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• W2078045122 abstract "Fragile X syndrome is a common inherited cause of mental retardation that results from loss or mutation of the fragile X mental retardation protein (FMRP). In this study, we identified the mRNA of the basic helix-loop-helix transcription factor human achaete-scute homologue-1 (hASH1 or ASCL1), which is required for normal development of the nervous system and has been implicated in the formation of neuroendocrine tumors, as a new FMRP target. Using a double-immunofluorescent staining technique we detected an overlapping pattern of both proteins in the hippocampus, temporal cortex, subventricular zone, and cerebellum of newborn rats. Forced expression of FMRP and gene silencing by small interference RNA transfection revealed a positive correlation between the cellular protein levels of FMRP and hASH1. A luciferase reporter construct containing the 5′-untranslated region of hASH1 mRNA was activated by the full-length FMRP, but not by naturally occurring truncated FMR proteins, in transient co-transfections. The responsible cis-element was mapped by UV-cross-linking experiments and reporter mutagenesis assays to a (U)10 sequence located in the 5′-untranslated region of the hASH1 mRNA. Sucrose density gradient centrifugation revealed that hASH1 transcripts were translocated into a translationally active polysomal fraction upon transient transfection of HEK293 cells with FMRP, thus indicating translational activation of hASH1 mRNA. In conclusion, we identified hASH1 as a novel downstream target of FMRP. Improved translation efficiency of hASH1 mRNA by FMRP may represent an important regulatory switch in neuronal differentiation. Fragile X syndrome is a common inherited cause of mental retardation that results from loss or mutation of the fragile X mental retardation protein (FMRP). In this study, we identified the mRNA of the basic helix-loop-helix transcription factor human achaete-scute homologue-1 (hASH1 or ASCL1), which is required for normal development of the nervous system and has been implicated in the formation of neuroendocrine tumors, as a new FMRP target. Using a double-immunofluorescent staining technique we detected an overlapping pattern of both proteins in the hippocampus, temporal cortex, subventricular zone, and cerebellum of newborn rats. Forced expression of FMRP and gene silencing by small interference RNA transfection revealed a positive correlation between the cellular protein levels of FMRP and hASH1. A luciferase reporter construct containing the 5′-untranslated region of hASH1 mRNA was activated by the full-length FMRP, but not by naturally occurring truncated FMR proteins, in transient co-transfections. The responsible cis-element was mapped by UV-cross-linking experiments and reporter mutagenesis assays to a (U)10 sequence located in the 5′-untranslated region of the hASH1 mRNA. Sucrose density gradient centrifugation revealed that hASH1 transcripts were translocated into a translationally active polysomal fraction upon transient transfection of HEK293 cells with FMRP, thus indicating translational activation of hASH1 mRNA. In conclusion, we identified hASH1 as a novel downstream target of FMRP. Improved translation efficiency of hASH1 mRNA by FMRP may represent an important regulatory switch in neuronal differentiation. The FMR1 gene encodes the fragile X mental retardation protein (FMRP), 6The abbreviations used are: FMRP, fragile X mental retardation protein; FMR1, fragile X mental retardation-1; CMV, cytomegalovirus; UTR, untranslated region; P2, postnatal day 2; HES1, Hairy and Enhancer of Split1; siRNA, small interference RNA. 6The abbreviations used are: FMRP, fragile X mental retardation protein; FMR1, fragile X mental retardation-1; CMV, cytomegalovirus; UTR, untranslated region; P2, postnatal day 2; HES1, Hairy and Enhancer of Split1; siRNA, small interference RNA. an RNA-binding protein, which is expressed among various tissues with the highest levels in neurons of the developing brain and in spermatogonia in adult testis (1Devys D. Lutz Y. Rouyer N. Bellocq J.P. Mandel J.L. Nat. Genet. 1993; 4: 335-340Crossref PubMed Scopus (633) Google Scholar, 2Bakker C.E. de Diego Otero Y. Bontekoe C. Raghoe P. Luteijn T. Hoogeveen A.T. Oostra B.A. Willemsen R. Exp. Cell Res. 2000; 258: 162-170Crossref PubMed Scopus (147) Google Scholar). Dysfunction of the fragile X mental retardation-1 (FMR1) gene transcription is associated with neuronal disorders, such as fragile X syndrome and fragile X-associated tremor/ataxia syndrome (3Oberle I. Rousseau F. Heitz D. Kretz C. Devys D. Hanauer A. Boue J. Bertheas M.F. Mandel J.L. Science. 1991; 252: 1097-1102Crossref PubMed Scopus (1309) Google Scholar, 4Kremer E.J. Pritchard M. Lynch M. Yu S. Holman K. Baker E. Warren S.T. Schlessinger D. Sutherland G.R. Richards R.I. Science. 1991; 252: 1711-1714Crossref PubMed Scopus (791) Google Scholar, 5Verkerk A.J. Pieretti M. Sutcliffe J.S. Fu Y.H. Kuhl D.P. Pizzuti A. Reiner O. Richards S. Victoria M.F. Zhang F.P. Cell. 1991; 65: 905-914Abstract Full Text PDF PubMed Scopus (2912) Google Scholar, 6Willemsen R. Hoogeveen-Westerveld M. Reis S. Holstege J. Severijnen L.A. Nieuwenhuizen I.M. Schrier M. van Unen L. Tassone F. Hoogeveen A.T. Hagerman P.J. Mientjes E.J. Oostra B.A. Hum. Mol. Genet. 2003; 12: 949-959Crossref PubMed Scopus (238) Google Scholar). FMRP binds to cis-regulatory mRNA elements that include G-quartet structures, poly-U sequences, and the so-called kissing complex (7Penagarikano O. Mulle J.G. Warren S.T. Annu. Rev. Genomics Hum. Genet. 2007; 8: 109-129Crossref PubMed Scopus (313) Google Scholar). Beside direct FMRP/RNA binding, FMRP is part of messenger ribonucleoprotein complexes by interaction with further RNA-binding proteins, i.e. the poly-A-binding protein, nucleolin, and others (8Bagni C. Greenough W.T. Nat. Rev. Neurosci. 2005; 6: 376-387Crossref PubMed Scopus (392) Google Scholar). At least 12 different FMR proteins are generated by alternative mRNA splicing (1Devys D. Lutz Y. Rouyer N. Bellocq J.P. Mandel J.L. Nat. Genet. 1993; 4: 335-340Crossref PubMed Scopus (633) Google Scholar, 9Verkerk A.J. de Graaff E. De Boulle K. Eichler E.E. Konecki D.S. Reyniers E. Manca A. Poustka A. Willems P.J. Nelson D.L. Hum. Mol. Genet. 1993; 2: 399-404Crossref PubMed Scopus (86) Google Scholar) and may contribute to the functional diversity of the FMR1 gene. FMRP has been suggested to play a role in synaptic development and plasticity through regulating mRNA transport and local protein synthesis at synapses (10Jin P. Warren S.T. Trends Biochem. Sci. 2003; 28: 152-158Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 11Jin P. Alisch R.S. Warren S.T. Nat. Cell Biol. 2004; 6: 1048-1053Crossref PubMed Scopus (275) Google Scholar, 12Bittel D.C. Kibiryeva N. Butler M.G. Genet. Med. 2007; 9: 464-472Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Accordingly, FMRP was found to gate the translation of a large set of mRNAs in dendrites that are involved in synaptic plasticity. Some of these transcripts are transported in mRNA granules together with FMRP. Activation of metabotropic glutamate receptors has been shown to stimulate synaptic translation of FMRP (13Weiler I.J. Irwin S.A. Klintsova A.Y. Spencer C.M. Brazelton A.D. Miyashiro K. Comery T.A. Patel B. Eberwine J. Greenough W.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5395-5400Crossref PubMed Scopus (544) Google Scholar, 14Hou L. Antion M.D. Hu D. Spencer C.M. Paylor R. Klann E. Neuron. 2006; 51: 441-454Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar). Since protein synthesis by activation of GluR was enhanced in the absence of FMRP (15Huber K.M. Gallagher S.M. Warren S.T. Bear M.F. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7746-7750Crossref PubMed Scopus (1078) Google Scholar), a model of mGluR-stimulated inhibition of local mRNA translation by FMRP has been developed (16Bear M.F. Huber K.M. Warren S.T. Trends Neurosci. 2004; 27: 370-377Abstract Full Text Full Text PDF PubMed Scopus (1274) Google Scholar). Reduced synaptic protein formation may explain at least some of the mental deficits in patients suffering from fragile X syndrome due to FMRP deficiency. FMRP may function not only as a translational repressor, but also as an activator of specific mRNA translation (8Bagni C. Greenough W.T. Nat. Rev. Neurosci. 2005; 6: 376-387Crossref PubMed Scopus (392) Google Scholar). Depending on its phosphorylation status (17Ceman S. O'Donnell W.T. Reed M. Patton S. Pohl J. Warren S.T. Hum. Mol. Genet. 2003; 12: 3295-3305Crossref PubMed Scopus (263) Google Scholar), FMRP co-localizes with actively translating polysomes (18Corbin F. Bouillon M. Fortin A. Morin S. Rousseau F. Khandjian E.W. Hum. Mol. Genet. 1997; 6: 1465-1472Crossref PubMed Scopus (206) Google Scholar, 19Feng Y. Absher D. Eberhart D.E. Brown V. Malter H.E. Warren S.T. Mol. Cell. 1997; 1: 109-118Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, 20Khandjian E.W. Huot M.E. Tremblay S. Davidovic L. Mazroui R. Bardoni B. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13357-13362Crossref PubMed Scopus (141) Google Scholar). In addition, FMRP shuttles between the nucleus and the cytoplasm (21Eberhart D.E. Malter H.E. Feng Y. Warren S.T. Hum. Mol. Genet. 1996; 5: 1083-1091Crossref PubMed Scopus (330) Google Scholar, 22Feng Y. Gutekunst C.A. Eberhart D.E. Yi H. Warren S.T. Hersch S.M. J. Neurosci. 1997; 17: 1539-1547Crossref PubMed Google Scholar, 23Menon R.P. Gibson T.J. Pastore A. J. Mol. Biol. 2004; 343: 43-53Crossref PubMed Scopus (85) Google Scholar). Thus, it has been proposed that FMRP might contribute to chromatin remodeling through the RNA interference pathway in the nucleus (8Bagni C. Greenough W.T. Nat. Rev. Neurosci. 2005; 6: 376-387Crossref PubMed Scopus (392) Google Scholar). Furthermore, FMRP has been implicated in mRNA splicing (24Willemsen R. Oostra B.A. Bassell G.J. Dictenberg J. Ment. Retard. Dev. Disabil. Res. Rev. 2004; 10: 60-67Crossref PubMed Scopus (94) Google Scholar), mRNA stabilization (25Zalfa F. Eleuteri B. Dickson K.S. Mercaldo V. De Rubeis S. di Penta A. Tabolacci E. Chiurazzi P. Neri G. Grant S.G. Bagni C. Nat. Neurosci. 2007; 10: 578-587Crossref PubMed Scopus (289) Google Scholar), and as a component of RNA granules like stress granules (26Mazroui R. Huot M.E. Tremblay S. Filion C. Labelle Y. Khandjian E.W. Hum. Mol. Genet. 2002; 11: 3007-3017Crossref PubMed Scopus (278) Google Scholar). Identification and validation of downstream targets may provide the key to understanding the role of FMRP in neuronal development and disease. Previous attempts to discover FMRP-regulated genes were based mostly on immunoprecipitation of FMRP in mouse tissue followed by extraction and micro-array analysis of co-precipitated RNA (8Bagni C. Greenough W.T. Nat. Rev. Neurosci. 2005; 6: 376-387Crossref PubMed Scopus (392) Google Scholar, 12Bittel D.C. Kibiryeva N. Butler M.G. Genet. Med. 2007; 9: 464-472Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 27Darnell J.C. Mostovetsky O. Darnell R.B. Genes Brain Behav. 2005; 4: 341-349Crossref PubMed Scopus (94) Google Scholar). Alternative approaches consisted in the use of random oligonucleotides linked to anti-FMRP antibody to reveal putative FMRP targets (28Miyashiro K.Y. Beckel-Mitchener A. Purk T.P. Becker K.G. Barret T. Liu L. Carbonetto S. Weiler I.J. Greenough W.T. Eberwine J. Neuron. 2003; 37: 417-431Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar) and in differential display analysis (29Zhong N. Ju W. Nelson D. Dobkin C. Brown W.T. Am. J. Med. Genet. 1999; 84: 268-271Crossref PubMed Google Scholar, 30Sung Y.J. Conti J. Currie J.R. Brown W.T. Denman R.B. Biochem. Biophys. Res. Commun. 2000; 275: 973-980Crossref PubMed Scopus (76) Google Scholar). While these investigations yielded important information on the RNA-binding properties of FMRP, little overlap among the proposed candidate targets was found (8Bagni C. Greenough W.T. Nat. Rev. Neurosci. 2005; 6: 376-387Crossref PubMed Scopus (392) Google Scholar). Furthermore, the precise molecular mechanisms of FMRP function remain elusive (31Zalfa F. Achsel T. Bagni C. Curr. Opin. Neurobiol. 2006; 16: 265-269Crossref PubMed Scopus (115) Google Scholar), and the question of how abnormal regulation of a single gene can result in such a diversity of neuronal dysfunctions remains to be answered (32Koukoui S.D. Chaudhuri A. Brain Res. Rev. 2007; 53: 27-38Crossref PubMed Scopus (52) Google Scholar). Thus, the goal of this study was to identify potential new FMRP targets, which play a pivotal role in gene regulation during neuronal development. Starting with an unbiased combined approach of gene ontology and motif search followed by extensive experimental analyses we characterize the basic helix-loop-helix transcription factor, human achaete-scute homologue-1 (hASH1 or ASCL1), as a new FMRP target and describe its molecular mechanism of regulation by FMRP in detail. Because hASH1 is crucial in generating neuronal diversity by regulating neuronal subtype specification and differentiation (33Bertrand N. Castro D.S. Guillemot F. Nat. Rev. Neurosci. 2002; 3: 517-530Crossref PubMed Scopus (1153) Google Scholar), our findings offer a new view of how FMRP influences neuronal development. Cell Culture—Human embryonic kidney (HEK)293 cells (ACC 305) were obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). The cells were maintained in Dulbecco's modified Eagle's medium (high glucose, PAA Laboratories GmbH, Cölbe, Germany), supplemented with 10% fetal calf serum (Biochrom KG, Berlin, Germany), 50 units/ml penicillin, 50 μg/ml streptomycin, 15 mm Hepes, and 2 mm glutamine, at 37 °C, 5% CO2. For primary hippocampal neurons animals were sacrificed according to the permit (LaGeSo, 0122/07) given by the Office for Health Protection and Technical Safety of the regional government of Berlin and in compliance with regulations laid down in the European Community Council Directive. Hippocampal cultures from E19 Wistar rats were prepared as previously described (34Eichler S.A. Kirischuk S. Juttner R. Legendre P. Lehmann T.N. Gloveli T. Grantyn R. Meier J.C. J. Cell Mol. Med. 2008; 12: 2848-2866Crossref PubMed Scopus (90) Google Scholar) and maintained for 8 days in vitro in B27- and 1% fetal calf serum-supplemented neurobasal medium (35Brewer G.J. Cotman C.W. Brain Res. 1989; 494: 65-74Crossref PubMed Scopus (326) Google Scholar). Plasmids—For expression of the full-length FMRP, a construct described earlier (36Darnell J.C. Jensen K.B. Jin P. Brown V. Warren S.T. Darnell R.B. Cell. 2001; 107: 489-499Abstract Full Text Full Text PDF PubMed Scopus (785) Google Scholar) (kind gift of J. Darnell) served as a template for re-cloning of the FMR1 cDNA into the CMV promoter based pEGFP-C1 vector (BD Biosciences, Clontech, Heidelberg, Germany). FMRP-variant expression vectors (also under control of a CMV-promoter) were purchased by Deutsches Ressourcenzentrum für Genomforschung GmbH (Berlin, Germany). For the generation of useful luciferase reporter constructs, the pGL3-promotor vector, which contains a constitutive SV40 promoter (Promega, Madison, WI), was modified as follows: The vector-specific 5′- and 3′UTRs of luciferase mRNA were replaced by the human hASH1 mRNA UTRs. The UTRs were amplified by PCR using a hASH1 full-length cDNA clone (Deutsches Ressourcenzentrum für Genomforschung GmbH) as template, and restriction sites were added by primer extension. The following cis-elements were deleted in the hASH1 5′UTR: U-rich element (positions 223–232), G-quartet motif (positions 304–330), and AU-rich element (positions 397–416) according to the ASCL1 sequence accession number gi:190343011 (NM_004316). The quality of processed vectors was confirmed by sequencing. Cell Transfection Experiments and Reporter Gene Assays—Cultured cells were grown to ∼70% confluence in 96-well plates (μClear Platte 96K, Greiner BIO-ONE GmbH, Frickenhausen, Germany) and transiently co-transfected with a reporter construct containing the firefly luciferase gene flanked by the 5′- and/or 3′UTRs of hASH-1 mRNA (pGL3-promoter vector; Promega), and the Renilla luciferase phRL-TK vector (Promega). A ratio (DNA:transfection reagent) of 1:3 was used with the FuGENE 6 Transfection Reagent (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's protocol. Transfection of HEK293 cells with empty pGL3-promoter vector served as control. Co-transfection with the Renilla luciferase reporter plasmid was performed for normalization of transfection efficiencies. The luciferase activities were measured in a luminometer (Labsystems Luminoscan RS, Helsinki, Finland) programmed with individual software (Luminoscan RII, Ralf Mrowka) 24 h after transfection as described in Ref. 37Mrowka R. Steege A. Kaps C. Herzel H. Thiele B.J. Persson P.B. Bluthgen N. Nucleic Acids Res. 2007; 35: 5120-5129Crossref PubMed Scopus (12) Google Scholar. Luciferase mRNA quantification was performed as described recently (38Ufer C. Wang C.C. Fähling M. Schiebel H. Thiele B.J. Billett E.E. Kuhn H. Borchert A. Genes Dev. 2008; 22: 1838-1850Crossref PubMed Scopus (80) Google Scholar). For forced expression of FMR proteins, cells were transfected in 6-well or 60-mm dishes for 36 h using the FuGENE 6 transfection reagent as described above. For transfection of siRNAs human neuroblastoma-derived Kelly cells (ACC 355) were obtained from the American Type Culture Collection and grown to ∼50% confluence in 60-mm dishes in RPMI medium (PAA Laboratories) supplemented with 10% fetal calf serum (Biochrom KG, Berlin, Germany), 100 units/ml penicillin, 100 μg/ml streptomycin, and 1% glutamate (all from Invitrogen). The siRNA for targeting the human FMR1 gene (NCBI accession number NM_002024) was synthesized by Dharmacon (Lafayette, CO). A pool of non-targeting siRNAs (Dharmacon, 200 pmol per dish) was used as a negative control. To achieve maximum efficiency of gene silencing the cells were transfected with a mixture of four different siRNAs, each at 50 pmol per dish. For this purpose the siRNAs were diluted at a 1:20 volume ratio in 0.2 ml of serum- and antibiotic-free RPMI. Likewise, the DharmaFECT® transfection reagent (Perbio Science, Bonn, Germany) was diluted (1:100) in 0.2 ml of serum- and antibiotic-free RPMI. The diluted siRNAs and the transfection reagent were combined and kept at room temperature for 20 min to allow complex formation. In the meantime the culture medium was removed from the cells and replaced with 1.6 ml of fresh RPMI/10% fetal calf serum. The siRNA transfection mixtures (0.4 ml) were carefully added to the cells, which were subsequently grown in the tissue culture incubator for 60 h. Determination of mRNA and Protein Levels—Total RNA was isolated from cultured cells with the TRIzol® reagent (Invitrogen). First-strand cDNA synthesis was performed with 2 μgof total RNA using oligo(dT) primers and superscript II reverse transcriptase (Invitrogen). mRNA quantification was performed as described previously (39Fähling M. Mrowka R. Steege A. Martinka P. Persson P.B. Thiele B.J. J. Biol. Chem. 2006; 281: 9279-9286Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The following intron bridging primers were used for the amplification reactions: hASH1-forward: 5′-CGACTTCACCAACTGGTTCT, hASH1-reverse: 5′-CCGTGAATGATTGGAGTGC, β-actin-forward: 5′-TGAAGTGGTACGTGGACATC, and β-actin-reverse: 5′-GTCATAGTCCGCCTAGAAGC. Cytosolic protein extracts were prepared from cultured cells as described previously (40Fähling M. Mrowka R. Steege A. Nebrich G. Perlewitz A. Persson P.B. Thiele B.J. J. Biol. Chem. 2006; 281: 26089-26101Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) and separated on a 10% polyacrylamide gel. The proteins were transferred onto polyvinylidene difluoride membranes (Amersham Biosciences) with the use of a semidry blotting apparatus (Bio-Rad). Immunodetection of hASH1 and FMRP was performed according to our routine protocol with the following primary antibodies: anti-Mash1 antibody diluted 1:500 (#556604, BD Biosciences), anti-FMR1, diluted 1:1000 (H-120, #sc28739, Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal antibody against β-actin in a 1:1000 dilution (#MAB1501R, Chemicon, Schwalbach/Ts, Germany). The anti-β-actin antibody was applied after stripping the membranes with 0.2 m NaOH at room temperature for 6 min to reveal possible differences in protein loading. Primary antibodies were detected with peroxidase-coupled secondary antibodies, and the reaction products were visualized with the enhanced chemiluminescence system (Amersham Biosciences). Antibodies, Immunofluorescence, and Quantification—Mash1 and FMRP were stained using mouse monoclonal (1:500, clone 24B72D11.1, #556604, BD Biosciences) and rabbit polyclonal (1:500, H-120, #sc28739, Santa Cruz Biotechnology) antibodies, respectively. Immunofluorescence staining was performed on primary hippocampal neurons and on horizontal sections of postnatal day 2 (P2) rat brain fixed with ice-cold mixture of paraformaldehyde and sucrose (4% both) in phosphate-buffered saline buffer. Primary neurons and 2- to 3-mm thick freshly isolated horizontal rat brain sections were fixed for 15 min at room temperature and 1 h at 4 °C, respectively. After fixation slices were washed three times with phosphate-buffered saline at room temperature and cryoprotected overnight at 4 °C in phosphate-buffered saline supplemented with 8% sucrose. Brain sections were embedded in O.C.T. TissueTek (Sakura Finetec), and 12-μm cryosections were obtained (CM1850, Leica Microsystems, Wetzlar, Germany) and mounted on Superfrost Plus microscope slides (Menzel GmbH, Braunschweig, Germany). Sections were post-fixed for 5 min with ice-cold paraformaldehyde/sucrose. Prior to incubation with antibodies (1 h at room temperature) primary neurons were permeabilized with 0.12% Triton X-100 for 4 min at room temperature. Antibody reaction with cryosections occurred overnight at 4 °C in phosphate-buffered saline-gelatin supplemented with 0.12% Triton X-100. Appropriate control experiments were performed using species-matched normal sera instead of specific primary antibodies. Secondary antibodies (1:200 each) were coupled to carboxymethyl indocyanine or fluorescein isothiocyanate (Jackson ImmunoResearch Laboratories, West Grove, PA). Preparations were mounted in 4′,6-diamidino-2-phenylindole-containing Vectashield medium (Vector Laboratories, Burlingame, CA). The slides were viewed under an epifluorescence microscope (Olympus BX51, Olympus Deutschland GmbH, Hamburg, Germany). Images were acquired with a 14-bit cooled charge-coupled device camera (Spot PURSUIT, Visitron Systems GmbH, Puchheim, Germany) and the software Metamorph (Universal Imaging Corp., Downingtown, PA). Cell-matched signals were quantified within circular, nucleus-centered, regions of interest (Mash and FMRP: diameter 50 and 150 pixels, respectively). Integrated fluorescence intensities were obtained from min/max-thresholded images using Metamorph. Correlation analysis was performed using the Spearman rank order algorithm. RNA-Protein Interaction Studies: UV Cross-linking—In vitro transcripts representing the 5′- or 3′UTR of hASH1 mRNA were radioactively labeled using [α-32P]uridine-, [α-32P]cytosine-, [α-32P]adenine-, or [α-32P]guanosine-5′-triphosphate (800 Ci/mmol, MP Biomedicals GmbH, Heidelberg, Germany). In vitro transcripts were purified by BD Chroma Spin™-100 (DEPC) columns (BD Bioscience). 1–2 ng of the [α-32P]NTP labeled in vitro transcripts (corresponding to 100,000 cpm) was incubated with 35 μg of cytosolic protein extract for 30 min at room temperature in 10 mm Hepes, pH 7.2, 3 mm MgCl2, 5% glycerol, 1 mm dithiothreitol, 150 mm KCl, and 2 units/μl RNaseOUT (Invitrogen Life Technologies) in the presence of rabbit rRNA (0.5 μg/μl) as competitor. The samples were exposed to UV light (255 nm, 1.6 Joule, UV-Stratalinker) on ice, then treated with RNase-A (30 μg/ml final concentration) and RNase-T1 (750 units/ml final concentration) for 15 min at 37 °C and subjected to 12% SDS-PAGE and subsequent autoradiography using the Phospho-Imager-System (Fujifilm FLA-3000). For cis-element analyses by label transfer, transcripts were separately labeled using the four nucleotides U-, C-, A- or GTP. The radioactive counts of all nucleotides were adjusted to comparable levels before in vitro transcription. Equal RNA concentrations of the resulting in vitro transcripts were used for the UV-cross-linking assay. Electrophoretic Mobility Shift Assay/Supershift Assay—Cytosolic extract (35 μg of protein) of HEK293 cells transiently transfected with either FMRP expression vector or empty vector control (mock) were incubated with hASH1 mRNA 5′UTR in vitro transcripts at 10,000 cpm. The binding reaction was carried out in 10 mm Hepes, pH 7.2, 3 mm MgCl2, 5% glycerol, 1 mm dithiothreitol, 150 mm KCl, 2 units/μl RNaseOUT (Invitrogen Life Technologies), 0.5 μg/μl rabbit rRNA for 30 min at room temperature. The formed RNA-protein complexes were incubated overnight at 4 °C with anti-FMRP antibody or anti-β-actin control antibody with gentle shaking. The complexes were separated by electrophoresis (0.5 × TBE buffer (50 mm Tris-borate, 2 mm EDTA)). Radioactive signals were detected using the Phospho-Imager-System (Fujifilm FLA-3000). Sucrose Gradient Centrifugation and Polysomal Profiles—Prior to lysis, cells were pre-treated with cycloheximide (100 μg/ml) for 10 min. Cells were lysed on ice for 10 min in a buffer containing 20 mm Tris (pH 7.4), 150 mm KCl, 30 mm MgCl2, 100 μg/ml cycloheximide, 1 mm dithiothreitol, 1× proteinase inhibitor mixture (Roche Diagnostics), 100 units/ml of RNase inhibitor (MBI Fermentas GmbH, St. Leon-Rot, Germany), and 0.5% Nonidet P-40. Cytosolic extracts were obtained after centrifugation at 10,000 × g for 10 min at 4 °C. The cytoplasmic supernatant was layered onto 11 ml of a linear 17–51% sucrose gradient (0.5–1.5 m sucrose, 20 mm Tris, pH 7.4, 150 mm KCl, 5 mm MgCl2, 1 mm dithiothreitol) and centrifuged for 2 h at 36.000 rpm using a Beckman SW-41 rotor. Following sedimentation, the gradient solution was pumped out from the bottom with a peristaltic pump. The ribosomal profile was continuously determined at an absorbance of 254 nm using a 2138 UVICORD-S UV monitor (LKB Bromma). Sucrose gradients were split into 12 subfractions each, starting with 1 (bottom) to 12 (top). For protein isolation trichloroacetic acid was supplemented to a 10% final concentration. Precipitated proteins were sedimented, washed twice with acetone, and solved in buffer (25 mm Tris, 1% SDS). RNA was isolated using the E.Z.N.A. RNA Total Kit (#OMEGR6834-02, VWR International, Darmstadt, Germany) according to the manufacturer's protocol. Selection of Potential FMRP Targets—We obtained UTR sequences from the 5′ and 3′ ends of the RNA from the ENSEMBL data base using the BIOMART tool. The underlying genomic data came from the human NCBI36 dataset representing 56,511 unique transcripts from 32,562 ENSEMBL genes. The dataset of the 3′UTRs and 5′UTRs consisted of 38,857 and 38,685 sequences, respectively. These numbers are smaller than the total number of transcripts, because some genes did not have a UTR prediction. These data were the basis for the motif search for poly-U stretches (U[8]) and G-quartet motifs according to the consensus [DWGG(N0–2)DWGG(N0–1)DWGG(N0–1)DWGG] (D = A/G/T, W = A/T, and N = A/T/C/G) (36Darnell J.C. Jensen K.B. Jin P. Brown V. Warren S.T. Darnell R.B. Cell. 2001; 107: 489-499Abstract Full Text Full Text PDF PubMed Scopus (785) Google Scholar) representing known FMRP binding sites. An additional filter step was the selection based on known molecular function. The prediction of molecular function was obtained with BIOMART from the ENSEMBLE data base. We used the Gene-Ontology classification identifiers for that purpose. The gene ontology is a rooted tree-like structured classification. The database entries in ENSEMBLE do contain the last tree children only, but not the complete ontology tree. Therefore we remapped the complete tree structure for each ontology entry. From all genes we selected those having a positive “GO:0007399: nervous system development” in their complete remapped ontology trees. Statistics—If not otherwise indicated, all values are presented as means ± S.D. Students' paired t test was performed to reveal statistical significances. p values <0.05 were considered significant. Selection of Potential FMRP Targets—To identify potential FMRP target mRNAs with a role in neuronal development we selected all genes belonging to the gene ontology class “nervous system development” and searched within their mRNA untranslated regions (UTRs) for poly-U stretches (U[≥8]) and G-quartet motifs according to the consensus [DWGG(N0–2)DWGG(N0–1)DWGG(N0–1)DWGG] (D = A/G/T, W = A/T, and N = A/T/C/G) (36Darnell J.C. Jensen K.B. Jin P. Brown V. Warren S.T. Darnell R.B. Cell. 2001; 107: 489-499Abstract Full Text Full Text PDF PubMed Scopus (785) Google Scholar). Both cis-elements are known FMRP binding sites. We retrieved 5545 unique genes that had a poly-U stretch or a G-quartet in their mRNA 5′-or 3′UTR. When all genes were filtered for the gene ontology “nervous system development” we obtained 336 genes. Within the dataset of all genes a total number of 682 transcription factors was identified. Appling all three criteria together, we ended with a list of 17 genes. The complete relationships between the sets are presented in the Venn diagram in Fig. 1. A summary of the genes matching all criteria is given in supplemental Table S1 together with detailed motif information. The hASH1 mRNA encoded by the ASCL1 gene was selected for detailed analysis of potential FMRP interaction, because the hASH1 mRNA UTRs ind" @default.
• W2078045122 created "2016-06-24" @default.
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• W2078045122 title "Translational Regulation of the Human Achaete-scute Homologue-1 by Fragile X Mental Retardation Protein" @default.
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