The Neuronal Splicing Factor Nova Co-Localizes with Target RNAs in the Dendrite

Nova proteins are neuron-specific RNA binding proteins targeted by autoantibodies in a disorder manifest by failure of motor inhibition, and they regulate splicing and alternative 3′ processing. Nova regulates splicing of RNAs encoding synaptic proteins, including the inhibitory glycine receptor α2 subunit (GlyRα2), and binds to others, including the GIRK2 channel. We found that Nova harbors functional NES and NLS elements, shuttles between the nucleus and cytoplasm, and that 50% of the protein localizes to the soma-dendritic compartment. Immunofluoresence and EM analysis of spinal cord motor neurons demonstrated that Nova co-localizes beneath synaptic contacts in dendrites with the same RNA, GlyRα2, whose splicing it regulates in the nucleus. HITS-CLIP identified intronic and 3′ UTR sites where Nova binds to GlyRα2 and GIRK2 transcripts in the brain. This led directly to the identification of a 3′ UTR localization element that mediates Nova-dependent localization of GIRK2 in primary neurons. These data demonstrate that HITS-CLIP can identify functional RNA localization elements, and they suggest new links between the regulation of nuclear RNA processing and mRNA localization.

[1]  C. Holt,et al.  Subcellular mRNA Localization in Animal Cells and Why It Matters , 2009, Science.

[2]  M. Visnapuu,et al.  Single-molecule imaging of DNA curtains reveals intrinsic energy landscapes for nucleosome deposition , 2009, Nature Structural &Molecular Biology.

[3]  J. Fak,et al.  Rescuing Z+ agrin splicing in Nova null mice restores synapse formation and unmasks a physiologic defect in motor neuron firing , 2009, Proceedings of the National Academy of Sciences.

[4]  K. Martin,et al.  mRNA Localization: Gene Expression in the Spatial Dimension , 2009, Cell.

[5]  P. Worley,et al.  The Immediate Early Gene Arc/Arg3.1: Regulation, Mechanisms, and Function , 2008, The Journal of Neuroscience.

[6]  S. Warren,et al.  Fragile X Syndrome: Loss of Local mRNA Regulation Alters Synaptic Development and Function , 2008, Neuron.

[7]  Eric T. Wang,et al.  Alternative Isoform Regulation in Human Tissue Transcriptomes , 2008, Nature.

[8]  Tyson A. Clark,et al.  HITS-CLIP yields genome-wide insights into brain alternative RNA processing , 2008, Nature.

[9]  Eric C Greene,et al.  Visualizing one-dimensional diffusion of proteins along DNA , 2008, Nature Structural &Molecular Biology.

[10]  J. Condeelis,et al.  Mechanisms and cellular roles of local protein synthesis in mammalian cells. , 2008, Current opinion in cell biology.

[11]  D. Reichman,et al.  Dynamic basis for one-dimensional DNA scanning by the mismatch repair complex Msh2-Msh6. , 2007, Molecular cell.

[12]  C. Holt,et al.  Local translation and directional steering in axons , 2007, The EMBO journal.

[13]  J. Richter,et al.  CPEB: a life in translation. , 2007, Trends in biochemical sciences.

[14]  M. Moore,et al.  The nuclear nurture and cytoplasmic nature of localized mRNPs. , 2007, Seminars in cell & developmental biology.

[15]  B. Blencowe,et al.  An RNA map predicting Nova-dependent splicing regulation , 2006, Nature.

[16]  E. Schuman,et al.  Dendritic Protein Synthesis, Synaptic Plasticity, and Memory , 2006, Cell.

[17]  O. Steward,et al.  Synaptic Regulation of Translation of Dendritic mRNAs , 2006, The Journal of Neuroscience.

[18]  R. Darnell,et al.  Paraneoplastic syndromes affecting the nervous system. , 2006, Seminars in oncology.

[19]  J. Ule,et al.  RNA binding proteins and the regulation of neuronal synaptic plasticity , 2006, Current Opinion in Neurobiology.

[20]  S. Kunes,et al.  Synaptic Protein Synthesis Associated with Memory Is Regulated by the RISC Pathway in Drosophila , 2006, Cell.

[21]  Jernej Ule,et al.  CLIP: a method for identifying protein-RNA interaction sites in living cells. , 2005, Methods.

[22]  J. Ule,et al.  Common Molecular Pathways Mediate Long-Term Potentiation of Synaptic Excitation and Slow Synaptic Inhibition , 2005, Cell.

[23]  Tyson A. Clark,et al.  Nova regulates brain-specific splicing to shape the synapse , 2005, Nature Genetics.

[24]  Daniel St Johnston,et al.  Moving messages: the intracellular localization of mRNAs , 2005, Nature Reviews Molecular Cell Biology.

[25]  Robert B Darnell,et al.  Nova autoregulation reveals dual functions in neuronal splicing , 2005, The EMBO journal.

[26]  A. Ephrussi,et al.  Splicing of oskar RNA in the nucleus is coupled to its cytoplasmic localization , 2004, Nature.

[27]  T. Kress,et al.  Nuclear RNP complex assembly initiates cytoplasmic RNA localization , 2004, The Journal of cell biology.

[28]  Jernej Ule,et al.  CLIP Identifies Nova-Regulated RNA Networks in the Brain , 2003, Science.

[29]  R. Singer,et al.  Localization of a β-Actin Messenger Ribonucleoprotein Complex with Zipcode-Binding Protein Modulates the Density of Dendritic Filopodia and Filopodial Synapses , 2003, The Journal of Neuroscience.

[30]  M. Ehlers,et al.  Activity-Dependent mRNA Splicing Controls ER Export and Synaptic Delivery of NMDA Receptors , 2003, Neuron.

[31]  R. Darnell,et al.  Paraneoplastic syndromes involving the nervous system. , 2003, The New England journal of medicine.

[32]  R. Darnell,et al.  Nova Regulates GABAA Receptor γ2 Alternative Splicing via a Distal Downstream UCAU-Rich Intronic Splicing Enhancer , 2003, Molecular and Cellular Biology.

[33]  I. Palacios.  RNA Processing: Splicing and the Cytoplasmic Localisation of mRNA , 2002, Current Biology.

[34]  W. Gu,et al.  A predominantly nuclear protein affecting cytoplasmic localization of β-actin mRNA in fibroblasts and neurons , 2002, The Journal of cell biology.

[35]  A. Krainer,et al.  Exon identity established through differential antagonism between exonic splicing silencer-bound hnRNP A1 and enhancer-bound SR proteins. , 2001, Molecular cell.

[36]  A. Ephrussi,et al.  Drosophila Y14 shuttles to the posterior of the oocyte and is required for oskar mRNA transport , 2001, Current Biology.

[37]  S. Moss,et al.  Constructing inhibitory synapses , 2001, Nature Reviews Neuroscience.

[38]  J. Yong,et al.  Pre-mRNA splicing imprints mRNA in the nucleus with a novel RNA-binding protein that persists in the cytoplasm. , 2000, Molecular cell.

[39]  R. Darnell,et al.  A brain-enriched polypyrimidine tract-binding protein antagonizes the ability of Nova to regulate neuron-specific alternative splicing. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[40]  H. Le Hir,et al.  Pre-mRNA splicing alters mRNP composition: evidence for stable association of proteins at exon-exon junctions. , 2000, Genes & development.

[41]  Robert B Darnell,et al.  Nova-1 Regulates Neuron-Specific Alternative Splicing and Is Essential for Neuronal Viability , 2000, Neuron.

[42]  R. Reed,et al.  Splicing is required for rapid and efficient mRNA export in metazoans. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[43]  A. Triller,et al.  Dendritic and Postsynaptic Protein Synthetic Machinery , 1999, The Journal of Neuroscience.

[44]  R. Darnell,et al.  The neuronal RNA-binding protein Nova-2 is implicated as the autoantigen targeted in POMA patients with dementia. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[45]  R. Darnell,et al.  The neuronal RNA binding protein Nova-1 recognizes specific RNA targets in vitro and in vivo , 1997, Molecular and cellular biology.

[46]  R. Singer,et al.  Actin messenger RNA localization and protein synthesis augment cell motility , 1997 .

[47]  RJ Buckanovich,et al.  The onconeural antigen Nova-1 is a neuron-specific RNA-binding protein, the activity of which is inhibited by paraneoplastic antibodies , 1996, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[48]  R. Huganir,et al.  Regulated subcellular distribution of the NR1 subunit of the NMDA receptor. , 1995, Science.

[49]  M. Morales,et al.  Aggregation of vasopressin mRNA in a subset of axonal swellings of the median eminence and posterior pituitary: light and electron microscopic evidence , 1994, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[50]  R. Darnell,et al.  Nova, the paraneoplastic Ri antigen, is homologous to an RNA-binding protein and is specifically expressed in the developing motor system , 1993, Neuron.

[51]  R. Singer,et al.  Determinants of mRNA localization. , 1992, Current opinion in cell biology.

[52]  G. Dreyfuss,et al.  Shuttling of pre-mRNA binding proteins between nucleus and cytoplasm , 1992, Nature.

[53]  M. Tohyama,et al.  Localization of glycine receptor α1 subunit mRNA-containing neurons in the rat brain: An analysis using in situ hybridization histochemistry , 1991, Neuroscience.

[54]  B. Marquèze-Pouey,et al.  Widespread expression of glycine receptor subunit mRNAs in the adult and developing rat brain. , 1991, The EMBO journal.

[55]  A. Kuryatov,et al.  Alternative splicing generates two isoforms of the α2 subunit of the inhibitory glycine receptor , 1991, FEBS letters.

[56]  M. Malim,et al.  HIV-1 structural gene expression requires the binding of multiple Rev monomers to the viral RRE: Implications for HIV-1 latency , 1991, Cell.

[57]  S. Palay,et al.  The Fine Structure of the Nervous System: Neurons and Their Supporting Cells , 1991 .

[58]  H. Betz,et al.  A single amino acid exchange alters the pharmacology of neonatal rat glycine receptor subunit , 1990, Neuron.

[59]  P. Seeburg,et al.  Glycine vs GABA receptors , 1987, Nature.

[60]  H. Korn,et al.  gamma-Aminobutyric acid-containing terminals can be apposed to glycine receptors at central synapses , 1987, The Journal of cell biology.

[61]  J. Lawrence,et al.  Intracellular localization of messenger RNAs for cytoskeletal proteins , 1986, Cell.

[62]  R. Wenthold,et al.  Identification of glycinergic synapses in the cochlear nucleus through immunocytochemical localization of the postsynaptic receptor , 1986, Brain Research.

[63]  H. Korn,et al.  Distribution of glycine receptors at central synapses: an immunoelectron microscopy study , 1985, The Journal of cell biology.

[64]  F. Pfeiffer,et al.  Monoclonal antibodies and peptide mapping reveal structural similarities between the subunits of the glycine receptor of rat spinal cord. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[65]  P. Greengard,et al.  Synapsin I (protein I), a nerve terminal-specific phosphoprotein. III. Its association with synaptic vesicles studied in a highly purified synaptic vesicle preparation , 1983, The Journal of cell biology.

[66]  P. Greengard,et al.  Synapsin I (protein I), a nerve terminal-specific phosphoprotein. I. Its general distribution in synapses of the central and peripheral nervous system demonstrated by immunofluorescence in frozen and plastic sections , 1983, The Journal of cell biology.

[67]  F. Bloom,et al.  Immunocytochemical localization, in synapses, of protein I, an endogenous substrate for protein kinases in mammalian brain. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[68]  F. Bloom,et al.  Widespread distribution of protein I in the central and peripheral nervous systems. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[69]  Donny D. Licatalosi,et al.  RNA processing and its regulation: global insights into biological networks , 2010, Nature Reviews Genetics.

[70]  E. Klann,et al.  Making synaptic plasticity and memory last: mechanisms of translational regulation. , 2009, Genes & development.

[71]  Robert B Darnell,et al.  CLIP: crosslinking and immunoprecipitation of in vivo RNA targets of RNA-binding proteins. , 2008, Methods in molecular biology.

[72]  R. Darnell,et al.  Developing global insight into RNA regulation. , 2006, Cold Spring Harbor symposia on quantitative biology.

[73]  R. Harvey,et al.  Differential agonist sensitivity of glycine receptor alpha2 subunit splice variants. , 2004, British journal of pharmacology.

[74]  P. Macdonald,et al.  Recognition of a bicoid mRNA localization signal by a protein complex containing Swallow, Nod, and RNA binding proteins. , 2003, Developmental cell.

[75]  K. Jaeckle.  Paraneoplastic Neurologic Disease , 2002 .

[76]  D. St Johnston,et al.  Getting the message across: the intracellular localization of mRNAs in higher eukaryotes. , 2001, Annual review of cell and developmental biology.

[77]  R. Singer,et al.  Neuronal RNA localization and the cytoskeleton. , 2001, Results and problems in cell differentiation.

[78]  O. Steward,et al.  Protein synthesis at synaptic sites on dendrites. , 2001, Annual review of neuroscience.

[79]  R. Darnell,et al.  Paraneoplastic neurologic disease antigens: RNA-binding proteins and signaling proteins in neuronal degeneration. , 2001, Annual review of neuroscience.

[80]  A. Triller,et al.  Cell-specific dendritic localization of glycine receptor alpha subunit messenger RNAs. , 1998, Neuroscience.

[81]  A. Triller,et al.  Dendritic and postsynaptic localizations of glycine receptor alpha subunit mRNAs. , 1997, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[82]  J. Elf,et al.  Supporting Online Material Materials and Methods Figs. S1 to S3 References Probing Transcription Factor Dynamics at the Single-molecule Level in a Living Cell , 2022 .