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J Biol Chem. 2002 May 17;277(20):18229-37. Epub 2002 Mar 11.

Inhibition of collagen alpha 1(I) expression by the 5' stem-loop as a molecular decoy.

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  • 1Department of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599, USA.


Collagen alpha1(I) mRNA is posttranscriptionally 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 alpha2(I) and alpha1(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 alpha1(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 alpha1(I) mRNA. The decoy decreases collagen alpha1(I) mRNA expression by 50-60% during activation of HSCs. It also decreases collagen alpha2(I) mRNA expression and collagen alpha1(III) mRNA expression. The cellular levels of collagen alpha1(I) propeptide and of disulfide-bonded collagen type I trimer are reduced by 70%. However, the p74WT decoy did not decrease alpha 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 alpha1(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.

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