Modulation of ribosome velocity as a means to rescue refractory CF-causing variants

项目摘要(摘要) 囊性纤维化是一种由囊性纤维化跨膜突变引起的致死性常染色体隐性遗传病 电导调节器(CFTR)。大多数慢性粒细胞白血病患者至少有一个F508del-CFTR拷贝 突变，导致蛋白质错误折叠和严重的多器官损伤。这项提议的首要目标是 是通过纠正基本的遗传缺陷来确定可以改善疾病表型的细胞靶点 来自F508del和不太流行的变体，如对电流不敏感的过早截断密码子(PTCs) 心理治疗。越来越明显的是，CFTR编码序列的改变不仅扰乱了最初的 蛋白质结构，但也扰乱核糖体动态，随后的mRNA利用，以及蛋白质 折叠/生物发生。在以前的研究中，酵母表型分析导致了核糖体蛋白(RP)的发现 模块作为F508del-CFTR贩运的效应器。在这方面，我们已经确定RPL12(UL11) 耗竭通过降低翻译起始和延伸率来挽救F508del-CFTR缺陷，从而 允许核糖体和/或相关的伴侣促进功能性蛋白质构象。发现 在本K99/R00中概述的结果表明，Rpl12抑制也纠正了罕见的PTC，W1282X-CFTR， 达到可能使临床患者受益的程度。因此，我们假设RP沉默改变了翻译 速度和/或核糖体保真度，部分挽救难治性CFTR变异体的合成和组装。我们 提出三个具体目标：(1)表征RP抑制对突变的cftr生物发生的影响(S)，(2) 确定RP沉默改变翻译动力学以挽救难治性CFTR变异体的机制， 以及(3)在转基因CF小鼠中确定RPL12中断的体内相关性。我们将利用多学科 细胞生物学、生物化学、分子遗传学和哺乳动物生理学方面的专业知识 从机制上解决了核糖体影响蛋白质折叠的新方式的基本假设。 这些研究旨在将翻译控制确立为CFTR过程中的一个新的和关键的检查点 处理，并确定除Rpl12外还介导这一途径的特定RP。这样的结果将会有所改善 了解囊性纤维性疾病的发病机制，在动物模型中建立抑制Rp12的安全性，以及 为测试该策略在其他遗传性人类疾病状态中的相关性提供了基础。在资助期间 在获奖期间，奥利弗博士将接受CFTR生化技术、核糖体图谱、RNA- SEQ，生物信息学，CF的小鼠模型，以及职业/职业发展。在这些领域的指导将 让她为奖项的独立(R00)阶段做好准备。埃默里大学提供了丰富的学习环境 职业发展，并利用高度协作的学术研究中心的最先进设施。 一旦奥利弗博士成功地完成了为她的K99所描述的研究，她将处于有利的地位 追求教员职位和她理想的职业生涯，成为一名独立的CF研究员。

PROJECT SUMMARY (ABSTRACT) Cystic fibrosis (CF) is a lethal autosomal recessive disorder caused by mutation of the CF transmembrane conductance regulator (CFTR). The majority of CF patients harbor at least one copy of the F508del-CFTR variant, which results in protein misfolding and severe multi-organ damage. An overarching goal of this proposal is to identify cellular targets that can ameliorate disease phenotype by correcting basic genetic defects resulting from F508del and less prevalent variants, such as premature truncation codons (PTCs) unresponsive to current therapy. It has become increasingly evident that CFTR coding sequence alterations not only disrupt primary protein structure, but also perturb ribosome dynamics, consequent mRNA utilization, and protein folding/biogenesis. In previous studies, yeast phenomic analyses led to discovery of ribosomal protein (RP) modules as effectors of F508del-CFTR trafficking. In this context, we have established that Rpl12 (uL11) depletion rescues the F508del-CFTR defect by reducing rates of translation initiation and elongation, thereby allowing the ribosome and/or associated chaperones to promote a functional protein conformation. Findings outlined in the present K99/R00 demonstrate that Rpl12 suppression also corrects a rare PTC, W1282X-CFTR, to a degree that may benefit patients in the clinic. Thus, we hypothesize that RP silencing alters translational velocity and/or ribosome fidelity to partially rescue synthesis and assembly of refractory CFTR variants. We propose three specific aims: (1) characterize the effect(s) of RP inhibition on mutant CFTR biogenesis, (2) ascertain the mechanism by which RP silencing alters translational kinetics to rescue refractory CFTR variants, and (3) determine in vivo relevance of RPL12 disruption in transgenic CF mice. We will utilize multidisciplinary expertise directed towards cellular biology, biochemistry, molecular genetics, and mammalian physiology to mechanistically address a fundamental hypothesis regarding new ways the ribosome influences protein folding. The studies are intended to establish translation control as a novel and critical checkpoint during CFTR processing, and identify specific RPs in addition to Rpl12 that mediate this pathway. Such results will improve understanding of cystic fibrosis disease mechanism, establish safety of repressing Rpl12 in animal models, and provide a basis for testing relevance of the strategy in other inherited human disease states. During the funding period of this award, Dr. Oliver will receive training in CFTR biochemical techniques, ribosome profiling, RNA- seq, bioinformatics, murine models of CF, and career/professional development. Mentorship in these areas will prepare her for the independent (R00) phase of the award. Emory University provides a rich environment for career advancement and leverages state-of-the-art facilities in a highly collaborative academic research center. Once Dr. Oliver has successfully completed the studies described for her K99, she will be well positioned to pursue a faculty position and her desired career as an independent CF researcher.