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Role of histone-modifying enzymes on developmental timing of Dictyostelium is presented.

Eukaryotic structural gene expression involves gene transcription and transport of processed mRNA to the cytoplasm. There are indications that the eukaryotic nucleus may be functionally compartmentalized, and a model whereby the expression of genes directs the reorganization of nuclear components is supported.

• 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]

Two arguments on RNA transportation from its production site to the cytoplasm is presented. One of these theories maintains that RNA synthesizing mechanisms are located at specific locations in the nucleus and RNA transcripts move on these defined pathways. The other argument contradicts the prediction made by the first theory that active RNA polymerase II splicing components and nuclear pores have defined and varied locations.

X-ray crystallography is used to determine the three-dimensional structure of She2p, which reveals that She2p is composed of a single globular domain with a five alpha helix bundle that forms a symmetric homodimer. After demonstrating potent, dimer-dependent RNA binding in vitro, the RNA binding surface of She2p is mapped to a basic helical hairpin in vitro and in vivo.

An inducible system to visualize gene expression at the levels of DNA, RNA and protein in living cells is developed. Using this system, changes are correlated in chromatin structure with the progression of transcriptional activation that allows obtaining a real-time integrative view of gene expression.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2008.08.020 Byline: Robert H. Singer (1) Abstract: 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. Author Affiliation: (1) Departments of Anatomy and Structural Biology, Cell Biology, and Neuroscience, Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, NY 10461, USA

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]

Fragile X syndrome (FXS) is a leading cause of inherited intellectual disability and autism. Whereas dysregulated RNA translation in Fmr1 knockout (KO) mice, a model of FXS, is well studied, little is known about aberrant transcription. Using single-molecule mRNA detection, we show that mRNA encoding the AMPAR subunit GluA2 (but not GluA1) is elevated in dendrites and at transcription sites of hippocampal neurons of Fmr1 KO mice, indicating elevated GluA2 transcription. We identify CPEB3, a protein implicated in memory consolidation, as an upstream effector critical to GluA2 mRNA expression in FXS. Increased GluA2 mRNA is translated into an increase in GluA2 subunits, a switch in synaptic AMPAR phenotype from GluA2-lacking, Ca2+-permeable to GluA2-containing, Ca2+-impermeable, reduced inhibitory synaptic transmission, and loss of NMDAR-independent LTP at glutamatergic synapses onto CA1 inhibitory interneurons. These factors could contribute to an excitatory/inhibitory imbalance—a common theme in FXS and other autism spectrum disorders. [Display omitted] • Transcription of GluA2 mRNA is elevated in Fmr1- deficient hippocampal neurons • CPEB3 and STAT5b are the upstream effectors critical to GluA2 mRNA expression • Increase in GluA2 underlies a switch in synaptic AMPAR phenotype in CA1 interneurons • A switch in AMPAR phenotype causes deficits in synaptic transmission and plasticity Using single-molecule FISH and patch-clamp recording, Hwang et al. show that dysregulation of GluA2 transcription is critical to synaptic function in an animal model of autism. The increase in GluA2 results in a switch in AMPAR phenotype and deficits in synaptic transmission and plasticity at synapses onto CA1 inhibitory interneurons. [ABSTRACT FROM AUTHOR]

Successive generations of mice doubly null for the tolemerase RNA (mTR) and the INK4a tumor suppressor genes were examined to determine the effect of telomere dysfunction on tumorigenesis. Results indicate that tumor growth in vivo and oncogenic potential in vitro in late generations of telomerase deficiency were slowed down. However, the reintroduction of mTR into the cells was not abe to fully stop tumor formation. It only restored the cell's oncogenic potential.

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

Summary: Localization of mRNA is a critical mechanism used by a large fraction of transcripts to restrict its translation to specific cellular regions. Although current high-resolution imaging techniques provide ample information, the analysis methods for localization have either been qualitative or employed quantification in nonrandomly selected regions of interest. Here, we describe an analytical method for objective quantification of mRNA localization using a combination of two characteristics of its molecular distribution, polarization and dispersion. The validity of the method is demonstrated using single-molecule FISH images of budding yeast and fibroblasts. Live-cell analysis of endogenous β-actin mRNA in mouse fibroblasts reveals that mRNA polarization has a half-life of ∼16 min and is cross-correlated with directed cell migration. This novel approach provides insights into the dynamic regulation of mRNA localization and its physiological roles. [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]

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]

Abstract: Studying single mRNA molecules has added new dimensions to our understanding of gene expression and the life cycle of mRNA in cells. Advances in microscopes and detection technology have opened access to single molecule research to most researchers interested in molecular biology. Here we provide an overview technique for single molecule studies of RNA in either fixed samples or in living cells. As part of a volume on mRNA turnover, it is increasingly relevant, because many of the recent advances in studies of mRNA turnover have suggested that there is non-homogeneous distribution of turnover factors in the cell. For this reason, understanding of spatial relationships between mRNA and mRNA turnover factors should enrich our understanding of this process. [Copyright &y& Elsevier]

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]

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]

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 transport and translation of dendritic mRNAs by RNA-binding proteins (RBPs) allows for spatially restricted gene expression in neuronal processes. Although local translation in neuronal dendrites is now well documented, there is little evidence for corresponding effects on local synaptic function. Here, we report that the RBP Sam68 promotes the localization and translation of Arc mRNA preferentially in distal dendrites of rodent hippocampal CA1 pyramidal neurons. Consistent with Arc function in translation-dependent synaptic plasticity, we find that Sam68 knockout (KO) mice display impaired metabotropic glutamate-receptor-dependent long-term depression (mGluR-LTD) and impaired structural plasticity exclusively at distal Schaffer-collateral synapses. Moreover, by using quantitative proteomics, we find that the Sam68 interactome contains numerous regulators of mRNA translation and synaptic function. This work identifies an important player in Arc expression, provides a general framework for Sam68 regulation of protein synthesis, and uncovers a mechanism that enables the precise spatiotemporal expression of long-term plasticity throughout neurons. • Sam68 promotes the localization and translation of Arc mRNA at distal dendrites • Distinct processes regulate Arc protein synthesis at the soma versus the dendrite • Sam68 regulates structural and functional plasticity exclusively at distal synapses • Interactome analysis reveals a role for Sam68 in translation initiation Although local translation in neuronal dendrites is well documented, there is little evidence for corresponding effects on local synaptic function. Klein et al. demonstrate that Sam68 is required for Arc protein synthesis at distal dendritic regions and is required for synaptic plasticity exclusively at distal dendrites of hippocampal pyramidal neurons. [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]

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]

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]

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]

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]

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

Unlike the core histones, which are incorporated into nucleosomes concomitant with DNA replication, histone H3.3 is synthesized throughout the cell cycle and utilized for replicationindependent (RI) chromatin assembly. The RI incorporation of H3.3 into nucleosomes is highly conserved and occurs at both euchromatin and heterochromatin. However, neither the mechanism of H3.3 recruitment nor its essential function is well understood. Several different chaperones regulate H3.3 assembly at distinct sites. The H3.3 chaperone, Daxx, and the chromatin-remodeling factor, ATRX, are required for H3.3 incorporation and heterochromatic silencing at telomeres, pericentromeres, and the cytomegalovirus (CMV) promoter. By evaluating H3.3 dynamics at a CMV promoter-regulated transcription site in a genetic background in which RI chromatin assembly is blocked, we have been able to decipher the regulatory events upstream of RI nucleosomal deposition. We find that at the activated transcription site, H3.3 accumulates with sense and antisense RNA, suggesting that it is recruited through an RNA-mediated mechanism. Sense and antisense transcription also increases after H3.3 knockdown, suggesting that the RNA signal is amplified when chromatin assembly is blocked and attenuated by nucleosomal deposition. Additionally, we find that H3.3 is still recruited after Daxx knockdown, supporting a chaperone-independent recruitment mechanism. Sequences in the H3.3 N-terminal tail and αN helix mediate both its recruitment to RNA at the activated transcription site and its interaction with double-stranded RNA in vitro. Interestingly, the H3.3 gain-of-function pediatric glioblastoma mutations, G34Rand K27M, differentially affect H3.3 affinity in these assays, suggesting that disruption of an RNA-mediated regulatory event could drive malignant transformation. [ABSTRACT FROM AUTHOR]

Recent developments in reagent design can address problems in single cells that were not previously approachable. We have attempted to foresee what will become possible, and the sorts of biological problems that become tractable with these novel reagents. We have focused on the novel fluorescent proteins that allow convenient multiplexing, and provide for a time-dependent analysis of events in single cells. Methods for fluorescently labeling specific molecules, including endogenously expressed proteins and mRNA have progressed and are now commonly used in a variety of organisms. Finally, sensitive microscopic methods have become more routine practice. This article emphasizes that the time is right to coordinate these approaches for a new initiative on single cell imaging of biological molecules. [Copyright &y& Elsevier]

Abstract: The Karpas-620 human myeloma cell line (HMCL) expresses high levels of Cyclin D1 (CCND1), but has a der(8)t(8;11) and a der(14)t(8;14), and not a conventional t(11;14). Fluorescent in situ hybridization (FISH) and array comparative genomic hybridization (aCGH) studies suggest that der(14)t(11;14) from a primary translocation underwent a secondary translocation with chromosome 8 to generate der(8)t(8;[14];11) and der(14)t(8;[11];14). Both secondary derivatives share extensive identical sequences from chromosomes 8, 11, and 14, including MYC and the 3′ IgH enhancers. Der(14), with MYC located ∼700kb telomeric to the 3′IGH enhancer, expresses MYC. By contrast, der(8), with both CCND1 and MYC repositioned near a 3′IGH enhancer, expresses CCND1, which is telomeric of the enhancer, but not MYC, which is centromeric to the enhancer. The secondary translocation that dysregulated MYC resulted in extensive regions from both donor chromosomes being transmitted to both derivative chromosomes, suggesting a defect in DNA recombination or repair in the myeloma tumor cell. [Copyright &y& Elsevier]

Summary: The function of local protein synthesis in synaptic plasticity and its dysregulation in fragile X syndrome (FXS) is well studied, however the contribution of regulated mRNA transport to this function remains unclear. We report a function for the fragile X mental retardation protein (FMRP) in the rapid, activity-regulated transport of mRNAs important for synaptogenesis and plasticity. mRNAs were deficient in glutamatergic signaling-induced dendritic localization in neurons from Fmr1 KO mice, and single mRNA particle dynamics in live neurons revealed diminished kinesis. Motor-dependent translocation of FMRP and cognate mRNAs involved the C terminus of FMRP and kinesin light chain, and KO brain showed reduced kinesin-associated mRNAs. Acute suppression of FMRP and target mRNA transport in WT neurons resulted in altered filopodia-spine morphology that mimicked the FXS phenotype. These findings highlight a mechanism for stimulus-induced dendritic mRNA transport and link its impairment in a mouse model of FXS to altered developmental morphologic plasticity. [Copyright &y& Elsevier]

After the export from the nucleus it turns out that all mRNAs are not treated equally. Not only is mRNA subject to translation, but also through RNA-binding proteins and other trans-acting factors, eukaryotic cells interpret codes for spatial sorting within the mRNA sequence. These codes instruct the cytoskeleton and translation apparatus to make decisions about where to transport and when to translate the intended protein product. Signaling pathways decode extra-cellular cues and can modify transport and translation factors in the appropriate cytoplasmic space to achieve translation locally. Identifying regulatory sites on transport factors as well as novel physiological functions for well-known translation factors has provided significant advances in how spatially controlled translation impacts cell function. [Copyright &y& Elsevier]

Active transport and localized translation of the ASH1 mRNA at the bud tip of the budding yeast Saccharomyces cerevisiae is an essential process that is required for the regulation of the mating type switching. ASH1 mRNA localization has been extensively studied over the past few years and the core components of the translocation machinery have been identified. It is composed of four localization elements (zipcodes), within the ASH1 mRNA, and at least three proteins, She1p/Myo4p, She2p and She3p. Whereas the movement of the RNA can be attributed to direct interaction with myosin, the regulation of the RNA expression is less well understood. Recent insights have revealed a role for translation that might have a key function in the regulation of Ash1 protein sorting. [Copyright &y& Elsevier]

Although it is generally accepted that nuclear architecture is an important determinant of nuclear activity, it is not clear whether cytoplasmic events, such as transcript localization and cell polarity, are affected by this architecture. Characterization of the nuclear architecture of the single-cell alga Chlamydomonas reinhardtii revealed a polarized nucleus, with nuclear pore complexes preferentially concentrated at the posterior side of the nucleus. Nuclear asymmetry was greatly exaggerated during the upregulation of genes encoding flagellar proteins, when nuclear pore complexes (NPCs) were observed to hyperpolarize to the posterior side of the nucleus while heterochromatin polarized to the anterior side. Interestingly, prior to deflagellation, the β2-tubulin gene was preferentially located in the posterior region of the nucleus, and following deflagellation, β2-tubulin transcripts accumulated posteriorly in polysome-rich cytoplasmic regions adjacent to the highest concentration of NPCs, suggesting a connection between nuclear architecture and cytoplasmic transcript localization. [Copyright &y& Elsevier]