Robust, high-throughput identification of RNA processing regulators and regulatory networks genome-wide

项目总结 RNA结合蛋白(RBP)与细胞内非编码的、前期的和成熟的RNA结合，以调节每一个 RNA加工的步骤，包括前mRNA选择性剪接，RNA的稳定性和定位，以及控制 翻译。已经清楚的是，改变的RNA处理在几乎所有研究中都扮演着关键角色 生物系统，最近的研究表明，相当一部分致病基因突变 影响RNA处理，包括导致家族性脊髓性肌萎缩症、肌萎缩侧索硬化症 硬化症和多种癌症类型。对A股下游监管网络的机械性理解 RBP对于研究并最终改善这些疾病是必不可少的；然而，仍然需要 稳健、公正的全基因组方法来表征RBP靶标和调节剂。在我们最近的基础上 关于增强交联和免疫沉淀(ECLIP)的发展，我建议将这项工作扩展到三个方面 独特的方向，每个方向都有助于我们获得RNA处理的全球高质量视图 整个转录本： 1.建立低样本和Tag-eCLIP方法，用于低浓度限制性商业惯例的体内可并行性分析。 输入样本，以及缺乏用于免疫沉淀的高质量天然抗体的限制性商业惯例。 2.证明转录组分析结合RBP靶标识别可以识别关键的 生物系统的调节器，使用人类诱导的多能干细胞的分化作为一种 模型系统 3.发展无偏识别非编码RNA上游功能调控因子的方法 以及以RNA为中心的RNA处理。 我在基因组学、计算生物学以及DNA和RNA结合研究方面的丰富专业知识 蛋白质使我成为进行上述研究的理想人选。这三个目标需要 不同的方法将结合在一种强大的能力中，以感兴趣的RBP开始并识别其 受管制的目标，或从感兴趣的RNA开始并确定调节剂限制性商业惯例，这将作为基础 作为一名独立教师候选人参加我的独立研究项目。加州大学圣迭戈分校的Yeo实验室是一个理想的选择 进行这项研究的环境并完成我的培训，以追求独立的学术 教师地位，因为它一直是开发实验和计算方面的领先者 方法对RBP的调控进行表征。此外，YEO实验室的位置靠近卓越 加州大学圣迭戈分校、索尔克研究所和拉霍亚的其他研究机构和生物技术公司的研究人员 将提供干细胞培养和分化方面的具体动手实验培训，以及充足的 在进行研究和开发独立研究计划方面接受指导培训的机会。

PROJECT SUMMARY RNA binding proteins (RBPs) bind to non-coding, pre-, and mature RNA within the cell to regulate each step of RNA processing, including pre-mRNA alternative splicing, RNA stability and localization, and control of translation. It has become clear that altered RNA processing plays critical roles in nearly every studied biological system, and recent work has suggest that a substantial fraction of disease-causing genetic mutations affect RNA processing, including mutations that cause familial Spinal Muscular Atrophy, Amyotrophic Lateral Sclerosis, and multiple cancer types. Mechanistic understanding of the downstream regulatory network of an RBP is essential to studying and, ultimately, ameliorating these diseases; however, there remains a need for robust, unbiased genome-wide methods to characterize RBP targets and regulators. Building upon our recent development of enhanced crosslinking and immunoprecipitation (eCLIP), I propose to extend this work in three unique directions that each contribute to our ability to gain global, high-quality views of RNA processing transcriptome-wide: 1. Develop low-sample and tag-eCLIP methods for highly parallelizable in vivo profiling of RBPs in low input samples, and for RBPs which lack high-quality native antibodies for immunoprecipitation. 2. Show that transcriptome profiling coupled with RBP target identification can identify critical regulators of a biological system, using differentiation of human induced pluripotent stem cells as a model system 3. Develop methods for unbiased identification of upstream functional regulators of non-coding RNAs and RNA processing in an RNA-centric manner. My extensive expertise in genomics, computational biology, and the study of DNA and RNA binding proteins makes me an ideal candidate to perform the research proposed above. These three aims take different approaches that will coalesce in a robust ability to begin either with an RBP of interest and identify its regulated targets, or begin with an RNA of interest and identify regulator RBPs, which will serve as the basis for my independent research program as an independent faculty candidate. The Yeo lab at UCSD is an ideal environment to perform this research and complete my training towards pursuit of an independent academic faculty position, as it has consistently been a leader in developing both experimental and computational methods to characterize RBP regulation. Additionally, the location of the Yeo lab proximal to outstanding researchers at UCSD, the Salk Institute, and other research institutes and biotechnology companies in La Jolla will provide specific hands-on experimental training in stem cell culture and differentiation, as well as ample opportunities for mentored training in performing research and developing an independent research program.