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: 10311645
• 负责人: David S White
• 金额: $ 6.56万
• 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 2024-07-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10311645
• 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（pre-mRNA）的选择性剪接极大地扩展了蛋白质的多样性，并且是一种重要的生物学功能。 真核生物中细胞活性的决定因素。每个剪接事件开始于U1小核 在一个实施方案中，所述重组子可将核糖核蛋白（snRNP）插入外显子-内含子边界处的5'剪接位点（5' SS）。在5 'SS和 序列或U1 snRNA可引起异常剪接，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仍然知之甚少。拟议的工作旨在适应和开发新的体外技术， 进行高通量生物化学，揭示U1 snRNP选择5 'SS的物理规律。在目标1中， 最近开发的大规模平行阵列上的RNA（RNA-MaP）方法将被应用于测量 U1 snRNP同时与数千个独特的5 'SS序列结合的热力学和动力学。 这些实验将导致第一个预测模型的5 'SS序列对U1的识别接地 生物化学的理解和揭示致病性5 'SS突变如何改变这些相互作用。在目标2中， 将研究U1 snRNA的5 'SS结合区中的致病性或治疗性突变 使用RNA-MaP。这些结果将揭示每个突变体驱动5 'SS选择的亲和力景观， 有助于设计拼接矫正疗法。技术目标1将把RNA-MaP技术扩展到 产生一种新的生物化学测定，测量蛋白质-RNA相互作用和剪接动力学， 数以千计的不同RNA序列。这项技术将被用来调查之间的关系， 5 'SS序列、内含子选择和剪接过程中的内含子去除。总的来说，这些目标将解决长期- 在该领域的常设问题，使新的测量，这将是至关重要的未来研究前mRNA 剪接及其在疾病中的改变。此外，这些目标为发展以下方面的专门知识提供了一个切入点： RNA生物化学和下一代测序，这将有助于申请人成功地过渡到一个 研究基因表达的生物物理基础。