Characterization of the interaction between αCP(2) and the 3′-untranslated region of collagen α1(I) mRNA

Activated hepatic stellate cells produce increased type I collagen in hepatic fibrosis. The increase in type I collagen protein results from an increase in mRNA levels that is mainly mediated by increased mRNA stability. Protein–RNA interactions in the 3′-UTR of the collagen α1(I) mRNA correlate with stabilization of the mRNA during hepatic stellate cell activation. A component of the binding complex is αCP(2). Recombinant αCP(2) is sufficient for binding to the 3′-UTR of collagen α1(I). To characterize the binding affinity of and specificity for αCP(2), we performed electrophoretic mobility shift assays using the poly(C)-rich sequence in the 3′-UTR of collagen α1(I) as probe. The binding affinity of αCP(2) for the 3′-UTR sequence is ∼2 nM in vitro and the wild-type 3′ sequence binds with high specificity. Furthermore, we demonstrate a system for detecting protein–nucleotide interactions that is suitable for high throughput assays using molecular beacons. Molecular beacons, developed for DNA–DNA hybridization, are oligonucleotides with a fluorophore and quencher brought together by a hairpin sequence. Fluorescence increases when the hairpin is disrupted by binding to an antisense sequence or interaction with a protein. Molecular beacons displayed a similar high affinity for binding to recombinant αCP(2) to the wild-type 3′ sequence, although the kinetics of binding were slower.