Long-term memory and synaptic plasticity require changes in gene expression and yet can occur in a synapse-specific manner. mRNA localization and regulated translation at synapses are thus critical for establishing synapse specificity. Using live cell microscopy of photoconvertible fluorescent protein translational reporters, we directly visualized local translation at synapses during long-term facilitation of Aplysia sensory-motor synapses. Translation of the reporter required multiple applications of serotonin, was spatially restricted to stimulated synapses, was transcript-and stimulus-specific, and occurred during long-term facilitation but not during longterm depression of sensory-motor synapses. Translational regulation only occurred in the presence of a chemical synapse and required calcium signaling in the postsynaptic motor neuron. Thus highly regulated local translation occurs at synapses during long-term plasticity and requires transsynaptic signals.Long-lasting learning-related synaptic plasticity requires transcription for its persistence (1-3) and yet can occur in a synapse-specific manner (4-7). One mechanism that has been proposed to mediate this spatial restriction of gene expression during neuronal plasticity involves regulated translation of localized mRNAs at stimulated synapses (8-10). Many findings support the existence of local translation at synapses. First, all of the machinery required for translation is present in neuronal processes, including polyribosomes (11,12), translation factors (13), and a select population of mRNAs (14-18). Second, studies using

mRNA localization and regulated translation provide a means of spatially restricting gene expression within each of the thousands of subcellular compartments made by a neuron, thereby vastly increasing the computational capacity of the brain. Recent studies reveal that local translation is regulated by stimuli that trigger neurite outgrowth/collapse, axon guidance, synapse formation, pruning, activity-dependent synaptic plasticity, and injury induced axonal regeneration. Impairments in the local regulation of translation result in aberrant signaling, physiology, and morphology of neurons, and are linked to neurological disorders. This review highlights current advances in understanding how mRNAs are translationally repressed during transport and how local translation is activated by stimuli. We address the function of local translation in the context of fragile X mental retardation.

A localized transcriptome at the synapse facilitates synapse-, stimulus- and transcript-specific local protein synthesis in response to neuronal activity. While enzyme-mediated mRNA modifications are known to regulate cellular mRNA turnover, the role of these modifications in regulating synaptic RNA has not been studied. We established low-input mA-sequencing of synaptosomal RNA to determine the chemically modified local transcriptome in healthy adult mouse forebrains and identified 4,469 selectively enriched mA sites in 2,921 genes as the synaptic mA epitranscriptome (SME). The SME is functionally enriched in synthesis and modulation of tripartite synapses and in pathways implicated in neurodevelopmental and neuropsychiatric diseases. Interrupting mA-mediated regulation via knockdown of readers in hippocampal neurons altered expression of SME member Apc, resulting in synaptic dysfunction including immature spine morphology and dampened excitatory synaptic transmission concomitant with decreased clusters of postsynaptic density-95 (PSD-95) and decreased surface expression of AMPA receptor subunit GluA1. Our findings indicate that chemical modifications of synaptic mRNAs critically contribute to synaptic function.

Functional cellular substrates for localized cell stimulation by small molecules provide an opportunity to control and monitor cell signalling networks chemically in time and space. However, despite improvements in the controlled delivery of bioactive compounds, the precise localization of gaseous biomolecules at the single-cell level remains challenging. Here we target nitric oxide, a crucial signalling molecule with site-specific and concentration-dependent activities, and we report a synthetic strategy for developing spatiotemporally controllable nitric oxide-releasing platforms based on photoactive porous coordination polymers. By organizing molecules with poor reactivity into polymer structures, we observe increased photoreactivity and adjustable release using light irradiation. We embed photoactive polymer crystals in a biocompatible matrix and achieve precisely controlled nitric oxide delivery at the cellular level via localized two-photon laser activation. The biological relevance of the exogenous nitric oxide produced by this strategy is evidenced by an intracellular change in calcium concentration, mediated by nitric oxide-responsive plasma membrane channel proteins.

Determination of subcellular localization and dynamics of mRNA is increasingly important to understanding gene expression. A new convenient and versatile method is reported that permits spatiotemporal imaging of specific non-engineered RNAs in living cells. The method uses transfection of a plasmid encoding a gene-specific RNA aptamer, combined with a cell-permeable synthetic small molecule, the fluorescence of which is restored only when the RNA aptamer hybridizes with its cognitive mRNA. The method was validated by live-cell imaging of the endogenous mRNA of β-actin. Application of the technology to mRNAs of a total of 84 human cytoskeletal genes allowed us to observe cellular dynamics of several endogenous mRNAs including arfaptin-2, cortactin, and cytoplasmic FMR1-interacting protein 2. The RNA-imaging technology and its further optimization might permit live-cell imaging of any RNA molecules.

Messenger RNA (mRNA) localization and regulated translation can spatially restrict gene expression to each of the thousands of synaptic compartments formed by a single neuron. Although cisacting RNA elements have been shown to direct localization of mRNAs from the soma into neuronal processes, less is known about signals that target transcripts specifically to synapses. In Aplysia sensory-motor neuronal cultures, synapse formation rapidly redistributes the mRNA encoding the peptide neurotransmitter sensorin from neuritic shafts into synapses. We find that the export of sensorin mRNA from soma to neurite and the localization to synapse are controlled by distinct signals. The 3′ UTR is sufficient for export into distal neurites, whereas the 5′ UTR is required for concentration of reporter mRNA at synapses. We have identified a 66-nt element in the 5′ UTR of sensorin that is necessary and sufficient for synaptic mRNA localization. Mutational and chemical probing analyses are consistent with a role for secondary structure in this process.M essenger RNA (mRNA) localization and regulated translation provide a means of spatially restricting gene expression within distinct subcellular compartments. In the brain, local protein synthesis is critical to the development and experience-driven refinement of neural circuits, playing roles in axon guidance, synaptogenesis, and synaptic plasticity (1, 2). A large but select population of transcripts localizes to axons and dendrites (3-8), indicating that local translation subserves diverse cell biological functions. Where studied, the localization of mRNAs to axons or dendrites has been shown to depend on cisacting localization elements (LEs) usually found in the 3′ UTR, although occasionally present in the coding sequence or 5′ UTR (1, 2, 9). These cis-acting mRNA LEs recruit specific transacting RNA binding proteins, and the resulting messenger ribonucleoproteins are packaged into RNA transport granules that interact with molecular motors to be delivered to their final subcellular destination (10-12).In situ hybridization studies in neurons indicate that localized mRNAs in neurons are targeted to distinct subcellular compartments and domains within neuronal processes. For example, MAP2 mRNA concentrates within proximal dendrites, whereas calcium-calmodulin dependent protein kinase IIα (CaMKIIα) mRNA extends to distal dendrites (13). mRNA localization also seems to be dynamically regulated during development and with activity. In mature neurons, β-actin mRNA localizes to dendrites, and its concentration to distal dendrites is stimulated by depolarization (14). Stimuli that activate NMDA or neurotrophic receptor tyrosine kinase 2 (TrkB) receptors drive specific BDNF mRNA isoforms into distal dendrites of hippocampal neurons (15). High-frequency stimulation of perforant path projections to the dentate gyrus has been shown to direct localization of the mRNA encoding the immediate-early gene Arc selectively and specifically to activated dendritic lamina (16) and to drive localizati...

DNA methylation is one of the most important epigenetic mechanisms to regulate gene expression, which is highly dynamic during development and specifically maintained in somatic cells. Aberrant DNA methylation patterns are strongly associated with human diseases including cancer. How are the cell-specific DNA methylation patterns established or disturbed is a pivotal question in developmental biology and cancer epigenetics. Currently, compelling evidence has emerged that long non-coding RNA (lncRNA) mediates DNA methylation in both physiological and pathological conditions. In this review, we provide an overview of the current understanding of lncRNA-mediated DNA methylation, with emphasis on the roles of this mechanism in cancer, which to the best of our knowledge, has not been systematically summarized. In addition, we also discuss the potential clinical applications of this mechanism in RNA-targeting drug development.

Iron is an essential transition metal species for all living organisms and plays various physiologically important roles on the basis of its redox activity; accordingly, the disruption of iron homeostasis triggers oxidative stress and cellular damage. Therefore, cells have developed sophisticated iron-uptake machinery to acquire iron while protecting cells from uncontrolled oxidative damage during the uptake process. To examine the detailed mechanism of iron uptake while controlling the redox status, it is necessary to develop useful methods with redox state selectivity, sensitivity, and organelle specificity to monitor labile iron, which is weakly bound to subcellular ligands. Here, we report the development of Mem-RhoNox to monitor local Fe(II) at the surface of the plasma membrane of living cells. The redox state-selective fluorescence response of the probe relies on our recently developed N-oxide strategy, which is applicable to fluorophores with dialkylarylamine in their π-conjugation systems. Mem-RhoNox consists of the N-oxygenated rhodamine scaffold, which has two arms, both of which are tethered with palmitoyl groups as membrane-anchoring domains. In an aqueous buffer, Ac-RhoNox, a model compound of Mem-RhoNox, shows a fluorescence turn-on response to the Fe(II) redox state-selectively. An imaging study with Mem-RhoNox and its derivatives reveals that labile Fe(II) is transiently generated during the major iron-uptake pathways: endocytotic uptake and direct transport. Furthermore, Mem-RhoNox is capable of monitoring endosomal Fe(II) in primary cultured neurons during endocytotic uptake. This report is the first example that identifies the generation of Fe(II) over the course of cellular iron-uptake processes.