Discovering and Validating Functional Elements in the Genome

描述（由申请人提供）：Pre-mRNA剪接是一个关键和受调节的加工事件，内含子被精确地从新生RNA转录物中切除。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是不完整的。本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素，并了解这些元素如何协同工作。虽然该方法广泛适用，但本建议仅关注PTB结合pre-mRNA的初步数据，并建立在从试点研究获得的初步数据基础上。这个项目有两个互补的部分。第一个目标描述了一种对RNA结合蛋白的数千种候选配体进行实验验证的方法。因子与这些配体的结合在一种新的大规模平行高通量结合试验中进行了测试。下一阶段是将我们从屏幕中学到的PTB绑定特征合并到概率模型中。然后，我们通过CLIP和功能测定对模型进行体内测试。

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.