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The ability of neurons to process and store salient environmental features underlies information processing in the brain. Long-term information storage requires synaptic plasticity and regulation of gene expression. While distinct patterns of activity have been linked to synaptic plasticity, their impact on immediate early gene (IEG) expression remains poorly understood. The activity regulated cytoskeleton associated (Arc) gene has received wide attention as an IEG critical for long-term synaptic plasticity and memory. Yet, to date, the transcriptional dynamics of Arc in response to compartment and input-specific activity is unclear. By developing a knock-in mouse to fluorescently tag Arc alleles, we studied real-time transcription dynamics after stimulation of dentate granule cells (GCs) in acute hippocampal slices. To our surprise, we found that Arc transcription displayed distinct temporal kinetics depending on the activation of excitatory inputs that convey functionally distinct information, i.e., medial and lateral perforant paths (MPP and LPP, respectively). Moreover, the transcriptional dynamics of Arc after synaptic stimulation was similar to direct activation of GCs, although the contribution of ionotropic glutamate receptors, L-type voltage-gated calcium channel, and the endoplasmic reticulum (ER) differed. Specifically, we observed an ER-mediated synapse-to-nucleus signal that supported elevations in nuclear calcium and, thereby, rapid induction of Arc transcription following MPP stimulation. By delving into the complex excitation-transcription coupling for Arc, our findings highlight how different synaptic inputs may encode information by modulating transcription dynamics of an IEG linked to learning and memory. [ABSTRACT FROM AUTHOR]

• Composition of mRNPs is dynamic across space and time. • mRNPs in dendrites and axons share common features of diffusion and directed transport. • HMM Bayes approach is a useful tool to analyze mRNA transport dynamics. • mRNA localization and local translation in response to synaptic activity are important for long-lasting plasticity. Neurons are highly polarized cells that can extend processes far from the cell body. As such, transport of messenger RNAs serves as a set of blueprints for the synthesis of specific proteins at distal sites. RNA localization to dendrites and axons confers the ability to regulate translation with extraordinary precision in space and time. Although the rationale for RNA localization is quite compelling, it is unclear how a neuron orchestrates such a complex task of distributing over a thousand different mRNAs to their respective subcellular compartments. Recent single-molecule imaging studies have led to insights into the kinetics of individual mRNAs. We can now peer into the transport dynamics of mRNAs in both dendrites and axons. [ABSTRACT FROM AUTHOR]

Highlights • The brain is characterized by the diversity of cell types. • Variability (i.e., noise) in the synthesis and intracellular movement of mRNAs is thought to play a crucial role in regulating the structure and function of the brain. • The dynamics of mRNA is observed in the brain of living mouse, where cellular identities are undisturbed, by intravital two-photon microscopy. • Context-dependent mRNA dynamics can be resolved by engineering viral construct for MBS-MCP. Abstract We describe a method for visualizing mRNAs in living mouse. Nascent transcripts and cytoplasmic mRNAs were labeled via lentiviral expression of MS2 coat protein (MCP) tagged with fluorescent protein (MCP-XFP) in knock-in mice whose β-actin mRNAs contained MCP binding stem loops (MBS). Then the mRNA molecules were imaged in the live cerebral cortex through an optical cranial window by intravital two-photon microscopy. By means of the controlled expression of MCP-XFP, single mRNA particles could be detected differentially in the nucleus and cytoplasm of a specific cell type. Consequently, this method is useful for investigating the cell-type-dependent dynamics of mRNAs underlying the structure and function of the brain. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

David Yaffe, PhD passed away on July 3, 2020 at the age of 91. His funeral and burial were held at his kibbutz in Israel, Givat Brenner. At the time of his death, he was emeritus professor of molecular cell biology at the Weizmann Institute of Science. If there is one word to describe David, it would be “pioneer”, Halutz in Hebrew. He participated in the birth of his country as a soldier during the War of Independence, contributed his efforts to expanding the kibbutz movement and the nascent scientific community in Israel. He was a major figure in creating the field of muscle cell biology by generating the immortalized cell lines that could be induced to differentiate into muscle fibers in tissue culture. This made myogenesis a prime candidate for studying cell differentiation, accessible to hundreds of scientists during the dawn of molecular cell biology. Many prominent scientists, among them students of David made their careers on studying the molecular events that occurred as the myoblast cell lines developed into the syncytial myofibers that twitched spontaneously. This opened the field to studies on muscle diseases, such as muscular dystrophy, that could then be addressed through a tractable model for molecular studies. David claimed he was never paid for his scientific activities; his salary at the Weizmann Institute went directly to the kibbutz in compensation for his time taken away from work there. He never knew what his salary was, he said, since it was negotiated between the kibbutz and the Weizmann. Even so, he had to take off weeks from his science occasionally to perform daily tasks, usually in the kibbutz dining hall washing dishes. Disconnected from the material aspects of life, David was driven solely by his love for nature and science, which he continued until the very end. David remains an inspiration to all of us. In this issue several of David Yaffe’s lab members, pupils, coauthors and colleagues meet at a “David Yaffe myogenesis distance meeting" to pay tribute with their collective reflections. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 infection remains a global burden. Despite intensive research, the mechanism and dynamics of early viral replication are not completely understood, such as the kinetics of the formation of genomic RNA (gRNA), sub-genomic RNA (sgRNA), and replication centers/organelles (ROs). We employed single-molecule RNA-fluorescence in situ hybridization (smRNA-FISH) to simultaneously detect viral gRNA and sgRNA and immunofluorescence to detect nsp3 protein, a marker for the formation of RO, and carried out a time-course analysis. We found that single molecules of gRNA are visible within the cytoplasm at 30 min post infection (p.i.). Starting from 2 h p.i., most of the viral RNA existed in clusters/speckles, some of which were surrounded by single molecules of sgRNA. These speckles associated with nsp3 protein starting at 3 h p.i., indicating that these were precursors to ROs. Furthermore, RNA replication was asynchronous, as cells with RNA at all stages of replication were found at any given time point. Our probes detected the SARS-CoV-2 variants of concern, and also suggested that the BA.1 strain exhibited a slower rate of replication kinetics than the WA1 strain. Our results provide insights into the kinetics of SARS-CoV-2 early post-entry events, which will facilitate identification of new therapeutic targets for early-stage replication to combat COVID-19. [ABSTRACT FROM AUTHOR]

RNA binding proteins (RBPs) regulate nearly all post-transcriptional processes within cells. To fully understand RBP function, it is essential to identify their in vivo targets. Standard techniques for profiling RBP targets, such as crosslinking immunoprecipitation (CLIP) and its variants, are limited or suboptimal in some situations, e.g. when compatible antibodies are not available and when dealing with small cell populations such as neuronal subtypes and primary stem cells. This review summarizes and compares several genetic approaches recently designed to identify RBP targets in such circumstances. TRIBE (targets of RNA binding proteins identified by editing), RNA tagging, and STAMP (surveying targets by APOBEC-mediated profiling) are new genetic tools useful for the study of post-transcriptional regulation and RBP identification. We describe the underlying RNA base editing technology, recent applications, and therapeutic implications. RNA editing regulates the splicing, coding, and metabolic features of RNAs. In this review, Xu et al. highlight how RNA editing enzymes are being used to identify RNA binding protein targets with high sensitivity and specificity and the implications of these technological advances for RNA editing-based therapeutics. [ABSTRACT FROM AUTHOR]

The tumor suppressor p53 protein activates expression of a vast gene network in response to stress stimuli for cellular integrity. The molecular mechanism underlying how p53 targets RNA polymerase II (Pol II) to regulate transcription remains unclear. To elucidate the p53/Pol II interaction, we have determined a 4.6 Å resolution structure of the human p53/Pol II assembly via single particle cryo-electron microscopy. Our structure reveals that p53's DNA binding domain targets the upstream DNA binding site within Pol II. This association introduces conformational changes of the Pol II clamp into a further-closed state. A cavity was identified between p53 and Pol II that could possibly host DNA. The transactivation domain of p53 binds the surface of Pol II's jaw that contacts downstream DNA. These findings suggest that p53's functional domains directly regulate DNA binding activity of Pol II to mediate transcription, thereby providing insights into p53-regulated gene expression. Liou et al. report a 4.6 Å resolution structure of the human p53/ RNA polymerase II assembly, using single particle cryoelectron microscopy. This study suggests that p53's functional domains regulate the DNA binding activity of RNA polymerase II, providing insights into p53-regulated gene expression. [ABSTRACT FROM AUTHOR]

This review focuses on imaging DNA and single RNA molecules in living cells to define eukaryotic functional organization and dynamic processes. The latest advances in technologies to visualize individual DNA loci and RNAs in real time are discussed. Single-molecule fluorescence microscopy provides the spatial and temporal resolution to reveal mechanisms regulating fundamental cell functions. Novel insights into the regulation of nuclear architecture, transcription, posttranscriptional RNA processing, and RNA localization provided by multicolor fluorescence microscopy are reviewed. A perspective on the future use of live imaging technologies and overcoming their current limitations is provided. [ABSTRACT FROM AUTHOR]

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 in situ 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 Drosophila melanogaster brains. Here, we present a FISH platform for high-throughput detection of eight common neurotransmitter phenotypes in Drosophila 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 coexpression, 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. [ABSTRACT FROM AUTHOR]

Full-length mRNAs transfer between adjacent mammalian cells via direct cell-to-cell connections called tunneling nanotubes (TNTs). However, the extent of mRNA transfer at the transcriptome-wide level (the 'transferome') is unknown. Here, we analyzed the transferome in an in vitro human-mouse cell co-culture model using RNA-sequencing. We found that mRNA transfer is non-selective, prevalent across the human transcriptome, and that the amount of transfer to mouse embryonic fibroblasts (MEFs) strongly correlates with the endogenous level of gene expression in donor human breast cancer cells. Typically,<1% of endogenous mRNAs undergo transfer. Non-selective, expression-dependent RNA transfer was further validated using synthetic reporters. RNA transfer appears contact-dependent via TNTs, as exemplified for several mRNAs. Notably, significant differential changes in the native MEF transcriptome were observed in response to co-culture, including the upregulation of multiple cancer and cancer-associated fibroblast-related genes and pathways. Together, these results lead us to suggest that TNT-mediated RNA transfer could be a phenomenon of physiological importance under both normal and pathogenic conditions. [ABSTRACT FROM AUTHOR]

The article cites a research study that describes a novel mouse model for imaging activity-dependent dynamics of endogenous activity-regulated cytoskeleton-associated protein (Arc) mRNA in live neurons. In the transgenic mouse model, the endogenous Arc gene is tagged in its 3' untranslated region, allowing visualization of individual mRNAs in real time.

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. [ABSTRACT FROM AUTHOR]

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-fieldmicroscopy 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 determinewhich 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

R370 --> 787.3 --> MS2 system is widely used to image mRNAs in living cells, where the spatial distribution of mRNAs regulates various aspects of cell biology, including translation and RNA decay. To image an mRNA of interest, the mRNA's 3' untranslated region (UTR) is genetically fused with an array of MS2 stem loops, which recruit fluorescently labeled MS2 coat proteins. And each mRNA molecule appears as a diffraction‐limited spot under fluorescent microscope. Here, we report that the MS2 system renders some of the tagged mRNAs less stable and being degraded by the nonsense‐mediated mRNA decay (NMD) pathway. To resolve this issue, we improved the MS2 system by fusing the MS2 coat protein with the translation termination factor eRF3, which protects the mRNA from being degraded through the NMD pathway. Our improved MS2 system faithfully reports the mRNAs' stability and localization, and will see wide applications in future studies of RNA biology. [ABSTRACT FROM AUTHOR]

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 zebra fish. By using this binary system to co-express 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 Zebra fish 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 Zebra fish. 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'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 zebra fish developmental processes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The heritability of chromatin states through cell division is a potential contributor to the epigenetic maintenance of cellular memory of prior states. The macroH2A histone variant has properties of a regulator of epigenetic cell memory, including roles controlling gene silencing and cell differentiation. Its mechanisms of regional genomic targeting and maintenance through cell division are unknown. Here, we combined in vivo imaging with biochemical and genomic approaches to show that human macroH2A is incorporated into chromatin in the G1 phase of the cell cycle following DNA replication. The newly incorporated macroH2A retargets the same large heterochromatic domains where macroH2A was already enriched in the previous cell cycle. It remains heterotypic, targeting individual nucleosomes that do not already contain a macroH2A molecule. The pattern observed resembles that of a new deposition of centromeric histone variants during the cell cycle, indicating mechanistic similarities for macrodomain-scale regulation of epigenetic properties of the cell. [ABSTRACT FROM AUTHOR]

Genes are transcribed in irregular pulses of activity termed transcriptional bursts. Cellular differentiation requires coordinated gene expression; however, it is unknown whether the burst fraction (i.e. the number of active phases of transcription) or size/intensity (the number of RNA molecules produced within a burst) changes during cell differentiation. In the ocular lens, the positions of lens fiber cells correlate precisely with their differentiation status, and the most advanced cells degrade their nuclei. Here, we examined the transcriptional parameters of the β-actin and lens differentiation–specific α-, β-, and γ-crystallin genes by RNA fluorescent in situ hybridization (FISH) in the lenses of embryonic day (E) E12.5, E14.5, and E16.5 mouse embryos and newborns. We found that cellular differentiation dramatically alters the burst fraction in synchronized waves across the lens fiber cell compartment with less dramatic changes in burst intensity. Surprisingly, we observed nascent transcription of multiple genes in nuclei just before nuclear destruction. Nuclear condensation was accompanied by transfer of nuclear proteins, including histone and nonhistone proteins, to the cytoplasm. Although lens-specific deletion of the chromatin remodeler SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 5 (Smarca5/Snf2h) interfered with denucleation, persisting nuclei remained transcriptionally competent and exhibited changes in both burst intensity and fraction depending on the gene examined. Our results uncover the mechanisms of nascent transcriptional control during differentiation and chromatin remodeling, confirm the burst fraction as the major factor adjusting gene expression levels, and reveal transcriptional competence of fiber cell nuclei even as they approach disintegration. [ABSTRACT FROM AUTHOR]

Activity-dependent expression of immediate early genes (IEGs) is critical for long-term synaptic remodeling and memory. It remains unknown how IEGs are maintained for memory despite rapid transcript and protein turnover. To address this conundrum, we monitored Arc , an IEG essential for memory consolidation. Using a knockin mouse where endogenous Arc alleles were fluorescently tagged, we performed real-time imaging of Arc mRNA dynamics in individual neurons in cultures and brain tissue. Unexpectedly, a single burst stimulation was sufficient to induce cycles of transcriptional reactivation in the same neuron. Subsequent transcription cycles required translation, whereby new Arc proteins engaged in autoregulatory positive feedback to reinduce transcription. The ensuing Arc mRNAs preferentially localized at sites marked by previous Arc protein, assembling a "hotspot" of translation, and consolidating "hubs" of dendritic Arc. These cycles of transcription-translation coupling sustain protein expression and provide a mechanism by which a short-lived event may support long-term memory. [Display omitted] • Reactivation of transcription drives cycling of the Arc gene in individual neurons • Feedback from new proteins reinduces Arc transcription in the next cycle • Arc mRNAs from later cycles localize to sites marked with previous Arc protein • Repetitive translation in hotspots consolidates dendritic Arc in selective hubs How are short-lived mRNAs and proteins maintained over time to impact long-term memory? Das et al. report that repetitive cycles of transcription and local translation of the immediate early gene Arc amplify a single burst stimulation over time to assemble Arc protein hubs, revealing a molecular mechanism that supports memory consolidation. [ABSTRACT FROM AUTHOR]

Cortex G: Cortex glia, Enshea G: Ensheathing glia, Astro G: Astrocyte glia, Surf G: Surface glia The original article has been corrected. A single-cell transcriptomic atlas of complete insect nervous systems across multiple life stages. [Extracted from the article]

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. [ABSTRACT FROM AUTHOR]

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 melanogaster shavenbaby (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. [ABSTRACT FROM AUTHOR]

Local translation of specific mRNAs is regulated by dynamic changes in their subcellular localization, and these changes are due to complex mechanisms controlling cytoplasmic mRNA transport. The budding yeast Saccharomyces cerevisiae is well suited to studying these mechanisms because many of its transcripts are transported from the mother cell to the budding daughter cell. Here, we investigated the translational control of ASH1 mRNA after transport and localization. We show that although ASH1 transcripts were translated after they reached the bud tip, some mRNAs were bound by the RNA-binding protein Puf6 and were non-polysomal. We also found that the DEAD-box helicase Dhh1 complexed with the untranslated ASH1 mRNA and Puf6. Loss of Dhh1 affected local translation of ASH1 mRNA and resulted in delocalization of ASH1 transcript in the bud. Forcibly shifting the non-polysomal ASH1 mRNA into polysomes was associated with Dhh1 dissociation. We further demonstrated that Dhh1 is not recruited to ASH1 mRNA co-transcriptionally, suggesting that it could bind to ASH1 mRNA within the cytoplasm. Of note, Dhh1 bound to the 5'-UTR of ASH1 mRNA and inhibited its translation in vitro. These results suggest that after localization to the bud tip, a portion of the localized ASH1 mRNA becomes translationally inactive because of binding of Dhh1 and Puf6 to the 5'- and 3'-UTRs of ASH1 mRNA. [ABSTRACT FROM AUTHOR]

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 synapsespecific 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, Zipcodebinding 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. [ABSTRACT FROM AUTHOR]

Recent advancements in single-cell and single-molecule imaging technologies have resolved biological processes in time and space that are fundamental to understanding the regulation of gene expression. Observations of single-molecule events in their cellular context have revealed highly dynamic aspects of transcriptional and post-transcriptional control in eukaryotic cells. This approach can relate transcription with mRNA abundance and lifetimes. Another key aspect of single-cell analysis is the cell-to-cell variability among populations of cells. Definition of heterogeneity has revealed stochastic processes, determined characteristics of under-represented cell types or transitional states, and integrated cellular behaviors in the context of multicellular organisms. In this review, we discuss novel aspects of gene expression of eukaryotic cells and multicellular organisms revealed by the latest advances in single-cell and single-molecule imaging technology. [ABSTRACT FROM AUTHOR]

Learning and memory rely on long-lasting, synapse-specific modifications. Although postsynaptic forms of plasticity typically require local protein synthesis, whether and how local protein synthesis contributes to presynaptic changes remain unclear. Here, we examined the mouse hippocampal mossy fiber (MF)-CA3 synapse, which expresses both structural and functional presynaptic plasticity and contains presynaptic fragile X messenger ribonucleoprotein (FMRP), an RNA-binding protein involved in postsynaptic protein-synthesis-dependent plasticity. We report that MF boutons contain ribosomes and synthesize protein locally. The long-term potentiation of MF-CA3 synaptic transmission (MF-LTP) was associated with the translation-dependent enlargement of MF boutons. Remarkably, increasing in vitro or in vivo MF activity enhanced the protein synthesis in MFs. Moreover, the deletion of presynaptic FMRP blocked structural and functional MF-LTP, suggesting that FMRP is a critical regulator of presynaptic MF plasticity. Thus, presynaptic FMRP and protein synthesis dynamically control presynaptic structure and function in the mature mammalian brain. [Display omitted] • Mossy fiber boutons (MFBs) contain ribosomes and synthesize protein locally • Local presynaptic translation is increased by in vitro and in vivo GC activity • MFB structural plasticity relies on de novo protein synthesis • Presynaptic FMRP is required for MF-CA3 structural and functional plasticity Monday, Kharod et al. report that neuronal activity regulates presynaptic FMRP function and the local synthesis of β-actin. In vitro activity and in vivo experience engage the FMRP-dependent spatiotemporal regulation of protein synthesis that is required for presynaptic remodeling and long-term changes in neurotransmitter release. [ABSTRACT FROM AUTHOR]

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 amino acids per second). In primary neurons, mRNAs are translated in proximal dendrites but repressed in distal dendrites and display "bursting" translation.This technology provides a tool with which to address the spatiotemporal translation mechanism of single mRNAs in living cells. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: The class IA PI 3-kinase regulatory subunit p85α dimerizes by mechanisms not fully understood. Results: p85α dimerization is driven by intermolecular interactions at both its N and C termini. A structural model of the p85α dimer reveals conformational diversity. Conclusion: p85α dimerization is poised to link the heterologous assemblies that regulate PI3K signaling. Significance: The p85α dimer is a dynamic participant in PI3K signaling. [ABSTRACT FROM AUTHOR]

A correction to the article "In the right place at the right time: visualizing and understanding mRNA localization" that was published in the 2015 issue is presented.

Summary Specific binding proteins are crucial for the correct spatiotemporal expression of mRNA. To understand this process, a method is required to characterize RNA-protein interactions in single living cells with subcellular resolution. We combined endogenous single RNA and protein detection with two-photon fluorescence fluctuation analysis to measure the average number of proteins bound to mRNA at specific locations within live cells. We applied this to quantify the known binding of zipcode binding protein 1 (ZBP1) and ribosomes to β-actin mRNA within subcellular compartments of primary fibroblasts and neurons. ZBP1-mRNA binding did not occur in nuclei, contrary to previous conclusions. ZBP1 interaction with β-actin mRNA was enhanced perinuclearly in neurons compared to fibroblasts. Cytoplasmic ZBP1 and ribosome binding to the mRNA were anti-correlated depending on their location in the cell. These measurements support a mechanism whereby ZBP1 inhibits translation of localizing mRNA until its release from the mRNA peripherally, allowing ribosome binding. [ABSTRACT FROM AUTHOR]

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 (3-actin mRNAs freely access the entire nucleus and fewer than 60% of mRNAs are more than 0.5 pm away from a nuclear pore, and we do so for the first time accounting for spatial inhomogeneity of nuclear organization. [ABSTRACT FROM AUTHOR]

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. Singlemolecule 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. [ABSTRACT FROM AUTHOR]

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 showhere a useful and generally applicable method to maintain the integrity of expressed genes, critical for the correct interpretation of probe readouts. [ABSTRACT FROM AUTHOR]

The article focuses on the development of a fluorescence microscopy method that reports on first translation events of individual messenger RNA molecules. It states the biosensor will allow scientists to examine spatiotemporal regulation of translation during normal growth and stress, as well as during oocyte development in Drosophila. It reports the study found messenger RNA wasn't translated in the cell nucleus and that translation is regulated by sequestration within P-bodies.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Transcription is a highly regulated biological process, initiated through the assembly of complexes at the promoter that contain both the general transcriptional machinery and promoter-specific factors. Despite the abundance of studies focusing on transcription, certain questions have remained unanswered. It is not clear how the transcriptional profile of a promoter is affected by genomic context. Also, there is no single cell method to directly compare transcriptional profiles independent of gene length and sequence. In this work, we employ a single genetic site for isolating the transcriptional kinetics of yeast promoters. Utilizing single molecule FISH, we directly compare the transcriptional activity of different promoters, considering both synthesis and cell-to-cell variability. With this approach, we provide evidence suggesting promoters autonomously encode their associated transcriptional profiles, independent of genomic locus, gene length and gene sequence. [ABSTRACT FROM AUTHOR]

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/. [ABSTRACT FROM AUTHOR]

The article presents the discussion on DNA surveillance pathway using the biomechanical link between supercoiling and transcription for homeostatically amplifying transcriptional fluctuations including differentiation of embryonic stem cells into neural ectodermal cells.

The process of intravasation involving transendothelial migration is a key step in metastatic spread. How the triple cell complex composed of a macrophage, Mena over-expressing tumor cell and endothelial cell, called the tumor microenvironment of metastasis (TMEM), facilitates tumor cell transendothelial migration is not completely understood. Previous work has shown that the physical contact between a macrophage and tumor cell results in the formation of invadopodia, actin-rich matrix degrading protrusions, important for tumor cell invasion and transendothelial migration and tumor cell dissemination. Herein, we show that the macrophage-induced invadopodium is formed through a Notch1/MenaINV signaling pathway in the tumor cell upon macrophage contact. This heterotypic tumor cell - macrophage interaction results in the upregulation of MenaINV through the activation of MENA transcription. Notch1 and MenaINV expression are required for tumor cell transendothelial migration, a necessary step during intravasation. Inhibition of the Notch signaling pathway blocked macrophage-induced invadopodium formation in vitro and the dissemination of tumor cells from the primary tumor in vivo. Our findings indicate a novel role for Notch1 signaling in the regulation of MenaINV expression and transendothelial migration and provide mechanistic information essential to the use of therapeutic inhibitors of metastasis. [ABSTRACT FROM AUTHOR]