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

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)。我们的长期目标是理解 细胞内产生胶原蛋白从而实现合理设计的翻译调节机制 治疗纤维化疾病的新药。拟议的工作将确定5‘非翻译区是如何 I型胶原信使RNA(MRNAs)通过与胶原蛋白相互作用调节胶原蛋白的合成。 RNA结合蛋白LARP6。我们的中心假设是LARP6重塑了一个关键的二级结构 该区域中的一个元件，一个鼓起的茎环(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。我们的理由是具体和有序的 胶原A1(I)mRNA的5‘非翻译区与LARP6之间的相互作用 胶原蛋白合成的调节。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结构对LARP6热力学的贡献 结合亲和力和2)确定LARP6对5‘SL胶原蛋白的链退火和解离动力学 伴侣活动调节翻译。在第一个目标中，我们将确定 胶原A1(I)和A2(I)5‘SLS，表征了对LARP6结合的焓和熵贡献，以及 确定静电和非静电对结合能的贡献。在第二个目标中，我们将 表征LARP6如何影响RNA链的退火和野生型5‘SLS的解离动力学 作为改变内部隆起的序列和预测结构的突变体。对当前的理解 LARP6介导的I型胶原表达几乎完全集中在该蛋白上。这项建议 该项目将具有重大意义，因为它将通过确定如何 主要分子靶标--胶原蛋白mRNAs的5‘非编码区的结构和热力学有助于 为了与蛋白质结合，启动密码子可及性，并随后调节I型胶原蛋白的表达。 最后，本申请中描述的目标与AREA计划的目标紧密结合， 这是为了1)支持本科生重点院校的有价值的研究，2)加强 国内机构的研究环境，以及3)为本科生提供机会 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.