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 â‰ƒ400 residues, whereas the C-terminus provides a nonspecific binding surface.
Adinolfi S., B.C. (1999). Dissecting FMR1, the protein responsible for fragile X syndrome, in its structural and functional domains. RNA, 5(9), 1248-1258.
|Tipologia:||Articolo su rivista|
|Citazione:||Adinolfi S., B.C. (1999). Dissecting FMR1, the protein responsible for fragile X syndrome, in its structural and functional domains. RNA, 5(9), 1248-1258.|
|IF:||Con Impact Factor ISI|
|Settore Scientifico Disciplinare:||Settore BIO/13|
|Revisione (peer review):||Sì, ma tipo non specificato|
|Digital Object Identifier (DOI):||http://dx.doi.org/10.1017/S1355838299990647|
|Stato di pubblicazione:||Pubblicato|
|Data di pubblicazione:||1999|
|Titolo:||Dissecting FMR1, the protein responsible for fragile X syndrome, in its structural and functional domains|
|Autori:||Adinolfi S., Bagni C., Musco G., Gibson T., Mazzarella L., Pastore A.|
|Appare nelle tipologie:||01 - Articolo su rivista|