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

分子伴侣介导的 mRNA 重塑对 I 型胶原蛋白表达的调节

• 批准号: 10582369
• 负责人: Eric Baggs
• 金额: $ 5.28万
• 依托单位: BOISE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10582369
• 关键词: 5' Untranslated Regions; Address; Affect; Affinity; Binding; Binding Proteins; Biomedical Research; Calorimetry; Cells; Cessation of life; Chronic; Code; Collagen; Collagen Type I; Developed Countries; Disease; Dissociation; Elements; Entropy; Fibrosis; Goals; Heart; Homeostasis; Initiator Codon; Intestines; Ions; Kidney; Kinetics; Lead; Liver; Luciferases; Lung; Mediating; Messenger RNA; Molecular; Molecular Chaperones; Molecular Target; Mutation; NMR Spectroscopy; Organ; Organ failure; Pathologic; Production; Proteins; RNA; RNA-Binding Proteins; Regulation; Reporter; Resolution; Skin; Structure; System; Testing; Thermodynamics; Tissues; Titrations; Translation Initiation; Translations; Up-Regulation; Work; base; experience; extracellular; mutant; novel therapeutics; rational design; repaired; stem; undergraduate student; wound healing

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.

项目摘要 胶原蛋白的合成和动态平衡对于所有组织的正常功能和修复都是不可或缺的。调控失调 胶原蛋白的产生是病理性纤维化的一个标志，它可以影响任何含有胶原的器官。 (包括肝、肺、肾、心脏、皮肤和肠道)。长期目标是了解这些机制 在细胞内产生胶原蛋白，从而使合理设计治疗纤维化疾病的新药。 这项拟议的工作将确定I型胶原信使RNA(MRNA)的5‘非翻译区是如何 通过与RNA结合蛋白LARP6相互作用来调节胶原蛋白的合成。我们的中央 假设LARP6重塑了该区域的一个关键的二级结构元件，一个鼓起的茎环 为了增加蛋白质编码序列起始密码子对细胞翻译的可及性 机械设备。我们的理论基础是，5‘非翻译区之间的特定和有序的相互作用 胶原α1(I)m RNA和LARP6在胶原合成的调节中起关键作用。我们将测试这个中心 通过以下特定目的进行假设：1)确定胶原α1(I)的5‘SL结构和 α2(I)对LARP6结合亲和力的热力学做出了贡献，2)定义了链退火和 LARP6分子伴侣对5‘SL胶原蛋白的解离动力学调节翻译。在第一个目标中，我们 将使用溶液核磁共振(核磁共振)光谱来确定高分辨率结构 胶原α1(I)和α2(I)5‘SLS。我们将表征对LARP6结合的焓和熵的贡献 并用等温滴定量热法对离子效应进行了分子热力学分析。在 第二个目标，我们将表征LARP6如何影响RNA链的退火和解离动力学 野生型5‘SLS以及改变预测的内部凸起的序列和结构的突变体。我们会 我还开发了一个基于荧光素酶的翻译报告系统来表征突变和LARP6的影响 翻译启蒙中的伴侣活动。业内对LARP6介导的I型胶原的认识 表达几乎完全集中在蛋白质上。这个拟议的项目将具有重大意义，因为它将填补 通过确定分子的结构和热力学，我们对这一机制的理解出现了关键的差距 主要的分子靶点是胶原蛋白mRNAs的5‘非编码区，有助于蛋白质结合，启动密码子可及性， 并随后上调I型胶原的产生。