Quantitative and Predictive Analysis of 5' Splice Site Recognition by U1 snRNP using Massively Parallel Arrays

使用大规模并行阵列对 U1 snRNP 5 剪接位点识别进行定量和预测分析

• 批准号: 10460136
• 负责人: David S White
• 金额: $ 6.72万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10460136
• 关键词: 5' Splice Site; Address; Affinity; Alternative Splicing; Area; Automobile Driving; Base Pairing; Binding; Biochemical; Biochemistry; Biological Assay; Biophysics; Cells; Complex; DNA Library; Defect; Disease; Elements; Eukaryota; Event; Excision; Exons; Fellowship; Foundations; Future; Gene Expression; Genes; Genomic approach; Human; In Vitro; Introns; Investigation; Kinetics; Knowledge; Label; Malignant Neoplasms; Maps; Measurement; Measures; Mediating; Mentorship; Methods; Modeling; Molecular; Mutation; Neuromuscular Diseases; Oligonucleotides; Pathogenicity; Positioning Attribute; Process; Proteins; RNA; RNA Biochemistry; RNA Splicing; RNA-Binding Proteins; RNA-Protein Interaction; Regulation; Regulatory Element; Research; Research Personnel; Site; Spectrum Analysis; Spliceosomes; Techniques; Technology; Therapeutic; Therapeutic Uses; Thermodynamics; Tissues; Training; U1 Small Nuclear Ribonucleoprotein; U1 small nuclear RNA; Variant; Work; biophysical analysis; biophysical model; career; design; experimental study; gene therapy; high throughput technology; human disease; insight; mRNA Precursor; mutant; next generation sequencing; predictive modeling; success; transcriptome

Project Summary Alternative splicing of precursor messenger RNA (pre-mRNA) greatly expands protein diversity and is a crucial determinant of cellular activity in eukaryotes. Each splicing event begins with the binding of the U1 small nuclear ribonucleoprotein (snRNP) to a 5' splice site (5'SS) at an exon-intron boundary. Mutations in both the 5'SS sequence or the U1 snRNA can cause aberrant splicing and are associated with numerous human diseases including cancer and neuromuscular disorders. Strikingly, our understanding of how a 5'SS is selected by U1 snRNP remains poorly understood. The proposed work aims to adapt and develop new in vitro techniques to perform high throughput biochemistry and uncover the physical rules of 5'SS selection by U1 snRNP. In Aim 1, the recently developed RNA on a massively parallel array (RNA-MaP) method will be applied to measure the thermodynamics and kinetics of U1 snRNP binding to thousands of unique 5'SS sequences simultaneously. These experiments will lead to the first predictive model of 5'SS sequence on U1 recognition grounded in biochemical understanding and reveal how pathogenic 5'SS mutations alter these interactions. In Aim 2, mutations in the 5'SS binding region of the U1 snRNA that are pathogenic or therapeutic will be investigated using RNA-MaP. These results will reveal the affinity landscape driving 5'SS selection by each mutant and will aid the design of splicing-corrective therapeutics. Technical Aim 1 will extend the RNA-MaP technique to generate a new biochemical assay that measures both protein-RNA interactions and splicing kinetics across thousands of sequences diverse RNAs. This technique will be used to investigate the relationships between 5'SS sequences, intron selection, and intron removal during splicing. Overall, these aims will address long- standing questions in the field and enable new measurements that will be vital for future studies of pre-mRNA splicing and its alteration in disease. In addition, these aims provide an entry point for developing expertise in RNA biochemistry and next-generation sequencing which will help the applicant successfully transition into an independent position to study the biophysical basis of gene expression.

项目摘要 前体信使RNA（前MRNA）的替代剪接大大扩展了蛋白质多样性，并且是至关重要的 真核生物中细胞活性的决定因素。每个剪接事件始于U1小核的结合 核糖核蛋白（SNRNP）到外显子内边界处的5'剪接位点（5'SS）。两个5's的突变 序列或U1 snRNA会引起异常的剪接，并与许多人类疾病有关 包括癌症和神经肌肉疾病。令人惊讶的是，我们对U1如何选择5'SS的理解 SNRNP仍然很了解。拟议的工作旨在适应和开发新的体外技术 执行高通量生物化学，并通过U1 SNRNP选择5'S选择的物理规则。在AIM 1中， 将在大规模平行阵列（RNA-MAP）方法上使用最近开发的RNA来测量 U1 SNRNP的热力学和动力学同时结合了数千个独特的5'S序列。 这些实验将导致基于U1识别的5's序列的第一个预测模型 生化理解并揭示致病性5'SS突变如何改变这些相互作用。在AIM 2中， 将研究具有致病性或治疗性的U1 snRNA的5'S结合区域的突变 使用RNA图。这些结果将揭示每个突变体选择5's选择的亲和力景观，将 帮助设计剪接验证治疗学。技术目标1将将RNA-MAP技术扩展到 生成一种新的生化测定法，该测定法既测量蛋白质-RNA相互作用又剪接动力学 数千个序列不同的RNA。该技术将用于调查 剪接过程中的5's序列，内含子选择和内含子去除。总体而言，这些目标将长期解决 在现场的常规问题，并实现新的测量，这对于将来的MRNA研究至关重要 剪接及其在疾病中的改变。此外，这些目标为发展专业知识提供了一个入口处 RNA生物化学和下一代测序将有助于申请人成功过渡到 研究基因表达的生物物理基础的独立位置。