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

项目总结 人类基因组编码数以万计的CircRNA，其中的翻译对于 调节重要的生物功能，如细胞的增殖、分化和迁移。干扰 CircRNA的翻译可以导致肿瘤的发生和转移。然而，目前还不清楚是如何 扰乱CircRNA的翻译可能会导致疾病。鉴定CircRNA的调控成分 翻译可以提供有价值的临床见解。然而，目前的技术不能区分CircRNA 有效地从线性RNA中分离出来，从而使调节CircRNA翻译的机制和分子组成 目前仍不清楚。因此，开发一种能够识别调节成分的技术势在必行 CircRNA翻译具有较高的敏感性和特异性。 我已经开发了一种高通量的记者筛选试验，可以系统地筛选和鉴定 特异性地启动CircRNA翻译的RNA序列。利用这项技术，我发现了两组 分别驱动CircRNA或线性RNA上的翻译的RNA序列，表明 线性RNA和CircRNA翻译之间的调节成分是不同的(Chen等人， 重新提交给《科学》杂志)。在这项拟议的研究中，我将进一步改编和应用这项新技术来(I) 识别和表征特定调控CircRNA翻译的序列和蛋白质成分，(Ii) 确定遗传变异、CircRNA翻译和疾病之间的联系，(Iii)表征CircRNA 系统地以组织依赖的方式进行翻译，以及(Iv)构建非整合和稳定的基因 表达水平和组织特异性可调的表达平台。这项拟议的研究将建立 为我今后的研究奠定基础，作为一名独立的研究员来调查规范和协调 在不同RNA之间的不同翻译机制之间(大写依赖翻译与独立翻译 物种(线形与环状RNA)。 这项工作将在斯坦福大学学院的Howard Chang博士的指导下进行 他是一位技术开发和RNA生物学方面的专家，在 将博士后研究员安排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现有的资源来实现 大学医学院和博士后事务办公室，提供了一个杰出的智力- 用一切成功所需的设施和资源来刺激环境。所有建议的培训都将 补充我之前在RNA生物学、技术开发和计算机编程方面的培训，并 促进我向独立研究人员的过渡，调查功能、法规和临床 CircRNA的含义。

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.