UC San Diego

The loss of expression of the fragile X mental retardation protein (FMRP) leads to Fragile X Syndrome, which is a genetically-linked condition resulting in intellectual disability. FMRP is an RNA-binding protein known to repress the translation of target mRNAs that encode pre- and post-synaptic proteins that are directly attributed to the phenotypes of the condition. Although hundreds of mRNAs have been shown to associate with FMRP, very few have actually been validated through biochemical studies. More importantly, the sequence specificities of the KH domains and the RGG box have not carefully been deciphered. Recently, Tuschl and co-workers proposed that the KH1 and KH2 domains of FMRP bind to WGGA (W=A/U) and ACUK (K=G/U) RNA recognition elements, respectively (Ascano et al., 2012). We developed a fluorescence anisotropy assay to quantitatively measure the binding affinity of N-terminally truncated Drosophila FMRP (NT- dFMRP) and functionally relevant mutants (I244N, I307N, [Delta]RGG) to PolyG₁₈-Fl. Our results show that NT-dFMRP, I244N, and I307N bind PolyG₁₈-Fl with similar affinities of 87 ± 18 nM, 105 ± 14 nM, and 52 ± 22 nM, respectively; while the [Delta]RGG mutant does not. Since circular dichroism spectroscopy demonstrates that PolyG₁₈- Fl folds into a standard parallel G-quadruplex structure, we propose that the RGG box is responsible for binding RNA substrates containing this structural feature. Our studies also show that NT-dFMRP does not bind to PolyC₁₈-Fl, PolyC₁₈(ACUU)-Fl, and PolyC₁₈(UGGA)-Fl RNAs, indicating that single sets of ACUK and WGGA sequences are not sufficient enough for NT-dFMRP binding