Fragile X syndrome is the result of the unstable expansion of a trinucleotide repeat in the 5'-untranslated region of the FMR1 gene. Fibroblast subclones from a mildly affected patient, each containing stable FMR1 alleles with 57 to 285 CGG repeats, were shown to exhibit normal steady-state levels of FMR1 messenger RNA. However, FMR protein was markedly diminished from transcript with more than 200 repeats. Such transcripts were associated with stalled 40S ribosomal subunits. These results suggest that a structural RNA transition beyond 200 repeats impedes the linear 40S migration along the 5'-untranslated region. This results in translational inhibition by trinucleotide repeat expansion.

Fragile X syndrome is caused by the transcriptional silencing of the FMR1 gene due to a trinucleotide repeat expansion. The encoded protein, Fmrp, has been found to be a nucleocytoplasmic RNA-binding protein containing both KH domains and RGG boxes that associates with polyribosomes as a ribonucleoprotein particle. RNA binding has previously been demonstrated with in vitro-translated Fmrp; however, it remained uncertain whether the selective RNA binding observed was an intrinsic property of Fmrp or required an associated protein(s). Here, baculovirus-expressed and affinity-purified FLAG-tagged murine Fmrp was shown to bind directly to both ribonucleotide homopolymers and human brain mRNA. FLAG-Fmrp exhibited selectivity for binding poly(G) > poly(U) > > poly(C) or poly(A). Moreover, purified FLAG-Fmrp bound to only a subset of brain mRNA, including the 3 untranslated regions of myelin basic protein message and its own message. Recombinant isoform 4, lacking the RGG boxes but maintaining both KH domains, was also purified and was found to only weakly interact with RNA. FLAG-purified I304N Fmrp, harboring the mutation of severe fragile X syndrome, demonstrated RNA binding, in contrast to previous suggestions. These data demonstrate the intrinsic property of Fmrp to selectively bind RNA and show FLAG-Fmrp as a suitable reagent for structural characterization and identification of cognate RNA ligands.Fragile X syndrome, a common mental retardation syndrome, results from an unstable CGG repeat expansion in the 5Ј-untranslated region of the FMR1 gene, leading to transcriptional silencing and the absence of Fmrp protein (1-4). Characterization of in vitro-translated Fmrp has shown it to be an RNA-associated protein with selectivity for homopolymer RNA and some human brain transcripts (5, 6). Fmrp contains nuclear localization and export sequences (7), as well as RGG box and KH domain motifs found in many RNA-binding proteins. The RGG box has been found in hnRNP 1 U, hnRNP A1, nucleolin, and fibrillarin and has been shown to autonomously bind homopolymeric RNA (8). However, the amino acid context surrounding the RGG box may influence the specificity and avidity of the RNA-nucleic acid interaction (8, 9). The KH domain is highly conserved in hnRNP K, yeast MER-1 splicing regulator, Sam68, and chicken vigillin, among other RNA-binding proteins (10).Fmrp expression is widespread but not ubiquitous. The majority of Fmrp is associated with polyribosomes and has been found to form an mRNP, which can be stripped from ribosomes by EDTA or RNase treatment (11)(12)(13)(14). A prevailing model suggests that Fmrp directly binds specific mRNAs in the nucleus as part of a hnRNP particle and then mediates its transport to the cytoplasm and delivery of the mRNP to the ribosome (4, 7).Homopolymer binding assays performed with in vitro-translated Fmrp and carboxyl-terminal truncated Fmrp proteins suggested a role for both the KH domain and RGG box in RNA binding (15). However, whether the observed RNA binding by Fmrp is mediated directly...