Regulation of Collagen Type I Expression by Chaperone-Mediated mRNA Remodeling

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Project Summary Collagen synthesis and homeostasis are integral to the proper function and repair of all tissues. Dysregulation of collagen production is a hallmark of pathological fibrosis, which can affect any organ that contains collagen (including the liver, lung, kidney, heart, skin, and intestine). The long-term goal is to understand the mechanisms within the cell that produce collagen and thereby enable rational design of novel drugs to treat fibrotic diseases. The proposed work will identify how the 5’ untranslated region of collagen type I messenger RNA (mRNA) regulates synthesis of the collagen protein through interactions with the RNA-binding protein LARP6. Our central hypothesis is that LARP6 remodels a critical secondary structural element in this region, a bulged stem-loop (termed “5’SL”), to increase the accessibility of the protein coding sequence start codon to the cellular translation machinery. Our rationale is that specific and ordered interactions between the 5’ untranslated region of the collagen α1(I) mRNA and LARP6 and are critical to regulation of collagen synthesis. We will test this central hypothesis through the following specific aims: 1) Determine how structures of the 5’SL of collagen α1(I) and α2(I) contribute to thermodynamics of LARP6 binding affinity and 2) Define how strand annealing and dissociation kinetics of the collagen 5’SL by LARP6 chaperone activity modulates translation. In the first aim, we will use solution nuclear magnetic resonance (NMR) spectroscopy to determine the high-resolution structures of the collagen α1(I) and α2(I) 5’SLs. We will characterize enthalpic and entropic contributions to LARP6 binding and conduct a molecular thermodynamic analysis of ion effects using isothermal titration calorimetry. In the second aim, we will characterize how LARP6 affects the RNA strand annealing and dissociation kinetics of the wildtype 5’SLs as well as mutants that alter the sequence and structure of the predicted internal bulge. We will also develop a luciferase-based translation reporter system to characterize effects of mutations and LARP6 chaperone activity on translation initiation. The field’s understanding of LARP6-mediated collagen type I expression has almost exclusively focused on the protein. This proposed project will be significant as it will fill a critical gap in our understanding of the mechanism by identifying how the structure and thermodynamics of the primary molecular target, the 5’UTR of collagen mRNAs, contributes to protein binding, start codon accessibility, and subsequent upregulation of collagen type I production.