Distinct domains in the CArG-box binding factor A destabilize tetraplex forms of the fragile X expanded sequence d(CGG)n

Abstract

Formation of hairpin or tetraplex structures of the FMR1 gene d(CGG)n sequence triggers its expansion, setting off fragile X syndrome. In searching for proteins that destabilize d(CGG)n secondary structures we purified from rat liver quadruplex telomeric DNA binding protein 42 (qTBP42) that disrupts G'2 bimolecular tetraplex d(CGG)n while paradoxically stabilizing the G'2 structure of the telomeric sequence d(TTAGGG)n. Based on peptide sequence homology of qTBP42 and mouse CArG-box binding factor A (CBF-A), we provide direct evidence that recombinant CBF-A protein is physically and immunochemically indistinguishable from qTBP42 and that it too destabilizes G'2 d(CGG)n while stabilizing G'2 d(TTAGGG)n. We inquired whether CBF-A employs the same or different domains to differentially interact with G'2 d(CGG)n and G'2 d(TTAGGG)n. Mutant CBF-A proteins that lack each or combinations of its five conserved motifs: RNP1(1), RNP1(2), RNP2(1), RNP2(2) and ATP/GTP-binding box were tested for their G'2 d(CGG)n destabilization and G'2 d(TTAGGG)n stabilization activities. We find that either RNP1(1) or the ATP/GTP motifs are necessary and sufficient for G'2 d(CGG)n destabilization whereas RNP2(1) suppresses destabilization by either one of these two motifs. Neither RNP1(1) nor the ATP/GTP motif are required for G'2 d(TTAGGG)n stabilization. Hence, CBF-A employs different domains to destabilize G'2 d(CGG)n or stabilize G'2 d(TTAGGG)n.

Cloning and Characterization of Porcine CArG Binding Factor A Expression in the Anterior Pituitary

“…Porcine CBF-A has two RNA recognition sites in the center of the molecule similar to rat CBF-A [20] (Fig. 1).…”

“…The amplified DNA was directly sequenced, and its identity was confirmed with that of cDNA (data not shown). On the other hand, the ATP/GTP binding motif in the carboxyl region (residue numbers 282-290: GSTNYGKSQ) [22,23] is important for disruption of the tetraplex helix structure of G'2 d(CGG)n disruption [20,21]. This motif is present just behind a tract of glycine residues, which is different than in the human, rat and mouse.…”

“…Confirmation of the extra repeats was performed by nucleotide sequence determination of the PCR product for porcine genomic DNA and subsequently verified (data not shown). According to a protein-protein BLAST search, amino acid numbers 87-159 and 171-208 showed identity to the putative RNA recognition motif [9] and the ATP-binding domain [20].…”

“…The amino acid sequences of putative RNA recognition motifs and core sequences (RNP11/RNA12 and RNP21/RNA22) [9]) are shaded and underlined. A putative ATP binding domain [20] is double underlined. …”

Magnesium in the Central Nervous System

“…The incidence of fragile X syndrome is estimated at 1/4000--1/6000 in males and half that for females (Wiesner et al, 2004). This disease is linked to the mutation of the FMR1 gene located on the X chromosome, characterized by expansion (CGG amplification) at the FRAXA site (Xq27.3) in the non--coding region of the first exon (Weisman--Shomer et al, 2002). These authors also showed that two factors were indispensable for the stabilization of the CGG tetraplex: magnesium and ATP.…”