Discovering and Validating Functional Elements in the Genome

描述(申请人提供)：前mRNA剪接是一个关键的和受调控的加工事件，内含子被精确地从新生的RNA转录本中删除。PTB是交替剪接的主要调控因子之一。然而，我们对PTB和许多其他RNA结合蛋白如何实现特异性的理解还不完全。这项研究计划的目标是开发有效的方法来识别控制基因组剪接的序列元件，并了解这些元件是如何协同工作的。虽然这种方法适用范围很广，但这项建议通过建立从先导性研究获得的初步数据，专门专注于PTB结合前-mRNA。这个项目有两个相辅相成的部分。第一个目标描述了一种对数千个RNA结合蛋白的候选配体进行实验验证的方法。因子与这些配体的结合是在一种新的大规模并行高通量结合试验中测试的。下一阶段是将我们从屏幕上了解到的PTB绑定功能合并到概率模型中。然后，我们通过夹闭和功能分析在体内测试该模型。 与公共卫生相关：绝大多数人类基因在被表达为蛋白质之前都要经过广泛的加工。大多数基因是在不同的组织间交替处理的。PTB是主要的剪接抑制因子，调节选择性剪接事件。该项目旨在确定人类细胞中PTB结合位点的序列、结构和更高阶的结构。

DESCRIPTION (provided by applicant): Pre-mRNA splicing is a critical and regulated processing event where introns are precisely excised from nascent RNA transcripts. PTB is one of the major regulators of alternate splicing. However our understanding of how PTB and many other RNA binding proteins achieve specificity is incomplete. The goal of this research proposal is to develop efficient methods to identify the sequence elements that govern splicing in the genome and to understand how these elements work together. While this method is broadly applicable, this proposal focuses exclusively on PTB binding pre-mRNA by building on preliminary data obtained from a pilot study. There are two complementary parts to this project. The first aim describes a means for the experimental validation of thousands of candidate ligands for RNA binding proteins. The binding of factors to these ligands are tested in a novel massively parallel high-throughput binding assay. The next phase is to incorporate the PTB binding features that we learn from the screen into a probabilistic model. We then test the model in vivo by CLIP and functional assays. PUBLIC HEALTH RELEVANCE: The vast majority of human genes undergo extensive processing before they are expressed as proteins. Most genes are alternatively processed across different tissues. PTB is the dominant splicing repressor that regulates alternative splicing events. This project seeks to define the sequence, structural and higher order architectures of PTB binding sites in human cells.