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

项目摘要 RNA结合蛋白（RBP）与细胞内的非编码RNA、前RNA和成熟RNA结合， RNA加工的步骤，包括前mRNA选择性剪接、RNA稳定性和定位以及 翻译.很明显，改变的RNA加工在几乎所有研究中都起着关键作用。 生物系统，最近的工作表明，相当一部分致病基因突变， 影响RNA加工，包括导致家族性脊髓性肌萎缩症、肌萎缩性侧索硬化症、 硬化症和多种癌症类型。一个基因的下游调控网络的机制性理解 RBP对于研究和最终改善这些疾病至关重要;然而，仍然需要 强大、公正的全基因组方法来表征RBP靶点和调节因子。基于我们最近 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加工的全球性，高质量的观点 全转录组： 1.开发低样品和tag-eCLIP方法，用于低浓度下RBP的高度平行化体内分析 输入样品，以及缺乏用于免疫沉淀的高质量天然抗体的RBP。 2.显示转录组分析加上RBP靶标鉴定可以识别关键的 生物系统的调节剂，使用人诱导多能干细胞的分化作为 模型系统 3.开发无偏鉴定非编码RNA上游功能调节子的方法 和以RNA为中心的RNA加工。 我在基因组学，计算生物学，以及DNA和RNA结合研究方面的广泛专业知识 蛋白质使我成为执行上述研究的理想候选人。这三个目标需要 不同的方法，将结合在一个强大的能力，开始与RBP的利益，并确定其 调节的靶点，或开始与感兴趣的RNA和鉴定调节RBP，这将作为基础 作为一名独立的教师候选人参加我的独立研究项目。加州大学圣地亚哥分校的Yeo实验室是一个理想的 环境进行这项研究，并完成我的训练，追求独立的学术 教师的位置，因为它一直是发展实验和计算的领导者 方法来表征RBP调节。此外，杨实验室的位置接近突出的 加州大学圣地亚哥分校、索尔克研究所和其他研究机构以及拉霍亚的生物技术公司的研究人员 将提供干细胞培养和分化的具体实践实验培训，以及充足的 在执行研究和开发独立的研究计划的指导培训的机会。

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.