5′ stem-loop of collagen α1(I) mRNA inhibits translation in vitro but is required for triple helical collagen synthesis in vivo

The 5' stem-loop is a conserved sequence element found around the translation initiation site of three collagen mRNAs, alpha1(I), alpha2(I), and alpha1(III). We show here that the 5' stem-loop of collagen alpha1(l) mRNA is inhibitory to translation in vitro. The sequence 5' to the translation initiation codon, as a part of the 5' stem-loop, is also not efficient in initiating translation under competitive conditions. This suggests that collagen alpha1(I) mRNA may not be a good substrate for translation. Since the 5' stem-loop binds protein factors in collagen-producing cells, this binding may regulate its translation in vivo. We studied in vivo translation of collagen alpha1(I) mRNA after transfecting collagen alpha1(I) genes with and without the 5' stem-loop into Mov 13 fibroblasts. The mRNA with the alpha1(I) 5' stem-loop wag translated into pepsin-resistant collagen, which was secreted into the cellular medium. This mRNA also produced more disulfide-bonded high molecular weight collagen found intracellularly. The mRNA in which the 5' stem-loop was mutated, but without affecting the coding region of the gene, was translated into pepsin-sensitive collagen and produced only trace amounts of disulfide-bonded collagen. This suggests that the 5' stem-loop is required for proper folding or stabilization of the collagen triple helix. To our knowledge this is the first example that an RNA element located in the 5'-untranslated region is involved in synthesis of a secreted multisubunit protein. We suggest that 5' stem-loop, with its cognate binding proteins, targets collagen mRNAs for coordinate translation and couples translation apparatus to the rest of the collagen biosynthetic pathway.