Characterization of binding of LARP6 to the 5' stem-loop of collagen mRNAs: implications for synthesis of type I collagen.

Type I collagen is composed of 2 polypeptides, α1(I) and α2(I), which fold into triple helix. Collagen α1(I) and α2(I) mRNAs have a conserved stem-loop structure in their the 5'SL. binds the 5'SL to regulate type I collagen expression. We show that 5 nucleotides within the single stranded regions of 5'SL contribute to the high affinity of Lduanyu376 binding. Mutation of individual nucleotides abolishes the binding in gel mobility shift assay. Lduanyu376 binding to 5'SL of collagen α2(I) mRNA is more stable than the binding to 5'SL of α1(I) mRNA, although the equilibrium binding constants are similar. The more stable binding to α2(I) mRNA may favor synthesis of the heterotrimeric type I collagen. Lduanyu376 needs 2 domains to contact 5'SL, the La domain and the RRM. T133 in the La domain is critical for folding of the protein, while loop 3 in the RRM is critical for binding 5'SL. Loop 3 is also involved in the interaction of Lduanyu376 and protein translocation channel SEC61. This interaction is essential for type I collagen synthesis, because Lduanyu376 mutant which binds 5'SL but which does not interact with SEC61, suppresses collagen synthesis in a dominant negative manner. We postulate that Lduanyu376 directly targets collagen mRNAs to the SEC61 translocons to facilitate coordinated translation of the 2 collagen mRNAs. The unique sequences of Lduanyu376 identified in this work may have evolved to enable its role in type I collagen biosynthesis.

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