion analyses revealed that PTC+ transcripts are specifically retained at the transcription site. Remarkably, the retained PTC+ transcripts are mainly unspliced, and this RNA retention is dependent upon two important NMD factors, UPF1 and SMG6, since their depletion led to the release of the PTC+ transcripts. Finally, ChIP analysis showed a physical association of UPF1 and SMG6 with both the PTC+ and the PTC- reporter genes in vivo. Collectively, our data support a mechanism for regulation of PTC+ transcripts at the transcription site.

}, keywords = {Cell Line, Codon, Nonsense, Cytoplasm, Gene Expression Regulation, Gene Order, Gene Silencing, Humans, Immunoglobulin mu-Chains, In Situ Hybridization, Fluorescence, Nonsense Mediated mRNA Decay, RNA Splicing, RNA, Messenger, Telomerase, Trans-Activators, Transcription, Genetic}, issn = {1469-9001}, doi = {10.1261/rna.02918111}, author = {de Turris, Valeria and Nicholson, Pamela and Orozco, Rodolfo Zamudio and Singer, Robert H and M{\"u}hlemann, Oliver} } @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 {46734, title = {Single-molecule mRNA decay measurements reveal promoter- regulated mRNA stability in yeast.}, journal = {Cell}, volume = {147}, year = {2011}, month = {2011 Dec 23}, pages = {1484-97}, abstract = {

Messenger RNA decay measurements are typically performed on a population of cells. However, this approach cannot reveal sufficient complexity to provide information on mechanisms that may regulate mRNA degradation, possibly on short timescales. To address this deficiency, we measured cell cycle-regulated decay in single yeast cells using single-molecule FISH. We found that two genes responsible for mitotic progression, SWI5 and CLB2, exhibit a mitosis-dependent mRNA stability switch. Their transcripts are stable until mitosis, when a precipitous decay eliminates the mRNA complement, preventing carryover into the next cycle. Remarkably, the specificity and timing of decay is entirely regulated by their promoter, independent of specific cis mRNA sequences. The mitotic exit network protein Dbf2p binds to SWI5 and CLB2 mRNAs cotranscriptionally and regulates their decay. This work reveals the promoter-dependent control of mRNA stability, a regulatory mechanism that could be employed by a variety of mRNAs and organisms.

}, keywords = {Cell Cycle, Cell Cycle Proteins, Cyclin B, Gene Expression Regulation, Fungal, In Situ Hybridization, Fluorescence, Kinetics, Promoter Regions, Genetic, Protein-Serine-Threonine Kinases, RNA Stability, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Transcription Factors, Transcription, Genetic}, issn = {1097-4172}, doi = {10.1016/j.cell.2011.11.051}, author = {Trcek, Tatjana and Larson, Daniel R and Mold{\'o}n, Alberto and Query, Charles C and Singer, Robert H} } @article {46738, title = {The survival of motor neuron (SMN) protein interacts with the mRNA-binding protein HuD and regulates localization of poly(A) mRNA in primary motor neuron axons.}, journal = {The Journal of Neuroscience}, volume = {31}, year = {2011}, month = {2011 Mar 9}, pages = {3914-25}, abstract = {

Spinal muscular atrophy (SMA) results from reduced levels of the survival of motor neuron (SMN) protein, which has a well characterized function in spliceosomal small nuclear ribonucleoprotein assembly. Currently, it is not understood how deficiency of a housekeeping protein leads to the selective degeneration of spinal cord motor neurons. Numerous studies have shown that SMN is present in neuronal processes and has many interaction partners, including mRNA-binding proteins, suggesting a potential noncanonical role in axonal mRNA metabolism. In this study, we have established a novel technological approach using bimolecular fluorescence complementation (BiFC) and quantitative image analysis to characterize SMN-protein interactions in primary motor neurons. Consistent with biochemical studies on the SMN complex, BiFC analysis revealed that SMN dimerizes and interacts with Gemin2 in nuclear gems and axonal granules. In addition, using pull down assays, immunofluorescence, cell transfection, and BiFC, we characterized a novel interaction between SMN and the neuronal mRNA-binding protein HuD, which was dependent on the Tudor domain of SMN. A missense mutation in the SMN Tudor domain, which is known to cause SMA, impaired the interaction with HuD, but did not affect SMN axonal localization or self-association. Furthermore, time-lapse microscopy revealed SMN cotransport with HuD in live motor neurons. Importantly, SMN knockdown in primary motor neurons resulted in a specific reduction of both HuD protein and poly(A) mRNA levels in the axonal compartment. These findings reveal a noncanonical role for SMN whereby its interaction with mRNA-binding proteins may facilitate the localization of associated poly(A) mRNAs into axons.

}, keywords = {Animals, Axons, Blotting, Western, Chick Embryo, Fluorescent Antibody Technique, HEK293 Cells, Hippocampus, Hu Paraneoplastic Encephalomyelitis Antigens, Humans, Immunoprecipitation, In Situ Hybridization, Fluorescence, Mice, Motor Neurons, Poly A, Poly(A)-Binding Proteins, Prosencephalon, Rats, SMN Complex Proteins}, issn = {1529-2401}, doi = {10.1523/JNEUROSCI.3631-10.2011}, author = {Fallini, Claudia and Zhang, Honglai and Su, Yuehang and Silani, Vincenzo and Singer, Robert H and Rossoll, Wilfried and Bassell, Gary J} } @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 {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} } @article {46737, title = {Variegated gene expression caused by cell-specific long-range DNA interactions.}, journal = {Nature Cell Biology}, volume = {13}, year = {2011}, month = {2011 Aug}, pages = {944-51}, abstract = {

Mammalian genomes contain numerous regulatory DNA sites with unknown target genes. We used mice with an extra β-globin locus control region (LCR) to investigate how a regulator searches the genome for target genes. We find that the LCR samples a restricted nuclear subvolume, wherein it preferentially contacts genes controlled by shared transcription factors. No contacted gene is detectably upregulated except for endogenous β-globin genes located on another chromosome. This demonstrates genetically that mammalian trans activation is possible, but suggests that it will be rare. Trans activation occurs not pan-cellularly, but in {\textquoteright}jackpot{\textquoteright} cells enriched for the interchromosomal interaction. Therefore, cell-specific long-range DNA contacts can cause variegated expression.

}, keywords = {Animals, beta-Globins, DNA, GATA1 Transcription Factor, Gene Expression, In Situ Hybridization, Fluorescence, Kruppel-Like Transcription Factors, Locus Control Region, Mice, Mice, Transgenic, RNA, Messenger, Transcriptional Activation}, issn = {1476-4679}, doi = {10.1038/ncb2278}, author = {Noordermeer, Daan and de Wit, Elzo and Klous, Petra and van de Werken, Harmen and Simonis, Marieke and Lopez-Jones, Melissa and Eussen, Bert and de Klein, Annelies and Singer, Robert H and de Laat, Wouter} } @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} }