title: Dissecting FMR1, the protein responsible for fragile X syndrome, in its structural and functional domains.
abstract: FMR1 is an RNA-binding protein that is either absent or mutated in patients affected by the fragile X syndrome, the most common inherited cause of mental retardation in humans. Sequence analysis of the FMR1 protein has suggested that RNA binding is related to the presence of two K-homologous (KH) modules and an RGG box. However, no attempt has been so far made to map the RNA-binding sites along the protein sequence and to identify possible differential RNA-sequence specificity. In the present article, we describe work done to dissect FMR1 into regions with structurally and functionally distinct properties. A semirational approach was followed to identify four regions: an N-terminal stretch of 200 amino acids, the two KH regions, and a C-terminal stretch. Each region was produced as a recombinant protein, purified, and probed for its state of folding by spectroscopical techniques. Circular dichroism and NMR spectra of the N-terminus show formation of secondary structure with a strong tendency to aggregate. Of the two homologous KH motifs, only the first one is folded whereas the second remains unfolded even when it is extended both N- and C-terminally. The C-terminus is, as expected from its amino acid composition, nonglobular. Binding assays were then performed using the 4-nt homopolymers. Our results show that only the first KH domain but not the second binds to RNA, and provide the first direct evidence for RNA binding of both the N-terminal and the C-terminal regions. RNA binding for the N-terminus could not be predicted from sequence analysis because no known RNA-binding motif is identifiable in this region. Different sequence specificity was observed for the fragments: both the N-terminus of the protein and KH1 bind preferentially to poly-(rG). The C-terminal region, which contains the RGG box, is nonspecific, as it recognizes the bases with comparable affinity. We therefore conclude that FMR1 is a protein with multiple sites of interaction with RNA: sequence specificity is most likely achieved by the whole block that comprises the first approximately 400 residues, whereas the C-terminus provides a nonspecific binding surface.
authors.affiliation: The National Institute for Medical Research, The Ridgeway, Mill Hill, London, United Kingdom.
authors.forename: S
authors.initials: S
authors.lastname: Adinolfi
authors.forename: C
authors.initials: C
authors.lastname: Bagni
authors.forename: G
authors.initials: G
authors.lastname: Musco
authors.forename: T
authors.initials: T
authors.lastname: Gibson
authors.forename: L
authors.initials: L
authors.lastname: Mazzarella
authors.forename: A
authors.initials: A
authors.lastname: Pastore
mesh_terms: D000595:Amino Acid Sequence; D015153:Blotting, Western; D002942:Circular Dichroism; D051860:Fragile X Messenger Ribonucleoprotein 1; D005600:Fragile X Syndrome / Q000235:genetics*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics*; D011485:Protein Binding; D017510:Protein Folding; D012313:RNA / Q000378:metabolism*; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
chemical_list: D051860:Fragile X Messenger Ribonucleoprotein 1; D009419:Nerve Tissue Proteins; D012313:RNA; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; C497683:FMR1 protein, human