Mechanisms of Enhancer Dependent Splice Site Activation

描述(申请人提供)：随着人类基因组计划的完成，基因的数量显然不能解释人类蛋白质组的复杂性。这一结论使我们对转录后基因调控机制的丰富性和重要性有了极大的了解。在已提出的几种机制中，选择性前mRNA剪接被认为是从单个基因产生多种蛋白质亚型的最有效和最广泛的途径之一。目前的估计表明，超过90%的人类基因经历了选择性剪接，从而极大地提高了我们基因组的编码潜力。这一更新应用侧重于了解剪接位点选择的机制，长期目标是生成剪接代码，允许基于序列分析进行不同的剪接预测。本申请中概述的实验建立在前一个资助期最令人兴奋的发现的基础上；绘制了替代剪接和顺式作用RNA剪接元件之间的高度显著的相关性，发现了一种描述剪接调节因子位置相关活动的机制，并发现剪接体组分沿着连接的外显子保留确保了高效的加工。该建议有三个主要目标：1)从实验和计算上研究外显子选择的复杂性。提出了一种系统的方法来系统地研究顺式作用的RNA剪接元件的组合如何影响剪接位点的选择，目的是生成允许基于序列分析的不同剪接预测的剪接代码。2)验证位置依赖的剪接激活或抑制是由于剪接调控复合体和剪接体成分之间的独特相互作用所致的假说。3)确定确保含有前mRNAs的多内含子高效处理的机制。由于剪接缺陷会导致许多人类遗传性疾病，包括一些与癌症有关的基因，因此这项研究将极大地提高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量化框架，它们将允许剪接预测对外显子突变进行分类，从而为设计抗击疾病的替代治疗方法铺平道路。 公共卫生相关性：替代的前信使核糖核酸剪接被认为是从单个基因产生多种蛋白质亚型的最有效和最广泛的途径之一。虽然许多不同的顺式作用RNA剪接元件已被证明影响选择性剪接，但目前尚不清楚它们如何相互影响中介外显子的包含或排除。在本申请中，我们建议使用系统的方法来定义一个剪接代码，该代码预测外显子被剪接体识别的概率，并确定确保多内含子前mRNAs有效处理的机制。

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.