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29 Janelia Publications

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    06/20/18 | A transgenic mouse for imaging activity-dependent dynamics of endogenous Arc mRNA in live neurons.
    Das S, Moon HC, Singer RH, Park HY
    Science Advances. 2018 Jun;4(6):eaar3448. doi: 10.1126/sciadv.aar3448

    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' 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.

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    05/20/18 | Imaging mRNA in vivo, from birth to death.
    Tutucci E, Livingston NM, Singer RH, Wu B
    Annual Review of Biophysics. 2018 May 20;47:85-106. doi: 10.1146/annurev-biophys-070317-033037

    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.

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    04/19/18 | Transvection Goes Live-Visualizing Enhancer-Promoter Communication between Chromosomes.
    Tsai A, Singer RH, Crocker J
    Molecular Cell. 2018 Apr 19;70(2):195-196. doi: 10.1016/j.molcel.2018.04.004

    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."

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    11/02/17 | Nuclear microenvironments modulate transcription from low-affinity enhancers.
    Tsai A, Muthusamy AK, Alves MR, Lavis LD, Singer RH, Stern DL, Crocker J
    eLife. 2017 Nov 02;6:. doi: 10.7554/eLife.28975

    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.

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    10/24/17 | Intercellular mRNA trafficking via membrane nanotube-like extensions in mammalian cells.
    Haimovich G, Ecker CM, Dunagin MC, Eggan E, Raj A, Gerst JE, Singer RH
    Proceedings of the National Academy of Sciences of the United States of America. 2017 Oct 24;114(46):E9873-E9882. doi: 10.1073/pnas.1706365114

    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.

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    09/21/17 | Genomic probes.
    Singer RH, Deng W, Lionnet T
    USPTO. 2017 Sep 21;A1:

    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.

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    06/05/17 | Quantitative mRNA imaging throughout the entire Drosophila brain.
    Long X, Colonell J, Wong AM, Singer RH, Lionnet T
    Nature Methods. 2017 Jun 05;14(7):703-6. doi: 10.1038/nmeth.4309

    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).

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    03/07/17 | Imaging mRNA and protein interactions within neurons.
    Eliscovich C, Shenoy SM, Singer RH
    Proceedings of the National Academy of Sciences of the United States of America. 2017 Mar 07;114(10):E1875-E1884. doi: 10.1073/pnas.1621440114

    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.

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    02/28/17 | RNP transport in cell biology: the long and winding road.
    Eliscovich C, Singer RH
    Current Opinion in Cell Biology. 2017 Feb 28;45:38-46. doi: 10.1016/j.ceb.2017.02.008

    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.

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    09/15/16 | Rapid dynamics of general transcription factor TFIIB binding during preinitiation complex assembly revealed by single-molecule analysis.
    Zhang Z, English BP, Grimm JB, Kazane SA, Hu W, Tsai A, Inouye C, You C, Piehler J, Schultz PG, Lavis LD, Revyakin A, Tjian R
    Genes and Development. 2016 Sep 15;30:2106-18. doi: 10.1101/gad.285395.116

    Transcription of protein-encoding genes in eukaryotic cells requires the coordinated action of multiple general transcription factors (GTFs) and RNA polymerase II (Pol II). A “step-wise” preinitiation complex (PIC) assembly model has been suggested based on conventional ensemble biochemical measurements, in which protein factors bind stably to the promoter DNA sequentially to build a functional PIC. However, recent dynamic measurements in live cells suggest that transcription factors mostly interact with chromatin DNA rather transiently. To gain a clearer dynamic picture of PIC assembly, we established an integrated in vitro single-molecule transcription platform reconstituted from highly purified human transcription factors and complemented it by live-cell imaging. Here we performed real-time measurements of the hierarchal promoter-specific binding of TFIID, TFIIA, and TFIIB. Surprisingly, we found that while promoter binding of TFIID and TFIIA is stable, promoter binding by TFIIB is highly transient and dynamic (with an average residence time of 1.5 sec). Stable TFIIB–promoter association and progression beyond this apparent PIC assembly checkpoint control occurs only in the presence of Pol II–TFIIF. This transient-to-stable transition of TFIIB-binding dynamics has gone undetected previously and underscores the advantages of single-molecule assays for revealing the dynamic nature of complex biological reactions.

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