Genomic Analysis of Alternative Splice-Site Selection

描述（由申请人提供）：随着人类基因组计划的完成，基因的数量不能解释人类蛋白质组的复杂性已经变得很清楚。这一结论导致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。在几种提出的机制中，选择性前体mRNA剪接被认为是从单个基因产生多种蛋白质异构体的最有效和最广泛的途径之一。目前的估计表明，超过60%的人类基因经历选择性剪接，从而大大增加了我们基因组的编码潜力。Hertel实验室（主要研究者）已经成功地使用定量方法来揭示前mRNA剪接反应的重要调控方面。Baldi实验室（合作者）在开发大型数据集分析所需的生物信息学方法方面发挥了重要作用。在这项提议中，两个小组的专业知识将结合起来，以确定选择性剪接位点激活的机制，这是选择性前mRNA剪接的最常见形式之一。生化分析将补充大型EST数据库的计算分析，以提供一个替代剪接位点激活的分子理解。这项研究提案的长期目标是了解剪接机制正确识别外显子并忠实地去除插入序列的机制。本申请中提出的实验有两个主要目标：表征可变5'剪接位点的活化（具体目标1）和表征可变3'剪接位点的活化（具体目标2）。对人类基因组的初步计算分析表明，在显性5'剪接位点上游或下游4个核苷酸处，5'剪接位点的交替激活频率异常高。然而，目前尚不清楚这些重叠剪接位点是否是随机激活的，或者它们是否是由调控元件诱导的。在具体目标1中，我们提出了生物化学实验和计算分析来评估这些可能性。此外，我们将检验重叠5'剪接位点的选择性剪接用于重建被额外剪接事件破坏的框架的阅读的假设。在具体目标2中，我们将进行一组类似的计算和生物化学实验，以确定为什么3'剪接位点激活3个核苷酸的上游或下游的主要剪接位点是最常见的形式的替代3'剪接位点的使用在人类基因组中。

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 60% of all human genes undergo alternative splicing, thus greatly increasing the coding potential of our genome. The Hertel laboratory (Principal Investigator) has successfully used quantitative approaches to uncover important regulatory aspect of the pre-mRNA splicing reaction. The Baldi laboratory (Collaborator) has been instrumental in the development of bioinformatics approaches required for the analysis of large datasets. In this proposal, the expertise of both groups will be combined to determine the mechanisms of alternative splice-site activation, one of the most frequent forms of alternative pre-mRNA splicing. Biochemical assays will be complemented by computational analyses of large EST databases to provide a molecular understanding for alternative splice-site activation. The long- term goal of this research proposal is to understand the mechanisms by which the splicing machinery correctly identifies exons and faithfully removes intervening sequences. The experiments proposed in this application have two major goals: To characterize the activation of alternative 5' splice sites (Specific Aim 1) and to characterize the activation of alternative 3' splice sites (Specific Aim 2). Preliminary computational analysis of the human genome demonstrated an unusually high frequency of alternative 5' splice-site activation 4 nucleotides upstream or downstream of the dominant 5' splice site. However, it is not clear whether these overlapping splice sites are activated stochastically or whether they are induced by regulatory elements. In Specific Aim 1 we propose biochemical experiments and computational analyses to evaluate these possibilities. Furthermore, we will test the hypothesis that alternative splicing of overlapping 5' splice sites serves to reestablish reading the frame disrupted by additional splicing events. In Specific Aim 2, we will perform a similar set of computational and biochemical experiments to determine why 3' splice-site activation 3 nucleotides upstream or downstream of the dominant splice site is the most frequent form of alternative 3' splice-site usage within the human genome.