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.Keywords: FMRP; fragile X syndrome; G quadruplex; MAP1B RNA; RGG boxThe lack of fragile X mental retardation protein (FMRP) results in the fragile X syndrome, the most prevalent inherited mental disorder (Crawford et al. 2001). The absence of FMRP is due to the transcriptional inactivation of the fmr1 gene, caused by an unstable expansion of a CGG trinucleotide repeat in its 59-untranslated region (UTR) (Jin and Warren 2000;O'Donnell and Warren 2002). The function of FMRP has been extensively studied, however its cellular role and how its loss causes mental retardation is still not fully understood. It is believed that FMRP is a translational regulator for specific messenger RNAs (mRNAs), and it has been shown that it is associated with actively translating ribosomes (Antar and Bassell 2003;Jin and Warren 2003;Massimiliano et al. 2004). Nucleic acid chaperone properties have also been attributed to this protein (Gabus et al. 2004). This 632 amino acid protein has two types of RNA binding domains: two K-homology (KH) domains and one arginine-glycine-glycine (RGG) box, suggesting that FMRP exerts its function through RNA binding (Siomi et al. 1993).Several studies reported that FMRP uses its RGG box to bind with high affinity to mRNAs rich in G content that could adopt more complex G quadruplex structures Darnell et al. 2001;Schaeffer et al. 2001;Ramos et al. 2003). Formed from four guanine residues, a G quartet has a planar conformation stabilized by Hoogsteen base pairs (Fig. 1A). Several such planar structures can stack to form G quadruplexes, which are stabilized by potassium or sodium cations, but are disrupted in the presence of lithium cations (Davis 2004;Hazel et al. 2004;Mergny et al. 2005). It has also been shown that FMRP uses its KH2 domain to bind to a synthetic RNA that harbors a loop-loop kissing complex (Darnell et al. 2005). However, despite these extensive efforts, the in vivo...

HIV-1 reverse transcriptase (RT) is a bi-functional enzyme, having both DNA polymerase (RNA- and DNA-dependent) and ribonuclease H (RNH) activities. HIV-1 RT has been an exceptionally important target for antiretroviral therapeutic development, and nearly half of the current clinically used antiretrovirals target RT DNA polymerase. However, no inhibitors of RT RNH are on the market or in preclinical development. Several drug-like small molecule inhibitors of RT RNH have been described, but little structural information is available about the interactions between RT RNH and inhibitors that exhibit antiviral activity. In this report, we describe NMR studies of the interaction of a new RNH inhibitor, BHMP07, with a catalytically active HIV-1 RT RNH domain fragment. We carried out solution NMR experiments to identify the interaction interface of BHMP07 with the RNH domain fragment. Chemical shift changes of backbone amide signals at different BHMP07 concentrations clearly demonstrate that BHMP07 mainly recognizes the substrate handle region in the RNH fragment. Using RNH inhibition assays and RT mutants, the binding specificity of BHMP07 was compared with another inhibitor, dihydroxy benzoyl naphthyl hydrazone. Our results provide a structural characterization of the ribonuclease H-inhibitor interaction and are likely to be useful for further improvements of the inhibitors.

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.

HIV-1 reverse transcriptase (RT) has been an attractive target for the development of antiretroviral agents. Although this enzyme is bi-functional, having both DNA polymerase and ribonuclease H (RNH) activities, there is no clinically approved inhibitor of the RNH activity. Here, we characterize the structural basis and molecular interaction of an allosteric site inhibitor, BHMP07, with the wild type (WT) RNH fragment. Solution NMR experiments for inhibitor titration on WT RNH showed relatively wide chemical shift perturbations, suggesting a long-range conformational effect on the inhibitor interaction. Comparisons of the inhibitor-induced NMR chemical-shift changes of RNH with those of RNH dimer, in the presence and absence of Mg2+, were performed to determine and verify the interaction site. The NMR results, with assistance of molecular docking, indicate that BHMP07 preferentially binds to a site that is located between the RNH active site and the region encompassing helices B and D (the “substrate-handle region”). The interaction site is consistent with the previous proposed site, identified using a chimeric RNH (p15-EC) [Gong, el (2011) Chem. Biol. Drug Des. 77, 39-47], but with slight differences that reflect the characteristics of the amino acid sequences in p15-EC compared to the WT RNH.

Fragile X syndrome, the most common form of inherited mental retardation is caused by the expansion of a CGG trinucleotide repeat in the fragile X mental retardation 1 (fmr1) gene. The abnormal expansion of the CGG repeat causes hypermethylation and subsequent silencing of the fmr1 gene, resulting in the loss of the fragile X mental retardation protein (FMRP). FMRP has been shown to use its arginine-glycine-glycine rich region (RGG box) to bind to messenger RNAs that form G quadruplex structures. Several studies reported that the G quadruplex RNA recognition alone is not sufficient for FMRP RGG box binding and that an additional stem and/or a G quadruplex-stem junction region may also be important in recognition. In this study we have used biophysical methods such as fluorescence, UV, CD and NMR spectroscopy to demonstrate that the recognition of the RNA G quadruplex structure per se, in the absence of a stem region, is sufficient for the FMRP high affinity and specific binding. These findings indicate that the presence of a stem structure in some of the FMRP G quadruplex forming mRNAs is not a requirement for protein recognition as previously believed, but rather for the proper formation of the correct RNA G quadruplex structure recognized by FMRP.