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

项目摘要 前体信使rna(pre-mrna)的选择性剪接极大地扩大了蛋白质的多样性，是一种至关重要的 真核生物中细胞活动的决定因素。每个剪接事件都始于U1小核的结合 核糖核蛋白(SnRNP)到外显子-内含子边界的5‘剪接点(5’SS)。两个5‘SS的突变 序列或U1 SnRNA可导致异常剪接，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 对SnRNP的了解仍然很少。拟议的工作旨在适应和开发新的体外技术，以 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， 最近开发的大规模并行阵列上的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。 这些实验将导致基于U1识别的第一个5‘SS序列预测模型 了解生物化学并揭示致病的5‘SS突变如何改变这些相互作用。在目标2中， 将研究U1单链RNA 5‘SS结合区的致病或治疗性突变 使用RNA-MAP。这些结果将揭示驱动每个突变体5‘SS选择的亲和力格局，并将 协助设计剪接矫正疗法。技术目标1将RNA-MAP技术扩展到 产生一种新的生化分析方法，测量蛋白质-RNA相互作用和剪接动力学 数以千计的序列不同的RNA。这项技术将被用来调查 剪接过程中的5‘SS序列、内含子选择和内含子移除。总体而言，这些目标将解决长期- 现场长期存在的问题，并使新的测量成为可能，这将对未来的Pre-mRNA研究至关重要 剪接及其在疾病中的变化。此外，这些目标还为开发以下方面的专业知识提供了切入点 RNA生物化学和下一代测序，将帮助申请者成功过渡到 独立位置研究基因表达的生物物理基础。

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.