Interactions of the G quartet forming semaphorin 3F RNA with the RGG box domain of the fragile X protein family

Abstract

Fragile X syndrome, the most common cause of inherited mental retardation, is caused by the transcriptional silencing of the fmr1 gene due to an unstable expansion of a CGG trinucleotide repeat and its subsequent hypermethylation in its 5' UTR. This gene encodes for the fragile X mental retardation protein (FMRP), an RNA-binding protein that has been shown to use its RGG box domain to bind to G quartet-forming RNA. In this study, we performed a detailed analysis of the interactions between the FMRP RGG box domain and one of its proposed RNA targets, human semaphorin 3F (S3F) RNA by using biophysical methods such as fluorescence, UV and circular dichroism spectroscopy. We show that this RNA forms a G quartet-containing structure, which is recognized with high affinity and specificity by the FMRP RGG box. In addition, we analyzed the interactions of human S3F RNA with the RGG box and RG cluster of the two FMRP autosomal paralogs, the FXR1P and FXR2P. We found that this RNA is bound with high affinity and specificity only by the FXR1P RGG box, but not by the FXR2P RG cluster. Both FMRP and FXR1P RGG box are able to unwind the G quartet structure of S3F RNA, however, the peptide concentrations required in this process are very different: a ratio of 1:6 RNA:FMRP RGG box versus 1:2 RNA:FXR1P RGG box.

Figure 1.

(A) Secondary structure of the human S3F mRNA (S3F-lg) fragment used in this study generated using the RNA structure 4.11 software (49). (B) CD spectrum of 10 μM S3F-lg RNA in 10 mM cacodylic acid containing 150 mM KCl, pH 6.5. (C) UV thermal denaturation profile of S3F-lg RNA (10 µM) containing 150 mM KCl. (D) 15% native gel electrophoresis performed in the presence of 75 mM KCl at different S3F-lg RNA concentrations: 1.5 μM (lane 1), 6 μM (lane 2), 10 μM (lane 3), 20 μM (lane 4), 30 μM (lane 5), 50 μM (lane 6).

Figure 2.

(A) Secondary structure of the S3F-M2 RNA fragment with the mutated base pairs highlighted in blue (49). (B) Proposed G quadruplex parallel structure of S3F-M2 RNA. The 2-AP insertion in S3F-M2_15AP is highlighted in red. (C) CD spectra showing the G quadruplex formation in S3F-M2 RNA by titrating increasing concentrations of KCl. (D) Fifteen percent of native gel electrophoresis of S3F-M2 RNA at various RNA concentrations: 3 μM (lane 1), 6 μM (lane 2), 10 μM (lane 3), 20 μM (lane 4), 30 μM (lane 5), 50 μM (lane 6), 100 μM (lane 7). (E) 1D ¹H NMR spectrum of S3F-M2 RNA (387 µM) in 10 mM Tris pH 7.5 and various KCl concentrations.

Figure 3.

UV thermal denaturation profile of S3F-M2 RNA (10 µM) containing either 150 mM KCl (A) or LiCl (B). (C) UV melting profiles of S3F-M2 RNA in 10 mM cacodylic acid, pH 6.5 containing 150 mM KCl at the following RNA concentrations: 10 μM (blue trace), 20 μM (gray trace), 50 μM (brown trace). (D) Plot of the melting temperature of the S3F-M2 RNA G quartet as a function of the RNA concentration. (E) UV melting profile of 10 µM S3F-M2 free RNA (blue trace) and in a 1:1 ratio with the FMRP RGG box (green trace).

Figure 4.

(A) EMSA of the FMRP RGG box binding to S3F-M2 (lanes 1 and 2) and S3F-M2_15AP RNA (lanes 3 and 4). The RNA concentration was 10 µM and the FMRP RGG box was used in a 1:1 ratio. (B) The 2-AP at the 15th position of the S3F-M2_15AP RNA is sensitive to the G quartet structure formation as indicated by the steady-state fluorescence of S3F-M2_15AP in 150 mM KCl (blue trace) or LiCl (black trace). (C) Increasing concentrations of FMRP RGG box were titrated into a solution of 150 nM S3F-M2_15AP in 10 mM cacodylic acid, pH 6.5 containing 150 mM KCl. Sc1-sh RNA, which forms a G quartet but does not have a stem in its structure, has been used as a negative control (42). The binding of FMRP RGG Box to the S3F-M2 in the presence of an 6-fold excess FXR2P RG peptide is also shown.(D) The association constant, K_obs = 1/K_d for the S3F-M2_15AP-FMRP RGG complex was determined as a function of temperature in the range 20–45°C. The binding thermodynamics results are summarized in Table 2. (E) The binding of FMRP to S3F-M2_15AP RNA measured at different salt concentrations: 150, 250, 400 and 1000 mM KCl.

Figure 5.

(A) EMSA of the binding of S3F-M2 RNA by the FMRP RGG box (lanes 1 and 2), FXR2P RG cluster (lanes 3 and 4) and FXR1P RGG box (lanes 5 and 6). The RNA concentration was 20 µM and the peptides were in a 1:2 ratio with the RNA. The primary sequences of the peptides used in this study are shown in the table. (B) Binding of the FXR1P RGG box to S3F-M2_15AP RNA in the absence (green triangles, K_d = 55.0 ± 3.8 nM) and presence of a 10-fold excess of Munc-13 site 1 RNA (orange circles, K_d = 55.1 ± 4.2 nM) or presence of a 6-fold excess of FXR2P RG cluster (blue circles, K_d = 53.8 ± 2.8 nM). (C) Binding of the FMRP RGG box to S3FM2_15AP in the absence (black triangles, K_d = 0.7 ± 0.3 nM) and presence (blue circles, K_d = 0.8 ± 0.5 nM) of a 10-fold excess of Munc-13 site 1 RNA or of a 6-fold excess of FXR2P RG cluster (red circles, K_d = 0.6 ± 0.3 nM). (D) UV melting profile of free 10 µM S3F-M2 RNA (blue trace) and in a 1:1 ratio with the FXR1P RGG box (red trace). (E) UV melting profile of free 10 µM S3F-M2 RNA (blue trace) and in a 1:1 ratio with the FXR2P RG (light green trace).

Figure 6.

CD spectra of 10 µM S3F-M2 RNA in the presence of increasing concentrations of the RGG boxes of FMRP (A) and FXR1P (B). 10 µM S3F-M2 RNA+ 100 µM FMRP RGG Box (C) and the 10 µM S3F-M2 RNA+ 100 µM FXR1P RGG Box (D) with proteinase K (1 µg) for 1 h at 25°C, to check for the degradation of the RNA.