Discovering and Validating Functional Elements in the Genome

描述（由申请人提供）：前体mRNA剪接是一个关键的和受调控的加工事件，其中内含子从新生RNA转录物中精确切除。PTB是选择性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是不完整的。这项研究计划的目标是开发有效的方法来识别基因组中控制剪接的序列元件，并了解这些元件如何协同工作。虽然这种方法是广泛适用的，但该建议通过建立从试点研究获得的初步数据，仅关注PTB结合前mRNA。这个项目有两个互补的部分。第一个目标描述了一种用于实验验证数千种RNA结合蛋白的候选配体的方法。在一种新的大规模并行高通量结合试验中测试因子与这些配体的结合。下一阶段是将我们从屏幕上学习到的PTB结合特征纳入概率模型。然后我们通过CLIP和功能测定在体内测试该模型。 公共卫生相关性：绝大多数人类基因在表达为蛋白质之前经历了广泛的加工。大多数基因在不同的组织中交替加工。 PTB是调节可变剪接事件的显性剪接阻遏物。 该项目旨在确定人类细胞中PTB结合位点的序列，结构和更高层次的结构。

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.