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. 2001 Sep 3;20(17):4803-13.
doi: 10.1093/emboj/20.17.4803.

The fragile X mental retardation protein binds specifically to its mRNA via a purine quartet motif

C Schaeffer  1 , B Bardoni, J L Mandel, B Ehresmann, C Ehresmann, H Moine
Affiliations

The fragile X mental retardation protein binds specifically to its mRNA via a purine quartet motif

C Schaeffer et al. EMBO J. .

Abstract

Fragile X syndrome is caused by the absence of protein FMRP, the function of which is still poorly understood. Previous studies have suggested that FMRP may be involved in various aspects of mRNA metabolism, including transport, stability and/or translatability. FMRP was shown to interact with a subset of brain mRNAs as well as with its own mRNA; however, no specific RNA-binding site could be identified precisely. Here, we report the identification and characterization of a specific and high affinity binding site for FMRP in the RGG-coding region of its own mRNA. This site contains a purine quartet motif that is essential for FMRP binding and can be substituted by a heterologous quartet-forming motif. The specific binding of FMRP to its target site was confirmed further in a reticulocyte lysate through its ability to repress translation of a reporter gene harboring the RNA target site in the 5'-untranslated region. Our data address interesting questions concerning the role of FMRP in the post-transcriptional control of its own gene and possibly other target genes.

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Figures

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Fig. 1. Competition experiments to determine the relative affinity of GST–FMRP for various RNA sequences. (A) RNA sequences from FMR1 mRNA or from various origins; their lengths are indicated. (B) Subdomains of FMR1 mRNA with an indication of the corresponding exons and domains in FMRP (upper line). Nucleotide positions are given with respect to the coding sequence start (+1). (C) Fraction of bound Iso-1 RNA (32P labeled), retained on immobilized GST–FMRP, plotted against competitor RNA concentration. Each point reflects at least four independent experiments for each RNA species. For clarity, RNA constructs displaying similar competition strengths and falling within the same standard deviation have been grouped and are represented by four types of symbols as indicated in (A) and (B).
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Fig. 2. Binding specificity of GST–FMRP for N19 RNA. (A) Labeled N19 RNA was incubated in the absence (lane 1) or presence of increasing amounts of recombinant GST–FMRP (lanes 2–6: 0.01, 0.05, 0.1, 0.2 and 0.5 pmol, respectively). A control is shown with GST alone (lane 7, 20 pmol). (B) Inhibition of the formation of an N19 RNA–GST–FMRP complex by monoclonal antibody 1C3. Lane 1 is without FMRP, lanes 2–8 are in the presence of 0.1 pmol FMRP, and antibodies were added at a concentration of 10, 50 and 150 µg/ml (lanes 3, 4 and 5 for anti-FMRP, and lanes 6, 7 and 8 for anti-mouse IgG). (C) Northwestern with GST–FMRP and N19 RNA. Lane 1, Coomassie staining of the GST–FMRP on a 10% SDS–polyacrylamide gel; lane 2, corresponding autoradiograph of the membrane hybridized with labeled N19 RNA. (D) Competition experiment to determine the relative binding strength of the various subfragments of FMR1 mRNA (Figure 1B) by gel retardation experiments. 32P-labeled N19* RNA was incubated with GST–FMRP (0.1 pmol) in the presence of increasing concentrations of unlabeled competitor. Lanes C, control without protein; lanes 0, control without competitor RNA; numbers are the log of competitor concentrations. The graph depicts the fraction of bound labeled N19 RNA plotted against unlabeled competitor RNA concentrations. Each point is the mean with standard deviation of at least three independent experiments. As in Figure 1C, the different RNA constructs are sorted into distinct classes represented by different symbols: N19 (filled circles), N5, N7, N18 and 3′-UTR (open squares), N6 and N8 (open circles), tRNA (open triangles).
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Fig. 3. Determination of the boundaries of the FBS. (A) Determination of the 5′ and 3′ boundaries on N19 RNA (left) with a control performed on N8 RNA (right). 5′ and 3′ = RNA 32P-labeled at its 5′ or 3′ end, respectively. Lanes 1 are RNAs statistically digested with alkali. Lanes 2 are RNAs statistically digested with alkali that have been retained on immobilized GST–FMRP. Lanes 3, same as lanes 2 but in the presence of 10–8 M competitor N19 RNA. Lane T1, RNA submitted to statistical RNase T1 digestion. 5′ and 3′ boundaries are indicated by arrows. (B) Densitometer trace of lanes 1 (thick line), 2 (gray line) and 3 (thin line); the ordinate is in arbitrary units, the abscissa gives nucleotide positions. (C) Effect of deletions or domain exchange in the FBS. 32P-labeled N19* RNA was incubated with GST–FMRP (0.1 pmol) in the presence of increasing concentrations of unlabeled RNA: N19-Δ35 (open circles), FBS (filled circles), N19–IGF (open triangles). The fraction of bound labeled N19* RNA, as visualized by gel shift assays (left), was plotted against competitor RNA concentrations (right). Each point is the mean with standard deviation of at least three independent experiments.
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Fig. 4. Indication of the presence of a quadruplex structure in the FBS. (A) Cation-dependent termination of reverse transcription at the purine-rich domain in the N19 subfragment (N19) and in the full-length Iso-1 RNA with its 3′-UTR (Iso1 + 3′ UTR). Strong pauses of reverse transcriptase are indicated by arrowheads with their position within the FMR1 mRNA sequence. Full-length extension products are indicated by open triangles. (B) Hydrogen bonding scheme for the G quartet alongside its schematic model. The bases are hydrogen bonded via Hoogsteen base pairs in a square-planar symmetric array. N7 positions are indicated by stars. The quadruplex is stabilized by coordination of a monovalent ion lying between or within the planes of the guanine tetrads (Laughlan et al., 1994). (C) Chemical probing of N7 positions of guanines of 3′-end labeled FBS RNA with DMS. RNA was incubated in buffer containing 150 mM KCl (K) or NaCl (Na) prior to treatment. Lane T1, statistical RNase T1 digestion; lanes C, controls without DMS; lanes 1, RNAs treated with DMS. The black line indicates an area of protected guanines in the presence of KCl; the asterisk indicates a hyper-reactivity. The densitometer traces of lanes 1 are shown alongside the autoradiograph (thick line is Na, thin line is K); the ordinate is in arbitrary units; the abscissa coincides with nucleotide positions on the autoradiograph. (D) Termination of reverse transcription at the beginning of the IGF II motif inserted in the chimeric N19–IGF RNA. The sequence of the inserted motif is indicated, with tracts of Gs forming the quadruplex in bold. The full-length product is indicated as in (A).
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Fig. 5. Cation-dependent binding of FMRP on its RNA-binding site. Labeled N19 RNA was incubated in the absence (lane 0) or presence of increasing amounts of GST–FMRP in binding buffer containing 150 mM KCl (filled squares), NaCl (open diamonds) or LiCl (open triangles). The percentage of bound RNA (as determined by phosphoimager quantification) is plotted against the amount of GST–FMRP (error bars are reflect at least three independent experiments).
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Fig. 6. GST–FMRP inhibits translation of a chimeric FBS-luc transcript in vitro. Translation reaction mixture (rabbit reticulocyte lysate) was incubated with luc, FBSΔ35-luc or FBS-luc mRNAs and increasing amounts of GST–FMRP. (A) Graph depicting the dose–response relationship between the amount of GST–FMRP and the luciferase activity (error bars reflect six independent experiments). Control with GST alone is shown. (B) Stability of the mRNA after translation in the presence or absence of GST–FMRP (0.5 pmol). Labeled mRNA substrates were extracted from the reticulocyte lysate mixture after translation and analyzed by denaturing gel electrophoresis.
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Fig. 7. Sequence alignment of the FBS purine-rich region from vertebrate FMR1 genes. The two arrows indicate the strong K+-dependent pauses of reverse transcriptase shown in Figure 4A. Protected N7 positions in the presence of K+ are indicated by dots (strong protection is represented by filled circles; moderate protection by open circles), enhanced reactivity towards DMS is indicated by a + sign. Conserved Gs in homologous FMR1 genes are depicted in bold. The sequence of RNA N19–IGF containing the insertion of the purine quartet region of IGF II mRNA (in lower case) is given. The four G tracts involved in G quartet formation are boxed.
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References

    1. Ashley C.T. Jr, Wilkinson,K.D., Reines,D. and Warren,S.T. (1993a) FMR1 protein: conserved RNP family domains and selective RNA binding. Science, 262, 563–566. - PubMed
    1. Ashley C.T., Sutcliffe,J.S., Kunst,C.B., Leiner,H.A., Eichler,E.E., Nelson,D.L. and Warren,S.T. (1993b) Human and murine FMR-1: alternative splicing and translational initiation downstream of the CGG-repeat. Nature Genet., 4, 244–251. - PubMed
    1. Bardoni B., Schenck,A. and Mandel,J.L. (1999) A novel RNA-binding nuclear protein that interacts with the fragile X mental retardation (FMR1) protein. Hum. Mol. Genet., 8, 2557–2566. - PubMed
    1. Bashkirov V.I., Scherthan,H., Solinger,J.A., Buerstedde,J.M. and Heyer,W.D. (1997) A mouse cytoplasmic exoribonuclease (mXRN1p) with preference for G4 tetraplex substrates. J. Cell Biol., 136, 761–773. - PMC - PubMed
    1. Brown V., Small,K., Lakkis,L., Feng,Y., Gunter,C., Wilkinson,K.D. and Warren,S.T. (1998) Purified recombinant Fmrp exhibits selective RNA binding as an intrinsic property of the fragile X mental retardation protein. J. Biol. Chem., 273, 15521–15527. - PubMed

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