Biophysical Study of the G Quadruplex Formed by FMRP mRNA and of its Interactions with the FMRP RGG Box

Abstract

Fragile X syndrome, the most common form of inherited mental retardation in humans, affects about 1 of 3000 males and 1 of 5000 females. It is caused by the loss of expression of the fragile X mental retardation protein (FMRP), due to a CGG trinucleotide repeat expansion in the 5’-untranslated region (UTR) of the fragile x mental retardation-1 (fmr1) gene. FMRP has been shown by biophysical methods to use its arginine-glycine-glycine (RGG) box RNA binding domain to bind with high affinity and specificity to G quadruplex forming mRNA sequences. The binding of FMRP to a proposed G quadruplex structure in the coding region of its own mRNA (100 nucleotide fragment named FBS) has been proposed to affect mRNA splicing events for isoforms 1 through 3. In this study we truncated the original 100 nt FMRP mRNA region rich in guanines to 67 nt and used biophysical methods such as UV thermal denaturation, CD spectroscopy, and 1H-NMR spectroscopy to directly demonstrate its folding into a G-quadruplex structure. We have also analyzed the binding properties of the FMRP RGG box domain in the context of different protein isoforms to this truncated FMRP FBS mRNA.