Characterization of the interaction between alphaCP(2) and the 3'-untranslated region of collagen alpha1(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 alpha1(I) mRNA correlate with stabilization of the mRNA during hepatic stellate cell activation. A component of the binding complex is alphaCP(2). Recombinant alphaCP(2) is sufficient for binding to the 3'-UTR of collagen alpha1(I). To characterize the binding affinity of and specificity for alphaCP(2), we performed electrophoretic mobility shift assays using the poly(C)-rich sequence in the 3'-UTR of collagen alpha1(I) as probe. The binding affinity of alphaCP(2) for the 3'-UTR sequence is approximately 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 alphaCP(2) to the wild-type 3' sequence, although the kinetics of binding were slower.
Lindquist
J N
JN
Department of Medicine and Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599-7038, USA.
Kauschke
S G
SG
Stefanovic
B
B
Burchardt
E R
ER
Brenner
D A
DA
D020413:3' Untranslated Regions / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016475:3T3 Cells; D000818:Animals; D020029:Base Pairing; D015374:Biosensing Techniques; D003094:Collagen / Q000235:genetics*; D015342:DNA Probes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005456:Fluorescent Dyes; D007700:Kinetics; D008103:Liver Cirrhosis / Q000235:genetics; D051379:Mice; D009691:Nucleic Acid Denaturation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011066:Poly C / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D020871:RNA Stability; D016601:RNA-Binding Proteins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics; D014157:Transcription Factors / Q000378:metabolism*
D020413:3' Untranslated Regions; D015342:DNA Probes; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D009838:Oligodeoxyribonucleotides; C501904:Pcbp2 protein, mouse; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D011066:Poly C; D003094:Collagen