Mechanisms of Enhancer Dependent Splice Site Activation

描述（由申请人提供）：随着人类基因组计划的完成，很明显，基因的数量不能解释人类蛋白质组的复杂性。这一结论使我们对基因调控的转录后机制的丰富性和重要性的认识急剧增加。在几种被提出的机制中，选择性前mrna剪接被认为是最有效和广泛传播的途径之一，可以从单个基因中产生多种蛋白质同种异构体。目前的估计表明，超过90%的人类基因经历了选择性剪接，从而大大增加了我们基因组的编码潜力。这个更新应用程序的重点是理解剪接位点选择的机制，其长期目标是生成一个剪接代码，允许基于序列分析的替代剪接预测。本申请中概述的实验建立在之前资助期间最令人兴奋的发现之上；绘制了选择性剪接和顺式作用RNA剪接元件之间高度显著的相关性，发现了描述剪接调节因子位置依赖性活性的机制，并发现剪接体成分沿连接外显子的保留确保了有效的加工。本研究有三个主要目标：1)通过实验和计算研究外显子选择的复杂性。提出了一种系统方法来系统地检查顺式作用RNA剪接元件的组合如何影响剪接位点的选择，目的是生成一个剪接代码，允许基于序列分析的替代剪接预测。2)验证位置依赖剪接激活或抑制是由于剪接调节复合体和剪接体组分之间独特的相互作用。3)确定确保高效处理含有多内含子的pre- mrna的机制。由于剪接缺陷导致许多人类遗传疾病，包括许多与癌症有关的基因，因此拟议的研究将极大地增强我们对调控基因表达和人类疾病的理解。特别是，这些研究的结果将为外显子识别提供一个定量框架，它们将允许剪接预测对外显子突变进行分类，从而为设计对抗疾病的替代治疗方法铺平道路。

DESCRIPTION (provided by applicant): With the completion of the human genome project, it has become clear that the number of genes cannot account for the complexity of the human proteome. This conclusion has lead to a dramatic increase in our appreciation of the abundance and importance of post-transcriptional mechanisms of gene regulation. Among several proposed mechanisms, alternative pre-mRNA splicing is considered to be one of the most efficient and wide spread avenues to generate multiple protein isoforms from individual genes. Current estimates indicate that over 90% of human genes undergo alternative splicing, thus greatly increasing the coding potential of our genome. This renewal application focuses on understanding the mechanisms of splice site selection with the long-term goal to generate a splicing code that permits alternative splicing predictions based on sequence analysis. The experiments outlined in this application build on the most exciting discoveries made during the previous funding period; charting highly significant correlations between alternative splicing and cis-acting RNA splicing elements, the discovery of a mechanism that describes position-dependent activities of splicing regulators, and the discovery that the retention of spliceosomal components along ligated exons ensures efficient processing. The proposal has three major goals: 1) To investigate experimentally and computationally the complexity of exon selection. A systems approach is proposed to examine methodically how the combination of cis-acting RNA splicing elements influences splice site selection with the objective to generate a splicing code that permits alternative splicing predictions based on sequence analysis. 2) To test the hypothesis that position dependent splicing activation or repression is due to unique interactions between splicing regulatory complexes and spliceosomal components. 3) To determine the mechanisms that ensure highly efficiency processing of multi-intron containing pre-mRNAs. Because defects in splicing lead to many human genetic diseases, including a number of genes that have been implicated in cancer, the proposed research will greatly enhance our understanding of regulated gene expression and human disease. In particular, the results from these studies will provide a quantitative framework for exon recognition and they will permit splicing predictions to classify exonic mutations, thus paving the way for the design of alternative therapeutic approaches to combat disease. PUBLIC HEALTH RELEVANCE: Alternative pre-mRNA splicing is considered one of the most efficient and widespread avenues to generate multiple protein isoforms from individual genes. While many different cis-acting RNA splicing elements have been shown to influence alternative splicing, it is currently unknown how they influence each other mediate exon inclusion or exclusion. In this application we propose to use a systems approach to define a splicing code that predicts the probability of exons to be recognized by the spliceosome and to determine the mechanisms that ensure efficient processing of multi-intron pre-mRNAs.