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Dendritic targeting of mrna FMRP-dependent mglur-induced dendritic targeting of MAP1b and CaMKII mrna IMP-dependent NMDAR-induced dendritic targeting of β-action mrna Discussion (12/15) RNA and protein targeting and translational regulation Discussion (12/17) Local protein turnover at the synapse

Figure 1. Developmental prevalence of FMRP at synapses Immature: 8 DIV Mature: 19 DIV DIV: days in vitro 5 μ FMRP Synapsin Actin Antar, L. N. et al. J. Neurosci. 2004;24:2648-2655 Copyright 2004 Society for Neuroscience

Figure 2. Localization of FMRP and Fmr1 mrna granules in dendritic compartments and their KCl-induced localization 10 μ EGFP-FMRP Antar, L. N. et al. J. Neurosci. 2004;24:2648-2655 Copyright 2004 Society for Neuroscience

Figure 3. FRAP analysis of increased EGFP-FMRP granule trafficking in response to KCl stimulation 0 3 5 min after photo-bleaching Control KCl Antar, L. N. et al. J. Neurosci. 2004;24:2648-2655 Copyright 2004 Society for Neuroscience

Figure 6. Model for FMRP and Fmr1 mrna localization in dendrites and at synapses FMRP binds KLC, KIF5 cargo-binding subunit, & mediates transport of MAP1b and CaMKII mrna to dendrites and spines upon mglur activation for proper spine formation Dev. Cell 14, 926, 2008 Antar, L. N. et al. J. Neurosci. 2004;24:2648-2655 Copyright 2004 Society for Neuroscience

FMRP associates with target mrnas in vivo in the absence of BC1 RNA Iacoangeli A. et.al. PNAS 2008;105:734-739 2008 by National Academy of Sciences

The N-terminal region of FMRP binds specifically to BC1 RNA RNA motif: I304N kissing complex G quartet Transcriptional silencing of Fmr1 due to CGG expansion or FMRP functional disruption via I304N mutation causes mental retardation Brown et al. Cell 107, 477-487, 2001 (MAP1B, SAPAP4, Munc13, FMR1 ) Kevin et al., Neuron 37, 417-421, 2003 (RGS5, CDK4 ) Darnell et al. Gen. Dev. 19, 903-918, 2005 Kissing complex RNAs mediate interaction between the Fragile-X mental retardation protein KH2 domain and brain polyribosomes

Magnesium-dependent binding suggests a loop-loop interaction for the KH2 RNA ligands Likely Mg 2+ binding sites Darnell J. C. et.al. Genes Dev. 2005;19:903-918 2005 by Cold Spring Harbor Laboratory Press

Compensatory mutation analysis of Watson-Crick interactions in selected KH2 RNA ligands Darnell J. C. et.al. Genes Dev. 2005;19:903-918 2005 by Cold Spring Harbor Laboratory Press

Mutational analysis of the non-watson-crick-paired regions of Δkc2 Darnell J. C. et.al. Genes Dev. 2005;19:903-918 R: A or G W: A or U N: any nucleotide S: Watson-Crick pair 2005 by Cold Spring Harbor Laboratory Press

kc2 RNA competes FMRP off of mouse brain polyribosomes in a dose-dependent manner Darnell J. C. et.al. Genes Dev. 2005;19:903-918 What might FMRP do to mrna in polysomes? Repression? RNAi effector complex (RISC) in polysomes & P-bodies 2005 by Cold Spring Harbor Laboratory Press

Identification of dfxr [the Drosophila homolog of the Fragile X Mental Retardation Protein (FMRP)], and VIG as RISC components Caudy A. A. et.al. Genes Dev. 2002;16:2491-2496 2002 by Cold Spring Harbor Laboratory Press

Identification of dfxr [the Drosophilahomolog of the Fragile X Mental Retardation Protein (FMRP)], and VIG as RISC components Caudy A. A. et.al. Genes Dev. 2002;16:2491-2496 2002 by Cold Spring Harbor Laboratory Press

dfxr [the Drosophila homolog of the Fragile X Mental Retardation Protein (FMRP)] and VIG associate with RISC Ion exchange column sirna Caudy A. A. et.al. Genes Dev. 2002;16:2491-2496 2002 by Cold Spring Harbor Laboratory Press

dfxr [the Drosophila homolog of the Fragile X Mental Retardation Protein (FMRP)] and VIG are required for efficient RNA interference in Drosophila S2 cells.drosophila S2 cells were transfected with double-stranded RNAs (dsrnas) corresponding to the indicated cdnas. Three days later, cells were transfected with dsrnas directed against luciferase (or GFP for control) and with expression vectors for luciferase. Values shown are luciferase expression levels in luciferase dsrna-transfected cells normalized to control. Caudy A. A. et.al. Genes Dev. 2002;16:2491-2496 2002 by Cold Spring Harbor Laboratory Press

dfxr- [the Drosophila homolog of the Fragile X Mental Retardation Protein (FMRP)] and VIG-based RISC complexes (~500 kd) contain micrornas (mirnas) mir13a mir2b Caudy A. A. et.al. Genes Dev. 2002;16:2491-2496 2002 by Cold Spring Harbor Laboratory Press

Figure 2 Stress-dependent granules in yeast and mammalian systems P-body RISC-mediated repression Biochemical Society Transactions www.biochemsoctrans.org Biochem.. Soc. Trans. (2008) 36, 648-652 652 Control of translation and mrna degradation by mirnas and sirnas Valencia-Sanchez, Liu, Hannon and Parker, Genes & Development (2006) 20, 515-524

Figure 4. Acute exogenous FMRP-GFP expression in Fmr1 knockout mice and endogenous FMRP reduce the number of synapses I304N linked to severe FXS abolishes FMRP interaction with kissing complex RNAs, reduces dimerization with other FMRP proteins and polyribosome association (Mol Cell 1, 109, 1997; Hum Mol Genet 10, 329, 2001; Genes Dev 19, 903, 2005) I304N I304N I304N Pfeiffer, B. E. et al. J. Neurosci. 2007;27:3120-3130 Copyright 2007 Society for Neuroscience

Figure 5. An intact KH2 RNA-binding domain of FMRP, but not an RGG box, is required to reduce synapse function I304N ΔRGG Pfeiffer, B. E. et al. J. Neurosci. 2007;27:3120-3130 Copyright 2007 Society for Neuroscience

Murine FMRP peptide 491-529 is the primary phosphopeptide and Serine 499 is the primary phosphorylation site Ceman, S. et al. Hum. Mol. Genet. 2003 12:3295-3305; doi:10.1093/hmg/ddg350 Copyright restrictions may apply.

Unphosphorylated FMRP is associated with actively translating polyribosomes and runs off more quickly after azide mediated inhibition of translation initiation polysome Flag-FMRP ribosome Azide blocks Initiation, not elongation Copyright restrictions may apply. Ceman, S. et al. Hum. Mol. Genet. 2003 12:3295-3305; doi:10.1093/hmg/ddg350

Phosphorylated FMRP remains associated with stalled heavy polysomes Ceman, S. et al. Hum. Mol. Genet. 2003 12:3295-3305; doi:10.1093/hmg/ddg350 Copyright restrictions may apply.

Synaptic stimulation can act via ionotropic glutamate receptors (AMPA/NMDA; 1a) and mglurs (1b) and can initiate translation (2) of locally synthesized proteins (3; shown in yellow), including regulators (underlined red text) and plastic structural elements (black) Grossman, A. W. et al. J. Neurosci. 2006;26:7151-7155 Copyright 2006 Society for Neuroscience

Figure 2. KCl induced ZBP1 granule localization to dendrites depends on NMDA receptor control KCl ZBP1 MAP2 Actin Proximal Middle Distal dendrite Tiruchinapalli, D. M. et al. J. Neurosci. 2003;23:3251-3261 Copyright 2003 Society for Neuroscience

Figure 4. KCl depolarization stimulates EGFP-ZBP1 dendritic transport 0 min 5 min 15 min 30 min in KCl Tiruchinapalli, D. M. et al. J. Neurosci. 2003;23:3251-3261 Copyright 2003 Society for Neuroscience

Figure 3. Reduced ZBP1 and β-actin mrna localization in dendrites after antisense knock-down of rzbp1 β-actin mrna ZBP1 control (reverse) antisense Eom, T. et al. J. Neurosci. 2003;23:10433-10444 Copyright 2003 Society for Neuroscience

Figure 6. BDNF-induced increase of dendritic protrusions but not spines is occluded by zipcode-containing constructs Eom, T. et al. J. Neurosci. 2003;23:10433-10444 Copyright 2003 Society for Neuroscience

Figure 7. Overexpression of EGFP-β-actin increases the density of filopodial synapses in a zipcode-dependent manner Eom, T. et al. J. Neurosci. 2003;23:10433-10444 Copyright 2003 Society for Neuroscience

Figure 8. Overexpression of EGFP constructs with the β-actin zipcode resultes in recruitment of endogenous β-actin mrna and ZBP1 Eom, T. et al. J. Neurosci. 2003;23:10433-10444 Copyright 2003 Society for Neuroscience

3xFLAG-IMP1 exhibits a behavior similar to endogenous IMP1 UV crosslink to H19 RNA Jonson, L. (2007) Mol. Cell. Proteomics 6: 798-811 IMP (insulin-like growth factor II mrna-binding protein): zip code-binding protein (ZBP) Copyright 2007 American Society for Biochemistry and Molecular Biology

IMP1 granules in relation to other RNP granules IMP co-ip with HuB IMP granules distinct from P-bodies and stress granules Distinct IMP granules & FMRP granules Jonson, L. (2007) Mol. Cell. Proteomics 6: 798-811 Copyright 2007 American Society for Biochemistry and Molecular Biology

Atomic force microscopy of IMP1 granules Control (FLAG-ERG1) IMP1 granule IMP1 monomer IMP1 dimer bound to igf2 3 -UTR Jonson, L. (2007) Mol. Cell. Proteomics 6: 798-811 Copyright 2007 American Society for Biochemistry and Molecular Biology

The protein composition of IMP1 granules Presence of 40S but not 60S ribosome, PABP & exon junction complex: untranslated mrna in granules Jonson, L. (2007) Mol. Cell. Proteomics 6: 798-811 Copyright 2007 American Society for Biochemistry and Molecular Biology

IMP1 granules are enriched in transcripts encoding proteins involved in metabolism and protein secretion Jonson, L. (2007) Mol. Cell. Proteomics 6: 798-811 Copyright 2007 American Society for Biochemistry and Molecular Biology

Schematic representation of the ubiquitin-proteasome pathway and the secretory pathway with the quality control machinery that dislocate misfolded proteins from the ER (transcripts found in IMP1 granules) Jonson, L. (2007) Mol. Cell. Proteomics 6: 798-811 Copyright 2007 American Society for Biochemistry and Molecular Biology