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

对全基因组 RNA 加工调节因子和调节网络进行稳健、高通量的鉴定

基本信息

批准号：
10364689
负责人：
Eric Lyman Van Nostrand
金额：
$ 24.9万
依托单位：
BAYLOR COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-05-06 至 2023-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10364689
关键词：
Address Affect Alternative Splicing Amyotrophic Lateral Sclerosis Antibodies Binding Binding Proteins Binding Sites Biological Models Biotechnology CRISPR screen Cardiac Myocytes Cell Culture Techniques Cell Differentiation process Cells Code Computational Biology Computing Methodologies Coupled DNA Binding DNA Sequence Alteration Data Data Set Development Disease Ectoderm Elements Endoderm Environment Event Faculty Genetic Transcription Genetic Variation Genomics High-Throughput Nucleotide Sequencing Human Immunoprecipitation Institutes Islets of Langerhans Location Mentors Mesoderm Methodology Methods Molecular Motor Neurons Mutation Phase Phenotype Play Positioning Attribute Proteins RNA RNA Computations RNA Processing RNA Stability RNA-Binding Proteins Regulation Research Research Institute Research Personnel Resources Role Sampling Spinal Muscular Atrophy System Techniques Tissue Sample Tissues Training Translating Translations Untranslated RNA Work biological systems cancer type cell type crosslink differential expression experimental study genome-wide human disease improved in vivo induced pluripotent stem cell interest knock-down mRNA Precursor overexpression programs protein profiling stem cells transcriptome

项目摘要

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.

项目概要 RNA 结合蛋白 (RBP) 与细胞内的非编码 RNA、前 RNA 和成熟 RNA 结合，调节每个 RNA RNA 加工步骤，包括前 mRNA 选择性剪接、RNA 稳定性和定位以及控制翻译。很明显，RNA 加工的改变在几乎所有研究中都发挥着关键作用。生物系统，最近的研究表明，很大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、肌萎缩侧索硬化症的突变硬化症和多种癌症类型。对下游监管网络的机制理解 RBP 对于研究并最终改善这些疾病至关重要；然而，仍然需要稳健、公正的全基因组方法来表征 RBP 靶标和调节因子。基于我们最近的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每个独特的方向都有助于我们获得 RNA 加工的全局、高质量视图的能力全转录组： 1. 开发低样本和标签 eCLIP 方法，以在低样本量下对 RBP 进行高度可并行的体内分析。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. 表明转录组分析与 RBP 靶标识别相结合可以识别关键的生物系统的调节剂，利用人类诱导多能干细胞的分化作为模型系统 3. 开发非编码RNA上游功能调节因子的公正鉴定方法以RNA为中心的方式进行RNA加工。我在基因组学、计算生物学以及 DNA 和 RNA 结合研究方面拥有丰富的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起形成强大的能力，从感兴趣的 RBP 开始并识别其监管目标，或从感兴趣的 RNA 开始并确定监管 RBP，这将作为基础作为一名独立教师候选人，我的独立研究项目。 UCSD 的 Yeo 实验室是一个理想的选择进行这项研究并完成我追求独立学术的培训的环境教职地位，因为它一直是实验和计算开发领域的领导者表征 RBP 调节的方法。此外，Yeo 实验室的位置靠近杰出的加州大学圣地亚哥分校、索尔克研究所以及拉霍亚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实践实验培训，以及充足的进行研究和开发独立研究计划的指导培训的机会。