Fragile X mental retardation protein interactions with the microtubule associated protein 1B RNA

Abstract

Fragile X mental retardation syndrome, the most common form of inherited mental retardation, is caused by the absence of the fragile X mental retardation protein (FMRP). FMRP has been shown to use its arginine-glycine-glycine (RGG) box to bind to a subset of RNA targets that form a G quadruplex structure. We performed a detailed analysis of the interactions between the FMRP RGG box and the microtubule associated protein 1B (MAP1B) mRNA, a relevant in vivo FMRP target. We show that MAP1B RNA forms an intramolecular G quadruplex structure, which is bound with high affinity and specificity by the FMRP RGG box. We determined that hydrophobic interactions are important in the FMRP RGG box-MAP1B RNA association, with minor contributions from electrostatic interactions. Our findings that at low protein:RNA ratios the RNA G quadruplex structure is slightly stabilized, whereas at high ratios is unfolded, suggest a mechanism by which the FMRP concentration variation in response to a neurotransmitter stimulation event could act as a regulatory switch for the protein function, from translation repressor to translation activator.

FIGURE 1.

(A) Structure of a G quartet. (B) Secondary structure of the MAP1B RNA fragment (34-mer) used in this study, which was generated using Zuker's algorithm (2003). The adenine at position 19 (circled) in the MAP1B RNA structure was replaced with 2-aminopurine in MAP1B-19AP RNA.

FIGURE 2.

Changes of the imino proton resonance region of the 1D ¹H NMR spectrum of MAP1B RNA (500 μM in 10 mM Tris, pH 7.5, 29°C) upon titrating increasing concentrations of KCl in the range of 0–10 mM.

FIGURE 3.

(A) The heating and cooling UV thermal denaturation profiles of MAP1B RNA (10 μM in 10 mM cacodylic acid, pH 6.5 and 10 mM KCl) (black diamonds 20°C–99°C, open triangles 99°C–20°C). (B) Plot of the MAP1B RNA G quadruplex melting temperature as a function of the RNA concentration. (C) Plot of ΔG⁰ as a function of the logarithm of K⁺ ion concentration. The number Δn of K⁺ ion equivalents released upon the unfolding of MAP1B RNA G quadruplex structure was determined from the slope ΔΔG⁰/Δ log [K⁺].

FIGURE 4.

CD spectra showing the type I G quadruplex formation by MAP1B RNA (10 μM RNA in 10 mM cacodylic acid, pH 6.5 in the presence of increasing concentrations of KCl ranging from 0 to 10 mM). The KCl concentrations are indicated in the figure inset.

FIGURE 5.

(A) Binding curves of the FMRP RGG box to MAP1B-19AP RNA in the absence (black squares) and presence of a 10-fold excess of a nonspecific Munc-13 site 1 RNA (black triangles) or of a nonspecific HCV peptide (black crosses) or of rComp4 RNA sequence (black circles). These experiments were carried out at 25°C. (B) Nonlinear van't Hoff plot, showing the temperature dependence of the association constant, K_obs = 1/K_d, for the MAP1B-19AP RNA:FMRP RGG complex, in the range of 20°C–45°C. The ΔC°_pobs = −(1.0 ± 0.2) kcal/ mol K, T _H (K) = (296 ± 2); T _S (K) = (307 ± 1) values were determined by fitting this plot with Equation 5. (C) Dependence of the entropy, enthalpy, and free energy of the MAP1B-19AP RNA:FMRP RGG complex formation on temperature. (D) The binding of FMRP RGG box to MAP1B-19AP was measured at 25°C in the presence of different salt concentrations [M⁺] (Salt composition: 10 mM KCl + variable mM LiCl mM) in the range 10 mM to 1000 mM [M⁺]. A value of (−∂logK_obs/∂log[M⁺]) of ∼0.2 was determined from the linear fit of the plot.

FIGURE 6.

(A) CD spectra of 10 μM MAP1B RNA in 10 mM cacodylic acid, pH 6.5, 25°C in the presence of increasing concentrations of the FMRP RGG box. (B) 10 μM MAP1B RNA+100 μM FMRP RGG box was incubated with proteinase K (1 μg) for 1 h at 25°C to check whether the addition of an excess of the FMRP RGG box causes the RNA degradation. (C) Imino proton resonance region of the 1D ¹H NMR spectra of MAP1B RNA in the presence of increasing concentrations of the FMRP RGG box.

FIGURE 7.

Proposed model for a regulatory switch of FMRP function from translation repressor to activator, in response to a neurotransmitter stimulation event. (A) In resting state when the FMRP:RNA ratio is low, FMRP represses the translation of specific mRNA targets by stabilizing a G quadruplex structure present in their 5′-UTR. (B) A stimulation of the mGluR by agonists leads to an increase in the FMRP concentration, unfolding the RNA G quadruplex structure, and allowing translation to occur.