Characterization of the interaction between alphaCP2 and the 3'-untranslated region of collagen alpha1(I) mRNA
Open Access
- 1 November 2000
- journal article
- research article
- Published by Oxford University Press (OUP) in Nucleic Acids Research
- Vol. 28 (21), 4306-4316
- https://doi.org/10.1093/nar/28.21.4306
Abstract
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 αCP2. Recombinant αCP2 is sufficient for binding to the 3′-UTR of collagen α1(I). To characterize the binding affinity of and specificity for αCP2, 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 αCP2 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 αCP2 to the wild-type 3′ sequence, although the kinetics of binding were slower.Keywords
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