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，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60%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。Hertel实验室（首席研究员）已经成功地使用定量方法揭示了前mrna剪接反应的重要调控方面。Baldi实验室（合作者）在开发分析大型数据集所需的生物信息学方法方面发挥了重要作用。在本提案中，这两个小组的专业知识将结合起来，以确定替代剪接位点激活的机制，这是替代pre-mRNA剪接最常见的形式之一。生化分析将由大型EST数据库的计算分析补充，以提供对可选剪接位点激活的分子理解。本研究计划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中间序列的机制。本应用程序中提出的实验有两个主要目标：表征可选5‘剪接位点的激活（Specific Aim 1）和表征可选3’剪接位点的激活（Specific Aim 2）。对人类基因组的初步计算分析表明，在显性5‘剪接位点的上游或下游4个核苷酸的选择性5’剪接位点激活频率异常高。然而，尚不清楚这些重叠剪接位点是随机激活的还是由调控元件诱导的。在具体目标1中，我们提出生化实验和计算分析来评估这些可能性。此外，我们将验证重叠5'剪接位点的替代剪接有助于重建被额外剪接事件破坏的阅读框架的假设。在Specific Aim 2中，我们将执行一组类似的计算和生化实验，以确定为什么3‘剪接位点激活在显性剪接位点的上游或下游的3个核苷酸是人类基因组中替代3’剪接位点使用的最常见形式。

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.