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 细胞内产生胶原蛋白的翻译调节机制，从而实现合理设计 治疗纤维化疾病的新药。拟议的工作将确定如何5'非翻译区的 I型胶原信使RNA（mRNA）通过与胶原蛋白的相互作用调节胶原蛋白的合成。 RNA结合蛋白LARP 6。我们的中心假设是LARP 6重塑了一个关键的二级结构， 在这个区域中的元件，一个凸出的茎环（称为“5'SL”），以增加蛋白质编码的可及性。 序列起始密码子到细胞翻译机器。我们的理由是， 胶原α 1（I）mRNA的5'非翻译区与LARP 6之间的相互作用， 调节胶原蛋白的合成。我们将通过以下具体目标来检验这一中心假设：1） 确定胶原a1（I）和a2（I）的5'SL的结构如何有助于LARP 6的热力学 结合亲和力和2）定义如何链退火和解离动力学的胶原5'SL的LARP 6 分子伴侣活性调节翻译。在第一个目标中，我们将确定的高分辨率结构的 胶原a1（I）和a2（I）5 ′ SL，表征对LARP 6结合的趋化和熵贡献，和 确定静电和非静电对结合能的贡献。第二个目标，我们将 表征LARP 6如何影响RNA链退火和野生型5'SL的解离动力学 作为突变体，改变序列和预测的内部凸起结构。目前了解的 LARP 6介导的I型胶原蛋白表达几乎完全集中在蛋白质上。这一拟议 该项目将是重要的，因为它将填补我们对该机制的理解的一个关键空白， 胶原蛋白mRNA的5'UTR这一主要分子靶点的结构和热力学， 蛋白质结合、起始密码子可及性和随后的I型胶原蛋白表达的调节。 最后，本申请中描述的目标与AREA计划的目标紧密一致， 这是1）支持在本科重点机构有价值的研究，2）加强 研究环境在家里的机构，和3）提供机会，本科生是 从事生物医学研究。

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.