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

The stability of collagen α1(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 αCP. When cultured in a three-dimensional gel composed of type I collagen, mouse fibroblasts had decreased collagen α1(I) mRNA steady-state levels, which resulted from a decreased mRNA half-life. In cells cultured in gel, hybrid mouse–human collagen α1(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 α1(I) mRNA. Protein binding to the 5′ stem–loop was reduced in cells grown in gel, which was associated with destabilization of the collagen α1(I) mRNA. In addition to the binding of a cytoplasmic protein, there was also a nuclear binding activity directed to the collagen α1(I) 5′ stem–loop. The nuclear binding was increased in cells grown in gel, suggesting that it may negatively regulate expression of collagen α1(I) mRNA. Binding of αCP, a protein involved in stabilization of collagen α1(I) mRNA, was unchanged by the culture conditions.