Discovery of novel mechanisms that impact CFTR translation and contribute to cystic fibrosis pathogenesis

发现影响CFTR翻译并导致囊性纤维化的新机制 发病机制 摘要 该项目旨在推进高通量酵母表型组学技术，并提供新的机制， 关于模型遗传性疾病囊性纤维化（CF）的信息。当临床上显著的突变在 CF跨膜传导调节因子（CFTR）在酵母同源物（YOR 1）中被工程化，全基因组 使用酵母缺失菌株文库（YDSL）的表型分析鉴定了影响CF的新途径 分子表型CF治疗的新实验靶点也来自这种类型的研究。下 更新的具体目标1，我们展示了全基因组酵母研究可以促进理解的方法 包括无义（1型）和严重折叠（2型）缺陷的CFTR变体， 目前存在调节剂治疗。我们还证明了表型组学可以用于阐明途径 需要药物救助。具体目标2采用前沿技术（核糖体分析， 基于修饰映射的深度测序（Psi-Seq）），以深入研究新的（和意想不到的） 基于哺乳动物翻译效率分析的CFTR生物合成的介导物。我们尤其 检查表型分析所描述的核糖体“碰撞”或“碰撞”的作用， 假尿苷化对CFTR翻译和蛋白质合成的影响。具体目标3，应用创新 CF细胞模型，以确定目的1和2的发现的临床意义。拟议的研究将测试 与CF发病机制相关的重要假设，提供了关于平移速度和 CFTR表达过程中mRNA的利用，并显示核糖体碰撞和假尿苷酸化 会导致囊性纤维化我们的研究结果还将有助于推进治疗难治性CF的新靶点。 从这些研究中吸取的经验教训可以应用于许多其他遗传性和严重的人类疾病。

Discovery of novel mechanisms that impact CFTR translation and contribute to cystic fibrosis pathogenesis Abstract This project is intended to advance high throughput yeast phenomics technology and provide new mechanistic information regarding a model genetic disease, cystic fibrosis (CF). When clinically significant mutations in the CF transmembrane conductance regulator (CFTR) are engineered in a yeast homolog (YOR1), genome-wide phenomic analysis using the yeast deletion strain library (YDSL) identifies novel pathways that impact CF molecular phenotype. New experimental targets for CF therapy also result from studies of this type. Under Specific Aim 1 of the renewal, we show ways in which genome-wide yeast studies can advance understanding of CFTR variants including nonsense (Type 1) and severe folding (Type 2) defects for which no “personalized” modulator treatment currently exists. We also demonstrate phenomics can be applied to elucidate pathways necessary for pharmacologic rescue. Specific Aim 2 engages leading-edge technology (ribosomal profiling, deep-sequencing based on modification mapping (Psi-Seq)) to delve deeply into novel (and unexpected) mediators of CFTR biogenesis based on analysis of mammalian translational efficiency. In particular, we examine the role of ribosomal “collisions” or “queueing” described by phenomic analysis, and mRNA pseudouridinylation for effects on CFTR translation and protein synthesis. Specific Aim 3, applies innovative CF cell models to establish clinical significance of findings from Aims 1 and 2. The proposed studies will test important hypotheses relevant to CF pathogenesis, provide new knowledge regarding translational velocity and mRNA utilization during CFTR expression, and show that ribosomal collisions and pseudouridinylation contribute to cystic fibrosis. Our findings will also help advance new targets for treating refractory forms of CF. Lessons learned from the studies can be applied to many other inherited and serious human illnesses.