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      The Central Dogma Decentralized: New Perspectives on RNA Function and Local Translation in Neurons

      review-article
        1 , , 2 , ∗∗
      Neuron
      Cell Press

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          Abstract

          The elaborate morphology of neurons together with the information processing that occurs in remote dendritic and axonal compartments makes the use of decentralized cell biological machines necessary. Recent years have witnessed a revolution in our understanding of signaling in neuronal compartments and the manifold functions of a variety of RNA molecules that regulate protein translation and other cellular functions. Here we discuss the view that mRNA localization and RNA-regulated and localized translation underlie many fundamental neuronal processes and highlight key issues for future experiments.

          Abstract

          Recent years have brought a revolution in our understanding of the diverse roles of RNA molecules (both within and outside the nervous system), as well in our view of neuronal compartments as distinct signaling units. Christine Holt and Eric Schuman review how the convergence of these two cell biological areas has changed our view on spatial signaling in neurons.

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          Most cited references105

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          HITS-CLIP yields genome-wide insights into brain alternative RNA processing

          Summary Protein-RNA interactions play critical roles in all aspects of gene expression. Here we develop a genome-wide means of mapping protein-RNA binding sites in vivo, by high throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP). HITS-CLIP analysis of the neuron-specific splicing factor Nova2 revealed extremely reproducible RNA binding maps in multiple mouse brains. These maps provide genome-wide in vivo biochemical footprints confirming the previous prediction that the position of Nova binding determines the outcome of alternative splicing; moreover, they are sufficiently powerful to predict Nova action de novo. HITS-CLIP revealed a large number of Nova-RNA interactions in 3′ UTRs, leading to the discovery that Nova regulates alternative polyadenylation in the brain. HITS-CLIP, therefore, provides a robust, unbiased means to identify functional protein-RNA interactions in vivo.
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            A translational profiling approach for the molecular characterization of CNS cell types.

            The cellular heterogeneity of the brain confounds efforts to elucidate the biological properties of distinct neuronal populations. Using bacterial artificial chromosome (BAC) transgenic mice that express EGFP-tagged ribosomal protein L10a in defined cell populations, we have developed a methodology for affinity purification of polysomal mRNAs from genetically defined cell populations in the brain. The utility of this approach is illustrated by the comparative analysis of four types of neurons, revealing hundreds of genes that distinguish these four cell populations. We find that even two morphologically indistinguishable, intermixed subclasses of medium spiny neurons display vastly different translational profiles and present examples of the physiological significance of such differences. This genetically targeted translating ribosome affinity purification (TRAP) methodology is a generalizable method useful for the identification of molecular changes in any genetically defined cell type in response to genetic alterations, disease, or pharmacological perturbations.
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              Cell-type-specific isolation of ribosome-associated mRNA from complex tissues.

              Gene profiling techniques allow the assay of transcripts from organs, tissues, and cells with an unprecedented level of coverage. However, most of these approaches are still limited by the fact that organs and tissues are composed of multiple cell types that are each unique in their patterns of gene expression. To identify the transcriptome from a single cell type in a complex tissue, investigators have relied upon physical methods to separate cell types or in situ hybridization and immunohistochemistry. Here, we describe a strategy to rapidly and efficiently isolate ribosome-associated mRNA transcripts from any cell type in vivo. We have created a mouse line, called RiboTag, which carries an Rpl22 allele with a floxed wild-type C-terminal exon followed by an identical C-terminal exon that has three copies of the hemagglutinin (HA) epitope inserted before the stop codon. When the RiboTag mouse is crossed to a cell-type-specific Cre recombinase-expressing mouse, Cre recombinase activates the expression of epitope-tagged ribosomal protein RPL22(HA), which is incorporated into actively translating polyribosomes. Immunoprecipitation of polysomes with a monoclonal antibody against HA yields ribosome-associated mRNA transcripts from specific cell types. We demonstrate the application of this technique in brain using neuron-specific Cre recombinase-expressing mice and in testis using a Sertoli cell Cre recombinase-expressing mouse.
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                Author and article information

                Journal
                Neuron
                Neuron
                Neuron
                Cell Press
                0896-6273
                1097-4199
                30 October 2013
                30 October 2013
                : 80
                : 3
                : 648-657
                Affiliations
                [1 ]Department of Physiology Development and Neuroscience, Anatomy Building, Downing Street, University of Cambridge, Cambridge CB2 3DY, UK
                [2 ]Max Planck Institute for Brain Research, Max von Laue Strasse 4, 60438 Frankfurt am Main, Germany
                Author notes
                []Corresponding author ceh33@ 123456cam.ac.uk
                [∗∗ ]Corresponding author erin.schuman@ 123456brain.mpg.de
                Article
                S0896-6273(13)00988-4
                10.1016/j.neuron.2013.10.036
                3820025
                24183017
                aaf090d4-7153-4b90-ac33-b9560ad0ae1d
                © 2013 ELL & Excerpta Medica.

                This document may be redistributed and reused, subject to certain conditions.

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                Neurosciences

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