Dissecting FMR1, the protein responsible for fragile X syndrome, in its structural and functional domains

Détails

ID Serval
serval:BIB_867EA3E5AC0E
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Titre
Dissecting FMR1, the protein responsible for fragile X syndrome, in its structural and functional domains
Périodique
RNA
Auteur⸱e⸱s
Adinolfi S., Bagni C., Musco G., Gibson T., Mazzarella L., Pastore A.
ISSN
1355-8382 (Print)
ISSN-L
1355-8382
Statut éditorial
Publié
Date de publication
09/1999
Volume
5
Numéro
9
Pages
1248-58
Notes
Adinolfi, S
Bagni, C
Musco, G
Gibson, T
Mazzarella, L
Pastore, A
eng
MC_U117584256/Medical Research Council/United Kingdom
1999/09/25
RNA. 1999 Sep;5(9):1248-58.
Résumé
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.
Mots-clé
Amino Acid Sequence, Blotting, Western, Circular Dichroism, Fragile X Mental Retardation Protein, Fragile X Syndrome/*genetics, Humans, Magnetic Resonance Spectroscopy, Models, Genetic, Molecular Sequence Data, Mutagenesis, Nerve Tissue Proteins/*chemistry/*genetics, Protein Binding, Protein Folding, RNA/*metabolism, *RNA-Binding Proteins, Recombinant Proteins/metabolism, Sequence Homology, Amino Acid, Structure-Activity Relationship
Pubmed
Création de la notice
06/03/2017 17:23
Dernière modification de la notice
20/08/2019 14:45
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