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

FMR1 is an RNA-binding protein that is either absent or mutated in, patient s affected by the fragile X syndrome, the most common inherited cause of me ntal retardation in humans. Sequence analysis of the FMR1 protein has sugge sted that RNA binding is related to the presence of two K-homologous (KH) m odules 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 diffe rential RNA-sequence specificity. In the present article, we describe work done to dissect FMR1 into regions with structurally and functionally distin ct properties. A semirational approach was followed to identify four region s: an :N-terminal stretch of 200 amino acids, the two KH-regions, and a C-t erminal stretch. Each region was produced as a recombinant protein, purifie d, and probed for its state of folding by spectroscopical techniques. Circu lar,dichroism and NMR spectra of the N-terminus show formation of secondary structure with a strong tendency to aggregate. Of the two homologous KH mo tifs, 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 expecte d from its amino acid composition, nonglobular. Binding assays were then pe rformed using the 4-nt homopolymers. Our results show that only the first K H domain but not the second binds to RNA, and provide the first direct evid ence 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 b ecause no known RNA-binding motif is identifiable in this region. Different sequence specificity was observed for the fragments: both the N-terminus o f the protein and KH1 bind preferentially to poly-(ra). The C-terminal regi on, 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 likel y achieved by the whole block that comprises the first approximate to 400 r esidues, whereas the C-terminus provides a nonspecific binding surface.