Inhibition of collagen α1(I) expression by the 5′ stem-loop as a molecular decoy

Abstract

Collagen α1(I) mRNA is posttranseriptionally regulated in hepatic stellate cells (HSCs). Binding of protein factors to the evolutionary conserved stem-loop in the 5′-untranslated region (5′ stem-loop) is required for a high level of expression in activated HSCs. The 5′ stem-loop is also found in α2(I) and α1(III) mRNAs. Titration of the 5′ stem-loop binding factors by a stably expressed RNA containing the 5′ stem-loop (molecular decoy) may decrease the expression of these collagen mRNAs. We designed a 108-nt RNA that is transcribed from the optimized mouse U7 small nuclear RNA gene and contains the 5′ stem-loop (p74WT decoy). This decoy accumulates in the nucleus and in the cytoplasm. When expressed in NIH 3T3 fibroblasts, the p74WT decoy decreased collagen α1(I) mRNA level by 60% and decreased collagen type I secreted into the cellular medium by 50%. We also expressed this decoy in quiescent rat HSCs by adenoviral gene transfer. Quiescent HSCs undergo activation in culture, resulting in a 60-70-fold increase in collagen α1(I) mRNA. The decoy decreases collagen α1(I) mRNA expression by 50-60% during activation of HSCs HSCS. It also decreases collagen α2(I) mRNA expression and collagen al (III) mRNA expression. The cellular levels of collagen α1(I) propeptide and of disulfide-bonded collagen type I trimer are reduced by 70%. However, the p74WT decoy did not decrease a smooth muscle actin protein or the mRNA levels of glyceraldehyde-3-phosphate dehydrogenase and interleukin-6. The p74WT decoy was also introduced into activated human HSCs. In these cells, the decoy decreased collagen α1(I) propeptide and disulfide-bonded collagen trimer by 50-60%. These results indicate that the 5′ stem-loop specifically regulates fibrillar collagen synthesis and represents a novel target for antifibrotic therapy. The molecular decoys provide a generalized method of assessing the functional significance of blocking the interactions of mRNA and proteins.