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

Published online by Cambridge University Press:  01 September 1999


SALVATORE ADINOLFI
Affiliation:
The National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom
CLAUDIA BAGNI
Affiliation:
Dipartimento di Biologia, via della Ricerca Scientifica, Tor Vergata, Italy
GIOVANNA MUSCO
Affiliation:
The National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom
TOBY GIBSON
Affiliation:
European Molecular Biology Laboratory, Meyerhofstr. 1, D-69012 Heidelberg, Germany
LELIO MAZZARELLA
Affiliation:
Dipartimento di Chimica, via Mezzocannone 4, I-80134 Napoli, Italy
ANNALISA PASTORE
Affiliation:
The National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom

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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 ≈400 residues, whereas the C-terminus provides a nonspecific binding surface.


Type
Research Article
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Copyright
© 1999 RNA Society

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