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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]

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. [ABSTRACT FROM AUTHOR]

The central dogma of molecular biology - DNA makes RNA makes proteins - is a flow of information that in eukaryotes encounters a physical barrier: the nuclear envelope, which encapsulates, organizes and protects the genome. Nuclear-pore complexes, embedded in the nuclear envelope, regulate the passage of molecules to and from the nucleus, including the poorly understood process of the export of RNAs from the nucleus. Recent imaging approaches focusing on single molecules have provided unexpected insight into this crucial step in the information flow. This review addresses the latest studies of RNA export and presents some models for how this complex process may work. [ABSTRACT FROM AUTHOR]

Export of messenger RNA occurs via nuclear pores, which are large nanomachines with diameters of roughly 120?nm that are the only link between the nucleus and cytoplasm. Hence, mRNA export occurs over distances smaller than the optical resolution of conventional light microscopes. There is extensive knowledge on the physical structure and composition of the nuclear pore complex, but transport selectivity and the dynamics of mRNA export at nuclear pores remain unknown. Here we developed a super-registration approach using fluorescence microscopy that can overcome the current limitations of co-localization by means of measuring intermolecular distances of chromatically different fluorescent molecules with nanometre precision. With this method we achieve 20-ms time-precision and at least 26-nm spatial precision, enabling the capture of highly transient interactions in living cells. Using this approach we were able to spatially resolve the kinetics of mRNA transport in mammalian cells and present a three-step model consisting of docking (80?ms), transport (5-20?ms) and release (80?ms), totalling 180?±?10 ms. Notably, the translocation through the channel was not the rate-limiting step, mRNAs can move bi-directionally in the pore complex and not all pores are equally active. [ABSTRACT FROM AUTHOR]

She2p is an RNA-binding protein that recognizes a zipcode on specific mRNAs necessary for the assembly of a protein complex that localizes them to the yeast bud tip. In this issue of Genes & Development, Shen and colleagues (pp. 1914-1926) demonstrate that She2p associates with RNAPII globally, but then recognizes the nascent chain only if it contains a zipcode. This demonstrates yet another case where the mRNA's cytoplasmic fate is determined by the RNAPII complex. [ABSTRACT FROM AUTHOR]

Analyzing the expression of single genes in single cells appears minimalistic in comparison to gene expression studies based on more global approaches. However, stimulated by advances in imaging technologies, single-cell studies have become an essential tool in understanding the rules that govern gene expression. This quantitative view of single-cell gene expression is based on counting mRNAs in single cells, monitoring transcription in real time, and visualizing single proteins. Parallel advances in mathematical models based on stochastic, discrete descriptions of biochemical processes have provided crucial insights into the underlying cellular mechanisms that control expression. The view that has emerged is rooted in a probabilistic understanding of cellular processes that quantitatively explains both the mean and the variation observed in gene-expression patterns among single cells. Thus, the close coupling between imaging and mathematical theory has established single-cell analysis as an essential branch of systems biology. [Copyright &y& Elsevier]

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]

Studies of the intracellular localization of mRNA have clearly demonstrated that certain subsets of mRNA are concentrated in discrete locations within the cytoplasm. Localization is one aspect of the post-transcriptional control of gene expression, and is intertwined with the translation and turnover of mRNA to achieve the goal of local protein production. Different mechanisms have been identified that enable localized mRNAs to target different subcellular compartments, and recent advances in understanding these pathways is reviewed here. [Copyright &y& Elsevier]

The combined use of the new technologies of multiphoton-based intravital imaging, the chemotaxis-mediated collection of invasive cells, and high sensitivity expression profiling has allowed the correlation of the behavior of invasive tumor cells in vivo with their gene expression patterns. New insights have resulted including a gene expression signature for invasive cells and the tumor microenvironment invasion model. This model proposes that tumor invasion and metastasis can be studied as a problem resembling normal morphogenesis. We discuss how these new insights may lead to a better understanding of the molecular basis of the invasive behavior of tumor cells in vivo, which may result in new strategies for the diagnosis and treatment of metastasis. [ABSTRACT FROM AUTHOR]

Reports on the transcription of messenger RNA (mRNA) molecules in the nucleus before they undergo export into the cytoplasm, where they are translated to produce proteins. Features of mRNA localization systems.

Understanding the different molecular mechanisms responsible for gene expression has been a central interest of molecular biologists for several decades. Transcription, the initial step of gene expression, consists of converting the genetic code into a dynamic messenger RNA that will specify a required cellular function following translocation to the cytoplasm and translation. We now possess an in-depth understanding of the mechanism and regulations of transcription. By contrast, an understanding of the dynamics of an individual gene''s expression in real time is just beginning to emerge following recent technological developments. [Copyright &y& Elsevier]

Technical advances in the field of live-cell imaging have introduced the cell biologist to a new, dynamic, subcellular world. The static world of molecules in fixed cells has now been extended to the time dimension. This allows the visualization and quantification of gene expression and intracellular trafficking events of the studied molecules and the associated enzymatic processes in individual cells, in real time. [ABSTRACT FROM AUTHOR]

Discusses the development of fluorescence in situ hybridization (FISH), the assay of choice for localization of specific nucleic acids sequences in native context. Methodologies and modifications that have introduced to optimize the detection of DNA and RNA; Pervasiveness of the technique; Basic principles of FISH; Computer image processing; Low-noise hybridization probes.

Background: mRNA localization in somatic cells is an important mechanism for gene expression regulation. In fibroblasts, the protein ZBP1 associates with the sequence that localizes β-actin mRNA to the leading edge of fibroblasts, augmenting motility. β-actin mRNA localizes in a cytoskeleton-dependent manner, depending on intact actin and myosin ATP-hydrolysis, and is largely bound to the actin cytoskeleton. The ZBP1 protein contains four KH RNA binding domains and a classic RBD RNA binding domain. It also contains a putative nuclear import and export sequence, suggesting a nuclear phase in this protein''s function.Results: Using high-speed imaging, we show here the targeting of this RNA binding protein to β-actin pre-mRNA transcripts in the nuclei of living cells and measure the residence time of the RNA-protein complex before it leaves the transcription site. Then, the RNA-protein particle is exported to the cytoplasm, where it localizes at velocities of 0.6 μm/s by using actin filaments and/or microtubules. This RNA-ZBP1 complex is required for cytoplasmic localization in fibroblasts; mislocalizing the protein also mislocalizes the RNA, and expressing the protein in a ZBP1-deficient cell line induces β-actin mRNA localization.Conclusions: This work demonstrates that the RNA-protein association, essential for cytoplasmic localization, begins as soon as the RNA is transcribed. The ZBP1 then forms a ribonucleoprotein particle and moves in a myosin-dependent fashion by using the cytoskeleton for directional transport. [Copyright &y& Elsevier]

RNA-binding proteins are involved in various aspects of RNA metabolism such as processing, translational control, stabilization, localization and transport. Many of these proteins bind several RNA targets and have multiple functions. In this review we focus on RNA-binding proteins that are implicated in RNA transport and localization and that also have a role in other aspects of RNA metabolism. These proteins might link nuclear events, such as RNA splicing, with the subsequent cytoplasmic localization of specific transcripts. [Copyright &y& Elsevier]

The localization of mRNAs is used by various types of polarized cells to locally translate specific proteins, which restricts their distribution to a particular sub-region of the cytoplasm. This mechanism of protein sorting is involved in major biological processes such as asymmetric cell division, oogenesis, cellular motility, and synapse formation. With the finding of localized mRNAs in the yeast Saccharomyces cerevisiae, it is now possible to benefit from the powerful yeast laboratory tools to explore the molecular basis of RNA localization. Because mRNA transport and localization in yeast share many features with RNA localization in higher eukaryotes, including the formation of a large ribonucleoprotein (RNP) localization complex, the requirement of a polarized cytoskeleton and molecular motors, and the role of nuclear RNA-binding proteins in cytoplasmic localization, the yeast can be used as a paradigm for unraveling the molecular aspects of this process. This review summarizes the current knowledge on RNP transport and localization in yeast. [ABSTRACT FROM AUTHOR]

Examines the physiological significance of beta-actin messenger RNA localization in determining cell polarity and directional motility. Treatment of chicken embryo fibroblasts using antisense oligonucleotides; Use of Dynamic Image Analysis System to quantify movement of cells; Differences in the sense-treated and antisense-treated cells.

Focuses on the expression of the eukaryotic structural genes. Question of whether there are underlying organizing principles of the eukaryotic nucleus required for proper gene expression; Examination of the nuclear pore complex and the nucleolus; Compartmentalization of factors involved in RNA biogenesis; Speckles as a result of gene expression.

Presents research which studied whether cell binding to the extracellular matrix (ECM) promotes formation of a cytoskeletal microcompartment specialized for translational control at the site of integrin binding. Role of the ECM; Formation of focal adhesion complexes (FAC); Cell adhesion to ECM; Control of gene expression; Use of high-resolution in situ hybridization; Implications of research for gene regulation by integrins.

Reports a study that shows ASH1 messenger RNA (mRNA) preferentially accumulates in daughter cells by a process that is dependent on actin and myosin. The study results suggesting that localization of mRNA may have been an early property of the eukaryotic lineage; References and notes.

Cytoplasmic polyadenylated RNA of myogenic cells was shown to decay with biphasic kinetics, suggesting the existence of two main populations of mRNA with respect to stability. In the present study, the stability of mRNA extracted from actinomycin-D-treated cultures of a myogenic cell line was tested by its capacity to direct protein synthesis in the wheat germ cell-free system, The products were analyzed by dodecylsulphate/polyacrylamide gel electrophoresis. All major radioactive bands found in gels used for analyzing the products of the cell-free system directed by polyadenylated RNA extracted from untreated cultures were also found in similar gels containing products of RNA extracted after many hours of application of actinomycin D. The capacity to code for specific protein bands decays with a half-life ranging between 11 and 40 h. No fast-decaying translatable mRNA could be detected by this method. Instead. it was found that during the first 4-6 h following application of actinomycin D. the capacity of RNA to stimulate incorporation of amino acids into total acidinsoluble material increased by 20-30%. The synthesis of specific products increased by up to 100%The possibility that the fast-decaying polyadenylated RNA or part of it is nontranslatable RNA is discussed. [ABSTRACT FROM AUTHOR]

Three independent methods of measurement showed that cytoplasmic polyadenylated RNA from the differentiating myogenic cell line L8 consists of two main populations with regard to stability, one with a half-life of less than 4 h and the other with a half-life of 17-54 h. Similar results were obtained in the presence and absence of actinomycin D. During the fusion of mononucleated myoblasts into multinucleated fibers, there was an increase in both the steady-state pool of the more stable polyadenylated RNA and the proportion of stable polyadenylated RNA synthesized in pulse labelling. [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]

We all know that gene expression occurs within cells, yet we do not think of expression in terms of its fundamental unit – a single cell. Instead, we understand the expression of genes in terms of a cell population as all of our information comes from samples containing millions of cells. From a complex mixture of cells, we attempt to infer the probable state of an average cell in the population. In truth, what we obtain is an averaged cell, a contrivance for representing biological knowledge beyond the limits of detection. We never know the variation among the members of the population that our methods average into a mean. Recent technological advances allow the precise measurement of single-cell transcriptional states to study this variability more rigorously. How genes are expressed in the population is strikingly different to what we have assumed from extrapolating to an average cell. Does the average cell actually exist? As we discuss, it is becoming increasingly clear that it doesn''t. [Copyright &y& Elsevier]

Discusses tracks from DNA to cytoplasm in RNA travel. Movement of a synthesized polypeptide through cytoplasmic secretion machinery; Prediction from the transport model; Concentration of splicing components in a number of discrete light microscopic subnuclear domains.

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]

Two new studies reveal the role of microtubule polarity in the asymmetric localization of mRNAs. In this issue of Cell, show that the asymmetric localization of oskar mRNA in fruit fly oocytes results from a slight bias in the direction of its transport. Meanwhile, reporting in Developmental Cell find a subpopulation of microtubules that is critical for the asymmetric distribution of Vg1 mRNA in frog oocytes. [Copyright &y& Elsevier]

In a recent issue of Molecular Cell, demonstrate directly that polymerase assembly kinetics regulate Pol I transcription. [Copyright &y& Elsevier]

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]

Discusses research which studied triplet-repeat diseases. What such diseases are; Aspects of each generation of a repeated sequence of three nucleotides; Gauging the severity of such diseases; Cause of such diseases related to genetic defect; Triplet repeats outside the coding region; Models to explain such diseases; Example of myotonic dystrophy; Repeats that result in a gain of function; Cases of two myotonic dystrophy patients.

Summary: 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. [Copyright &y& Elsevier]

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]

Translational repression during mRNA transport is essential for spatial restriction of protein production. In the yeast Saccharomyces cerevisae, silencing of ASH1 mRNA before it is localized to the bud cortex in late anaphase is critical for asymmetric segregation of Ash1p to the daughter cell nucleus. Puf6p, an ASH1 mRNA-binding protein, has been implicated in this process as a translational repressor, but the underlying mechanism is unknown. Here, we used yeast extract-based in vitro translation assays, which recapitulate translation and phosphorylation, to characterize the mechanism of Puf6p-mediated translational regulation. We report that Puf6p interferes with the conversion of the 48S complex to the 80S complex during initiation, and this repression by Puf6p is mediated through the general translation factor eIF5B (Fun12p in S. cerevisiae). Puf6p interacts with Fun12p via the PUF domain, and this interaction is RNA-dependent and essential for translational repression by Puf6p. This repression is relieved by phosphorylation of the N-terminal region of Puf6p mediated by protein kinase CK2 (casein kinase II). Inhibition of phosphorylation at Ser31, Ser34, and Ser35 of Puf6p increases its translational repression and results in ASH1 mRNA delocalization. Our results indicate that Puf6p suppresses the translation initiation of ASH1 mRNA via interaction with Fun12p during its transport, and this repression can be released by CK2 phosphorylation in the N-terminal region of Puf6p when the mRNA reaches the bud tip. [ABSTRACT FROM AUTHOR]

RNA localization during development is required for proper sorting of developmental determinants. Direct visualization of endogenous transcribed RNA is now possible and should provide new insights into how this process occurs. [Copyright &y& Elsevier]

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]