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

项目摘要 胶原蛋白的合成和体内平衡是所有组织的正常功能和修复不可或缺的。失调 胶原蛋白的产生是病理性纤维化的标志，它可以影响任何含有胶原蛋白的器官。 （包括肝、肺、肾、心脏、皮肤和肠）。长期目标是了解 在细胞内产生胶原蛋白，从而能够合理设计治疗纤维化疾病的新药。 这项工作将确定I型胶原信使RNA（mRNA）的5 '非翻译区是如何被转录的。 通过与RNA结合蛋白LARP 6的相互作用调节胶原蛋白的合成。我们的中央 假设LARP 6重塑了该区域的一个关键二级结构元件，一个凸出的茎环 （称为"5'SL"），以增加蛋白质编码序列起始密码子对细胞翻译的可及性。 机械.我们的基本原理是，特定的和有序的相互作用之间的5 '非翻译区的 胶原α 1（I）mRNA和LARP 6，对调节胶原合成至关重要。我们将测试这个中央 1）确定胶原α 1（I）和α 2（II）的5'SL的结构是如何形成的， α 2（I）有助于LARP 6结合亲和力的热力学，以及2）定义链退火和 胶原5’SL通过LARP 6伴侣活性的解离动力学调节翻译。第一个目标，我们 将使用溶液核磁共振（NMR）光谱来确定高分辨率的结构， 胶原α 1（I）和α 2（I）5 'SL。我们将描述对LARP 6结合的熵和熵贡献 并使用等温滴定量热法进行离子效应的分子热力学分析。在 第二个目标，我们将描述LARP 6如何影响RNA链的退火和解离动力学。 野生型5'SL以及改变预测的内部凸起的序列和结构的突变体。我们将 还开发了一个基于翻译报告系统，以表征突变和LARP 6的影响 分子伴侣对翻译起始的活性。本领域对LARP 6介导的I型胶原蛋白的理解 表达几乎完全集中在蛋白质上。这个项目将是重要的，因为它将填补一个 关键差距，我们的理解机制，通过确定如何结构和热力学的， 主要的分子靶点，胶原mRNA的5'UTR，有助于蛋白质结合，起始密码子可及性， 以及随后I型胶原蛋白产生的上调。

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.