The 5' stem-loop regulates expression of collagen alpha1(I) mRNA in mouse fibroblasts cultured in a three-dimensional matrix

The stability of collagen alpha1(I) mRNA is regulated by its 5' stem-loop, which binds a cytoplasmic protein in a cap-dependent manner, and its 3'-untranslated region (UTR), which binds alphaCP. When cultured in a three-dimensional gel composed of type I collagen, mouse fibroblasts had decreased collagen alpha1(I) mRNA steady-state levels, which resulted from a decreased mRNA half-life. In cells cultured in gel, hybrid mouse-human collagen alpha1(I) mRNA with a wild-type 5' stem-loop decayed faster than the same mRNA with a mutated stem-loop. When the 5' stem-loop was placed in a heterologous mRNA, the mRNA accumulated to a lower level in cells grown in gel than in cells grown on plastic. This suggests that the 5' stem-loop down-regulates collagen alpha1(I) mRNA. Protein binding to the 5' stem-loop was reduced in cells grown in gel, which was associated with destabilization of the collagen alpha1(I) mRNA. In addition to the binding of a cytoplasmic protein, there was also a nuclear binding activity directed to the collagen alpha1(I) 5' stem-loop. The nuclear binding was increased in cells grown in gel, suggesting that it may negatively regulate expression of collagen alpha1(I) mRNA. Binding of alphaCP, a protein involved in stabilization of collagen alpha1(I) mRNA, was unchanged by the culture conditions.