Functional RNA elements in the human genome

描述（由申请人提供）：前体mRNA剪接对于高等真核生物基因组中的适当基因表达至关重要，因为绝大多数基因含有必须准确识别和去除的内含子。近年来的研究表明，脊椎动物中90%以上的基因发生了选择性剪接，这被认为是导致不同细胞类型和组织中蛋白质组复杂性的重要因素，大量证据表明剪接改变导致了多种人类疾病。尽管基于对模型小基因的生化解剖对剪接机制有广泛的了解，但我们对有多少基因参与选择性剪接的调控以及功能性RNA元件嵌入人类基因组的位置知之甚少。基于我们在当前获奖期间的富有成效的研究，我们现在提出了一个大胆的计划，以系统地攻击我们对选择性剪接调控的知识的关键差距。我们将通过利用最新和创新的基因组学技术进行三大研究。(1)我们将使用我们实验室最近开发的一个新的自动化平台来分析人类基因组中每个注释基因的数百个保守的选择性剪接事件。这种无偏见的方法将产生前所未有的信息，发现新的剪接调节和推导调节剪接的途径。(2)我们将专注于参与个别调控途径的RNA结合蛋白，通过绘制它们与表达的RNA的物理相互作用来阐明调控剪接的分子基础。为此目的，我们将构建基于FLP-In 293细胞的一大组细胞系，以在C末端表达单个RNA结合蛋白作为V5标记的蛋白，这将允许在类似和优化的一组条件下通过CLIP-seq（交联免疫沉淀，随后进行高通量测序）对RNA-蛋白相互作用进行大规模作图。(3)我们的第三个目标是使用所提出的映射和功能研究产生的信息，通过使用机器学习和图形模型来开发一个整合的框架，用于从头预测剪接调控。这项研究有可能从根本上改变我们对剪接控制及其对人类疾病的贡献的看法。

DESCRIPTION (provided by applicant): Pre-mRNA splicing is essential for proper gene expression in higher eukaryotic genomes, as the vast majority of genes contain introns that have to be accurately recognized and removed. Recent studies have revealed that >90% of the genes undergo alternative splicing, which is believed to contribute to the complexity of the proteome in different cell types and tissues in vertebrates and abundant evidence suggests that altered splicing causes a variety of human diseases. Despite extensive knowledge on the splicing mechanism based on biochemical dissection of model minigenes, we know little about how many genes are involved in the regulation of alternative splicing and where the functional RNA elements are embedded in the human genome. Built on our productive research in the current award period, we now propose a bold plan to systematically attack the critical gap of our knowledge about the regulation of alternative splicing. We will pursue three major lines of research by utilizing the latest and innovative genomics technologies. (1) We will use a new, automated platform recently developed in our lab to profile hundreds of conserved alternative splicing events against every annotated genes in the human genome. This unbiased approach will generate unprecedented information to uncover novel splicing regulators and deduce pathways in regulated splicing. (2) We will focus on RNA binding proteins involved in individual regulatory pathways to elucidate the molecular basis for regulated splicing by mapping their physical interactions with expressed RNA. For this purpose, we will construct a large panel of cell lines based on FLP-In 293 cells to express individual RNA binding proteins as a V5-tagged protein at the C- terminus, which will permit large-scale mapping of RNA-protein interactions by CLIP-seq (CrossLinking ImmunoPrecipitation followed by high throughput sequencing) under a similar and optimized set of conditions. (3) Our third goal is to use the information generated from the proposed mapping and functional studies to develop an integrated framework for de novo prediction of splicing regulation by using machine-learning and graphical models. This research has the potential to fundamentally change our view on splicing control and its contribution to human disease.