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

点击翻译按钮获取中文摘要

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 of translation regulation 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 a1(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 a1(I) and a2(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 determine the high-resolution structures of the collagen a1(I) and a2(I) 5’SLs, characterize enthalpic and entropic contributions to LARP6 binding, and determine the electrostatic and non-electrostatic contributions to binding energy. In the second aim, we will characterize how LARP6 affects RNA strand annealing and dissociation kinetics of the wildtype 5’SLs as well as mutants that alter the sequence and predicted structure of the internal bulge. The current 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 regulation of collagen type I protein expression. Finally, the goals described in this application are tightly aligned with the objectives of the AREA program, which are to 1) support meritorious research at an undergraduate focused institution, 2) strengthen the research environment at the home institution, and 3) provide opportunities for undergraduate students to be engaged in biomedical research.