@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 {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 {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 {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 {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 {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 {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 {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 {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 {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} }