Hostname: page-component-848d4c4894-xm8r8 Total loading time: 0 Render date: 2024-07-05T15:13:31.003Z Has data issue: false hasContentIssue false

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
Get access

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
Information
RNA , Volume 5 , Issue 9 , September 1999 , pp. 1248 - 1258
Copyright
© 1999 RNA Society

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)