Thermodynamics of the Fragile X Mental Retardation Protein RGG Box Interactions with G Quartet Forming RNA

Abstract

Fragile X syndrome, the most common form of inherited mental retardation, is the result of an unstable expansion of a CGG trinucleotide repeat in the 5' UTR of the fragile X mental retardation-1 (FMR1) gene. The abnormal hypermethylation of the expanded CGG repeats causes the transcriptional silencing of the FMR1 gene and, consequently, the loss of the fragile X mental retardation protein (FMRP). FMRP is an RNA binding protein that binds to G quartet forming RNA using its RGG box motif. In this study we have performed a thermodynamic analysis of the interactions between the FMRP RGG box domain and Sc1, an RNA molecule which had been previously shown to be bound with high affinity by both the full-length FMRP and by its RGG box domain. We have determined that the association between the FMRP RGG box and Sc1 RNA is dominated by hydrophobic and hydrogen bond interactions, with minor contributions from electrostatic interactions, and that the FMRP RGG box binding increases the stability of the G quartet RNA structure significantly. Interestingly, we found that the G quartet recognition is necessary but not sufficient for the FMRP RGG box binding to this RNA target, indicating that additional interactions of the peptide, possibly with the stem and/or stem-G quartet junction region, are required. Our results also indicate that the G quartet RNA recognition is not a general feature of the RGG box motif but rather carries some sequence, protein and/or RNA, specificity.

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

“…The Kd values measured for Shank1a_18AP and Shank1b_24AP RNA: FMRP RGG box complexes are larger than the dissociation constants reported previously for the FMRP RGG box binding to other G-quadruplex forming mRNA targets, which ranged between 1-20 nM. 13,43,45,46 This could be due to the fact that both Shank1a and Shank1b G-quadruplexes are predicted to consist of four G quartet planes surrounded by an extended longer loop of 8»10 nucleotides and a short one of 2»3 nucleotides ( Fig. 4A and B), whereas the previous G-quadruplexes investigated are formed by two-plane G-quartet surrounded by shorter loops containing between 1-3 nucleotides.…”

Structural Diversity of Triplet Repeat RNAs

“…The physiological importance of RNA G-tetraplexes, which are more stable and are more easily formed due to the single-stranded character of RNA than their corresponding DNA structures, was postulated in a number of reports (49 -52). Such structures have been implicated in specific protein binding and modulation of gene expression by regulating splicing, polyadenylation, translation, and transcript turnover (18,(53)(54)(55)(56)(57)(58). For example, short tetraplex-forming sequences from 5Ј-UTR of the NRAS and ZIC-1 genes decreased the translation of reporter genes several times when inserted into their 5Ј-UTRs (18,57).…”

Features of Double-stranded RNA-binding Domains of RNA Helicase A Are Necessary for Selective Recognition and Translation of Complex mRNAs

“…A similar RG-rich domain is observed in nucleolin and heterogeneous nuclear ribonucleoprotein A1 and interacts with protein cofactors that tether target mRNA (10 -12). Moreover, the RG-rich domain of fragile X mental retardation protein directly engages target RNA at G quartets (13,14). These findings suggest the possibility that the RG-rich domain of RHA is important for interaction with target mRNAs.…”

“…RHA-dependent translation activity is conferred by orientation-dependent activity of the ϳ150-nucleotide 5Ј terminus, which is designated the post-transcriptional control element (PCE) (13,14). PCE activity is attributable to two functionally redundant stem-loop structures (A and C) (2,21).…”