Decoding global RNP topologies in splicing regulation

解码拼接调节中的全局 RNP 拓扑

• 批准号: 10636541
• 负责人: Zhipeng Lu
• 金额: $ 56.75万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-15 至 2027-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10636541
• 关键词: 3-Dimensional; Acylation; Alternative Splicing; Amino Acids; Antisense Oligonucleotides; Base Pairing; Benchmarking; Binding; Binding Proteins; Biochemical; Cell Differentiation process; Cell Line; Cell Lineage; Cells; Chemicals; Chemistry; Chromatin; Chromatin Interaction Analysis by Paired-End Tag Sequencing; Classification; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Complex; Data; Development; Disease; Elements; Event; Exons; Formaldehyde; Free Energy; Future; Genes; Genetic; Genetic Diseases; Genome; HepG2; Human; Hydroxyl Radical; K-562; Laws; Ligation; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Measures; Mediating; Methods; Modeling; Molecular; Molecular Biology; Molecular Conformation; Muscular Dystrophies; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neuronal Differentiation; Phylogenetic Analysis; Play; Process; Protein Isoforms; Proteins; Proteome; Publishing; RNA; RNA Binding; RNA Folding; RNA Processing; RNA Splicing; RNA-Binding Proteins; RNA-Protein Interaction; RNA-targeting therapy; Regulation; Resources; Role; Site; Spinal Muscular Atrophy; Structure; System; Technology; Testing; Thermodynamics; Variant; brain cell; cell type; computational pipelines; computerized tools; crosslink; empowerment; experimental study; genome editing; high dimensionality; high throughput technology; human disease; in vivo; induced pluripotent stem cell; invention; mRNA Precursor; messenger ribonucleoprotein; multiple omics; nervous system disorder; neurodevelopment; new technology; precision medicine; predictive modeling; programs; protein complex; protein function; protein protein interaction; stem; targeted treatment; therapeutic target; three dimensional structure; three-dimensional modeling; tool; transcriptome

Alternative splicing (AS) is a major mechanism that generates the vast transcriptome and proteome diversity from the limited genome. Spatial and temporal regulation of AS contributes to cell differentiation and lineage determination. Mutations that disrupt splicing cause a wide range of diseases including neurodegeneration, muscular dystrophies, and cancer. Therefore, understanding and targeting splicing is essential to the future of precision medicine. In the past 3 decades, AS studies have focused on cis elements and their associated trans factors, such as RNA binding proteins (RBPs). The laws of thermodynamics dictate that RNAs fold into low free energy structures in the context of RNA-protein complexes (RNPs), however, very little is known about how pre-mRNA structures in large RNPs control splicing, and various RNA processing events in general. Our lab invented several chemical crosslink-ligation based methods that enabled direct analysis of transcriptome-wide protein-independent RNA 2D and 3D structures in vivo (PARIS and SHARC). In this proposal, in Aim 1, we develop and benchmark a high throughput technology, SHARCLIP, to directly capture all RNA structures, RNA-RNA, RNA-protein, and protein-protein interactions together. SHARCLIP is conceptually similar to methods that analyze protein-mediated chromatin conformations, e.g., ChIA-PET and hiChIP, and will bring 1D RNP interaction studies to higher dimensions. In aim 2, applying PARIS and SHARC to chromatin associated RNAs, and the new method SHARCLIP to key splicing regulators, we will build transcriptome-wide RNA-structure and RNA-protein interaction models and deconvolve their dynamics in 2 ENCODE cell lines HepG2 and K562, as well as 6 brain cell lineages from a human iPS differentiation system. In aim 3, further integrating cell type specific AS programs and disease variants implicated in splicing, we will test whether mutations act by altering the structures. With the structure models as a guide, we will use a combination of CRISPR genome editing and structure-perturbing antisense oligos to test the roles of specific structures in regulating splicing in neuronal differentiation. Together, this proposal will produce a new technology that simultaneously capture multi-valent RNA-protein interactome and RNA structurome in vivo, establish a structure-based splicing code, provide an important resource to enable mechanistic studies of RNA processing, and pave the way for future therapeutic targeting of splicing.

选择性剪接（alternative splicing，AS）是产生转录组和蛋白质组多样性的主要机制 从有限的基因组。AS的时空调控有助于细胞分化和谱系 保持战略定力破坏剪接的突变会导致广泛的疾病，包括神经变性， 肌肉萎缩症和癌症。因此，理解和靶向剪接对未来的生物学研究至关重要。 精准医疗在过去的30年里，AS的研究主要集中在顺式元件及其相关的反式 RNA结合蛋白（RBP）。热力学定律决定RNA折叠成低游离态， 然而，在RNA-蛋白质复合物（RNP）的背景下，对能量结构知之甚少。 大的RNP中的前mRNA结构控制剪接和一般的各种RNA加工事件。我们实验室 发明了几种基于化学交联连接的方法，能够直接分析转录组范围内的 蛋白质非依赖性RNA 2D和3D结构（巴黎和SHARC）。在本提案的目标1中，我们 开发并测试高通量技术SHARCLIP，以直接捕获所有RNA结构， RNA-RNA、RNA-蛋白质和蛋白质-蛋白质相互作用。SHARCLIP在概念上类似于 分析蛋白质介导的染色质构象的方法，例如，ChIA-PET和hiChIP，并将带来 一维RNP相互作用研究更高的维度。在目标2中，将巴黎和SHARC应用于染色质， 相关RNA，以及针对关键剪接调节因子的新方法SHARCLIP，我们将构建全转录组 RNA-结构和RNA-蛋白质相互作用模型及其在2个ENCODE细胞系中的动力学解卷积 HepG 2和K562，以及来自人iPS分化系统的6种脑细胞谱系。在目标3中，进一步 整合细胞类型特异性AS程序和涉及剪接的疾病变体，我们将测试是否 突变通过改变结构来起作用。以结构模型为指导，我们将使用以下组合 CRISPR基因组编辑和结构干扰反义寡核苷酸，以测试特定结构在 调节神经元分化中的剪接。总之，这项提案将产生一种新技术， 在体内同时捕获多价RNA-蛋白质相互作用组和RNA结构组， 基于结构剪接编码为RNA加工的机制研究提供了重要的资源， 并为未来的剪接治疗靶向铺平道路。