Systematic identification of RNA sequences and protein components regulating circular RNA translation

项目摘要 人类基因组编码数万个circRNA，其中的翻译是必不可少的， 调节重要的生物学功能，如细胞增殖、分化和迁移。干扰 与circRNA翻译相关的基因可能导致肿瘤的发生和癌症的转移。然而，目前还不清楚， 干扰circRNA的翻译会导致疾病。识别circRNA的调控成分 翻译可以提供有价值的临床见解。然而，目前的技术无法区分circRNA， 从线性RNA有效地，这样的机制和分子成分调节circRNA翻译 仍然不清楚。因此，必须开发一种技术，可以识别调节 circRNA翻译具有更高的灵敏度和特异性。 我已经开发了一种高通量的报告筛选试验，可以系统地筛选和识别 特异性启动circRNA翻译的RNA序列。利用这项技术，我发现了两组 分别特异性驱动circRNA或线性RNA翻译的RNA序列，表明 线性RNA和circRNA翻译之间的调节成分是不同的（Chen等， 重新提交给科学）。在这项拟议的研究中，我将进一步调整和应用这项新技术，以（i） 鉴定和表征特异性调节circRNA翻译的序列和蛋白质组分，（ii） 确定遗传变异、circRNA翻译和疾病之间的关联，（iii）表征circRNA 以组织依赖的方式系统地翻译，以及（iv）构建非整合和稳定的基因 具有可调表达水平和组织特异性的表达平台。这项研究将建立 作为一个独立的研究者，我的未来研究的基础，调查的监管和协调 不同RNA之间的不同翻译机制（帽依赖性翻译与独立翻译） 物种（线性与环状RNA）。 这项工作将在斯坦福大学学院的霍华德·张博士的指导下进行 他是技术开发和RNA生物学方面的专家， 将博士后研究员安置到领先机构的独立学术职位。进一步培训 科学和专业技能将通过利用斯坦福大学提供的资源来实现 大学医学院和博士后事务办公室，提供了一个杰出的智力- 激励的环境与成功所需的所有设施和资源。所有拟议的培训将 补充了我之前在RNA生物学、技术开发和计算机编程方面的培训， 促进我过渡到一个独立的研究人员，调查的职能，法规和临床 circRNA的意义

PROJECT SUMMARY The human genome encodes tens of thousands of circRNAs, of which the translation is essential for regulating important biological functions, such as cell proliferation, differentiation, and migration. Interference with circRNA translation can lead to tumorigenesis and metastasis of cancers. It is unclear, however, how disrupting circRNA translation can cause the diseases. Identifying the regulatory components of circRNA translation can provide valuable clinical insights. However, current technologies cannot distinguish circRNAs from linear RNAs efficiently, such that the mechanism and molecular components regulating circRNA translation remain unclear. Thus, it is imperative to develop a technology that can identify the components regulating circRNA translation with higher sensitivity and specificity. I have developed a high-throughput reporter screening assay that can systematically screen and identify the RNA sequences that initiate circRNA translation specifically. Utilizing the technology, I discovered two groups of the RNA sequences that specifically drive translation on either circRNA or linear RNA, respectively, indicating that the regulatory components between linear RNA and circRNA translation are different (Chen et al., resubmitted to Science). In this proposed research, I will further adapt and apply this novel technology to (i) identify and characterize the sequence and protein components that regulate circRNA translation specifically, (ii) identify the association between genetic variation, circRNA translation, and disease, (iii) characterize circRNA translation in a tissue-dependent manner systematically, and (iv) build a non-integrative and stable gene expression platform with tunable expression level and tissue specificity. This proposed research will build the foundation of my future research as an independent researcher to investigate the regulation and the coordination between different translation machinery (cap-dependent vs. independent translation) among different RNA species (linear vs. circular RNAs). This work will be performed under the mentorship of Dr. Howard Chang at the Stanford University School of Medicine, an expert in technology development and RNA biology who has highly-successful track records of placing postdoctoral fellows into independent academic positions at leading institutions. Further training in scientific and professional skills will be achieved by utilizing the resources available through the Stanford University School of Medicine and the Office of Postdoctoral Affairs, which provide an outstanding intellectually- stimulating environment with all facilities and resources necessary for success. All proposed training will complement my previous training in RNA biology, technology development, and computer programming and facilitate my transition to an independent researcher, investigating the functions, regulations and clinical implications of circRNA.