Analysis of the Fragile X mental retardation protein isoforms 1, 2 and 3 interactions with the G-quadruplex forming semaphorin 3F mRNA

Abstract

Fragile X syndrome, the most prevalent inheritable mental retardation, is caused by the loss of fragile X mental retardation protein (FMRP) expression. FMRP is an RNA-binding protein with nucleo-cytoplasmic shuttle activity, proposed to act as a translation regulator of specific mRNAs in the brain. It has been shown that FMRP uses its arginine-glycine-glycine (RGG) box domain to bind a subset of mRNA targets that form a G-quadruplex structure. FMRP has also been shown to undergo the post-translational modifications of arginine methylation and phosphorylation, as well as alternative splicing, resulting in multiple isoforms. The alternative splice isoforms investigated in this study, isoform 1 (ISO1), isoform 2 (ISO2), and isoform 3 (ISO3), are created by the alternative splicing acceptor site at exon 15. FMRP ISO2 and ISO3 are truncated by 12 and 13 residues, respectively, relative to the longest FMRP isoform ISO1. These truncations, which are in the close proximity of the RGG box domain, preserve the integrity of the RGG box in all three isoforms, but eliminate the in vivo phosphorylation sites, present only on FMRP ISO1. We have expressed and purified recombinant FMRP ISO1, ISO2 and ISO3 in Escherichia coli, free of post-translational modifications, and by using fluorescence spectroscopy, we show that each FMRP isoform binds G-quadruplex RNA, albeit with different binding affinities, suggesting that naturally occurring sequence modifications in the proximity of the RGG box modulate its G-quadruplex RNA binding ability.