FMRP interacts with G-quadruplex structures in the 3'-UTR of its dendritic target Shank1 mRNA - PubMed (original) (raw)

FMRP interacts with G-quadruplex structures in the 3'-UTR of its dendritic target Shank1 mRNA

Yang Zhang et al. RNA Biol. 2014.

Abstract

Fragile X syndrome (FXS), the most common cause of inherited intellectual disability, is caused by the loss of expression of the fragile X mental retardation protein (FMRP). FMRP, which regulates the transport and translation of specific mRNAs, uses its RGG box domain to bind mRNA targets that form G-quadruplex structures. One of the FMRP in vivo targets, Shank1 mRNA, encodes the master scaffold proteins of the postsynaptic density (PSD) which regulate the size and shape of dendritic spines because of their capacity to interact with many different PSD components. Due to their effect on spine morphology, altered translational regulation of Shank1 transcripts may contribute to the FXS pathology. We hypothesized that the FMRP interactions with Shank1 mRNA are mediated by the recognition of the G quadruplex structure, which has not been previously demonstrated. In this study we used biophysical techniques to analyze the Shank1 mRNA 3'-UTR and its interactions with FMRP and its phosphorylated mimic FMRP S500D. We found that the Shank1 mRNA 3 ' -UTR adopts two very stable intramolecular G-quadruplexes which are bound specifically and with high affinity by FMRP both in vitro and in vivo. These results suggest a role of G-quadruplex RNA motif as a structural element in the common mechanism of FMRP regulation of its dendritic mRNA targets.

Keywords: FMRP; Fragile X syndrome; G-quadruplex; RGG box; Shank1 mRNA.

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Figures

Figure 1.

1H NMR spectra of the imino proton region of Shank1a (A) and Shank1b (B) RNA in the presence of increasing concentrations of KCl in the range 0–50 mM.

Figure 2.

Native polyacrylamide gel electrophoresis (PAGE) of 15 μM Shank1a (A) and Shank1b (B) RNAs at various KCl concentrations: 0 mM (lane 1), 5 mM (lane 2), 10 mM (lane 3), 25 mM (lane 4) and 50 mM (lane 5). The CD spectra of 10 μM Shank1a (C) and Shank1b (D) RNAs prepared in 10 mM cacodylic acid at various KCl concentrations showing the presence of a parallel G-quadruplex structures stabilized by KCl.

Figure 3.

The UV thermal denaturation profiles of Shank1a RNA at 2.5 mM KCl (A) and Shank1b RNA at 10 mM KCl (B) in the presence of 10 mM cacodylic acid, pH 6.5. The melting temperatures of Shank1a (C) and Shank1b (D) G-quadruplexes were plotted versus the RNA concentrations showing that the melting temperature is independent of the RNA concentration. The UV hypochromic transitions of Shank1a RNA at 2.5 mM KCl (E) and Shank1b RNA at 10 mM KCl (F) were fitted equation 3 (materials and methods) that assumes a two state model.

Figure 4.

Proposed G-quadruplex structures adopted by Shank1a (A) and Shank1b RNA (B). The fluorescently labeled Shank1a_18AP and Shank1b_24AP RNAs were constructed by replacing the adenine at position 18 and position 24, respectively. The FMRP RGG box was titrated into 200 nM of the fluorescently labeled Shank1a_18AP (C) and Shank1b_24AP (D) in the presence of 1 μM HCV peptide. The steady-state fluorescence data was fitted with equation 4 (materials and methods). The reported Kd values represent the average of three Kd values in the triplicates.

Figure 5.

The FMRP ISO1 and FMRP S500D were titrated into 200 nM of the fluorescently label Shank1a_18AP (A, B) and Shank1b_24AP (C, D) RNAs in the presence of 1 μM BSA. The reported Kd value represents the average of three Kd values in the triplicates.

Figure 6.

FMRP recognizes the Shank1 G-quadruplex structures in vivo. 5’ biotin-labeled Shank1a, Shank1b and HCV RNA probes were incubated with E17 mouse brain lysate. Probes were precipitated with Neutravidin agarose beads and co-purified FMRP and SMN protein was assessed by immunoblot.

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FMRP interacts with G-quadruplex structures in the 3'-UTR of its dendritic target Shank1 mRNA - PubMed (original) (raw)